Glyco25, XXV International Symposium on Glycoconjugates

SCIENTIFIC COMMITTEE

• Richard D. Cummings

National Center for Functional Glycomics, Harvard Medical School, Boston, USA

• Carol de la Motte

Department of Pathobiology, Cleveland Clinic

• Domenico Garozzo

CNR IPCB, 95126 Catania, Italy

• Jin-ichi Inokuchi

Division of Glycopathology, Tohoku Medical and Pharmaceutical University, Japan

• Makoto Ito

Bio-Architechture Center, Kyushu University, Fukuoka, Japan

• Daniel Kolarich

Griffith University, Gold Coast campus

• Fu-Tong Liu

Academia Sinica, Taipei, Taiwan

• Antonio Molinaro

Department of Chemical Sciences, University of Napoli Federico II, Napoli, Italy

• Annamaria Naggi

Ronzoni Institute, Milano, Italy

• Francesco Nicotra

University of Milano-Bicoccca, Milano, Italy

• Chihiro Sato

Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya JAPAN

• Ronald L. Schnaar

Departments of Pharmacology and Neuroscienze, Johns Hopkins University MD, USA

• Naoyuki Taniguchi

Osaka University, Japan

• David Vocadlo

Departments of Chemistry and Molecular Biology and Biochemistry, Simon Fraser University. Burnaby, BC, Canada

ABSTRACTS

Title: Glyco Abstract Speakers

1. Understanding microbial glycosylation at the host-pathogen interface: how the glycoproteomes of pathogens shape infections

N. Scott¹; ¹The Department of Microbiology and Immunology, The University of Melbourne, Victoria, Australia

Protein glycosylation is increasingly recognised as an essential requirement for effective microbial infections. Within microbial pathogen’s protein glycosylation is used for both defensive and offensive purposes; enabling pathogens to fortify themselves against the host immune response or to disarm the host’s ability to resist infection. Although microbial protein glycosylation systems have been recognised for decades only recently has the true extend of protein glycosylation within microbes begun to be appreciated by the wider community. A key enabler for this conceptual shift has been the development and application of modern approaches for the characterisation of glycosylation. Over the last decade my research has focused on the development and application of proteomic tools to probe microbial glycosylation. By developing workflows which use glycopeptide enrichment and MS identification I have demostrated that microbial glycosylation events can easily be identified withour prior knowledge of the glycan used for protein glycosylation. These tools now make it possible to characterise the glycoproteomes of microbial species in a high-throughput manner and to track glycosylation at a site specific resolution. By coupling these approaches to quantitative proteomic techniques these technologies give researchers the means to measure changes in glycosylation providing new insights into microbial glycosylation systems. By developing tools to explore microbial glycosylation my research program seeks to understand the function of microbial glycosylation and substrates of these systems. To date my research has led to the characterisation of the N-linked glycosylation systems across the Campylobacter genus and identification of protein O-linked glycosylation in A. baumannii, R. solanacearum and B. cenocepacia. These studies have established that bacterial glycosylation is widespread, that glycan microheterogeneity is common place and that an extensive array of glycans are used to decorate protein compared to Eukaryotic glycosylation systems.

Excitingly the approaches developed to characterise bacterial glycosylation systems are equally amenable to studying newly discovered forms of microbial glycosylation such as Arginine glycosylation as well as glycosylation within the parasitic eukaryotic organisms T. gondii and P. falciparum. Using MS based approaches to probe these systems has provided critical insight into the host targets of Arginine glycosylating enzymes and the substrate specificity of O-fucosylation systems within eukaryotic parasites. Using MS based approaches glycosylation events can now be characterised and quantitively tracked across proteomes revealing new insights into microbial glycosylation systems. Looking forward the long-term goal of this work is to leverage the insights gained into microbial glycosylation to produce novel glycoconjugates that can bolster human immunity or control microbial pathogens.

2. Lectin-driven and glycosphingolipid-dependent construction of endocytic pits for polarized protein distribution by trafficking via the retrograde route

L. Johannes¹; ¹Institut Curie

Several endocytic processes do not require the activity of clathrin, and it has been a major question in membrane biology to know how the plasma membrane is bent and cargo proteins are sorted in these cases. Our previous studies have allowed us to propose the GL-Lect hypothesis: Nanodomain construction by GlycoLipid-binding cellular or pathological Lectins induces membrane curvature changes and drives the formation of tubular endocytic pits from which clathrin-independent carriers are generated for the cellular uptake of glycosylated membrane proteins (CD44, integrins…), pathogens (polyoma viruses, norovirus), or pathogenic factors (Shiga and cholera toxins). We are now analyzing how cortical actin dynamics contributes to the clustering of glycosphingolipid-lectin complexes on active membranes, thereby facilitating the nucleation of endocytic tubules exploiting membrane fluctuation force and asymmetric lipid compaction mechanisms that had not been linked before to endocytosis. Furthermore, we are identifying ways by which the GL-Lect mechanism is acutely controlled by growth factor signaling. Finally, we study how GL-Lect domain construction at the plasma membrane programs the intracellular distribution of cargo proteins via the retrograde transport route, thereby exploiting the polarized secretion capacity of the Golgi apparatus for the distribution of these cargoes to specialized plasma membrane domains in migrating cells (leading edge), epithelial cells (apico-basal sorting and transcytosis), and lymphocytes (immunological synapse). These studies are performed using a combination of cell biological (lattice light sheet microscopy), biochemical (membrane protein purification and reconstitution), and structural biology (cryo-EM) techniques on model membranes, in cells, and living organisms.

3. Sialic acid metabolism by gut bacteria

N. Juge¹; ¹Quadram Bioscience Institute

The gastrointestinal (GI) tract is inhabited by a diverse microbial community (gut microbiota) that has a major impact on human health. The mucus layer covering the GI tract plays a key role in maintaining a homeostatic relationship with our gut microbiota and protecting us against pathogen infection. Mucins are glycosylated with a diverse and complex array of O-glycan structures containing N-acetylgalactosamine (GalNAc), galactose (Gal) and N-acetylglucosamine (GlcNAc), and usually terminated by fucose and sialic acid [N-acetylneuraminic acid (Neu5Ac)] residues. There is an emerging paradigm that mucin glycans are key molecular determinants of gut homeostasis by providing binding sites and/or nutrients to the bacteria which have adapted to the mucus niche. However, the molecular mechanisms underpinning mucin glycan interactions in the gut remain poorly defined.

Ruminococcus gnavus is a prominent gut symbiont with a disproportional representation in many diseases. We have undertaken structural and functional investigations of how R. gnavus strains adapt to the mucosal environment focusing on glycoside hydrolases involved in mucin glycan degradation, highlighting the importance of strain-specific intramolecular trans-sialidases. Neu5Ac is a much-coveted target for both gut pathogenic and commensal bacteria. Using a combination of in silico and molecular and biochemical assays, we unravelled a novel sialic acid pathway which provides R. gnavus with a competitive nutritional advantage over other gut microbes including pathogens such as Salmonella or Clostridum difficile. We confirmed the role of this metabolic pathway in colonisation in vivo through competition experiments using germ-free mice colonised with R. gnavus wild-type and deletion mutant strains.

This work has opened up potential avenues for alternative strategies to reduce bacterial infection or inflammatory and metabolic diseases in humans.

4. Modifying the cell glycocalyx with synthetic glycolipids

N. Bovin², S. Henry¹; ¹Centre for Kode Technology Innovation, School of Engineering, Computer and Mathematical Sciences, Faculty of Design and Creative Technologies, Auckland University of Technology, Auckland, New Zealand, ²Institute of Bioorganic Chemistry, Russian Academy of sciences, Moscow

The main challenge of cell surface (glyco)engineering remains to rationally modify the cell without harming the cell or compromising its function. There are essentially three major approaches: genetic modification, chemical conjugation and passive insertion of glycolipids. Genetic modification is generally complex, while most chemical approaches damage existing functional components of the cell. In contrast, passive modification with glycolipids is simple and harmless. Our technology uses synthetic glycolipids (also known as function-spacer-lipid, FSL constructs) to attach bioactive material to cells. The glycan moiety of the FSL construct can also be peptide, or a plethora of different bioactive compounds and tags. The spacer (S) is able to confer the construct some important new features; primarily, optimize the antigen presentation of the glycan head away from the cell membrane. Many different variations of spacers were designed, including short, long, branched, clustered and fluorescently labeled, each of which can be additionally optimized. It should be emphasized, that the spacer is constructed as a rigid hinged moiety, which allows positioning of a glycan up to 20 nm from the membrane bilayer. Thus, a selected spacer can place a cell-modifying glycan within the glycocalyx or on its periphery. The time required for insertion of FSL is about 30 min and although labeling is temporary significant material is still observed at 24 h; depending on the cell type and the nature of the environment. The recognition of embedded glycans by antibodies and galectins depends on their location within the glycocalyx.

5. Membrane lipids and the activity of sphingolipid-cleaving hydrolases

K. Sandhoff¹; ¹LIMES c/o Kekulé.-Institute, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany

Sphingolipids (SLs) are amphiphilic components of cellular plasma membranes. Their catabolism is catalyzed by soluble hydrolases, mainly in the acidic lysosomal compartment. The catabolic rate of SL catabolism is strongly regulated by factors in the microenvironment of the enzymatic reaction at the surface of the SL-carrying membranes.

Digestion of SLs and other membrane lipids occurs mainly at intralysosomal luminal vesicles (ILVs). SAPs (SL activator proteins, which are lipid-binding and transfer proteins) are essential cofactors for physiological SL degradation. They present membrane bound SLs to water-soluble hydrolases for catabolism. Inherited defects of hydrolases or SAPs can cause fatal lysosomal lipid storage diseases.

Besides well known factors like pH value, ionic strength, SAPs etc., also lipids of the SL-carrying membranes and other low molecular weight factors like chondroitin sulfate strongly regulate and modify SL catabolism.

At the level of late endosomes, a maturation process removes lipids inhibiting lysosomal catabolism from the ILVs. Sphingomyelin is hydrolysed by acid sphingomyelinase, facilitating cholesterol export to the cytosol by NPC2 and NPC1 proteins. Their defects lead to an endolysosomal accumulation of cholesterol and a secondary increase of glycosphingolipids in Niemann Pick disease type C. Sphingomyelin and cholesterol poor ILVs serve as platforms for SL degradation in the lysosomes. Negatively charged BMP, which stimulates SL degradation, increases during endocytosis. Anionic lipids (phosphatidic acid, phosphatidylglycerol, BMP) generate a negative surface potential of ILVs, which attracts electrostatically protonated and positively charged hydrolases and SAPs to the site of SL catabolism. They and ceramide stimulate NPC-2 mediated cholesterol transfer whereas SM inhibits. Several anionic membrane lipids also activate sphingomyelin degradation by acid sphingomyelinase facilitating cholesterol export by NPC-2.

Anionic lipids also strongly enhance ganglioside GM1, GM2, sphingomyelin and glucosylceramide catabolism, whereas stabilizing lipids of the neuronal plasma membrane are often inhibitory. Sphingomyelin, the primary storage compound in Niemann-Pick disease A and B, inhibits cholesterol transfer by NPC2, catabolism of ganglioside GM2 and glucosylceramide strongly, causing a secondary accumulation of cholesterol and glycosphingolipids. Cholesterol and chondroitin sulphate also inhibit ganglioside GM2 degradation, triggering a secondary accumulation of GM2 and other glycolipids in NPC disease and some mucopolysaccharidoses, whereas ceramide stimulates cholesterol transfer by NPC2 and glucosylceramide hydrolysis. It is suggested that lipid and other low molecular modifiers affect the genotype-phenotype relationship observed in patients with lysosomal diseases.

6. Filling the gaps to solve the extensin plant glycoprotein puzzle

E. Marzol¹, D.R. Rodriguez Garcia¹,J. Estevez¹; ¹Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET). Av. Patricias Argentinas 435, Buenos Aires CP C1405BWE, Argentina

Extensins (EXTs) are highly repetitive plant O-glycoproteins that require several post-translational modifications (PTMs) to become functional in plant cell walls. First, they are hydroxylated on contiguous proline residues; then, they are O-glycosylated on hydroxyproline (Hyp) and serine. After secretion into the apoplast, O-glycosylated EXTs form a tridimensional network organized by inter- and intra-Tyr linkages. Recent studies have made significant progress in identifying the enzymatic machinery required to process functional EXTs, which includes prolyl 4-hydroxylases (P4Hs), glycosyltransferases (GTs), KDEL-cysteine endopeptidases (CEPs), and peroxidases (PERs). EXTs are abundant components of plant tissues, and are particularly important in rapidly expanding root hairs and pollen tubes, which grow in a polar manner. Small changes in EXT PTMs affect fast-growing cells, although the molecular mechanisms underlying this regulation are unknown. In this work, I will be highlighting recent advances in our understanding of EXT modifications throughout the secretory pathway, EXT assembly in the cell walls, and possible sensing mechanisms triggered by the Catharanthus roseus cell surface sensor receptor-like kinases (CrRLK1Ls) located at the interface between the apoplast and the cytoplasmic face of the plasma membrane.

7. Gold nanoparticles to interrogate immunological properties of carbohydrates in the context of conjugate-vaccines

F. Chiodo¹; ¹Amsterdam Infection and Immunity Institute, Amsterdam University Medical Center, Amsterdam, The Netherlands

Gold nanoparticles offer the possibility to combine different components in a well-controlled multivalent manner on a well-defined surface. During this lecture, I will present the potentiality of carbohydrate-coated gold nanoparticles as synthetic multivalent tool to interrogate and investigate immunological properties of carbohydrates, especially in the context of conjugate-vaccines.

Gold nanoparticles have been explored as a tool to study the molecular interactions between S. pneumoniae polysaccharides and the human immune system. We were able to reveal currently unknown mechanisms that directly link the nature of the carbohydrates to the overall immunological effects of conjugate-vaccines. In collaboration with Finlay Institute in Havana, we have made two key observations:

Firstly, we have demonstrated that bacterial polysaccharide nanoparticles can be used to elicit a functional immune response demonstrating that the spatial orientation of the polysaccharides on the gold nanoparticles is a key factor in triggering antibody production in vivo.

Secondly, we have recently demonstrated using in vitro and in vivo experiments, that several S. pneumoniae polysaccharides are actively involved in triggering innate immune responses through interactions with host antigen-presenting cells.

Our recent results are unveiling new immunological properties of bacterial polysaccharides, exploring gold nanoparticles as chemical tool to interrogate nature, helping in the design of better vaccination strategies.

8. Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in metabolic disorders

J. Inokuchi¹; ¹Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan

Innate immune signaling via toll-like receptor 4 (TLR4) plays crucial roles in both host defense and chronic inflammation in metabolic disorders. However, critical mechanisms underlying the homeostatic and pathogenic roles of TLR4, regulated by versatile exogenous/ endogenous ligands, are not completely understood. We demonstrates that GM3 ganglioside molecular species differing in acyl-chain structures and modifications act as pro- and anti-inflammatory endogenous TLR4 modulators. Very long-chain and α-hydroxy GM3 species amplified TLR4 activation, whereas long-chain and unsaturated GM3 species counteracted this effect and maintained homeostasis. GM3 species were recognized via myeloid differentiation protein-2 (MD2) and basic residues on TLR4, thereby inducing super-oligomer formation of TLR4/MD-2 complex. GM3-targeted lipidomic analyses of serum and adipose tissue revealed that imbalances between pro- and anti-proinflammatory GM3 species promote progression of metabolic disorders from presymptomatic phase via chronic inflammation. Our findings, taken together, indicate that GM3 species control the homeostatic and pathogenic roles of TLR4, and may provide useful diagnostic and therapeutic targets for treatment of chronic inflammation.

9. Cellular glycosylation: biosynthesis and functional implications in cancer

C.A. Reis¹; ¹i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto; Ipatimup – Institute of Molecular Pathology and Immunology of the University of Porto, Portugal

Alterations of glycosylation are common on the cell surface during the carcinogenesis process and are associated with cancer progression and poor prognosis of the patients. This presentation will provide a general overview on glycosylation in human cells, the biosynthesis of glycans and their functions in normal conditions and in cancer.

Cancer is a heterogeneous disease that requires multidisciplinary treatment. Current targeted therapy depend on patient stratification based on molecular features of the tumor. This presentation will reports the latest glycomic and glycoproteomic analyses in human gastrointestinal cancer cells providing novel relevant information with clinical implications. These results show that alterations of glycosylation impact the activation of tyrosine kinase receptors, such as MET (HGFR) and RON (MSPR), in gastric cancer cells, leading to the activation of downstream intracellular signaling pathways and the induction of cancer cell aggressive phenotypes. We also disclosed novel glycosylation features of epidermal growth factor receptor 2 (ErbB2) in gastric cancer. The analysis of the cellular- and receptor-specific glycan profiling of ErbB2-expressing gastric cancer cells unveiled a heterogeneous glycosylation pattern harboring the tumor-associated sialyl Lewis A (SLeA) antigen. Finally, it will be presented the finding that the Thomsen-Friedenreich (TF) antigen, the simple O-glycan is highly associated with Microsatellite instability, a molecular feature of a distinct molecular subtype of gastric cancer. These results disclose novel functional aspects of glycosylation modifications occurring in key proteins in gastric cancer and highlights their potential as cancer biomarkers for patient stratification, personalize medicine and improved therapeutic intervention.

References:

[1] Pinho SS, Reis CA. Nature Rev. Cancer 2015, 15, 540-555.

[2] Mereiter S, et al. Biochim. Biophys. Acta 2016, 1860, 1795-1808.

[3] Duarte, HO. et al. Int J Mol Sci. 2017, 18(11).

[4] Mereiter S, et al. J Clin Med. 2018, 7(9).

[5] Rodrigues JG, et al. Cell Immunol. 2018; pii: S0008-8749(18)30121-7.

[6] Freitas D, et al. EBioMedicine. 2019 Feb;40:349-362.

10. Protein n-glycosylation is sex-related in nilaparvata lugens

F. Scheys³, K. Gevaert¹, G. Smagghe³, E.J. Van Damme²; ¹Department of Biomolecular Medicine, Faculty of Medicine, Ghent University, Ghent, Belgium, ²Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium, ³Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

Although the process of N-glycosylation is highly conserved differences between species and between life stages of an organism have been reported. Several studies in literature support that protein glycosylation is essential for insect survival, growth and development. This paper focuses on recent glycomics studies for Nilaparvata lugens, the brown planthopper, being one of the most notorious pest insects of cultured rice. Important differences in the glycan repertoire have been detected between male and female insects.

The N-glycome of N. lugens was explored throughout post-embryonic development and reproductive stages, and revealed differential protein N-glycosylation events between adult sexes. A comprehensive investigation of the N-glycosylation sites for the adult stages enabled a qualitative and quantitative comparison between sexes at the glycopeptide level. Male and female specific N-glycosylation sites have been identified, and several sex-specific N-glycosites were attributed to proteins with a putative role in insect reproduction. Transcript analyses confirmed the sex-related N-glycosylation of proteins, and expression of glycoproteins in reproductive tissues.

Studies on the differential N-glycosylation process between male and female insects generate a better understanding of their biology and physiology, and can contribute to the elaboration of innovative pest management strategies.

Scheys, F., De Schutter, K., Shen, Y., De Pauw, E., Yu, N., Smargiasso, N., Van Damme, E.J.M., Smagghe, G., 2019. The N-glycome of the hemipteran pest insect Nilaparvata lugens reveals unexpected sex differences. Insect Biochemistry and Molecular Biology, 107, 39-45.

Scheys, F., Van Damme, E.J.M., Smagghe, G., 2019. Let’s talk about sexes: Sex-related N-glycosylation in ecologically important invertebrates. Glycoconjugate Journal, in press.

11. Physiological glycation by glucose and reactive dicarbonyl metabolite, methylglyoxal – targets, hotspots and engagement with the unfolded protein response

P. Thornalley¹; ¹Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Doha

Glycation of proteins occurs in physiological systems to low extent; typically 1 – 10 mol percent. Glycation by glucose forms N-terminal and lysyl side chain N-1-deoxyfructosyl residues or fructosamines. This is exploited clinically for assessment of glycemic control by measurement of glycated hemoglobin A1C. Fructosamine residues are repaired enzymatically by fructosamine 3-kinase (FN3K). Knockout of FN3K was without pathophysiological effect, suggesting glycation by glucose has limited functional impact in vivo. A further major type of protein glycation is by methylglyoxal (MG) – a reactive dicarbonyl metabolite formed mainly by trace level degradation of triosephosphate glycolytic intermediates. MG modifies proteins mainly on arginine residues forming hydroimidazolone MG-H1, inducing protein misfolding and inactivation. MG is mainly metabolized by glyoxalase 1 (Glo1) of the glyoxalase system in the cytosol of all cells. Homozygous mutation of Glo1 is considered embryonically lethal in human subjects; and also in mice unless compensatory increase in MG-metabolizing aldoketo reductases and dehydrogenases is induced. Cytosolic protein domain hotspots of MG modification are: chaperonin T-complex polypeptide 1 and GroEL chaperonins, phosphoserine and phosphothreonine binding sites of 14-3-3 proteins, proteasome alpha/beta subunits and conserved sites of aminoacyl-tRNA synthases. Reactome pathways enriched with proteins susceptible to MG modification were: protein folding, protein synthesis, glycolysis and gluconeogenesis. Silencing of Glo1 induced increased MG, MG protein modification, heat shock protein expression and activation of inflammatory signaling by the unfolded protein response. Increased MG concentration, dicarbonyl stress, occurs in obesity, diabetes and cancer chemotherapy linked to insulin resistance, diabetic vascular complications and anticancer therapeutic response, respectively.

12. Tumor-associated glycan structures: friend or foe in immunity to cancer?

L.A. Cornelissen¹, A. Blanas¹, J.C. Van Der Horst¹, L. Kruijssen¹, A. Zaal¹, Y. Van Kooyk¹, S.J. Van Vliet¹; ¹Amsterdam UMC, Vrije Universiteit Amsterdam, department of Molecular Cell Biology and Immunology, Cancer Center Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam, Netherlands

Malignant transformation is often accompanied by dramatic changes in the cellular glycome, resulting in de novo expression of the Tn antigen or elevated levels of sialylated epitopes. Sialoglycans can interact with specific lectins on immune cells , such as the sialic acid recognizing Ig-like lectins (Siglecs). Most Siglecs contain tyrosine-based inhibitory motifs in their cytoplasmic tail, which, upon recognition of endogenous levels of sialic acids, dampen unwanted immune activation to healthy cells. Therefore, the current dogma states that tumors exhibit hypersialylation as a form of enhanced-self to initiate immune evasion. Indeed, prior work from our group demonstrated that a partial loss of sialic acids in melanoma reduced tumor growth through augmented tumor destruction by NK and CD8⁺ T cells and inhibition of regulatory T cell differentiation. To study whether the same principle would uphold in other tumor types, we generated a panel of glyco-engineered colorectal cancer (CRC) cell lines to decipher the in vivo role of sialylated glycans during CRC development. Using CRISPR/Cas9 technology we created Sia^null variants of the murine CRC cell line MC38 by knocking out the N-acylneuraminate cytidylyltransferase (CMAS) gene, encoding the enzyme responsible for CMP-sialic acid synthesis. Surprisingly and in contrast to the melanoma tumors, MC38-Sia^null tumors displayed enhanced growth rates in vivo, which we could attribute to a lack of viable cytotoxic CD8⁺ T cells in the tumor microenvironment. As systemic CD8⁺ T cell numbers were normal, this hinted to a local and tumor site-specific phenomenon. Indeed, we could identify a soluble factor secreted by MC38-Sia^null cells, that was able to induce CD8⁺ T cell apoptosis in an antigen-independent manner. Our in vivo findings were furthermore recapitulated in human CRC, where low levels of the CMAS gene are associated with a reduced recurrence-free survival. In summary, we are the first to show the detrimental consequences of complete tumor desialylation, indicating that the influence of sialoglycans on cancer immunity is more complex than originally thought. Moreover, our results have strong implications for ongoing efforts aimed at abrogating tumor sialyation to promote anti-tumor immunity.

13. Acute stress-induced change of polysialic acid expression mediated by sialidase in mouse brain

C. Sato¹; ¹Bioscience and Biotechnology Center, Nagoya University

Stress is one of the environmental factors that influence human behavior and that cause several mental disorders. It has been shown that impairments of polysialic acid (polySia/PSA) structure caused by genetic alterations in ST8SIA2 encoding a polySia-synthesizing enzyme, are related to certain mental disorders (1). However, it remains unknown if those environmental factors, like stress, which cause mental disorders, impair the polySia structure. In this study, we analyzed the effects of acute stress on polySia expression and found that both the quantity and quality of polySia were declined in the olfactory bulb (OB) and prefrontal cortex (PFC) even on a short-term exposure to acute stress. Using sialidase inhibitors, we found that these declinations were due to a transient action of sialidase from microglia and astrocytes in OB and PFC, respectively. All these data suggest that the sialidase dynamically regulates polySia expression in a brain region-specific manner.

(1) Sato C and Hane M (2018) Glycoconjugate J. 35, 353-373

14. Synthesis and biofunctional studies of immunomodulating glycoconjugates

K. Fukase¹; ¹Department of Chemistry, Graduate School of Science, Osaka University

We have studied the synthesis of various microbial glycoconjugates such as peptidoglycans and lipopolysaccharides (LPS) to investigate their immunological functions. Here, we report on the application of their immunomodulatory effects to the developments of immune adjuvants and cancer vaccines. We found that LPS from Alcaligenes faecalis, a symbiotic bacterium inhabiting in dendric cells of Peyer’s Patches, shows low inflammatory but effectively promotes antibody production.[1] The structural determination of A. faecalis LPS and its active entity lipid A and the synthesis and biological studies of A. faecalis lipid A will be reported.

Self-adjuvanting vaccines consisting in antigens and adjuvants have been investigated to improve vaccine efficacy and safety. We synthesized self-adjuvanting cancer vaccines composed of a trimeric tumor-associated carbohydrate antigen Sialyl-Tn (STn), a TLR2 agonist Pam3CSK4 as an adjuvant, and a T-helper epitope, since clustered STn antigens are highly expressed on many cancer cells. Immunization of vaccines in mice induced the anti-triSTn IgG antibodies, which recognized triSTn-expressing cell lines PANC-1 and HepG2.[3]

Humans do not have α-gal trisaccharide but have natural antibodies (Ab) against α-gal. The reaction of anti-Gal Abs with α-gal causes hyperacute rejection in xenogeneic organ transplantation. In this study, α-gal-Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α-gal and anti-Gal Abs.[3]

[1] Shibata, N. et al. Mucosal Immunol. 2018, 11, 693-702.

[2] Chang, T.C. et al. Angew. Chem. Int. Ed. Engl. 2018, 57, 8219-8224.

[3] Sianturi, J. et al. Angew. Chem. Int. Ed. Engl. 2019, 58, 4526-4530.

15. Stress inducible plant lectins

M. Dubiel¹,J. Lambin¹, S. Demirel Asci¹, M. Tsaneva¹, E. Van Damme¹; ¹Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, Belgium

Plant lectins are a fascinating group of proteins that can specifically recognize and bind to endogenous (plants-own) or exogenous (e.g. from pathogens) glycans. The interaction of lectins with glycan structures is reversible and does not alter the structure of the glycan moiety. Due to the vast diversity in protein structures, carbohydrate recognition domains and binding specificities, plant lectins constitute a diverse class of proteins. Most classical lectins locate to the vacuole or the cell wall, and are expressed in high levels at some stage of development. In contrast, the lectins present in the nucleocytoplasmic compartment occur in very low quantities, but their expression is enhanced in response to different stress factors or changing environmental conditions.

Our work focuses on the family of Euonymus-related lectins (EUL) which represents a group of stress inducible lectins found in all terrestrial plants, suggesting that these proteins might play an important role. Arabidopsis thaliana contains a single EUL gene encoding a protein that preferentially interacts with galactosylated glycans. Expression of the lectin is strongly upregulated in response to both abiotic (e.g. drought and abscisic acid treatment) and biotic stress factors (e.g. bacterial infection). Rice contains a family of EUL genes that are differentially expressed after biotic and abiotic stresses. A better knowledge of these stress inducible lectins will enable to better understand their role in plant development, and can contribute to applications in agriculture, such as the elaboration of plants with better performance under changing climatic conditions.

16. Congenital disorders of glycosylation (CDG): an overview

J. Jaeken¹; ¹Center for Metabolic Disease, Department of Development and Regeneration, KU Leuven, Leuven, Belgium

Congenital defects of glycosylation (CDG) are inherited diseases due to hypoglycosylation of glycoproteins and glycolipids, and to defective biosynthesis of glycosylphosphatidylinositol anchors. The first clinical report appeared in 1980. Since then, the number of published CDG has shown an exponential expansion. Hundred thirty-five CDG are known. They can be divided into four groups: 1. disorders of protein N-glycosylation; 2. disorders of protein O-glycosylation; 3. disorders of lipid glycosylation; 4. disorders of multiple glycosylation pathways; and 5. disorders of glycosylphosphatidylinositol anchor biosynthesis. The actual CDG nomenclature, introduced in 2009, consists of the official gene symbol (unitalicized) followed by ‘-CDG’. Most CDG show brain disease besides variable involvement of most other organs. Some 10 % CDG are mono-organ diseases (in brain, cartilage, erythrocytes, liver, retina, skeletal muscles and skin). As a rule, CDG are autosomal-recessive diseases. Exceptions to this rule are ALG13-CDG, ATP6AP1-CDG, MAGT1-CDG, PIGA-CDG, SLC35A2-CDG, SSR4-CDG (X-linked) and EXT1/EXT2-CDG, GANAB-CDG, POFUT1-CDG, POGLUT1-CDG, PRKCSH-CDG (autosomal dominant). Serum transferrin isoelectrofocusing (or similar techniques such as capillary zone electrophoresis) is the method of choice for the diagnosis of protein N-glycosylation disorders associated with sialic acid deficiency. Serum apolipoprotein C-III isofocusing is a screening method for the detection of core 1 mucin type O-glycans. Only for two CDG, a more or less efficient treatment is available: oral mannose for MPI-CDG and oral uridine for CAD-CDG. About 2 % of the human genome is involved in glycosylation; the majority of CDG are thus still ‘in the waiting room’. Next generation sequencing techniques (panel analysis, whole exome sequencing and whole genome sequencing) are becoming the predominant diagnostic tools. The greatest challenges in the CDG field are the elucidation of their pathophysiology and the elaboration of efficient treatments.

References

Francisco R, Marques-da-Silva D, Brasil S et al. The challenge of CDG diagnosis. Mol Genet Metab 2019; 126: 1-5

Jaeken J, Péanne R What is new in CDG? J Inherit Metab Dis 2017; 40: 569-586

Joshi HJ, Hansen L, Narimatsu Y et al. Glycosyltransferase genes that cause monogenic congenital disorders of glycosylation are distinct from glycosyltransferase genes associated with complex diseases. Glycobiology 2018; 28: 284-294

Ng BG, Freeze HH. Perspectives on glycosylation and its congenital disorders. Trends Genet 2018; 34: 466-476

Witters P, Cassiman D, Morava E Nutritional therapies in congenital disorders of glycosylation (CDG). Nutrients 2017 doi: 10.3390/nu9111222

17. Carbohydrate-specific computational methods for the refinement, validation and representation of structures of glycan-lectin complexes

J. Agirre¹; ¹University of York

The introduction of intuitive graphical software has enabled structural biologists who are not experts in crystallography to build complete protein or nucleic acid models rapidly. In contrast, carbohydrates are in a completely different situation: scant automation exists, and users building models manually frequently trip over legacy issues such as incorrect dictionaries or non-standard atom naming, which evidence a historical lack of methodological support for carbohydrates. Sugars are stereochemically complex and, as pyranose rings, have clear conformational preferences. And despite this, all refinement programs may produce high-energy conformations at medium to low resolution, without any support from the electron density; this problem renders the affected structures unusable in glyco-chemical terms. Bringing structural glycobiology up to ‘protein standards’ is thus requiring a total methodological overhaul. Time is of the essence, as the community is steadily increasing the production rate of glycoproteins and protein-carbohydrate complexes, and electron cryo-microscopy has just started to image them in precisely that resolution range where crystallographic methods falter most. In this talk, I will introduce our latest methodological developments, designed to streamline and automate hitherto error-prone processes, effectively aiding crystallographers and electron microscopists alike in producing correct atomic models of lectin-carbohydrate complexes with confidence.

18. β-Prism fold lectins: structure, carbohydrate specificity and their therapeutic potential

A. Surolia¹; ¹Molecular Biophysics Unit, Indian Institute of Science, Bangalore

It wasn’t long ago when carbohydrates were thought of merely as sources of food, energy and structural materials; today, they are known to provide cells with their individuality and recognition patterns that crucially regulate cellular social behavior including their growth and differentiation. This “glycocode” at the cell surface is decoded by lectins that bind carbohydrates selectively to mediate cell-cell adhesion, cell signalling, immune response etc. making them subjects of intense scientific scrutiny. There are five families of plant lectins - jelly roll, haevin domains, β-prism I and II, β-trefoil and cyanovirin folds. β-prism I lectins are the most abundant family of lectins, ubiquitous in plant kingdom, are distributed virtually in all parts of the plant and occur in a variety of oligomeric forms. My talk will focus on features of carbohydrate recognition by lectins exhibiting the β-prism I fold. Most of them are glucose or mannose binding lectins but there are instances of galactose specificity E.g.: Jacalin. While the specificity for the interactions is provided by hydrogen bonding between the sugar atoms and the amino acid side-chains in the combining site of the lectins, water mediated hydrogen bonds, van der waals, stacking and OH-π interactions contribute significantly to their binding affinities. The occurrence of post-translational proteolysis and distortion of the pyranose ring in the bound conformation are unprecedented in most other lectins. Finally, the influence of these studies in the development of lectins for hematopoietic stem cell differentiation and treatment of HIV and other viral infections will be discussed.

19. A genetic dissection approach to functional glycomics

H. Clausen¹, Y. Narimatsu¹, H. Joshi¹, Y. Chen¹, W. Tian¹, K.K. Schjoldager¹, C. Steentoft¹, Z. Yang¹, S. Vakhrushev¹; ¹Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

The structural diversity of the human glycome is vast and poses challenges for exploring biological functions and dissecting specific structure-function relationships. The true functional diversity of the glycome is, however, predicted to be lower when grouping structures with common or repeated features. The human glycome is orchestrated by over 200 distinct glycosyltransferase genes, and our knowledge of the properties of these and their roles in known glycosylation pathways in cells is relatively advanced. Currently some 170 glycosyltransferase genes can be assigned to rather specific roles in biosynthetic steps for the human glycome, although for many of the isoenzyme families our understanding of the unique functions of individual enzymes is limited. Current knowledge of assembly of the human glycome suggests that it is simpler to explore and dissect the glycome by a genetic entry point rather than from a structural entry point. Emergence of nuclease-based gene-editing tools enabling highly specific and facile knockout and site-directed knockin of glycosylation-related genes have led to wide use of the genetic approach to glycomics and new tools for the field. Our group has extensively adopted the genetic approach for dissection and discovery of biological functions of protein glycosylation, and explored the options for custom design of glycosylation of recombinant glycoprotein therapeutics as well as using cell libraries for display of the glycome. An overview of these efforts will be presented.

20. Functional analysis of mucin-type O-glycans using drosophila melanogaster

S. Nishihara¹; ¹Department of Bioinformatics, Graduate School of Engineering, Soka University, Tokyo

Drosophila is one of the most important model organisms in the fields of molecular genetics as well as developmental biology. Three main structures of mucin-type O-glycans, i.e., Tn antigen (GalNAcα1-Ser/Thr), T antigen (Galß1-3GalNAcα1-Ser/Thr), and glucuronylated T antigen (GlcAß1-3Galß1-3GalNAcα1-Ser/Thr), have been identified in Drosophila.

The Drosophila orthologue of core 1 ß1,3-galactosyltrasferase 1 (C1GalT1), i.e., dC1GalT1 (CG9520), exhibits extremely strong core 1 ß3GalT activity. We previously reported that the dC1GalT1 is involved in the synthesis of T antigen in a subset of hemocytes and in the central nervous system in embryos. Moreover, dC1GalT1 mutant larvae show reduced numbers of embryonic hemocytes. Further, we also showed that T antigen, supplied by mature hemocytes in the lymph gland (the larval hematopoietic organ), is an essential factor responsible for maintaining the capacity of the extracellular environment to support filopodial extensions from niches that maintain hematopoietic stem cells. In a recent study, we reported that mucin-type core 1 glycans modulate the localization of neuromuscular junction (NMJ) boutons, NMJ synaptogenesis, and establishment of muscle cell architecture. In this way, the functions of T antigen in blood cells and the neurons have been analyzed for dC1GalT1 mutants.

In this study, in order to clarify the physiological role of glucuronylated T antigen, we created ß1,3-glucuronyltransferase-P (dGlcAT-P) (CG6207) null mutants and examined whether they displayed phenotypes similar to those observed in dC1GalT1 mutants. We found two phenotypes, a partial loss of basement membrane components and mislocalization of NMJ boutons at muscle 6/7 boundary in dGlcAT-P null mutant larvae. These phenotypes were identical to phenotypes observed in dC1GalT1 mutants. In addition, dGlcAT-P null mutants displayed fewer NMJ branches on muscles 6/7. Moreover, there was a genetic interaction between dGlcAT-P and dC1GalT1. These data clearly showed that glucuronylated core 1 glycans produced by dGlcAT-P regulate the formation of basement membranes, localization of NMJ boutons, and arborization of NMJs on larval muscles. In addition, we also showed that dGlcAT-P loss resulted in ultrastructural defects in NMJ boutons.

21. Pseudaminic acid plays a critical role in the conjugate vaccine against acinetobacter baumannii 54149 with high antigenicity

S. Wu², I. Lee², C. Wu¹; ¹Gemomics Research Center, ²Institute of Biological Chemistry, Academia Sinica, Taiwan

Similar to sialic acid, pseudaminic acid belongs to nonulosonic acids, a structural class of 9-carbon carbohydrate monomers. Pseudaminic acid (Pse) has been known for participating in crucial bacterial virulence and thus is an attractive target in the development of glycoconjugate vaccine. Particularly, this therapeutic alternative was suggested to be a potential solution against multi-drug resistant Acinetobacter baumannii that poses a serious global health threat. Also, Pse was found to be involved in the exopolysaccharide (EPS) of mild antibiotic resistant A. baumannii strain 54149 (Ab-54149) of which specific glycosyl linkage can be depolymerized by phage ΦAB6 tailspike protein (ΦAB6TSP). Furthermore, we found that the antibodies induced by Ab-54149 EPS was capable of recognizing a range of EPS of other clinical A. baumannii strains, and deemed as a great potential material for vaccination. To efficiently acquire homogeneous EPS-derived oligosaccharide with significant immunogenic activity for the production of glycoconjugate, we used the ΦAB6TSP for the fragmentation of Ab-54149 EPS instead of chemical methods. Moreover, insight into the ligand binding characterization of ΦAB6TSP suggested the branched Pse on the Ab-54149 EPS served as a recognition site of ΦAB6TSP. The serum boosted by ΦAB6TSP-digested product and carrier protein CRM197 conjugate complex displayed specific sensitivity toward Ab-54149 EPS with bacterial killing activity. Strikingly, Pse is an ideal epitope with strong antigenicity, profiting the application of the probe for pathogen detection and glyco-based vaccine.

Reference

I-M. Lee, F-L. Yang, T-L. Chen, K-S. Liao, C-T. Ren, N-T. Lin, Y-P. Chang, C-Y. Wu,* S-H. Wu* (2018) Pseudaminic Acid on Exopolysaccharide of Acinetobacter baumannii Plays a Critical Role in Phage-Assisted Preparation of Glycoconjugate Vaccine with High Antigenicity J. Am. Chem. Soc. 140, 8639−8643

22. Glycoproteomic identification of enterovirus A71 receptors

C. Chuan-Fa¹, H. Nien-En¹, L. Zheng-Xun¹, L. Yi-Chen¹, C. Wei-Ting¹, S. Pei-Yi¹; ¹National Cheng Kung University

Enterovirus 71 (EV-A71) is a major causative agent of hand-foot-and-mouth disease (HFMD), and EV-A71 infection of the central nerve system (CNS) would result in a high mortality in the infected children. However, the mechanisms of virus attachment, invasion and virulence determinants of EV-A71 are still not clear. Our previous studies indicated that sialic acids participated in the attachment and infection of EV-A71 to host cells. Hence, we used glycoproteomic approached to investigate EV-A71 interacting glycoproteins. Several proteins were identified and nucleolin (NCL), selenoprotein s (SELS), and plasminogen (PLG) showed strong binding with EV-A71. Based on the results form immunoprecipitation, ELISA assay, and Surface plasmon resonance (SPR), we found that EV-A71 could interact with NCL, SELS, and PLG directly. Anti-NCL, anti-SELS, or anti-PLG antibody could abolish the binding and replication of EV-A71 to host cells. The bindings of EV-A71 to NCL, SELS, and PLG knockdown cells were significantly reduced. In addition, we also found that NCL, SELS, or PLG interacted with SCARB-2, a known EV-A71 receptor, post EV-A71 infection. To evaluate the effects of hNCL in EV-A71 infection in vivo, we challenged 7-day-old wild type and hNCL transgenic mice with MP4 virus intraperitoneally. Significantly higher clinical scores could be observed in hNCL transgenic mice 4-10 days p.i. Severe paralysis was also observed in hNCL transgenic mice 5 days p.i. Finally, 90% of hNCL transgenic mice were dead by 10 days after infection, but all of the wild type mice were alive. We then analyzed the tissue section of EV-A71 infected wild type/hNCL-Tg mice. We found that the muscle of hNCL-Tg mice showed greater damage and significant destruction than wild type. Higher virus staining was also observed on brain stem and spinal cord section of hNCL-Tg mice. In addition, we also evaluated the cytokine expression in serum, brain stem and spinal cord of EV-A71 infected wild type/hNCL-Tg mice. The expression of MCP-1, IL-1ß, IL-6, TNF-α, IL-10, and IL-13 were significant higher in hNCL-Tg mice. Based on these findings, it is suggested that NCL should play important roles in the attachment and infection of human enterovirus species A viruses to host cells. These results also suggested that hNCL-Tg mice may be good animal model for the study of EV-A71 induced cytokine storm or pulmonary edema.

23. Glycoconjugate vaccines: present and future

R. Adamo¹; ¹GSK, Via Fiorentina 1, 53100 Siena, Italy

The cell wall of a large number of bacterial pathogens is surrounded by a dense layer composed of fibrous polysaccharides and globular glycoproteins, named glycocalyx containing anti-phagocytic properties. Carbohydrates forming this complex and sophisticated structure have been proven optimal targets for vaccines development¹. Unconjugated polysaccharide based vaccines failed to induce strong and long lasting immune response and adequate protection in high-risk groups (i.e. neonates and children under 2 years of age)². Conjugation to an appropriate carrier protein³ overcomes these limitations, triggering a T -cell dependent immune response to the polysaccharide moiety. Glycoconjugate vaccines Haemophilus influenzae type b (Hib), Streptococcus pneumoniae (23 serotypes), Neisseria meningitidis (A, C, W135 and Y) and Salmonella typhi, results in greatly improved immunogenicity, protective efficacy in very young children as well as elder population and immunological memory.

This presentation will show how advancements in the field of glycoconjugate vaccine design and production (chemo-enzymatic approaches for carbohydrate construction, modern conjugation methods, glycoengineering, and nanoparticles design) can support the development of next generation vaccines.

It will present, how emerging technologies hold the potential to address the future challenges of glycoconjugate vaccines which are: i) extending the coverage of existing vaccine by incorporating additional serotypes, ii) rendering vaccination accessible also in low-medium income countries,⁴ and iii) addressing unmet medical needs, particularly emerging antibiotic resistant pathogens.

1. F. Micoli, P. Costantino and R. Adamo, FEMS Microbiol. Rev., 2018, 42, 388-423.

2. D. Pace, Expert Opin. Biol. Ther., 2013, 13 11-33.

3. F. Micoli, R. Adamo and P. Costantino, Molecules, 2018, 23.

4. C. A. MacLennan, Seminars in Immunology, 2013, 25, 114-123.

24. Gut microbiota lipopolysaccharides: reverting the concept from bad to good

A. Molinaro¹; ¹Department of Chemical Sciences, University of Naples Federico II, via cintia 4, Naples, Italy, molinaro@unina.it

The Gut Microbiota (GM) is an essential actor in the modern concept of human health driving many host physiological and pathological processes, including immune system modulation. Accumulating evidences highlighted that studies of the immune system during health or disease cannot ignore our GM.[1] Initial sensing of microbes by the host immunity is mediated by the recognition of microbial-associated molecular patterns, such as lipopolysaccharides (LPS), which are highly conserved among bacteria, thus shared by both commensals and pathogens. The LPS structure strongly influences the biological effects on the host immune system. Defined LPS structures can act as potent agonists on the immune receptors whereas other can operate as antagonists reducing or inhibiting the cytokine production otherwise induced by toxic LPSs.[2] Thus, a crucial question to address is how the immune system distinguishes between permanently established commensals LPS and pathogens LPS.

The elucidation of the structure and the immunological activity of LPS isolated from gut microbes will bear new advances in the medicinal chemistry and in the field of search of new molecules able to antagonize pathogens LPS effect, as well as of GM LPS-inspired molecules able to prevent uncontrolled host immune response against our microbiota. This will also shed light on the structure-activity relationship of LPS itself, which is an open question in immunology field. In particular, this will improve the knowledge of the still poorly investigated GM world, giving insights in the host-microbe interaction mechanisms both at intestinal and systemic level furnishing, in parallel, information about the elicitation/modulation of immune response triggered by pathogens and commensals LPS.

In this communication, I will show some very recently elucidated GM LPS structures and their immunological properties that revealed to express unique and interesting features.

[1] J. Chow, S. M. Lee, Y. Shen, A. Khosravi, S. K. Mazmanian, Adv. Immunol. 2010, 107, 243-74.

[2] A. Molinaro, O. Holst, F. Di Lorenzo, M. Callaghan, A. Nurisso, G. D’Errico, A. Zamyatina, F. Peri, R. Berisio, R. Jerala, J.Jiménez-Barbero, A. Silipo, S. Martín-Santamaría, Chem. Eur. J. 2015, 21, 500-519.

25. Harness heparan sulfate biosynthetic pathway to prepare homogeneous oligosaccharides

J. Liu¹; ¹University of North Carolina

Biosynthesis of heparan sulfate consists of a cascade of enzymatic modifications. We use the biosynthetic enzymes to develop a chemoenzymatic method for synthesizing structurally homogeneous heparan sulfate oligosaccharides. The availability of heparan sulfate oligosaccharides has significantly accelerated the effort to investigate the contribution of unique saccharide sequences to the biological activities. In one example, we have synthesized several oligosaccharides that display the anticoagulant activity. The oligosaccharides are synthesized in high efficiency and can potentially replace animal-sourced low-molecular weight heparin. Each oligosaccharide has unique pharmacological properties to treat thrombotic disorders. Synthetic heparin will also eliminate the safety concerns that burden regulatory agencies around the world.

Another example is to exploit new biological/pharmacological properties of heparan sulfate in addition to the anticoagulant activity. We discover a heparan sulfate octadecasaccharide (18-mer) is able to protect against acetaminophen-induced acute liver injury in a murine model. The in-depth mechanistic studies indicate that the 18-mer neutralizes the pro-inflammatory activity of high mobility group box 1 (HMGB1) protein. Acetaminophen (also known as Paracetamol) overdose is the leading cause for drug-induced liver injury in US and Europe. Our findings add a potential new method to treat drug-induced liver injury patients. Overall, the availability of heparan sulfate oligosaccharide library offers opportunities to develop the next generation of heparin-based medicines.

26. Inhibitors of the human neuraminidase enzymes as probes in glycobiology

C.W. Cairo¹; ¹University of Alberta

The human neuraminidase enzymes (NEU) are a family of four isoenzymes (NEU1, NEU2, NEU3, and NEU4) which have wide variability in their substrate preferences as well as cellular and tissue localization. Specific functions of this enzyme family continue to be investigated, and the development of isoenzyme-selective inhibitors is providing new tools for in vitro and in vivo studies. Due to a lack of structural data on several isoenzymes, development of new inhibitors requires the synthesis of libraries and screening for activity. Furthermore, the development of computational models of these enzymes has aided in directing the synthesis of new, more selective compounds. Our group has developed a panel of selective inhibitors, many with nanomolar potency, which we are using as tools to investigate systems where NEU play important roles.

Previous investigations in our group have examined the role of NEU enzymes in integrin-mediated cell migration. We found evidence that β1 integrins were positively regulated by the NEU3 isoenzyme. Our current work is examining the role of this isoenzyme in β2 integrin–mediated cell adhesion and migration. Applications of selective inhibitors to cell migration and T cell adhesion will be discussed. We propose that these compounds will be essential tools to reveal the specific roles of NEU isoenzymes in important pathologies, including cancer, atherosclerosis, and inflammation.

27. Emerging roles of mammalian neuraminidases in CNS development and metabolic regulation

A.V. Pshezhetsky¹; ¹Sainte-Justine Hospital Research Center, Departments of Pediatrics and Biochemistry and Molecular Medicine, University of Montreal, McGill University, Department of Anatomy and Cell Biology, Montreal, CANADA

The sialylated glycoconjugates (SGC) are found in abundance on the surface of mammalian cells, where they form a dense array of glycans mediating cell/cell and cell/protein recognition in numerous physiological and pathological processes. In the CNS, they are essential for division and differentiation of neurons, axon growth, integrity and myelination, synaptic transmission and plasticity. Metabolic genetic blocks in processing and catabolism of SGC result in development of severe storage disorders, dominated by CNS involvement including marked neuroinflammation and neurodegeneration which pathophysiological mechanism is still discussed. SGC composition is cell and organelle-specific, dynamic and maintained by highly coordinated processes of their biosynthesis, trafficking, processing and catabolism. Three mammalian neuraminidases, neuraminidase 1 (Neu1), neuraminidase 3 (Neu3) and neuraminidase 4 (Neu4) residing on lysosomal, endosomal and plasma membranes are major candidates for processing cell surface SGC.

To elucidate the physiological functions of Neu1, Neu3 and Neu4 we studied gene-targeted mouse strains deficient in one or several enzymes. In my presentation I will summarize emerging data that point to crucial roles of neuraminidases in metabolic regulation and CNS development. Neu1 plays major role in processing sialic acid residues of surface glycoproteins, altering their structure, activity and interaction patterns. In particular it desialylates insulin receptor and activates insulin signaling. Both Neu1 and Neu3 desialylate circulating low density lipoprotein particles inducing their uptake by macrophages and play major role in pathology of atherosclerosis. In the CNS, Neu3 and Neu4 are mainly responsible for desialylation of brain gangliosides participating in degradation of GM3 in lysosomes of microglia and pericytes. In addition, Neu4 generates membrane-associated GM1 ganglioside in neurons, essential for axon growing and neuritogenesis. Together our data provide evidence that the sialoglycome composition is modulated by neuraminidases which “trim off” sialic residues from SGC at the required site and time in response to external or internal stimuli.

28. Regulation of heparan sulfate biosynthesis - enough is enough

U. Lindahl¹; ¹Department of Medical Biochemistry and Microbiology, University of Uppsala, Uppsala, Sweden

Heparan sulfate proteoglycans (HSPGs) regulate a multitude of processes, of physiological and pathophysiological importance, by binding and thus modulating functions of a variety of proteins (growth factors, morphogens, cytokines etc). The specificity behind these interactions appears variable, and the underlying HS structure/function relations are largely unclear. Beyond the few known examples of protein-binding HS epitopes depending on rare sugar residues (such as 3-O-sulfated glucosamine residues), there is no clear indication of distinct sequence specificity based on the precise distribution of the more abundant, “common” sulfate groups that constitute binding motifs for the great majority of protein ligands. In fact, fully sulfated heparin analogues can often substitute for endogenous HS ligands of signaling proteins in vitro (1, 2). Some of these interactions are undoubtedly nonspecific. On the other hand, HS biosynthesis generates polysaccharide populations of varied yet strictly regulated composition (3). Conceivably, the probability of generating a particular constellation of sulfate groups, required for interaction with a specific protein, through a process of essentially stochastic 2-O- and 6-O-sulfation within a N-sulfated domain will increase with increasing levels of sulfate substitution. The various sulfation reactions may be independently adjusted to accommodate the relative need for different sulfate groups, without any requirement for generation of strictly predetermined sequences. Notably, such a process may enable gradual modulation of HS-protein interactions. These notions will be considered in relation to functional effects observed following genetic ablation of selected HS biosynthesis enzymes (4).

(1) Jastrebova N., Vanwildemeersch M., Lindahl U., Spillmann D., 2010. Heparan sulfate domain organization and sulfation modulate FGF-induced cell signaling. J Biol Chem 285, 26842-51.

(2) Lindahl U., Li J-p., 2009. Interactions between heparan sulfate and proteins – design and functional implications. Intern Rev Cell Mol Biol 276, 105-159.

(3) Ledin J., Staatz W., Li J-p., Gotte M., Selleck S., Kjellén L., et al., 2004. Heparan sulfate structure in mice with genetically modified heparan sulfate production. J Biol Chem 279, 42732-41.

(4) Qiu H., Shi S., Yue, J., Xin M., Naim A.V., Lin L., et al., 2018. A mutant-cell library for systematic analysis of heparan sulfate structure-function relationships. Nat Methods 15, 889-99.

29. ST6Gal-I sialyltransferase has tumor-promoting activity through its role in imparting a cancer stem cell phenotype

A. Chakraborty¹, C. Britain¹, R. Jones¹, L. Smythies¹, S. Bellis¹; ¹University of Alabama at Birmingham

Cancer cells upregulate distinct glycosyltransferases and their cognate glycan structures, however the functional role of glycans in tumor cell behavior is not fully understood. One such glycosyltransferase is ST6Gal-I, a sialyltransferase which adds α2-6 sialic acids to select N-glycosylated receptors. Through modulating receptor activity, ST6Gal-I imparts cancer stem cell (CSC) characteristics including tumor-initating potential, cell invasiveness and apoptosis-resistance. ST6Gal-I is overexpressed in many cancers including pancreatic ductal adenocarcinoma (PDAC), and animal studies by our group have confirmed ST6Gal-I’s tumor-driver function. Forced expression of ST6Gal-I in PDAC cells impels tumor growth in xenograft models, and likewise, ST6Gal-I promotes carcinogenesis in genetically-engineered mouse (GEM) models. Specifically, we developed a pancreas-specific ST6Gal-I knock-in mouse and crossed this to the “KC” murine PDAC model, which harbors oncogenic K-ras in the pancreas. Mice with both ST6Gal-I knock-in and oncogenic K-ras (“KSC” mice) exhibit greatly accelerated PDAC initiation, metastatic progression, and mortality as compared with KC mice (Kras alone). Given that ST6Gal-I confers CSC features, we hypothesized ST6Gal-I would contribute to PDAC initiation. One of the earliest events in PDAC development is acinar to ductal metaplasis (ADM), where pancreatic acinar cells trans-differentiate into stem-like, ductal cells which are primed for neoplastic transformation. IHC staining of the GEM models revealed that ductal markers are abnormally expressed in the non-malignant acinar cells of mice with ST6Gal-I knock-in, suggesting that ADM had occurred. Moreover, using organoids derived from the GEM models, along with single cell RNASeq of cells dissociated from GEM pancreata, we determined that ST6Gal-I activity increases the expression of ductal and stem cell genes, while suppressing hallmark acinar genes. ST6Gal-I activity also potentiates the formation and viability of organoid cultures, consistent with promoting stemness. These results were mirrored by studies using the ADM cell model, 266-6 cells. Ectopic expression of ST6Gal-I in 266-6 cells induces an upregulation in ductal and stem genes, but downregulation in acinar genes, while the inverse was noted in 266-6 cells with ST6Gal-I knock-down. Taken together, these data support a role for ST6Gal-I in ADM and PDAC pathogenesis, and further implicate ST6Gal-I as a promising therapeutic target.

30. Spreading the neus: the role of sialidases in the pulmonary innate immune response

A. Cross¹, S. Goldblum², E. Lillehoj³, C. Feng¹; ¹Center for Vaccine Development and Global Health, Department of Medicine, University of Maryland School of Medicine, Baltimore, ²Department of Medicine, University of Maryland School of Medicine, Baltimore, ³Department of Pediatrics, University of Maryland School of Medicine, Baltimore

The respiratory tract is the interface between the external environment and the internal host milieu. Epithelial cells lining the airways regulate interactions between numerous exogenous and endogenous agonists and multiple host signaling systems. The human respiratory epithelial and human lung microvascular endothelial cell surfaces are highly sialylated. Superimposed on the highly sialylated surfaces are specific receptors that are themselves sialylated. Pulmonary epithelial and endothelial cells express sialidase activity which regulate receptor-ligand, cell-cell and host-pathogen interactions. Human lung microvascular endothelia express catalytically active NEU1 and NEU3, the former regulating endothelial remodeling in response to injury. Catalytically active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31 and restrains endothelial cell migration in wound healing assays. Human airway epithelia also express catalytically active NEU1 and NEU3 sialidases. Neu1 activity in airway epithelia modulates the activity of receptors that respond to epidermal growth factor (EGF) and to danger signals, thereby offering an additional level of regulation over airway epithelial responses to ligands, pathogens and injurious stimuli.

Host sialidase activity plays a critical role in the pulmonary response to the constant exposure to microbial pathogens. We previously reported that host chemokines (IL-8) recruited PMNs to the lung and that surface expression of sialidase was a prerequisite for PMN recruitment in vivo. Desialylation of the β2-integrin on PMNs and its counterligand, ICAM-1, on activated endothelial cells were critical to leukocyte arrest that precedes PMN transmigration. In addition, removal of sialyl residues from the TLR4 receptor complex facilitates the pulmonary inflammatory response. Neu1 also is critical to MUC1 activity important to the clearance of Pseudomonas aeruginosa. Thus endogenous host sialidases play a critical role in both lung physiology and antimicrobial host defenses. However, many respiratory pathogens, such as influenza and S. pneumoniae, express neuraminidase (NA)/sialidase as important virulence determinants that may potentiate the pulmonary inflammatory response. NA potentiated LPS-induced lung injury through enhanced PMN recruitment, pulmonary edema formation and endothelial and myeloid cell apoptosis. A similar “reprogramming” of the immune response leading to severe lung injury may occur during respiratory infection with NA-expressing microbes such as influenza. For example, desialylation of airway epithelial cells during influenza virus infection increases pneumococcal adhesion that may facilitate post-influenzal bacterial superinfection. Given that human sialidase activity is essential to the human immune response, the ability to modulate sialidase activity with either NA inhibitors or possibly vaccine-induced NA antibodies may compromise human sialidase activity critical to the human immune response. Alternatively, sialidase inhibitors (e.g. zanamivir) might prove useful in the treatment of hyperinflammatory conditions that may lead to acute lung injury. We conclude that a more expanded study of the impact of sialidase inhibitors on pulmonary immune responses is warranted.

31. Single-molecule imaging study of regulation mechanisms of EGF receptor activity by gangliosides

K.G.N. Suzuki¹; ¹G-CHAIN, Gifu University, Japan

Raft domains have been drawing extensive attention as signaling platforms. However, raft structure and function are still enigmatic. This may be partially due to lack of true lipid probes which behave like the parental molecules in rafts. Here, we developed fluorescent probes of representative raft lipids, gangliosides, which behave like the parental molecules.

By single fluorescent-molecule imaging of the ganglioside probes, the dynamic organization of rafts has been investigated. Virtually, all of the observed ganglioside probes are mobile, and continually formed transient (100-300 ms lifetime) homodimers based on glycan-glycan interactions. We further found that ganglioside homodimers recruited other raft-associated molecules such as sphingomyelin, and were stabilized by raft-lipid interactions. Therefore, we call these entities “ganglioside homodimer rafts”. Under higher physiological expression conditions, the homodimers of gangliosides coalesced to form the homo-oligomer rafts by raft-lipid interactions with shorter lifetimes.

Furthermore, we found that GM3 homodimer rafts interacted with EGF receptors (EGFRs), which resulted in inhibition of the EGFR dimerization and activation, while GM3 monomers did not. The transient ganglioside homodimer rafts are likely one of the basic units for the organization and function of raft domains.

32. Implacts of glycoshingolipids and their autoantibodies on neurological disorders

T. Mutoh¹, S. Shima¹, A. Ueda¹, H. Watanabe¹, Y. Mizutani¹, H. Akiyama², Y. Hirabayashi²; ¹Department of Neurology and Neuroscience, Fujita Health University School of Medicine; ²Riken

Previous studies have indicated that GM1 ganglioside (GM1) enhances nerve growth factor-induced Trk neurotrophin receptor (Trk) autophosphorylation by the direct mutual interaction (PNAS 1995), whereas anti-GM1 antibody that is related to Guillain-Barré syndrome exerts inhibitory effects on this process (Mol Cell Neurosci 2010). Moreover, our newly discovered anti-lactosylceramide (LacCer) antibody found in whole nervous system involving encephalomyeloradiculoneuropathy (EMRN) (Neurology 2014) also exhibits negative effects on Trk activities by disturbing its proper positioning in membrane rafts.

Here, we report the effects of anti-GM1 antibody on neutral sphingomyelinase (nSMase) action and the results of lipids analyses of cerebrospinal fluid (CSF) from EMRN patients and their immunological characteristics. The data indicated that 1) anti-GM1 antibody inhibits nSMase activities in both plasma membrane and rafts fractions resulting in the significant accumulation of sphingomyelin (SM) in the membrane fraction but not whole cells. Moreover, anti-GM1 antibody induced disappearance of nSMase protein from membrane fraction. 2) Lipids analysis of CSF with LC-MS/MS clearly indicated significant accumulation of LacCer and glucosylceramide (GlcCer) in EMRN patients. This increase in turn resulted in a significant activation of complement C5 in patients’ sera.

Thus, these data clearly suggested that autoantibodies against rafts resident GSL provoke profound effects on the rafts function and perhaps immune functions as well.

33. Development of genetically encoded, liquid Glycan array technology to probe glycan recognition ex vivo and in vivo

R. Derda¹; ¹University of Alberta

In this talk, I will describe genetically-encoded platform technology termed Liquid Glycan Array (LiGA). LiGA enables profiling of interactions of glycans with receptors on the surface of prokaryotic, and eukaryotic cells ex vivo and in vivo in specific tissues, organs, and cells from these organs.

The major barrier to studying the role of glycans and glycan binding proteins (GBPs) in vivo is the fundamental lack of one-to-one correspondence between sequence of DNA and structures of carbohydrates. Investigations of carbohydrates, thus, cannot rely on DNA sequencing directly. Glycan arrays, made by coupling of carbohydrates to glass or bead surfaces are the workhorse tools to identification of structures of carbohydrates that bind to a GBP. These technologies cannot study glycans and GBPs in their natural environment on the surface of the immune cells, on cells in tissues in vivo. To solve this challenge, a LiGA technology developed by Derda Lab introduces one-to-one correspondence between DNA sequence and carbohydrate structure by simple chemical attachment of carbohydrates to bacteriophage (phage) particles. Genetically encoded, monodisperse carriers based on 700 nm long M13 phage particles display 100-1500 copes of glycans on their surface. The identity and presentation (density) of glycans are encoded by the DNA barcode inside the phage genome. Simple pull down or flow-cytometry isolation of cells of interest followed by next generation sequencing identifies both the type and presentation of glycan interacting the target GBP or cell types. Simplicity, scalability and broad utility of LiGA platform make it an attractive glycan profiling tool for glycobiology community to complement existing technologies (solid and bead based arrays).

34. Synthetic plant glycans as tools for cell wall biology

F. Pfrengle¹; ¹Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Potsdam, Germany

Plant cells are surrounded by a polysaccharide-rich matrix that constitutes the cell wall of all higher plants. These structurally complex polysaccharides provide an important resource for food, renewable materials, and the generation of bioenergy. We have prepared oligosaccharides derived from different classes of plant cell wall polysaccharides using automated glycan assembly. For each class of polysaccharide a collection of oligosaccharide samples were synthesized from a limited set of monosaccharide building blocks. The assembled glycans were subsequently printed as microarray to investigate cell wall-directed antibodies and biosynthetic enzymes. Using our synthetic plant glycan array, we were able to determine the epitopes of 81 antibodies that recognize plant cell wall glycans and determine the acceptor substrate specificities of several glycosyltransferases that are involved in plant cell wall biosynthesis.

35. Novel ways to regulate heparan sulfate assembly

R.J. Weiss², P.N. Spahn⁴, A.G. Toledo², A.W. Chiang⁴, B.P. Kellman⁴, J. Li², C. Benner³, C.K. Glass², P.L. Gordts³, N.E. Lewis¹, J.D. Esko²; ¹Department of Bioengineering, University of California, San Diego, San Diego, CA 92093-0687, ²Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, CA 92093-0687, ³Department of Medicine, University of California, San Diego, San Diego, CA 92093-0687, ⁴Department of Pediatrics, University of California, San Diego, San Diego, CA 92093-0760

Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and in the extracellular matrix. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N- and O-sulfated. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites for protein ligands, and binding regulates important biological processes including cell proliferation and development. While most of the enzymes involved in HS biosynthesis have been studied extensively, much less information exists regarding the specific mechanisms that give rise to the variable composition and binding properties of HS. We have taken a two-prong approach towards uncovering these control systems. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to a dramatic increase in expression of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase. A genome-wide CRISPR/Cas9-mediated screen also was developed. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human melanoma cell line. High-throughput screening assays were adapted to identify lentiviral-encoded sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs and that reduce binding of HS-dependent ligands. Top hits from the screens were characterized and categorized based on their predicted gene functions and are currently being individually validated and examined for their potential involvement in the regulation of HS biosynthesis.

36. Over view: organization and physiological functions of glycolipid-enriched membrane domains

K. Iwabuchi¹; ¹Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate school of Medicine, Urayasu; Infection Control Nursing, Juntendo University Graduate School of Health Care and Nursing, Urayasu

Cellular membranes consist of several kinds of glycoproteins, glycolipids and other lipids, such as cholesterol, glycerophospholipids and sphingomyelin. According to their physicochemical properties, they may form various domains and/or segregate from each other. Asymmetry is another well-established characteristic of plasma membranes which is dependent on highly different lipid compositions of the two leaflets of the membrane bilayer. It is widely accepted that small membrane nano/meso domains, so called lipid rafts in which glycolipids, sphingolipids and cholesterol are enriched, play key roles in many cellular functions. For instance, human neutrophils highly express lactosylceramide (LacCer) and phosphatidylglucoside (PtdGlc) on plasma membranes. LacCer forms Lyn-associated lipid rafts and mediates phagocytosis. In contrast, PtdGlc forms Fas-associated lipid rafts and is involved in spontaneous apoptosis. However, we cannot yet fully explain how these glycolipids form their different nano/meso domains or mediate their specialized functions. The present symposium intends to give an overview of the current understanding on raft structures, mechanisms of facilitation and regulation of membrane receptor signaling in rafts. I addition, we will introduce state-of-the-art technologies to unravel these issues. In my talk, I will also introduce some important new concepts about roles of LacCer, PtdGlc and their metabolites in immunological functions.

37. Indication of the involvement of a golgi sphingomyelin-enriched domain in the innate-immunity signalling

T. Taguchi¹; ¹Tohoku University

STING, also known as MITA, ERIS, MPYS, or TMEM173 is an ER-localized transmembrane protein that is essential for the innate immune response against cytosolic DNA or cyclic dinucleotides (CDNs) from pathogens. Cytosolic DNA activates a DNA-binding protein cyclic GMP-AMP (cGAMP) synthase, leading to the generation of a CDN cGAMP in the cytosol. Other CDNs including cyclic di-GMP and cyclic di-AMP are secreted by intracellular bacteria following infection. CDNs bind to STING and activate STING to trigger the type I interferon and inflammatory responses. Chronic activation of STING has been implicated in the pathogenesis of monogenic autoinflammatory conditions, such as STING-associated vasculopathy with onset in infancy. In addition, accumulating evidence suggests that cytosolic self-DNA activates STING, which triggers autoinflammatory diseases, cellular senescence, and the activation of T-cell response against cancer.

We have shown that STING activation required the exocytic membrane traffic from the ER to the Golgi (1, 2) and palmitoylation of STING at the Golgi (1, 3). In the present talk, I will present our recent data that suggest that the sphingomyelin (SM)-enriched domain at the Golgi is essential for the STING activation.

References:

(1) K. Mukai et al., Nat. Commun. 2016

(2) E. Ogawa et al., Biochem. Biophys. Res. Commun. 2018

(3) A. Hansen et al., Proc. Natl. Acad. Sci. USA. 2018

38. Regulation of glycosylphosphatidylinositol biosynthesis

T. Kinoshita¹; ¹Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

Glycosylphosphatidylinositols (GPIs) and glycosphingolipids (GSLs) are two major glycolipids in mammalian cells. GPIs are synthesized in the ER and are mainly transferred by GPI transamidase to many proteins to act as their membrane anchors. Some GPIs are expressed on the cell surface as free glycolipids. GPI-anchored proteins and GSLs interact with each other in the plasma membrane, making lipid microdomains. It is unclear whether their interaction initially occurs during their biosynthesis. GSLs are synthesized in the Golgi. Both GPI-anchored proteins and free GPIs undergo maturation reactions in the Golgi such as side-chain modification. We aimed to identify an enzyme that elongates N-acetylgalactosamine (GalNAc) side-chain by galactose and found that GM1 synthase encoded by B3GALT4 gene also catalyzes galactose transfer to GalNAc side-chain of GPI. We also found that galactose transfer to GPI side-chain is inefficient in the absence of lactosylceramide synthesis. These results show that biosynthesis pathways for GPIs and GSLs are more closely related than expected. In cells defective in GPI transamidase, all GPIs remain as free GPIs and potentially accumulate in the ER. We found that disruption of ERAD-related genes in cells defective in GPI transamidase resulted in up to 10-fold increase in GPI biosynthesis. Therefore, accumulation of GPI is prevented by ERAD-mediated suppression of GPI biosynthesis.

39. What do chemokines see on the cell surface heparan sulfate

B. Connell¹, R. Sadir¹, P. Rueda², C. Laguri¹, F. Arenzana-Seisdedos²,H. Lortat-Jacob¹; ¹Institut de Biologie Structurale, UMR 5075, Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes (UGA), Grenoble, France, ²Institut Pasteur, INSERM U819, Paris, France

Chemokines, a group of proteins promoting oriented cell migration, have two ligands: G protein-coupled receptors (GPCR) through which they signal cells, and glycosaminoglycans (GAGs) such as heparan sulfate (HS). Binding to GAGs has been thought as providing positional information within tissues in such form as immobilized gradients along which cells can migrate directionally. Such a mechanism implies that chemokines can simultaneously recognize GAGs and their signaling receptor; however, the relationships between these two ligands have not been fully addressed. To investigate how GAGs can regulate chemokine functions, we used CXCL12, a chemokine existing in different isoforms, all signaling through CXCR4 but featuring distinct GAG binding domains. We observed that mice carrying a Cxcl12 gene mutation that precludes interactions with GAGs, while not affecting CXCR4-dependent cell signaling, display a marked impaired capacity to support revascularization following acute ischemia, demonstrating the necessity of GAG binding to elicit a functional response. Using cellular assays, we confirm that in the absence of GAGs, the CXCL12γ isoform only displayed reduced signaling and chemotactic activities although it binds to CXCR4 with very high affinity. This is caused by the HS-binding sequence, which mediated high-affinity, but nonproductive, binding to CXCR4. HS prevents this non-functional presentation of CXCL12γ to its receptor, thus strongly promoting the activity of the chemokine. Finally, we developed a chemo-enzymatic approach to generate ¹³C-labeled HS and characterize the structural attributes of the HS-CXCL12 complex using multidimensional NMR spectroscopy. Our data identify important sulfo-group for the interaction and showed that the HS-dependent regulation of chemokine functions directly modulates receptor ligation and activation, thus extends beyond the simple process of immobilization.

40. Glycosphingolipid-lipid interactions and domain formation in model bilayer membranes

S. Hanashima¹, M. Murata¹; ¹Department of Chemistry, Graduate School of Science, Osaka University

Sphingomyelin (SM), glycosphingolipids (GSLs) and cholesterol (Cho) are the important lipids for the formation of the biologically functional membrane domains, lipid rafts. We had examined the lipid interactions of SM, glycosphingolipids and Cho in model membranes using solid state ²H NMR. The data using SM with position selective deuterations revealed that the SM domains can be formed through both SM-SM interactions at the interface of the membranes and SM-Cho interactions in the interior of the bilayers [1].

The distribution of ganglioside GM1 in SM domain was examined using fluorescent microscopy and spectroscopies in model membranes. SM and GM1 are the typical raft lipids both share an identical ceramide lipid but the headgroup structures are quite different. Therefore, the difference of the Cho interactions with SM or GM1 in SM-GM1 mixed bilayer could be deeply related to the domain formation in membrane bilayers. When GM1 concentration was low (SM/GM1 < 10), the fluorescent GM1 probe and SM probe, both highly mimic the membrane properties of the original lipid, localized together in SM domains. In contrast, increase of the GM1 ratio not only reduced distribution of the GM1 probe in SM domains, but also diminished the domains size. Interestingly, increase of the GM1 ratio enhanced membrane affinity of cholestatrienol, which is a structurally similar Cho analogue having fluorescence. When GM1 methyl-ester was used instead of the native GM1, the domain size was not significantly altered. Therefore, the negative charge of GM1 would play an important role to regulate GM1 localization and domain size in model membrane as well as in the biological membranes.

[1] Yano, Y.; Hanashima, S.; Yasuda, T.; Tsuchikawa, H.; Matsumori, N.; Kinoshita, M.; Al Sazzad, M. A.; Slotte, J. P.; Murata M. Biophys. J. 2018, 115, 1530-1540.

41. Establishment of a novel glycan antibody for fucosylated haptoglobin and its possible implication of clinical investigation

E. Miyoshi¹; ¹Osaka University Graduate School of Medicine, Department of Molecular Biochemistry & Clinical Investigation

Fucosylation is one of the most important glycosylations involved in cancer and inflammation. We previously found that the serum level of fucosylated haptoglobin (Fuc-Hpt) was significantly increased in pancreatic cancer patients. To delineate the mechanism underlying this increase and develop a simple detection method, we set out to generate a novel glycan monoclonal antibody specific for Fuc-Hpt. After multiple screenings by enzyme-linked immunosorbent assay (ELISA), a 10-7G mAb was identified as being highly specific for Fuc-Hpt generated in a cell line as well as for Hpt derived from a pancreatic cancer patient. As a result from affinity chromatography with 10-7G mAb, followed by Western blot using lectin and mass spectrometry analyses, it was found that 10-7G mAb predominantly recognized both Fuc-Hpt and prohaptoglobin (proHpt), which was also fucosylated. We also identified an epitope of 10-7G mAb, which recognizes both haptoglobin and its glycan changes. In immunohistochemical analyses, hepatocytes surrounding metastasized cancer cells were stained by the 10-7G mAb, but neither the original cancer cells themselves nor normal hepatocytes exhibited positive staining, suggesting that metastasized cancer cells promote Fuc-Hpt production in adjacent hepatocytes. Serum level of Fuc-Hpt determined with newly developed ELISA system using the 10-7G mAb, was increased in patients of pancreatic and colorectal cancer. In this novel ELISA system, the specificity for diagnosis of pancreatic cancer was increased and the sensitivity was decreased, compared to conventional lectin-antibody ELSA we developed. Deletion of Hpt1-1 phenotype increased AUC value for diagnosis of pancreatic cancer as a result from ROC analysis. The epitope of 10-7G mAb was determined with inhibition assay and alanine substitution. Interestingly, dramatic increases in Fuc-Hpt levels were observed at the stage IV of colorectal cancer, especially in cases of liver metastasis. These results indicate that the 10-7G mAb is a promising antibody for cancer-associated haptoglobin, which recognizes Fuc-Hpt as well as proHpt and could be a useful diagnostic tool for detecting liver metastasis of cancer even if the metastasis is not diagnosed with imaging test.

42. His career, work, and wishes

A. Suzuki¹; ¹Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University

Tamio Yamakawa passed away on Oct. 7, 2018 at the age of 96, just 13 days before his 97th birthday. Amazingly, his mind remained sharp even just before hospitalization for a critical fall in his living room.

He started his laboratory work in the chemistry department of the Institute for Infectious Diseases, University of Tokyo, in the summer of 1946 when he was a third-year medical student. This training was terminated by an explosion produced by himself in the process of an oxidation reaction. He suffered life-threatening burns but did not abandon chemical approach for his further medical investigation. After graduation he joined the chemistry department of the same institute and began work on the chemical synthesis, purification, and structural determination of fatty acid derivatives. Then, his belief that medical research must employ pure substances with defined structures by quantitative determination was fortified.

At the age of 30, in 1951, he published a paper describing the isolation of a glycolipid with the structure, Cer-Gal-Gal-prehemataminic acid, which he named hematoside, from horse erythrocyte membranes. Hematoside was later confirmed to be GM3, and prehemataminic acid turned out to be sialic acid. Klenk named a glycolipid of human brain ganglioside and its acidic component neuraminic acid in 1941, and hematoside would be acknowledged as the first demonstration that glycolipids are localized at the cell membrane. Yamakawa greatly respected Klenk and they remained friends until Klenk’s death. In 1952, Yamakawa isolated a glycolipid from human erythrocyte membranes and named it globoside, Gb4Cer. This work proceeded the finding of ABO blood group antigen activities in erythrocyte glycolipids.

The distinct structure difference in major erythrocyte glycolipids among mammalians caused his wish to understand why this species difference is produced. His second wish was to understand how the structure difference between brain gangliosides and erythrocyte glycolipids is produced. His wishes affected the choice of research direction of my group and aided the identification of CMP-NeuNAc hydroxylase and several mouse genes responsible for tissue-specific expression. He profoundly influenced large numbers of people with his generous and unique character.

43. Analysis of electrostatic interaction between ganglioside GM3 and insulin receptor

K. Kabayama¹; ¹Department of Chemistry, Graduate School of Science, Osaka University

In recent years, some examples have been reported in which gangliosides control the activity of membrane proteins, particularly receptor tyrosine kinases. Insulin receptor is one of them and is known to interact with ganglioside GM3, resulting in the suppression of signal transduction. This interaction has attracted interest because it gives rise to type 2 diabetes by causing insulin resistance. However, there was no way to demonstrate the actual mechanism involved. Therefore, we constructed a model system that can analyze the interaction between insulin receptor and GM3 using liposome. Investigation of lipid ratio of liposome and 3D imaging made enables us to observe slight differences of peptide localization. This model would be useful for detailed analysis of electrostatic interactions.

44. Probing glycan-glycan binding protein interactions using a chemoenzymatic approach

P. Wu¹; ¹The Scripps Research Institute

Today, most glycan microarrays are fabricated by coupling individual sugars synthesized chemically to N-hydroxysuccinimide ester functionalized glass slides. The construction of such microarrays is time-consuming and requires highly specialized experts. It is difficult for scientists who are interested in studying the biological functions of glycans to fabricate such microarrays in their own labs. In addition, glycans attached to solid supports do not fully recapitulate their natural arrangements on the cell surface. To address these problems, we have developed a fast and cost-effective method for the assembly of cell-based glycan arrays to probe glycan-glycan-binding protein interactions directly on the cell surface. In this talk, I will discuss the design and applications of such an array platform.

45. Processing of GPI-anchors in the endoplasmic reticulum

M. Fujita¹; ¹Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University

Most secretory and membrane proteins are synthesized in the endoplasmic reticulum (ER), where they are post-translationally modified, folded and oligomerized. Before exiting from the ER, proteins are monitored by a quality control system that ensures correct folding. In the ER, a number of proteins are modified by a glycolipid, named glycosylphosphatidylinositol (GPI). Soon after GPI is attached to proteins, an acyl chain on the GPI-inositol is removed by PGAP1 (post-GPI attachment to proteins 1), a GPI-inositol deacylase. This is crucial for switching GPI-anchored proteins (GPI-APs) from protein folding to transport states. We performed haploid genetic screens to identify factors regulating GPI-inositol deacylation, identifying 7 genes. In particular, calnexin cycle impairment caused inefficient GPI-inositol deacylation. Calnexin was specifically associated with GPI-APs, dependent upon N-glycan and GPI moieties, and assisted efficient GPI-inositol deacylation by PGAP1. Under chronic ER stress, caused by misfolded GPI-APs, inositol-acylated GPI-APs were exposed on the cell surface. These results indicated that N-glycans participate in quality control and temporal ER retention of GPI-APs, ensuring their correct folding and GPI processing before exiting from the ER. Once the system is disrupted by ER stress, unprocessed GPI-APs become exposed on the cell surface.

46. Plasma membrane β-glucocerebrosidases in neuronal differentiation and senescence

M. Aureli¹, M. Samarani¹, E. Ferrari¹, G. Lunghi¹, N. Loberto¹, S. Prioni¹, E.V. Carsana¹, A. Prinetti¹, S. Sonnino¹; ¹Department of Medical Biotechnology and Translational Medicine, University of Milan

In mammalian cells two different enzymes actively involved in the homeostasis of the glycosphingolipid glucosylceramide were described so far. β-glucocerebrosidase 1 (GCase) is an enzyme mainly associated with lysosomes and only partially with the plasma membrane; whereas the non-lysosomal β-glucosylceramidase (NLGase) is considered a cell surface enzyme.

Interestingly, in neurons, differently from the other cells, we found that during the differentiation, the activities of the plasma membrane associated GCase and NLGase increase and, in fully differentiated neuron and mature brain, NLGase becomes the main β-glucocerebrosidase activity.

Despite it is nowadays clear that mutations loss of function in one of these two enzymes induce glucosylceramide accumulation with the onset of neuronal degeneration, scant are the information related to the molecular mechanism involved. We recently described that lysosomal accumulation of catabolized materials induces an aberrant increase in the plasma membrane of glycohydrolases, especially NLGase, responsible for the ectopic production of ceramide that causes the onset of cell damage. In particular, in case of GCase loss of function, we found that the glucosylceramide accumulation is not limited to lysosomes but occurs also at the plasma membrane level. In addition, we have demonstrated that cells react to GCase deficiency by up-regulation of NLGase. The existence at the plasma membrane of high levels of NLGase and its substrate glucosylceramide let to speculate to the production of ectopic cytotoxic ceramide. These data suggest a mechanistic link between GCase loss of function and the onset of neuronal degeneration.

Supported by Cariplo Foundation grant 2015-1017 to MA

47. Formation of tubules and helical ribbons by galactosylceramide and ceramide phosphoethanolamine-containing membranes

T. Kobayashi³, T. Inaba², M. Murate³, N. Tomishige³, Y. Lee², F. Hullin-Matsuda¹, N. Humbert³, B. Pollet³, Y. Mely³, Y. Sako², P. Greimel²; ¹INSERM, ²RIKEN, ³Strasbourg University

The vertebrate nervous system is characterized by ensheathment of axons with myelin, a multilamellar membrane enriched in galactosylceramide (GalCer). GalCer is one of the few natural lipids that forms helical ribbons in aqueous solution. These helical ribbon structures have been postulated to be stabilized by intermolecular hydrogen bonds between GalCer molecules. Although the detailed molecular mechanism is not fully understood, it has been speculated that planar lipid sheets roll up into cochleate cylinders to form myelin.

Unlike vertebrates, invertebrates such as Drosophila do not have myelin, but specialized glial cells that ensheath individual axons and fascicles of axons. Recently, ceramide phosphoethanolamine (CPE) was identified as a key player in axonal wrapping by glia in Drosophila. CPE is a phosphosphingolipid analog of sphingomyelin featuring a phosphoethanolamine instead of a phosphocholine headgroup Similar to GalCer, CPE:phosphatidylcholine (PC) mixtures tend to form tubules and helical ribbons. The formation of helical structures is related to strong intermolecular hydrogen bonds between CPE molecules and the hydrophobic mismatch between CPE and PC acyl chains.

Our results suggest that the ensheathment of axons in mammals and Drosophila is based on similar physical processes with different lipids.

48. Bacteriophage internalization into eukaryotic cells by binding to polysialic acid

T.A. Lehti¹, M.I. Pajunen¹, M.S. Skog¹, J. Finne¹; ¹Molecular and Integrative Biosciences, University of Helsinki, Finland

There are observations which suggest that interdomain gene exchange from bacteria to eukaryotes has occurred during evolution, but the cellular and molecular mechanisms have remained obscure. Bacteriophages are the most numerous replicating entities in the biosphere and are powerful vehicles for horizontal gene transfer in bacteria. Glycoconjugates on the surface of bacteria often serve as receptors for bacteriophages and have sometimes parts which are structurally similar to those of eukaryotic cells. The structurally shared glycans may therefore potentially serve as binding sites for phages on the eukaryotic cell surface. The question arises whether bacteriophages could exploit the binding sites on the cell surface for the penetration into the cells, and be potentially available for gene exchange phenomena. The eukaryotic cell membrane separating the prokaryotic and eukaryotic gene pools has, however, generally been thought not to allow the passing of phages inside the cell. Polysialic acid is a surface carbohydrate of some pathogenic bacteria and a cell surface component of some mammalian cells. We found that bacteriophages using polysialic acid as their bacterial binding target bind in addition to their E. coli hosts also to the cell surface of human neuroblastoma cells expressing polysialic acid. The bound bacteriophages were internalized via the endolysosomal route inside the cells and remained infective for up to one day, without affecting the viability of the cells. Ultimately the nucleic acid of the phages became exposed and was degraded. The bacteriophage–cell interaction discovered demonstrates a previously unrecognized concept that bacteriophages can penetrate eukaryotic cell membranes and gain access into the interior of the cells. This may suggest a potential mechanism to explain prokaryote–eukaryote gene flow with an important impact in evolution. Cell surface carbohydrates may serve a role in this exchange by presenting structures shared by glycoconjugates of bacteria and eukaryotes and used by bacteriophages as binding sites.

49. Cytosolic gelectins as sensors for endo-lysosmal damage

F. Liu¹; ¹Academia Sinica, Taipei, Taiwan

Galectins are β-galactoside-binding lectins containing one or two conserved carbohydrate-recognition domains (CRD). They do not contain a classical leader sequence and are found mostly inside the cells, although they can be released by cells. Both extracellular and intracellular functions have been demonstrated for these proteins. The intracellular functions have largely been linked to the proteins interacting with other intracellular partners in a carbohydrate-independent fashion. However, a picture that has emerged recently is galectins can bind to glycans on endosomes or lysosomes that become exposed to the cytosol when these organelles are damaged. Galectin-8 has been shown to bind to Salmonella-damaged vesicles and target these vesicles for autophagy, resulting in degradation of the bacteria. We found galectin-3 was accumulated around Listeria monocytogenes escaping from phagosomes, through binding to host N-glycans on the membrane of damaged phagosomes that contained the bacteria. Moreover, through this mechanism, galectin-3 suppressed autophagic activation induced by the bacterial infection. We also observed appearance of intracellular galectin-8 aggregates in the gastric epithelial line AGS exposed to Helicobacter (H.) pylori. Here, cytosolic galectin-8 accumulates around damaged lysosomes in response to H. pylori infection in a host O-glycan-dependent manner. In the case of infection of macrophages by Listeria monocytogenes, we also noted treatment of cells with sialidase before the infection resulted in increased galectin-3 accumulation and decreased galectin-8 recruitment at damaged phagosomes, and led to a reduced autophagic response. Thus, cytosolic galectins may sense changes in glycosylation on the cell surface and modulate the cellular response through differential recognition of glycans on damaged organellar membranes.

50. Role of laccer in LPS-mediated innate immunological functions

H. Nakayama², N. Yokoyama¹, K. Iwabuchi²; ¹Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, ²Laboratory of Biochemistry, Juntendo University Faculty of Health Care and Nursing, Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine.

Host innate immune cells identify invading pathogens through pattern-recognition receptors (PRRs), which mediate a diverse array of immunological functions and display gene homology across species. Therefore, a large number of studies <of these innate responses> using mice have been conducted. However, critical differences exist among species and/or cell types. For instance, human neutrophils abundantly express Lactosylceramide (LacCer, CDw17) on their surfaces, whereas mice neutrophils slightly express it. LacCer forms lipid rafts associated with palmitoylated signal transduction molecules, such as Lyn and Gαi, and these domains work as not only PRRs but signal transduction platforms for other PRRs. Toll-like receptor 4 (TLR4), which binds lipopolysaccharide (LPS) and activates through MyD88, highly expresses on the plasma membranes of human monocytes and mice phagocytes. In contrast, TLR4 is hardly detected on the cell surface of human neutrophils. CD14 and CD11b/CD18-integrin are also well known to transduce the LPS-mediated signaling in human neutrophils, and are abundantly expressed on the plasma membranes. However, how CD14 and CD11b/CD18 coordinate LPS binding signals remains unclear because these molecules are devoid of any catalytic motif in the cytoplasm region. We recently found that LacCer-enriched lipid rafts are involved in LPS-mediated neutrophil phagocytosis through both CD14 and CD11b/CD18. On the other hand, CD14 was necessary and sufficient to transduce monomeric LPS binding signaling through Lyn-coupled LacCer-enriched lipid rafts. Moreover, LacCer-enriched lipid rafts regulate not only CD14-dependent LPS signaling but bind directly to Salmonella minnesota R595-derived LPS. In addition, LPS induced a certain chemokine from human neutrophils, and the chemokine production was CD14-dependent but MyD88-independent. In my talk, I will discuss how LacCer-enriched lipid rafts involve LPS-mediated neutrophil immunological functions and propose a novel biological mechanism that explains how human neutrophils regulate LPS signaling.

51. When O-glcnac meets the clock

P. Lefebvre¹; ¹Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1011- EGID, F-59000 Lille, France

The cellular clock is phasing cell activities with a roughly 24-hour period and is synchronized by external cues such as light and food intake. O-GlcNAcylation is a reversible protein post-translational modification integrating nutritional and metabolic signals to regulate protein dynamics and activities. While there are clear evidence that O-GlcNAcylation regulates the cellular clock by targeting molecular component of this cellular clock, whether a reciprocal regulation exists is unclear. REV-ERBα is a cyclically expressed heme-regulated nuclear receptor regulating hepatic lipid metabolism and also a component of the clock machinery. An interactomic approach showed that O-GlcNAc transferase (OGT) is a REV-ERBα interactant which protects cytoplasmic OGT from proteasomal degradation while increasing nuclear OGT stability and activity. Consequently, the cyclic expression of REV-ERBα imposed a cyclical O-GlcNAcylation on a variety of proteins, including the cytoplasmic insulin-regulated protein kinase AKT/PKB and the nuclear ten-of-eleven translocation (TET) enzymes. The cyclical regulation of this two pathways is hypothesized to concur to the proper regulation of the lipogenic Srebf1 gene during the fasting/feeding transition, by controlling cellular insulin sensitivity and epigenomic events.

52. Application of glycated proteins and related analytes in clinical diagnosis. beyond glycated hemoglobin and diabetes

N. Rabbani¹; ¹University of Warwick

Assay of protein glycation has found clinical use in diabetes for assessment of glycemic control by measurement of glycated hemoglobin A1C, glycated albumin and serum fructosamine. This exploits the link of early-stage glycation adducts of glucose, fructosamines, with increased glucose exposure. Later-stage adducts are advanced glycation endproducts (AGEs), formed by the degradation of fructosamines and by glycation of proteins by reactive dicarbonyl metabolites such as glyoxal, methylglyoxal and 3-deoxyglucosone (3DG). Major AGEs in vivo are: methylglyoxal-derived hydroimidazolone (MG-H1), 3DG-derived hydroimidazolones (3DG-H), Nε-carboxymethyl-lysine (CML), and glucosepane (GSP). The trace-level fluorescent AGE, pentosidine, is also widely studied. Glycated proteins undergo cellular proteolysis with release of glycated amino acids, also called glycation free adducts, into plasma which are excreted in urine. Recent research has suggested wider application in clinical diagnosis.

GSP is derived from fructosamine, is stable and provides a better cumulative assessment of increased glucose exposure. MG-H1 is a major AGE quantitatively and reports on exposure to increased levels of MG. Measurement of plasma protein MG-H1 and plasma MG-H1 free adduct has been linked to the diabetic and non-diabetic vascular disease.

Increased proteolytic release of AGE free adducts from arthritic joints into plasma, linked in diagnostic algorithms with hydroxyproline – a biomarker to the bone remodeling, provided the basis of a blood test for early-stage detection and classification of arthritis with 91 – 99% accuracy.

Autism Spectrum Disorder (ASD) is a developmental disorder that affects communication and behavior. Clinical chemistry tests for ASD are currently unavailable. Algorithms to discriminate between ASD and healthy controls gave strong diagnostic performance with features: plasma protein AGEs - CML, 3DG-H and Nε-carboxymethyl-arginine, and oxidative damage marker, dityrosine; accuracy 88%.

53. Resolving unusual N-glycans in lower eukaryotes

K. Paschinger¹, A. Hykollari¹, S. Yan², B. Eckmair¹, I. Wilson¹; ¹Department für Chemie, Universität für Bodenkultur, Wien, ²Institut für Parasitologie, Veterinärmedizinische Universität Wien, Wien

Sugars cover the surfaces of all cells and a major class of these glycoconjugates are the asparagine-linked oligosaccharides attached to glycoproteins (N-glycans). The classical view has been that N-glycans in ‘lower’ eukaryotes (protists, fungi, plants and invertebrates) are simple. However, recent data show that, other than plants, the diversity of N-glycan modifications in non-vertebrate species is extremely high. From our own studies on insect and nematode species, a wide range of new glycan modifications have been demonstrated on enzymatically-released N-glycans. Using solid phase extraction, fluorescent labelling and RP- and NP-HPLC for isobaric and isomeric glycan separation prior to MALDI-TOF MS, we reveal a variety of ‘charged’ modifications of N-glycans from insects, such as glucuronic acid, sulphate phosphoethanolamine and phosphorylcholine; in addition, unusual core mannosylation as well as up to three branches increase the degree of glyco-structural ‘polymorphism’. In nematodes, a range of unusual highly-modified core and bisected structures are to be found in the model organism Caenorhabditis elegans, whereas in Dirofilaria immitis anionic N-glycans (specifically carrying glucuronic acid) were found for the first time in this phylum. Complementary to the MS analyses, example natural N-glycan pools were immobilised and tested for binding to antibodies, lectins and pentraxins. Our data show that the concept of ‘simple’ organisms possessing only ‘simple’ glycans is not a true reflection of the actually complex biosynthetic glycosylation capacity of invertebrates; thereby, the genomic and proteomic code of these organisms is extended by another layer of biological information due to their glycomes.

54. O-Glcnacylation on xiap inhibits colon cancer cell growth and invasion via proteasomal degradation of ogt

H.G. Seo¹,J.W. Cho¹; ¹Glycosylation Network Research Center, Yonsei University

O-GlcNAc transferase (OGT) catalyzes O-GlcNAc modification of nucleocytoplasmic proteins and this enzyme is highly expressed in many types of cancer. However, the mechanism regulating OGT expression in cancer cells is not well understood. Here, we demonstrate that OGT is a substrate of the E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) which has a broad role in cancer biology. Interestingly, OGT catalyzes the O-GlcNAc modification of XIAP at serine 406 and this modification is required for the E3 ubiquitin ligase activity of XIAP toward specifically OGT. Moreover, O-GlcNAcylation on XIAP suppresses colon cancer cell growth and invasion through promoting the proteasomal degradation of OGT. Therefore, our study provides a novel molecular mechanism for cancer growth and invasion regulated by OGT and O-GlcNAc modification.

55. The WclY gene of escherichia coli serotype o117 encodes an alpha1,4-glucosyltransferase with strict acceptor specificity but broad donor specificity

A. Kocev¹, V. Torgov², L. Danilov², V. Veselovsky², I. Brockhausen¹; ¹Queen’s University, Kingston, Canada, ²Zelinsky Institute of Organic Chemistry, Moscow, Russia

The O antigen of Escherichia coli serotype O117 consists of repeating units with the structure [D-GalNAc(β1-3)-L-Rha(α1-4)-D-Glc(α1-4)-D-Gal(β1-3)-D-GalNAc]n. The O antigen biosynthesis gene cluster reveals the presence of four putative glycosyltransferase genes, wclW, wclX, wclY and wclZ, as well as wzy and wzx suggesting that the O117 repeating unit is sequentially assembled via the polymerase-dependent pathway.

Many of the glycosyltransferases that synthesize O-antigenic oligosaccharides have an absolute requirement for the diphosphate-lipid moiety of the acceptor substrate. We have chemically synthesized a natural acceptor analog for the third step of the O117 repeating unit assembly catalyzed by putative glucosyltransferase WclY: P1-(11-phenoxyundecyl)-P2-(2-acetamido-3-O-β-D-galactopyranosyl-2-deoxy-α-D-galactopyranosyl) diphosphate (Galβ1-3GalNAc-PP-PhU). We showed that this compound was highly active as an acceptor substrate for WclY using UDP-Glc as the donor substrate. In contrast, Galβ1-3GalNAcα-O-benzyl was not active as a WclY acceptor substrate, indicating the need for the diphosphate moiety. The full length His6-tagged WclY protein was expressed in Escherichia coli Lemo21 (DE3) cells and showed characteristics of retaining glycosyltransferases of the GT4 family. A number of mutants of WclY were created that showed various activities with UDP-glucose or UDP-GlcNAc, or both, as well as with UDP-Gal and UDP-GalNAc, suggesting broad donor specificity. Mutation of Arg194 to Cys allows high GlcNAc-transferase activity. Mutants of the Glu263 and Glu271 residues of the EX7E motif were inactive, whereas mutations of the Asp residue of the DxD motif had no effect on activity. Characterization of this enzyme opens the door for the testing of inhibitors and potential therapeutic agents for bacterial infections. The technology for the enzymatic assembly of repeating units can be a basis for vaccine development.

This work was supported by NSERC and GlycoNet.

56. Siglecs as checkpoints in regulation of immune responses

J.C. Paulson¹; ¹The Scripps Research Institute

The sialic acid-binding immunoglobulin-type lectin family of cell adhesion receptors called Siglecs comprise 14 members in humans and 9 members in mice that are predominately expressed on white blood cells of the immune system. Many siglecs carry regulatory motifs that act as checkpoint inhibitors that regulate immune responses, and help the immune system distinguish between self and non-self. We have developed a liposomal nanoparticle platform that exploits the inhibitory functions of siglecs for suppression of antigen induced immune cell activation. Key to this platform are synthetic ligands that bind with high avidity and high specificity for a single Siglec. The tolerogenic nanoparticles display an antigen and a ligand of an inhibitory Siglec expressed on the cells that recognize the antigen. When an immune cell receptor recognizes the antigen, the Siglecs are recruited to the immunological synapse, suppressing the immune response and effectively inducing tolerance to the antigen. These siglec ligand-decorated antigenic nanoparticles show potential for suppression of antigen-mediated immune responses of B cells and mast cells. (NIH grants AI050143, AI099141, HL107151).

57. Liver-dependent sialylation and immune regulation

D. Oswald¹, M. Jones¹, B. Cobb¹; ¹Case Western Reserve University School of Medicine, Cleveland, OH

Changes in the glycans among plasma glycoproteins have been recognized as biomarkers of disease for many years, with loss of galactosylation being a known hallmark of ongoing inflammation. In our recently published work, we have also found that specific changes in these glycans can predict the increased risk of developing cardiac disease among HIV-positive individuals. Here, we have created a hepatocyte-specific knockout of ST6Gal1 (HcKO), the enzyme responsible for adding sialic acid to underlying galactose residues in an α2,6-linked fashion. We have found that this single glycan change leads to other unanticipated glycosylation changes among plasma glycoproteins which are not recapitulated by changes in the expression of any glycosylation-associated gene, and an age-dependent development of fatty liver disease. Interestingly, changes in IgG glycosylation were also not as predicted, and glycome changes in plasma glycoproteins, including decreased fucosylation and increased bisecting GlcNAc, precede liver pathology suggesting that these alterations may play an active role in disease. Moreover, we have found that HcKO mice have increased susceptibility to T cell-dependent, but not innate or passively-induced inflammatory disease. These data support a central role for hepatocytes in regulating certain immunologic functions, and that glycosylation changes often associated with inflammatory disease may be active participants in disease progression rather than merely biomarkers.

58. Tamio yamakawa award

N. Taniguchi¹; ¹Osaka International Cancer Institute

The Japan Consortium for Glycobiology and Glycotechnology (JCGG) was established a program intended to facilitate the exchange of research information, to promote research cooperation among glycoscientists, and to encourage young scientists to become interested in this field of research. In 2015 JCGG started the Tamio Yamakawa Award, to honor the distinguished career of Prof. Tamio Yamakawa and his outstanding contributions to the field of Glycoscience. The award is based on major funding from a donation from Sialic Acid Society managed by Prof. Haruo Ogura. The award is presented every two years to a glycoscientist who has made widely recognized and major contributions to the field of Glycoscience and is, in principle, a currently active researcher. JCGG announced that Dr. Markus Aebi, Professor of Mycology, Institute of Microbiology, the Swiss Federal Institute of Technology (ETH Zurich) is the recipient of the 2016 Tamio Yamakawa Award. The award ceremony was held on Nov. 1 on the occasion of the 14th JCGG annual symposium where he delivered the Tamio Yamakawa Lecture entitled “The Making of N-Glycoproteins”.

Although Dr. Yamakawa unfotunately passed away and he could not show up in the award ceremony, Tamio Yamakawa Award, the second award, went to Dr. Gerald W. Hart, Prof. of Johns Hopkins University School of Medicine, currently Professor and Georgia Research Alliance Eminent Scholar, University of Georgia, who found O-GlcNAc modification of proteins in the cytoplasma and created a novel research field in Glycoscience. The Award ceremony and Tamio Yamakawa Award Lecture was held in the 16th JCGG Symposium scheduled November 26-27, 2018 in Hongo, Tokyo University Hongo Campus.

59. Glycosylation in cancer

N. Taniguchi¹; ¹Osaka International Cancer Institute

Over the past 30 years, our group has purified and cloned a number of glycosyltransferases that are responsible for the biosynthesis of branching in N-glycans. We have identified the target glycoproteins of those enzymes, and explored the structural and functional significance of a series of glycosyltransferase genes (glycogenes) such as GnT-III, IV, V, VI, IX(Vb) and Fut8. We have also found that N-glycosylation abnormalities are one of the causative agents for cancer, Alzheimer’s disease and COPD (chronic obstructive pulmonary disease). In this symposium the roles of GnT-III, GnT-V and Fut8 which catalyze the biosynthesis of bisecting GlcNAc, β1-6 GlcNAc branching and core fucose, respectively will be mainly discussed in relation to cancer. GnT-III and GnT-V modulate adhesion molecules such as E-cadherin and integrins and regulate EMT(epithelial to mesenchymal transition) and cancer metastasis. In addition, they modulate also growth factor receptors such as EGFR and their signaling pathway and play pivotal roles in cancer metastasis. Addition of bisecting GlcNAc to N-glycans controls the additional elongation of glycans by GnT-II, IV and GnT-V etc. by competing for substrate specificity and steric hindrance.

High levels of the expression of a 1,6 Fucosyltransferase (Fut8) are now being used as a biomarker for primary hepatomas, pancreatic cancer and non-small lung cancer as well as others. The lack of a core fucose results in growth retardation and emphysematous changes in the lung due to a lack of TGF-β-signaling. The significance of core fucose units is also well known because of its importance in ADCC (antibody dependent cellular cytotoxicity) in antibody therapy. Core fucose is also involved in T-cell function. It is now well known that PD-1(programed cell death 1) is highly implicated in T cell immunity. This is one of the topics in check point therapy in cancer. Inhibiting the activity of Fut8 by genetic ablation or pharmacologic inhibition reduced the cell-surface expression of PD-1 and enhanced T cell activation, suggesting a possible strategy for efficiently inhibiting tumors. In conclusion aberrant glycosylation caused by these glycan genes related to N-glycan branching may play key roles in the prevention of, the diagnosis of and therapeutics for cancer.

60. Compartmentalization of class I-α-mannosidases ensures ordered N-glycan processing and degradation of misfolded glycoproteins

R. Strasser¹; ¹Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria

Arabidopsis thaliana has a family of five class I α-mannosidases (MNS1 to MNS5) involved in N-glycan processing and protein quality control. Failure of mannose trimming from oligomannosidic N-glycans in the endoplasmic reticulum (ER) and cis/medial-Golgi leads to a defect in root development. Interestingly, the root growth phenotype that is linked to MNS1 to MNS3 deficiency can be suppressed by alg mutants with distinct defects in the biosynthesis of the lipid-linked oligosaccharide precursor. Genetic and biochemical data indicate that the defect is uncoupled from the glycan-dependent ER-associated degradation (ERAD) pathway that removes misfolded glycoproteins with distinct oligomannosidic N-glycans from the ER. ERAD of a misfolded variant of the heavily glycosylated brassinosteroid receptor BRI1 requires an N-glycan signal that is generated by the two ER-resident proteins MNS4 and MNS5. These two α-mannosidases are also essential for the degradation of numerous topologically diverse ERAD substrates carrying different misfolded domains. By contrast, the N-glycan processing enzymes MNS1 to MNS3 are stringently located in the Golgi to prevent the interference with ERAD and the uncontrolled degradation of misfolded glycoproteins.

61. Biological functions of siglecs expressed on myeloid cells

G. Wu¹, G. Murugesan¹, M. Nagala¹, E. Sands¹,P.R. Crocker¹; ¹University of Dundee

We have used mouse models to investigate the biological functions of siglecs, focusing in particular on siaoladhesin (Sn, siglec-1), an adhesion receptor expressed exclusively by cells of the mononuclear phagocyte system, and siglec-E, an inhibitory receptor expressed constitutively on neutrophils and upregulated on LPS-stimulated macrophages. Sialic acid-containing ligands for these siglecs are strongly induced on CD4 T cell populations following T cell receptor triggering. Studies with Sn KO mice have shown that interactions between Sn and ligands on regulatory T cells (Treg) can suppress Treg activation and promote autoimmune inflammatory responses. To understand the nature of glycoprotein counter-receptors recognised by Sn, we have used a proximity-labelling method coupled to mass spectrometry and integrated our findings with proteomics and transcriptomics datasets to gain insights into how T cells upregulate siglec binding activity following activation.

62. Synthesis and immunomodulative functions of symbiotic bacterial lipid A

A. Shimoyama⁴, F. Di Lorenzo¹, K. Mizote⁴, T. Uto⁴, H. Yamaura⁴, N. Shibata², Y. Fujimoto³, J. Kunisawa⁵, H. Kiyono⁶, A. Molinaro¹, K. Fukase⁴; ¹Department of Chemical Sciences, University of Naples Federico II, ²Faculty of Science and Engineering, Waseda University, ³Faculty of Science and Technology, Keio University, ⁴Graduate School of Science, Osaka University, ⁵National Institutes of Biomedical Innovation, Health and Nutrition, ⁶The Institute of Medical Science, The University of Tokyo

The gut microbiota contribute to the maintenance of homeostasis through complicated interactions with host. Recently, human gut Peyer’s patch resident Alcaligenes faecalis was found by Kiyono et al. [1] and its cell surface component lipopolysaccharide (LPS), which is a representative bacterial-derived immune regulator, was suggested to be an important factor in the gut homeostasis [2]. In this study, we characterized A. faecalis LPS and synthesized its active principle lipid A for the elucidation of its immunomodulative functions and development of novel vaccines adjuvants.

At first, we identified A. faecalis LPS and found that its component is lipooligosaccharide (LOS) with shorter oligosaccharide. Although representative Escherichia coli LPS shows high inflammatory and lethal toxicity, A. faecalis LOS showed very low toxicity and weak inflammatory activity but high potency of antibody induction, and was found to be a promising immune adjuvant candidate.

We then determined the chemical structure of LOS by using NMR and MS to be a glycolipid composed of nona-saccharide and multiple fatty acids. Furthermore, we have accomplished the chemical synthesis of A. faecalis lipid A and its derivatives. Synthesized A. faecalis lipid A showed weaker IL-6 and IL-10 inducing activity than E. coli LPS with bell-shaped concentration-dependency. Interestingly, in the presence of E. coli LPS, A. faecalis lipid A promoted the production of anti-inflammatory IL-10 in a concentration-dependent manner. These results suggested that A. faecalis LOS/lipid A is associated with maintenance of homeostasis.

Reference

[1]. Obata, T., Kunisawa, J., Kiyono, H., et al. Proc. Natl. Acad. Sci. USA. 2010, 107, 7419-24.

[2]. Shibata, N., Kunisawa, J., Shimoyama, A., Fukase, K., Kiyono, H. et al. Mucosal Immunology 2018, 11, 693-702.

63. Identifying a constellation of sulfated glycotopes at the omics level by advanced mass spectrometry-based analytical workflows

K. Khoo¹; ¹Institute of Biological Chemistry, Academia Sinica, Taiwan

Despite the well-recognized importance of sulfation on several terminal glycotopes of N- and O-glycans in mediating cell adhesion and other biological process, the full range of the sulfated glycome other than the glycosaminoglycans remains under-explored. We have been devoting our effort to establish a highly sensitive, non-biased sulfoglycomic workflow that would balance throughput with precision and structural details gained. In particular, we have focused on comprehensive LC-MS/MS data acquisition coupled with glycotope dependent MS³ to enable confident assignment of detected sulfated glycotopes down to the level of location of the sulfate. Over the years, we found that certain cell types/tissues carry more varied sulfated glycotopes at higher level than others. The induced pluripotent stem cells and brain tissues are 2 prime examples that continue to spring surprises as we dig deeper into their respective enriched sulfated glycomes. For the brain tissues, we not only identified various Gal-3-O-, Gal-6-O- versus GlcNAc-6-O-monosulfated LacNAc and Le^X glycotopes, but also their previously unreported disulfated counterparts, as well as a few other truly unexpected ones. Our rapid and highly sensitive data acquisition and analysis pipeline now allows us to meaningfully dissect the sulfoglycomic changes induced by genetical manipulations and/or disease onsets. For example, we demonstrated that GlcNAc6ST1 contributed most among other GlcNAc6STs to the expression of GlcNAc-6-O-sulfated glycotopes on N-glycans by comparative sulfoglycomic mapping of samples derived from various sulfotransferase KO mice. When coupled with the use of endo-ß-galactosidase, our concerted workflow further allows us to map the keratan sulfate-like multiply sulfated, branched and unbranched polyLacNAc chains, found to be prominent in the particular iPS cells analyzed. Although moving from sulfoglycomics to sulfoglycoproteomics remains technically daunting, progress is being made also in various complementary mass spectrometry workflows. We can now conclude that the mammalian glycome is selectively layered with an extra complexity of sulfation on virtually all known glycotopes awaiting a full in-depth mapping that is hitherto not feasible.

64. Plasma membrane-associated glycosyltransferases: a new paradigm in the cellular control of glycolipid expression

J.L. Daniotti¹; ¹CIQUIBIC (UNC-CONICET), Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Glycosphingolipids are complex molecules consisting of a ceramide lipid moiety linked to a glycan chain of variable length and structure; they include gangliosides, which are sialosylated glycosphingolipids ubiquitously distributed on the outer layer of the vertebrate plasma membrane. The de novo synthesis of gangliosides starts at the endoplasmic reticulum and continues by a combination of glycosyltransferase activities at the Golgi complex, followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological and pathological processes. A number of enzymes for ganglioside anabolism and catabolism recently shown to be associated with the plasma membrane are able to exert their enzymatic activities on substrates residing on the same surface (cis-catalytic activity) and on that of neighboring cells (trans-catalytic activity). In particular, it was observed that ST8Sia-I is expressed at the plasma membrane (ecto-ST8Sia-I) and synthetizes GD3 ganglioside (cis-catalytic activity). Moreover, it was found in epithelial cells that ecto-ST8Sia-I displayed enzymatic activity towards GM3 ganglioside substrate exposed in the plasma membrane of neighboring cells (trans-catalytic activity) where endogenous ST8Sia-I is not expressed. More recently, we observed in a macrophage cell line that endogenous ecto-ST8Sia-I is present in the Golgi complex and at the cell surface, where the expression was markedly reduced in bacterial lipopolysaccharide-stimulated macrophages. These enzymatic activities, together with those exerting the opposite reactions (i.e. sialidase Neu3), emerge as new mediators for structural changes of plasma membrane-associated gangliosides, thus modifying the membrane organization and function.

65. Plasma membrane-associated sialidase neu3 as a crucial signaling regulator

T. Miyagi², K. Yamamoto⁵, K. Takahashi¹, K. Shiozaki⁴, K. Yamaguchi³; ¹Cell Recognition Study, Tohoku Medical and Pharmaceutical University, ²Division of Cancer Chemotherapy, Miyagi Cancer Center Research Institute, ³Division of Molecular and Cellular Oncology, Miyagi Cancer Center Research Institute, ⁴Faculty Fisheries, Kagoshima University, ⁵Faculty of Health, Saitama Medical University

The plasma membrane-associated sialidase NEU3, a key enzyme for ganglioside degradation, is involved in many cell surface phenomena. Although NEU3 has been considered as a crucial signaling regulator due to its catalytic activity and co-localization at cell surfaces, mechanisms underlying NEU3 activation are not fully understood. In this presentation, the possible mechanisms and functional significance of NEU3 activation and translocation at cell surface will be briefly introduced.

We previously demonstrated the remarkable up-regulation of NEU3 in various human cancers with augmented malignant properties, through activation of EGFR signaling. One of the mechanisms of NEU3 upregulation in cancer may be attributed to transcriptional regulation by Sp1/Sp3 factors, as documented to play critical roles in regulating the transcription of genes involved in cell growth control and tumorigenesis. Second, for detection of the catalytic activity in vitro, chemical detergents, such as Triton X-100, have been used, but recently we discovered phosphatidic acid to be a possible natural activator for the sialidase at neutral pH, and also a regulator of its translocation to and functions at plasma membranes. Third, using human lung cancer tissues and non-small cell lung cancer (NSCLC) cells, we found an evidence of a possible mechanism for regulating the activation of EGFR signaling. NEU3 was up-regulated in lung cancer tissues, compared with non-cancerous tissues, and enhanced the phosphorylation of EGFR at Tyr-845 in NSCLC cells. Interestingly, when the catalytic activity of the sialidase was measured, the cells with EGFR mutations, H1650 and H1975, showed higher endogenous sialidase activity towards gangliosides as compared to wild type A549 cells, indicating that NEU3 contributes to the activation of EGFR signaling through Src-mediated phosphorylation, leading to potentiation of tumorigenesis and then acceleration of malignant phenotype of the lung cancer cells in a positive feedback manner. NEU3 inhibitor may play a pivotal role on regulating EGFR signaling, subsequent to reduction of malignant phenotype of cancer cells.

66. The Golgi architecture and cell sensing

P. Gleeson¹; ¹The Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010,

In contrast to invertebrates, the Golgi apparatus in vertebrate cells consists of individual Golgi stacks laterally fused together in a continuous ribbon structure. The function of the ribbon structure of the Golgi has been enigmatic. Notably, recent advances have identified a role for the Golgi ribbon in regulation of a diverse range of cellular processes, such as mitosis, DNA repair, autophagy, metabolism and proliferation. Key questions under investigation center on the mechanism(s) by which the Golgi regulates these cell pathways and the potential contribution of Golgi glycosylation. Fragmentation of the Golgi ribbon results in modulation of a number of these cellular pathways. Various diseases and disorders, including cancer and neurodegeneration, are associated with the loss of the Golgi ribbon and the appearance of a dispersed fragmented Golgi. Here I will discuss the emerging theme of the Golgi as a cell sensor and highlight the relationship between the morphological status of the Golgi in vertebrate cells and the modulation of cell networks. In some cases, fragmentation of the Golgi has been shown to result in specific changes in glycosylation of membrane proteins which influence the metabolic status of the cell. Until recently the experimental approaches for fragmenting the Golgi ribbon were reliant on the use of drugs such as nocodazole which is a relatively blunt approach and induces multiple cellular effects. More recent approaches have targeted structural proteins of the Golgi which has begun to reveal the complexity of pathways influenced by the Golgi apparatus. Our laboratory has developed a cell system to explore the function of the Golgi ribbon by exploiting a finding that the membrane tether of the trans-Golgi network, GCC88, regulates the balance between Golgi mini-stacks and the Golgi ribbon in a variety of cell types.

67. Galectins as targets for clinical therapy and diagnostics

H. Leffler², U.J. Nilsson², A. Mackinnon³, F. Zetterberg¹, A. Pedersen¹, H.T. Schambye¹; ¹Galecto Biotech AB, Copenhagen, Denmark, ²Lund University, Sweden, ³University of Edinburgh, GB and Galecto Biotech AB, Copenhagen, Denmark

Galectins are small soluble, often abundant, proteins with affinity for extracellular/intravesicular glycoproteins and glycolipids, but also have cytosolic and nuclear activities. Studies in animal disease models, including KO mice, and expression levels in human tissues have suggested rate limiting roles in cancer and inflammatory disease like fibrosis. Experiments in cell culture have provided some possible mechanisms of action, including regulation of receptor organization and residence time at the cell surface and effects on cell migration.

These findings have inspired development of galectin inhibitors as therapeutics, and galectins as part of diagnostics. Galectins have a well characterized about 135 amino acid carbohydrate recognition domain folded as a ß-sheet. Galectins are druggable in the sense that it has been possible to design small molecule very potent inhibitors, even if the carbohydrate-binding site is relatively shallow. Such galectin inhibitors have shown promising effects in mouse models of cancer and also lung fibrosis. One molecule, TD139, is in phase II clinical development as an inhaled drug for idiopathic lung fibrosis.

The roles of galectins in disease can to some degree be related back to their roles in the cell, but not completely or in a simple way. This creates a challenge for defining mechanism of action of galectin inhibitors, and relevant aspects of pharmaco-kinetics and pharmaco- dynamics (PK/PD). At the same time galectin inhibitors can be seen as tools for deeper understanding of galectin function and related cell biology.

As diagnostics the level of galectin-3 in blood has found widespread use as a marker of cardiac insufficiency. Its level in thyroid tissue is also used to distinguish malignant from benignant tumors there. However, the level of specific galectin-binding glycoforms of common glycoproteins in serum may be an even more interesting diagnostic marker, and reflection of galectin function.

68. Development of liver fibrosis diagnostics, M2BPGi, using glycoscience technologies

H. Narimatsu¹; ¹RCMG Inc., National Institute of Advanced Industrial Science and technology (AIST)

Here, we organized a translational research session based on Glycoscience as a session of ACGG. We have been developing disease biomarkers utilizing our own developed glycoscience technologies. The fundamental technologies featured for development of these markers are: 1. Lectin microarray, 2. IGOT-LC/MS and Glyco-Ridge-LC/MS, and 3. Bioinformatics. The lectin microarray is used for selection of the probing lectins that can discriminate the diseased from non-diseased regions on a histopathological specimen. The IGOT-LC/MS technology enables identification of the glycoproteins that bound to the selected lectins from the cell culture supernatants and pathological and serum samples of patients. Similar analysis is conducted using samples from healthy controls to identify the disease-specific glycoproteins. The selected candidate proteins, the number of which is about a few hundreds at this point, are prioritized by bioinformatics. The candidates are then assessed for the feasibility as a biomarker by conventional methods in the order of the given priority. Finally, the lectin-antibody sandwich ELISA system consisted of the most appropriate set of the lectin and glycoprotein is established in consideration of practical applicability.

As a successful example of the markers already established and verified, clinical usefulness, WFA-M2BP (M2BPGi), the liver fibrosis marker, will be introduced in detail. M2BPGi was developed and optimized for simple, easy, and automated assay, and has been approved by the Japanese Pharmaceutical Law for marketing. More than 100 papers describing clinical usefulness of M2BPGi have been published during these 4 years. This is a great success fully supported by glycoscience from the development of basic technologies and their application to the establishment of the clinically significant biomarker.

69. A drosophila model for investigating the neurological defects associated with congenital disorder of glycosylation type IIE

A. Frappaolo², S. Sechi², T. Kumagai¹, R. Fraschini³, A. Karimpour Ghahnavieh², M. Tiemeyer¹, M.G. Giansanti²; ¹Complex Carbohydrate Research Center, University of Georgia, Athens, USA, ²Consiglio nazionale delle Ricerche, Istituto di Biologia e Patologia Molecolari, Università Sapienza di Roma, Roma, Italy, ³Dipartimento di Biotecnologie e bioscienze, Università degli studi di Milano Bicocca, Milano, Italy

Congenital Disorders of Glycosylation (CDG) are caused by mutations in genes required for synthesis of glycoconjugates. Over 100 distinct forms of CDGs have been identified and most of these diseases are associated with severe neurological deficit. CDGs comprise two large groups. Type I CDGs affect the synthesis of the dolichol-linked Glc₃Man₉GlcNac₂ oligosaccharide, or its transfer to acceptor proteins. Type II CDG diseases disrupt either the processing of N-linked glycans, the biosynthesis of O-linked oligosaccharides or the addition of glycans to lipids. The Conserved Oligomeric Golgi (COG) complex mediates tethering of vesicles carrying glycosylation enzymes across the Golgi cisternae. Mutations affecting human COG1, COG2, COG4-COG8 cause monogenic forms of inherited, autosomal recessive, CDGs-II. Patients suffering from COG7-CDG display neurological defects, including psychomotor delay, epileptic seizures and failure to thrive. Yet the correlation between defective glycosylation and the neuropathology is unknown.

We have generated a Drosophila COG7-CDG model, which closely parallels the pathological characteristics of COG7-CDG patients, including pronounced neuromotor defects and altered N-glycome profiles with hyposialylation. Multi-dimensional ion trap mass spectrometry, used to profile N-linked glycoprotein glycans in fly heads from Cog7 mutants, revealed an increased abundance of high mannose, paucimannose and difucosylated N-glycans. Similar to the hyposialylation reported in COG7 patients, the least abundant and most complex sialylated N-glycan detected among the adult head glycans, was decreased in two Cog7 allelic combinations compared to wild type. We have started dissecting the interactions of COG7 protein with Golgi trafficking proteins in Drosophila and human cells. GOLPH3 proteins are essential for proper localization of several Golgi glycosyltransferases. In addition human GOLPH3 interacts with sialyltransferases and affects α2,6 sialylation of N-glycans. Based on these data, we reasoned that GOLPH3 might cooperate with the COG complex in Golgi retention and proper functioning of glycosyltransferases. Co-immunoprecipitation and glutathione S-transferase pull-down experiments indicated that GOLPH3 interacts with the COG complex. Consistent with these data, we found Drosophila Cog7 protein in the list of molecular interactors identified by affinity purification coupled with mass spectrometry (AP-MS) using GOLPH3-RFP, as bait. Moreover the list of potential molecular partners of GOLPH3, identified by AP-MS, included many glycosyltransferases and some proteins involved in neuronal function.

The Drosophila Cog7-CDG disease model, that we developed, offers unique opportunities to clarify the molecular mechanisms underlying COG-CDGs and help identify novel therapeutic strategies.

70. Sighting of unusual N-glycans by mass spectrometry

D. Garozzo¹; ¹CNR Istituto per i Polimeri, Compositi e Biomateriali IPCB

All glycoproteomic investigation methods are based on the knowledge of the synthetic process in the cells. In particular, N-glycosylation is a highly ordered, sequential process that encompasses different cellular compartments. The nascent protein is glycosylated in the endoplasmic reticulum, where the precursor oligosaccharide Glc3Man9GlcNAc2 is first transferred en bloc to the polypeptide chain, and then processed in the Golgi. All the resulting N-linked glycans share the common pentasaccharide core structure of Man3-GlcNAc2 and are distinguished in complex, hybrid, and high-mannose types. The huge variability of N-glycan structures basically relies on the type and position of attached sugars and branching.

Basing on this biosynthetic process and on the knowledge of the enzymes present in the different organisms, the N-glycan analysis by mass spectrometry is thus less complex, so that it is often sufficient to acquire the molecular mass to delineate the structure.

Here we will present three examples dealing with the first description of unconventional N-glycan structures synthesized by totally or partially different biosynthetic pathways.

Primarily, it will be presented the N-glycans characterization of Paramecium Bursaria Chlorella Virus 1 (PBCV-1) major capsid protein (MCP) Vp54. Chloroviruses have a long evolutionary history, probably forgoing the eukaryotes development, thus it has been hypothesized that they could own a different glycosylation machinery. The structures of the four N-linked glycans attached to PBCV-1 MCP consist of a set of oligosaccharides not previously found in all the three domains of life. Very interestingly, these glycan structures are not located in a typical N-X-(T/S) consensus site.

A second example will regard the characterization of serum N-glycans of a patient with ALG12 deficiency (ALG-12 CDG), a CDG type 1 defect. Intact serum transferrin showed, as expected, underoccupancy of N-glycosylation site. Surprisingly, total serum proteins and IgG N-glycans showed some peculiar alterations, consisting in accumulating amount of specific and unusual high-mannose and hybrid structures.

Finally, it will be described very strange case of a patient with multiple genetic mutations that, perhaps interacting, lead to generation of hypersialylated N-glycans structures, never reported before in human serum.

71. Diagnostic significance of serum IgG glycosylation in cancers

S. Ren¹, R. Qin¹, W. Qin¹, J. Han¹, Y. Gu¹, J. Gu¹; ¹School of Basic Medical Sciences, Fudan University, Shanghai, China

Improvements in timely and effective diagnosis of cancer are urgently needed. Most serum proteins are glycosylated, and deregulation of glycosylation has been reported to be associated with a wide range of diseases including cancer. Immunoglobin G (IgG) is a highly abundant glycoprotein in serum and known to mediate a variety of blood immune responses. Aberrant glycosylation has been reported associated with many diseases including cancer. In our project, based on high throughput analytical method with mass spectrometry for relative quantitative analysis of IgG glycans we developed, we demonstrated that the alteration of IgG galactosylation exhibited a common feature in multiple cancer types compared to non-cancer controls by assessing the distribution of IgG galactosylation (referred to as Gal-ratio) in 12 types of cancers using large-size samples collected from multiple hospitals, which indicated that IgG Gal-ratio could be a pan-cancer biomarker for cancer diagnosis. In addition, we found that IgG Gal-ratio had a great performance in detection of pancreatic carcinoma (PC) and could be used to assist CA19-9 in improving diagnosis performance through early stage detection, differentiation from benign pancreatic disease, and PC diagnosis with CA19-9-negative level. Furtherly, we developed a simple and reliable N-glycome quantitation method by providing Bionic Glycome as internal standards which can be used to assess the relative alteration of IgG glycans in their quantity among individual samples. Therefore, the information of percentage and relative amount of IgG glycans can be compared and combined to assess their diagnosis significance in cancers as well as provide more insights into underlying mechanism.

72. Sorting determinants for differential ER exit of GPI-anchored proteins

S. Rodriguez-Gallardo⁴,S. Sabido-Bozo⁴, M. Nakano³, K. Funato³, V. Zoni², S. Vanni², K. Kazuo¹, A. Nakano¹, M. Muñiz⁴; ¹RIKEN, ²University of Fribourg, ³University of Hiroshima, ⁴University of Seville

Protein sorting upon vesicular transport in the secretory pathway is crucial to maintain cellular compartmentalization and homeostasis. We have investigated the underlying mechanism by which, in yeast, GPI-anchored proteins (GPI-APs), a glycolipid-linked type of cell surface proteins, are segregated from other plasma membrane proteins in the ER and sorted into distinct ER exit sites. In my talk, I will present direct evidence that ER sorting of GPI-APs depends on the acyl chain length of the ceramide lipid and requires a specific modification on the GPI-glycan. Our results have implications for understanding the role of glycolipids in protein sorting.

73. Recognition of bacterial glycolipids via host immune receptors

Y. Sho¹; ¹Research Institute for Microbial Diseases (RIMD), Immunology Frontier Research Center (IFReC), Osaka University, Japan

Mycobacterium tuberculosis, a causative agent of tuberculosis, has long been known to possess uniquely potent immunostimulatory activities. Indeed, complete Freund’s adjuvant (CFA), which efficiently elicits acquired immunity, is composed of heat-killed M. tuberculosis. However, the precise molecular mechanisms by which M. tuberculosis exerts adjuvanticity have not been clearly understood for a few decades. C-type lectin receptors (CLRs) are recently identified to be pattern recognition receptors (PRR) for pathogens as Toll-like receptors (TLRs), Nod-like receptors (NLRs) and RIG-I-like receptors (RLRs). Among them, we found that a series of ITAM-coupled CLRs, Mincle (Clec4e), MCL (Clec4d), Dectin-2 (Clec4n) and DCAR (Clec4b1), act as pattern recognition receptors (PRRs) for mycobacteria. Characteristic mycobacterial glycolipids known as adjuvant, such as trehalose dimycolate (TDM), lipoarabinomannan (LAM) and phosphatidylinositol mannosides (PIM), were identified as ligands for these receptors. These receptors appear to have arisen by gene duplication and clustered within the same genomic locus. Thus, the engagement of these clustered CLRs via mycobacterial ligands synergistically shapes robust innate and acquired immune responses against this life-threatening pathogen. These findings shed light on CLRs as novel immune receptor family for mycobacteria, and thus CLRs could be attractive targets for the development of novel vaccine adjuvants.

74. GFAT1 promotes stemness and chemoresistance of pancreatic cancer via activating β-catenin pathway

Y. Ruan¹, J. Gu¹; ¹School of Basic Medical Sciences, Fudan University, Shanghai

Pancreatic cancer is one of the most lethal of all types of cancer worldwide, with the 5-year survival rate ranging only at 6–7%. High resistance to chemotherapy is one of the main causes for the poor prognosis of pancreatic cancer patients. Cancer stem cells are cancer cells that possess characteristics associated with normal stem cells, and are crucial for the tumor chemoresistance and relapse. Glutamine-fructose-6-phosphate amidotransferase 1 (GFAT1) is the rate-limiting enzyme of hexosamine biosynthesis pathway (HBP), and modulates the protein glycosylation through controlling the synthesis of HBP endproduct UDP-GlcNAc. Our previous study revealed that GFAT1 was up-regulated in pancreatic cancer, and was positively correlated with lymph node metastasis, TNM stage and poor outcome of pancreatic cancer patients. However, whether GFAT1 contributed to the tumorigenesis and progression of pancreatic cancer remains unknown. In this study, we found that high expression of GFAT1 was positively correlated with tumor stemness in pancreatic cancer tissues. In vitro and in vivo studies confirmed that GFAT1 promoted the stemness and chemoresistance of pancreatic cancer cells. Gene set enrichment analysis revealed that GFAT1 was positively associated with β-catenin activation. Overexpression of GFAT1 could stabilize β-catenin protein and induce its nuclear translocation. Mechanism studies revealed that GFAT1 triggered AKT/GSK-3β phosphorylation to prevent β-catenin degradation. Overexpression of GFAT1 also enhanced the direct O-GlcNAcylation of β-catenin for activation. Besides, GFAT1 stimulated the de-localization of E-cadherin, leading to the release of β-catenin from membrane and its accumulation in cytoplasm/nucleus. Together, our research demonstrates a pivotal role of GFAT1 in modulating protein glycosylation and tumor progression in pancreatic cancer, and suggests GFAT1 as a potential therapeutic target for the treatment of pancreatic cancer patients.

75. Mycobacterium tuberculosis inhibits human innate immune responses via the production of TLR2 antagonist glycolipids

J. Nigou¹; ¹Department of Tuberculosis & Infection Biology, Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, Toulouse, France

Mycobacterium tuberculosis (Mtb) is a major human pathogen that is able to survive inside host cells and resist immune clearance. Most particularly, it inhibits several arms of the innate immune response, including phagosome maturation or cytokine production. In order to better understand the molecular mechanisms by which Mtb circumvents host immune defenses, we used a transposon mutant library generated in a virulent clinical isolate of Mtb to infect human macrophages, utilizing a cell line derivative of THP-1 cells expressing a reporter system for activation of the transcription factor NF-κB, a key regulator of innate immunity. We identified several Mtb mutants inducing a NF-κB activation stronger than that of the wild-type strain. One of these mutants was found to be deficient for the synthesis of cell envelope glycolipids, namely sulfoglycolipids, suggesting that the latter can interfere with innate immune responses. Using natural and synthetic molecular variants, we determined that sulfoglycolipids inhibit NF-κB activation and subsequent cytokine production or co-stimulatory molecule expression by acting as competitive antagonists of Toll-like Receptor-2, thereby inhibiting the recognition of Mtb by this receptor. Our study reveals that producing glycolipid antagonists of Pattern Recognition Receptors is a strategy used by Mtb to undermine innate immune defense. Sulfoglycolipids are major and specific lipids of Mtb, considered for decades as virulence factors of the bacilli. Our study uncovers a mechanism by which they may contribute to Mtb virulence.

76. Oligosaccharide based semi-synthetic conjugate vaccines

C. Pereira¹; ¹Vaxxilon

Glycoconjugates vaccines have been around for a few decades now. They have proven to be very effective against H. influenzae, N. meningitidis and S. pneumoniae. The most recent addition to this family has been S. typhi. With the current scenario in antimicrobial resistance and with few antibiotics in clinical development, the discovery of new targets is becoming even more important to control the various pathogens. Innovative vaccines based on glycans can be a powerful tool and an important alternative for the fight against such deadly pathogen.

The technology platform of Vaxxilon is based on rational design on a molecular level of the natural glycan repeating unit (RU). There are very many variables associated with the design of a successful glycoconjugate vaccine and Vaxxilon is using the power of organic chemistry to design epitopes of the RU which carry the same traits of the natural polysaccharide but are both immunogenic and protective. Such rationally designed epitopes help us to better understand on a molecular level what makes a particular glycan protective or non-protective. Having a better understanding then helps to custom design oligosaccharide epitopes that are also more stable than their parent polysaccharide, less variability between the batches, better solubility, efficient conjugation consistency, no loss of protective epitopes and better yields. In some cases they become the only alternative as some of the pathogens are difficult to grow, and in others harvesting enough quantities of the polysaccharides to develop a viable glycoconjugate vaccine becomes a challenge or some polysaccharides may not be immunogenic.

Overall the approach of Vaxxilon is one of the emerging novel vaccine technologies leading to well characterized, highly stable, potent oligosaccharide antigens being part of improved new rationally designed semi-synthetic glycoconjugate vaccines for potentially any glycan containing pathogen including those for which the conventional approach has so far failed to deliver.

77. Antithrombin-binding heparin sequences: alternative structures for the same function

M. Guerrini¹; ¹Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni

Heparin is a highly-sulfated glycosaminoglycan (GAG) of animal origin used as anticoagulant and antithrombotic drug. These properties are principally based on the binding and activation of antithrombin (AT), an endogenous inhibitor of serine proteases in the coagulation cascade. The interaction occurs through the pentasaccharide sequence GlcNAc/NS,6S-GlcA-GlcNS,3,6S-IdoA2S-GlcNS,6S (AGA*IA) which binding, inducing a conformational change in AT, enhances by several hundred-folds the rate of proteinases inhibition.

Different structural antithrombin binding (ATB) site variants have been isolated from heparin and low molecular weight heparins (LMWHs) and their characterization pointed out how structural peculiarities of the pentasaccharide, including its reducing and nonreducing extension as well as its position along the heparin chain, affect the strength of binding and thus the power of inhibition.

In the last decade studies, NMR was proved to be the most versatile technique to characterize protein ligand interactions under near physiological conditions, enabling the determination of the structure and conformation of the binding epitope of ligands in the bound state. In this framework, the “plasticity” of iduronic acid moiety was revealed to favour the interaction of oligosaccharides with AT and more generally of iduronic acid containing glycosaminoglycans with other heparin-binding proteins.

Recently, through a novel chemoenzymatic method several new ATB oligosaccharides have been synthetized, confirming the fundamental role of iduronic acid and the possible existence of alternative sequences able to bind and activate AT. Moreover, the characterization of heparin fractions endowed of high affinity to AT isolated from different animal sources modified and extended the concept of specificity of such an interaction.

78. GMMA as alternative carrier for polysaccharides supporting the development of multitarget vaccines

F. Micoli², R. Alfini², R. Di Benedetto², F. Necchi², F. Schiavo², F. Mancini², M. Carducci², C. Balocchi¹, G. Gasperini², B. Brunelli¹, D. Oldrini¹, O. Pitirollo², P. Costantino¹, D. Piccioli¹, R. Adamo¹, A. Saul²; ¹GSK Vaccines, ²GSK Vaccines Institute for Global Health

Nanoparticle systems are being explored for the display of carbohydrate antigens supporting the development of novel and more effective glycoconjugate vaccines. Special physico-chemical properties of nano-sized particles are combined to the presentation of multiple saccharide epitopes. In this context, we are testing GMMA (Generalized Modules for Membrane Antigens) as alternative carrier systems for polysaccharides. GMMA are Outer Membrane Vesicles (OMVs) naturally released from Gram-negative bacteria engineered to produce large quantities of OMVs in a detergent free process. GMMA combine antigen presentation to the immunopotentiator effect of the Toll-like receptor agonists naturally present on these systems. In addition, this approach allows to obtain immunization against antigens contained in GMMA.

We have developed different conjugation approaches for linking saccharides to GMMA surface. Chemical conjugation to GMMA strongly enhances the humoral immune response to the linked antigen, providing improved immunogenicity compared to the corresponding traditional glycoconjugate vaccines. This effect was observed by immunizing mice with a broad range of polysaccharides (e.g. Salmonella Typhi Vi, serogroup A and C meningococcal oligosaccharides, Haemophilus influenzae type b oligosaccharides, Streptococcus Group A Carbohydrate) conjugated to GMMA derived from different pathogens (Neisseria meningitidis, Salmonella, Shigella). Display of the antigen on GMMA surface had a major impact on the quality and functionality of the response.

Also, the impact of conjugation parameters such as saccharide length, saccharide density and attachment site to GMMA on the immune response induced was investigated, supporting the design of improved GMMA-based vaccines.

Interestingly, different polysaccharides can be simultaneously presented on the same GMMA particle with no immune interference and no major detrimental effect on the immune response to GMMA, supporting the use of GMMA technology for the development of effective multitarget vaccines against multiple diseases.

79. Ganglioside functions in the human brain and peripheral nervous system

R. Schnaar¹; ¹Departments of Pharmacology & Neuroscience, Johns Hopkins School of Medicine, Baltimore, Maryland, USA

Gangliosides, sialylated glycosphingolipids, are expressed at varying concentrations and complexities on all vertebrate cells and tissues, but are particularly abundant in the nervous system. They are the predominant glycans of nerve cells, where they are expressed primarily (but not exclusively) on the plasma membrane with their ceramide lipids firmly embedded in the phospholipid bilayer and their glycans extending outward into the extracellular space. Although there are hundreds of different ganglioside structures based on their glycan sequences, and even more when differences in the ceramide lipid are considered, the quantitatively major gangliosides of the mammalian brain are a well-defined and well conserved set of closely related structures. Studies from several laboratories have revealed that gangliosides function as molecular recognition and regulation molecules in two modes: cis and trans. In the cis mode, they engage laterally with molecules on their own cell surfaces (such as protein tyrosine kinases) to regulate downstream signaling. In the trans mode, they act as ligands for glycan binding proteins outside of the cell including soluble ganglioside-binding molecules (such as bacterial toxins) and ganglioside binding proteins on other cells (such as myelin-associated glycoprotein). The importance of gangliosides in human development and physiology is informed by two major congenital disorders resulting from mutations in two ganglioside biosynthetic genes, ST3GAL5 and B4GALNT1. Mutations in ST3GAL5, also known as GM3 synthase, result in profound developmental deficits, multi-organ dysfunction, deafness, visual impairment, severe intellectual disability, irritability, and disrupted sleep patterns. Mutations in B4GALNT1, also known as GM2/GD2 synthase, result in a less severe disorder, hereditary spastic paraplegia marked by progressive peripheral neuropathy of the axonal type. These patients also suffer intellectual disability, but of a milder form. Analyses of the structures, biosynthesis, and functions of gangliosides and their disruption in human congenital disorders and related experimental mutations in mice may enhance understanding of these major nervous system glycans and provide insights into human diseases and potential therapies.

80. Polysaccharides as tumor therapeutics, 1868-2019.

R.A. Laine¹; ¹Louisiana State University

Busch (1868) and Fehleisen (1880) showed sarcoma patients with nosocomial or induced erysipelas skin infections had tumor regression or elimination. Spronck (1891) showed that heat treated “Streptococcus erysipelatos” (now S. pyogenes) cultures, injected into sarcoma patients would likewise cause tumor regression. Heat stability was the first indication that polysaccharides might be the active pharmaceutical ingredient (API). Coley (1891, 1910) used a mixture of bacteria-free, heat treated culture medium of S. erysipelatos and Bacillus prodigiosa (Serratia marsescens) to successfully treat sarcoma patients. “Coley’s Toxin” was produced by Pfizer for 20 years. Coley applied the toxins to sarcomas and included many case studies (Coley, 1910b). Around 1920 the American Cancer Association recommended using radiation therapy instead of Coley’s Toxin for cancer, and Coley’s toxin fell into disuse, despite its potent effects. A Coley’s Toxin review was published by Nauts, et al., 1980, a table reproduced in Novotny’s review (1985) showing 5 year survival times of nearly 50% in 897 patients with 18 different cancers, treated with Coley’s toxin. Shear continued work on the Serratia component of Coley’s Toxin, and in 1943, Hartwell and Shear reported the Serratia API was a polysaccharide, containing lipid and phosphate, that caused tumor specific hemorrhage in mice. In the 1980’s Hellerqvist, Sundell, et al. independently, without knowledge of Coley’s Toxin worked at Vanderbilt on isolating the active toxin principle from Group B Streptococcus. They isolated a 270kDa polysaccharide from the GBS culture filtrate, with a lipid and phosphate attached. This GBS Toxin was remarkable in causing tumor specific capillary damage, hemorrhage in tumors, and regression of tumors in rodent models. A successful Phase I clinical trial in volunteer stage 4 humans showed 33% effectivity (DeVore, et al. 1997). We have recently repeated Shear and Hartwell’s work with modern chromatographic techniques and found the polysaccharide to be 250,000 in molecular weight by SEC and HPLC. We will report on partial structure and linkages of the Serratia polysaccharide. We believe these two polysaccharides from Streptococcus and Serratia are the API’s of Coley’s Toxin.

81. Distinct heparan sulfates modulate wnt signaling in a context-dependent manner

K. Saied-Santiago¹, C. Díaz-Balzac¹, H. Bülow¹; ¹Albert Einstein College of Medicine

Wnts are class of secreted morphogens in metazoans that function through Frizzled receptors to control wide range of developmental processes. Wnts are often expressed in overlapping patterns and, in vitro, most Wnts can bind to a range of Frizzled receptors. How selectivity of a Frizzled receptor for its cognate Wnt ligand is established in a cell type specific manner in vivo remains incompletely understood. Previous genetic studies in Caenorhabditis elegans showed that migration of the hermaphrodite specific neuron HSN and axonal migration of the PDB neuron functions through the heparan sulfate proteoglycan SDN-1/Syndecan and the EGL-20/Wnt–MIG-1/Frizzled and LIN-44/Wnt–LIN-17/Frizzled receptor pairs, respectively. Here we show that both the MIG-1/Fz and LIN017/Fz receptors are expressed in HSN and PDB neurons, respectively. Surprisingly, both the EGL-20/Wnt and LIN-44/Wnt ligands can both form a biochemical complex with the heparan sulfate proteoglycan SDN-1/Syndecan and the MIG-1/Frizzled receptor. Moreover, distinct combinations of intracellular signaling components of the Wnt signaling pathway act in a cell-specific manner downstream of the receptors in HSN and PDB neurons. Genetic experiments further suggest that SDN-1/Syndecan bears HS glycans with distinct modification patterns to control EGL-20/Wnt and LIN-44/Wnt signaling through their cognate receptors in HSN and PDB, respectively. The EGL-20/Wnt and LIN-44/Wnt ligands are known to be expressed in overlapping patterns in a region where HSN neurons and PDB axons originate. Thus, we propose that specifically modified HS on SDN-1/Syndecan provides cell-specific selectivity of Frizzled receptors for defined Wnt ligands in a context-dependent manner.

82. VPS13 proteins: lipid transporters localized at membrane contact sites and implicated in neurodegeneration

M. Leonzino¹; ¹Departments of Neuroscience and Cell Biology, Yale University Medical School

VPS13 is an evolutionarily conserved very large protein encoded by one gene in yeast and four genes in mammals. Mutations in each of the four human proteins cause severe neurodegenerative or neurodevelopmental diseases, but the localization and function of this family of proteins remained elusive until very recently. In yeast, Vps13 was shown to be localized at contacts between the vacuole and either mitochondria or the ER and to have a partially redundant function with the ERMES, a protein complex with lipid transport properties that bridges the ER to mitochondria. We have shown that the N-terminal portion of Vps13 has lipid harboring and transport abilities and that it contains a large hydrophobic cavity that can accommodate multiple lipids and thus account for these properties. We have also shown that VPS13A and VPS13C, whose mutations result in Neuroacanthocytosis and Parkinson’s disease, respectively, bind to the ER via an interaction with VAP (an intrinsic membrane protein of the ER) and tether it to other organelles. These findings identify VPS13A and VPS13C as lipid transporters at contact sites, implicating disruption of intracellular lipid homeostasis in neurodegenerative disorders resulting from their mutations. Interestingly, the N-terminal portion of Vps13 shares homology with the N-terminal portion of the autophagy protein ATG2 and, accordingly, ATG2 has now been reported to also have lipid transport properties. The peculiar rod-like shape of the lipid transport portion of these proteins suggest that they may act as hydrobobic channels connecting two membranes.

VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites. Kumar N*, Leonzino M*, Hancock-Cerutti W**, Horenkamp FA**, Li P, Lees JA, Wheeler H, Reinisch K***, and De Camilli P***, J Cell Biol. 217:3625-3639 Epub 2018 Aug 9. PMID: 30093493

83. Link between Golgi manganese homeostasis and congenital disorders of glycosylation

F. Foulquier¹; ¹CNRS, Université de Lille, UMR8576 Structural and Functional Glycobiology Unit, FRANCE

Within the secretory pathway, ionic homeostasis of each organelle is unique in terms of H⁺, Ca²⁺ and Mn²⁺ concentrations. Many transporters and pumps finely regulate these homeostasis in the Golgi compartment, known to be crucial for many cellular processes such as the vesicular fusion event, the secretion of proteins but also the activities of Golgi glycosyltransferases and glycosidases. Many pumps and transporters are involved in this tight regulation. Defects in TMEM165, SLC39A8 and recently in SLC10A7, were discovered to be associated to strong Golgi glycosylation abnormalities leading to Congenital Disorders of Glycosylation (CDG). In these deficiencies, disruptions in the Golgi Ca²⁺/ Mn²⁺ homeostasis are thought to be the molecular cause leading to glycosylation defects. This talk will first summarize the molecular machineries involved in the Golgi manganese homeostasis regulation and then the associated diseases resulting from defects in Golgi ion homeostasis.

84. An outlook on condensed phase simulations of glycolipid systems

M. Sega¹; ¹Helmholtz Institute Erlangen-Nürnberg, Forschungszentrum Jülich

Glycolipids are one of the most varied classes of lipids present in mammalian cells and take part in a large number of cellular functions, yet investigations by means of molecular simulations to determine their microscopic properties, at full-atomistic or at coarse-grained level, have been flourishing only recently, also thanks to technological advances in forcefield development and manipulation tools and, increasing available computational power. Here, I provide an overview of recent simulation results on biological and artificial glycolipid-containing membranes, including topics such as the influence of glycolipids on the thermodynamics of their host lipid membranes, rafts formation, membrane cohesion, reception function, showing how the simulations of lipid membranes have become ever more complex and predictive, and an invaluable tool in the investigation of glycolipids.

85. Antiviral lectins from natural products extracts: from discovery to the clinic

B. O’keefe¹; ¹Molecular Targets Program, Center for Cancer Research and Natural Products Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702 (USA).

Abstract:

The small organic solvent-soluble molecular constituents of natural product extracts have been productively exploited for centuries. Their positive effect on human health has and continues to be beyond question. More recently, researchers have begun to evaluate the macromolecular constituents of natural product extracts for their potential utility as drugs. This talk will discuss the development of novel lectins from natural sources for their use in human health. Several examples will be provided of proteins identified from natural sources including those with antiviral activity. In particular, the antiviral lectin griffithsin will be discussed including its discovery, determination of its mechanism of action and carbohydrate binding, and its development as an anti-HIV agent currently in Phase I clinical trials.

Keywords: proteins, lectins, griffithsin, antiviral, HIV

86. Using 1k synthetic glycomics library to search for protein binding

G.P. Wang¹; ¹Baylor College of Medicine

Understanding the structures and functions of glycans is essential to the understanding all processes in living systems. Theoretically, due to the variable and multiple connectivity, five monosaccharides can form a pentasaccharide with over half a billion possible structures. On the other hand, in any real biological system, much smaller numbers of glycan sequences than the theoretical number may exist since glycans are synthesized by glycosyltransferases (or other enzymes) with limited variations in sugar to sugar connections. For example, it is estimated that 20,000 to 30,000 glycan backbone structures may cover most mammalian glycomes. Thus, majority of biologically important glycan backbone sequences can be synthesized by chemo-enzymatic appraoches. Post-glycosylational modification on the glycan backbones will further produce the diversity and complexity of glycans.

Here we will discuss Core Synthesis/Enzymatic Extension (CSEE) approach which offers a practical solution to large glycoconjugates and synthetic glycomes. CSEE includes 1) convergent chemical synthesis of very limited numbers of small core structures, and 2) extension of the cores via enzymatic catalyzed reactions. As a test try, a library of N-glycans were prepared via CSEE in mg scales (1). Starting with 5 chemically prepared building blocks, 7 N-glycan core structures containing one or two terminal N-acetyl-D-glucosamine (GlcNAc) residue(s) were chemically synthesized via consistent use of oligosaccharyl thioethers as glycosylation donors in a convergent fragment coupling strategy. Each GlcNAc residue was then extended to 5 more structures (enzymatic extension I) by enzymatic reactions catalyzed by 4 robust glycosyltransferases, yielding a library of 73 N-glycans. Further work enables the synthesis of over 40 structures starting from one GlcNAc residues (enzymatic extension II), which may generate thousands of glycan structures from a few cores. Similary, current synthesis of complex O-glycan libraries, glycoaminoglycans (GAG), lipolysaccharides (LPS) conjugates, glycopeptides and glycoproteins will also be discussed.

87. Towards automated, high-throughput workflows for glycomic and glycoproteomic analysis by mass spectrometry

M. Wuhrer¹, G. Lageveen-Kammeijer¹, G. Vreeker¹, M. Bladergroen¹, D. Falck¹, S. Nicolardi¹, V. Dotz¹, B. Heijs¹, Y. Van Der Burgt¹, N. De Haan¹; ¹Leiden University Medical Center

We developed high-throughput mass spectrometric workflows to profile glycosylation in a glycomic and glycoproteomic manner. One workflow focuses on the profiling of the complex glycomes of various biological samples (e.g., plasma, serum, cell lines or tissues) based on the analysis of released glycans by MALDI-MS (high-throughput) or CE-ESI-MS (in-depth). The other workflow concentrates on the protein- and site-specific glycosylation profiling of antibodies, based on the analysis of glycopeptides by LC-ESI-MS.

The sample preparation for the glycomics workflow is for a large part robotized, additionally the data processing in both workflows is automated by the development of innovative software packages for targeted data extraction. The methods are applied on large clinical cohorts, including hundreds to thousands of samples. Aberrant plasma N-glycan profiles were found in diabetes, colorectal cancer, rheumatoid arthritis and inflammatory bowel disease. IgG glycopeptides were studied in the context of various autoimmune and alloimmune diseases. Additionally, using the glycopeptide method, we were able to study IgG and IgA derived from both plasma and saliva.

Overall, our workflows which are optimized regarding sample preparation, mass spectrometry and data processing enable the glyco(prote)omic and glycomic characterization of complex biological processes.

88. Sterical constraints and osmotic pressure in self-assembled systems containing saccharides

T. Zemb¹; ¹CEA Marcoule

Molecular forces and free energy of interaction between bilayers of glycolipids and nano-crystalline cellulose can be quantified once mechanical, entropic and hydrogen-bounded first water layer are considered with the same units, i.e, osmotic pressure for forces or free energy of deformation and interactions in KJ/mol.

I show this on three examples:

-The swelling dominated by repulsive hydration forces

-The packing of glyco- surfactants and lipids in aggregates

-The non-equilibirum de-swelling followed by ion-specifc swelling of the wood cell wall

These examples will illustrate the profound qualitive differences between phospo- and glyco- lipids in self assembly and behaviour when in contact with a reservoir fluid: these are mainly due to resistance to deformation of hydrated saccharidess as well as driving the structure of water most often as a cosmotropic head-group.

89. Molecular basis of broad spectrum n-glycan specificity and processing of IgG antibodies by endoglycosidase S2

M. Guerin¹; ¹Structural Biology Unit - CIC bioGUNE Technological Park of Bizkaia - Ed 801A 48160, Derio, Vizcaya SPAIN

Therapeutic immunoglobulin G (IgG) antibodies are a prominent and expanding class of drugs used for the treatment of several human disorders including cancer, autoimmunity, and infectious diseases. IgG antibodies are glycoproteins containing a conserved N-linked glycosylation site at residue Asn297 on each of the constant heavy chain 2 (CH2) domains of the fragment crystallizable (Fc) region. The presence of this N-linked glycan is critical for IgG function contributing both to Fc γ receptor binding and activation of the complement pathway. The precise chemical structure of the N-linked glycan modulates the effector functions mediated by the Fc domain. IgG antibodies including those produced for clinical use typically exist as mixtures of more than 20 glycoforms, which significantly impacts their efficacies, stabilities and the effector functions. To better control their therapeutic properties, the chemoenzymatic synthesis of homogeneously N-glycosylated antibodies has been developed.

Streptococcus pyogenes secretes two multidomain antibody-specific endoglycosidases, EndoS and EndoS2, which are central to these pathways. EndoS2 has a broader substrate specificity compared to EndoS, hydrolyzing not only biantennary complex type N-glycans, but also high-mannose, hybrid, and bisecting complex type N-glycans on IgG. Glycosynthase mutants of EndoS2 have also been developed to engineer antibodies with a more diverse set of N-glycans than similar EndoS mutants are capable of creating. In this work, we carried out a detailed study of the structure and function of EndoS and EndoS2, and elucidated the molecular mechanism by which EndoS2 recognizes an expanded repertoire of N-glycans compared to EndoS. Strikingly, this mechanism involves the action not only of the glycoside hydrolase domain, but also that of a carbohydrate binding module, which proved to be essential for IgG recognition and catalysis. These works certainly set the foundation for engineering enzymes to carry out customizable antibody glycosylation reactions for the diagnosis and treatment of human diseases.

References

1. Klontz, EH, et al. ACS Cent. Sci. 5:524-538 (2019). F1000Prime article.

2. Trastoy B, et al. Nat. Commun. 9:1874 (2018).

90. Advanced experimental techniques and biomimetic aggregates: radiation and neutron investigation

E. Del Favero¹; ¹BIOMETRA Department, University of Milan

Scattering techniques are well suited for studying the physico-chemical properties of nanoparticles and aggregates in solution, meanwhile being largely non-invasive. Different probing radiations allow to access different structural and dynamical parameters on different lengthscales, spanning from the size of particles (10 -1000 nm) to the very local internal structure (0.1-1 nm). Moreover, experiments can be designed to enhance the visibility of selected regions of the aggregates (like glyco-chains) without significative chemical drawback. The combined use of laser light, X-ray and neutrons techniques will be presented as powerful tool to probe the structural properties of different systems containing glycoconjugate molecules, ganglioside aggregates, vesicles and complex model membranes. Also, scattering and reflectometry investigations will be shown, addressing the structural response of model membranes to inserting proteins or peptides, like ion channels or amyloids, and approaching enzymes, like sialidase.

91. Pathological implications of glucosylceramide-enriched membrane domains

A.E. Ventura¹, S. Pokorna², A.H. Futerman²,L.C. Silva¹; ¹iMed.ULisboa, Faculdade de Farmácia, Universidade de Lisboa, ²Weizmann Institute of Science, Department of Biomolecular Sciences

Glucosylceramide (GlcCer), one of the simplest glycosphingolipids, is an important mediator of cell function. The molecular mechanisms underlying the biological action of this membrane lipid are yet to be elucidated. However, evidence suggests that GlcCer participates in the formation of membrane domains that can potentially regulate cellular events. Therefore, impairment in GlcCer metabolism can significantly affect membrane structure and organization, eventually disrupting cellular homeostasis. This might be particularly relevant in Gaucher disease (GD), where lysosomal accumulation of GlcCer is observed. To understand the biophysical implications of GlcCer accumulation we used multiple fluorescence-based methodologies and a combination of artificial membranes and diverse genetic and chemical cell models of GD. Data obtained from synthetic membranes showed that GlcCer decreases the fluidity of the membranes due to formation of ordered raft-like domains or gel phase domains, depending on membrane lipid composition and GlcCer levels. Similar observations were obtained in GD cellular models, where accumulation of GlcCer was accompanied by a decrease in membrane fluidity. Together, we demonstrate that elevated GlcCer levels change the biophysical properties of cellular membranes, which might compromise membrane-associated cellular events and be of relevance for understanding the pathological mechanisms leading to GD.

Acknowledgments: Fundação para a Ciência e Tecnologia (FCT), Portugal: PTDC/BBB-BQB/3710/2014, PTDC/BBB-BQB/6071/2014, PTDC/BIA-BFS/29448/2017, and Investigador FCT IF/ 00437/2014 to LCS.

92. Genetics of CDG: a plethora of features and findings

G. Matthijs¹; ¹Center for Human Genetics, University of Leuven, Belgium

Congenital Disorders of Glycosylation (CDG) are a clinically and genetically heterogeneous group of metabolic diseases. Today, over 100 different types have been described.

PMM2-CDG, caused by a phosphomannomutase deficiency remains by far the most frequent type of CDG, MAN1B1-CDG and several COG deficiencies are relatively frequent, while all other types are quite rare. Almost all CDG are recessive disorders, either autosomal or X-linked. However, the collection of CDG harbours a number of genetically interesting observations, including a few autosomal and even X-linked dominant diseases.

A few types will be highlighted. COG4-CDG is known as a typical CDG. Recently, it was shown that a recurrent de novo COG4 mutation causes Saul-Wilson syndrome. Defects in the X-linked SLC35A2 gene are dominant, with severe, early onset presentations in girls. De novo mosaics were described in male patients, also severely affected, while somatic mutations in the brain have been shown to cause severe intractable epilepsy. A careful screening of patients with a skeletal dysplasia with amelogenesis imperfecta, yet another CDG phenotype, revealed null alleles in SLC10A7, which have not been explained molecularly. Interestingly, a 5’ upstream GGC expansion of the XYLT1 gene is present either in a homozygous or in a compound heterozygous state in a large majority of patients with Baratela-Scott syndrome. Mutations in the X-linked ALG13 gene seem to affect boys and girls differently. The molecular diagnostics of ALG1-CDG is complicated by the occurrence of a number of highly identical pseudogene.

Hence, the clinical and genetic diagnosis of CDG remains challenging, while from a research standpoint, fantastic contributions have been made to understanding glycosylation by studying the clinical, cell biological and glycobiological features in patients.

93. Interaction of peptides and proteins with mimes of membrane domains.

L. Cantu’¹; ¹University of Milano. Dept. Medical Biotechnologies and Translational Medicine.

Biological membranes are complex systems where the different components cooperate to allow for many functions that are usually categorized as physical, biochemical and biological. Nonetheless, these different aspects are correlated to each other, both in the physiological state and in pathological conditions, or in the presence of external agents, as therapeutics or pollutants. The structural interference between biomimetic complex membranes and approaching or embedded bodies, like proteins and peptides, constitutes a physical basis underlying system adaptation, modulation and function. We show that the use of advanced experimental techniques and the synergic coupling of methods typical of the different approaches helps in unveiling these correlations.

(Work partially supported by grant n. 695078 - noMAGIC, H2020-ERC-2015)

94. Organelle contact sites: what are they, what are they good for and how can they be bad?

Q. Feng¹, S. Helle¹, A. Michel², S. Van Schie¹, A. John Peter¹, C. Gaebelein¹, T. Zambelli¹, J. Vorholt¹; ¹ETH Zurich, ²University of Oxford

Intracellular organelles constitute dense and branched membrane networks that are under constant remodeling. My lab is interested in how these organelle networks are generated, distributed and regulated. We also investigate how this networked morphology is related to the organelle’s activity.

The extended morphology of several organelles might allow them to contact each other to exchange lipid molecules.

These highly extensive and dynamic networks cohabit in the extremely crowded cytoplasmic space. This situation leads to unwanted collisions and entanglements that needs to be resolved. We show that, in the case of mitochondria, these collisions and entanglements can be resolved by mitochondrial fission. Mechanical forces applied to mitochondrial tubules lead to the recruitment and activation of the mitochondrial fission machinery, leading to the resolution of entanglements. These results imply that a biochemical response can be triggered by a mechanical stimulus and that forces within the cells participate in the shaping of organelles.

95. From Golgi to glycome to function: the GOLPH3/COPI complex drives intra-golgi cisternal maturation-linked recycling of specific enzymes to direct glycosphingolipid assembly and cell proliferation

A. Luini¹, R. Rizzo¹, D. Russo¹, G. D’angelo²; ¹Institute of Protein Biochemistry - National Research Council, Naples, ²Institute of Protein Biochemistry - National Research Council, Naples and EPFL, Lausanne

The mechanisms by which the Golgi complex assists glycans assembly on cargo proteins and lipids during traffic, thereby shaping the glycome and regulating cell functions, are poorly understood. To address this major yet long-neglected issue, we have analyzed the role of GOLPH3, a Golgi enzyme adapter with oncogenic properties, in glycan assembly at the Golgi complex. We find that GOLPH3 acts as a component of the cisternal maturation mechanism and drives the intra-Golgi recycling of a subset of glycosyltransferases of the glycosphingolipid metabolic pathway involved in cell growth control. GOLPH3 overexpression, by exaggerating the recycling of theses glycosyltransferases reduces their lysosomal degradation and enhances their levels, thereby reprogramming glycosphingolipid metabolism and hence enhancing cell proliferation. These findings explain how intra-Golgi enzyme dynamics assist the assembly of specific glycans to regulate cell functions; shed light on the role and organization of cisternal maturation in physiology and pathology; and unravel a novel growth-control mechanism.

96. Galectins as targets for clinical therapy and diagnostics

H. Leffler³, U.J. Nilsson³, A. Mackinnon⁴, F. Zetterberg¹, A. Pedersen¹, T. Sethi², H.T. Schambye¹; ¹Galecto Biotech AB, Copenhagen, Denmark, ²King’s College London, UK, ³Lund University, Sweden, ⁴University of Edinburgh, UK and Galecto Biotech AB, Copenhagen, Denmark

Galectins are small soluble, often abundant, proteins with affinity for extracellular/intravesicular glycoproteins and glycolipids, but also have cytosolic and nuclear activities. Studies in animal disease models, including KO mice, and expression levels in human tissues have suggested rate limiting roles in cancer and inflammatory disease like fibrosis. Experiments in cell culture have provided some possible mechanisms of action, including regulation of receptor organization and residence time at the cell surface and effects on cell migration.

These findings have inspired development of galectin inhibitors as therapeutics, and galectins as part of diagnostics. Galectins have a well characterized about 135 amino acid carbohydrate recognition domain folded as a ß-sheet. Galectins are druggable in the sense that it has been possible to design small molecule very potent inhibitors, even if the carbohydrate-binding site is relatively shallow. Such galectin inhibitors have shown promising effects in mouse models of cancer and also lung fibrosis. One molecule, TD139, is in phase II clinical development as an inhaled drug for idiopathic lung fibrosis.

The roles of galectins in disease can to some degree be related back to their roles in the cell, but not completely or in a simple way. This creates a challenge for defining mechanism of action of galectin inhibitors, and relevant aspects of pharmaco-kinetics and pharmaco- dynamics (PK/PD). At the same time galectin inhibitors can be seen as tools for deeper understanding of galectin function and related cell biology.

As diagnostics the level of galectin-3 in blood has found widespread use as a marker of cardiac insufficiency. Its level in thyroid tissue is also used to distinguish malignant from benignant tumors there. However, the level of specific galectin-binding glycoforms of common glycoproteins in serum may be an even more interesting diagnostic marker, and reflection of galectin function.

97. The GM1 ganglioside oligosaccharide-trka interaction as starting biochemical information for the developing of a new therapy for the treatment of parkinson’s disease.

E. Chiricozzi², G. Lunghi², E. Di Biase², M. Fazzari², M. Valsecchi², L. Mauri², S. Alselehdar¹, R.W. Ledeen¹, S. Sonnino²; ¹Rutgers New Jersey Medical School, ²University of Milano

Recent studies and functional data suggest the existence of a positive loop between the age dependent GM1 deficiency and α-synuclein (α-syn) accumulation involved in the onset of neurodegeneration of sporadic Parkinson’s disease (PD). This loop is triggered by the plasma membrane GM1 deficiency, which leads to a failure of trophic signalling and to the α-syn accumulation, increasing the susceptibility to neuronal death. The consequence of insufficient GM1 find the rational in a newly presented mouse model of sporadic PD based on partial deletion of this ganglioside due to heterozygous disruption of B4galnt1 gene required for GM1 biosynthesis leading to motor impairments, α-syn aggregates and dopaminergic neuron damage, that constitute a PD pathological hallmarks. Accordingly, GM1 ganglioside replacement therapy showed effectiveness, although accompanied by strong pharmacokinetic limitation, due to the GM1 amphiphilicity.

Since we recently demonstrated that GM1 neurotrophic and neuroprotective properties depends on the direct interaction between its oligosaccharide chain (II³Neu5Ac-Gg₄, OligoGM1) and TrkA receptor at the plasma membrane, we decide to investigate whether OligoGM1 could rescue behavioral symptoms and biochemical features in B4galnt1+/- mice.

Importantly, we found that the GM1 oligosaccharide, systemically administered, reaches the brain and completely rescues the physical symptoms, reduces α-syn aggregates, restores nigral tyrosine hydroxylase expression and striatal neurotransmitter levels, overlapping the wild-type condition.

This preliminary in vivo study supported the idea of impaired OligoGM1–plasma membrane protein interaction as main cause underling PD onset related to aged GM1 decline and provides the basis for developing a new PD therapy.

98. COG and GARP vesicle tethering complexes control the fate of golgi glycosylation enzymes

Z. D‘souza¹, J. Blackburn¹, T. Kudlyk¹, I. Pokrovskaya¹, V. Lupashin¹; ¹Department of Physiology and Biophysics, University of Arkansas for Medical Sciences

The Golgi is an essential hub in the secretory pathway where cargo is delivered to undergo modifications before targeting to final destination. In order for Golgi’s functions to be carried out properly, correct localization of the Golgi enzymes within the Golgi cisternae is crucial. The Conserved Oligomeric Golgi (COG) complex, a multi-subunit (COG1-8) tethering complexes controls membrane trafficking and ensures Golgi homeostasis by orchestrating retrograde vesicle trafficking within the Golgi. Human COG defects lead to severe multi-systemic diseases known as COG-Congenital Disorders of Glycosylation (COG-CDG). The four subunit (VPS51-54) Golgi Associated Retrograde Protein (GARP) complex controls the retrograde transport from endosomes to the trans-Golgi network.

The CRISPR approach was utilized to generate a complete set of HEK293T cells deficient for individual COG and GARP subunits as well as for several key double knock-down (DKO) combinations. COG KO cells show an extensive fragmentation of the Golgi cisternae and a significant enlargement of late endosomal compartment, defects in retrograde trafficking, sorting and glycosylation. GARP KO cells show defects in retrograde trafficking and, surprisingly, in both N- and O-glycosylation. Glycosylation and trafficking defects were even more severe in COG/GARP DKO cells, while the enlargement of endolysosomal compartment was partially suppressed, indicating that the GARP activity is required for formation of aberrant endosomal compartment.

To gain better understanding of tethering complex deficiency, flow cytometry, EM and superresolution microscopy was used to compare single and double KO mutants with wt and rescued cells. We found that stability of both cis/medial (MGAT1) and trans-Golgi (B4GALT1 and ST6GAL1) enzymes was compromised in both COG and GARP KO cells, indicating that these complexes are essential for the maintenance of Golgi glycosylation machinery. To gain a better understanding of Golgi enzyme fate in COG and GARP deficient cells we have utilized a RUSH pulse-chase approach, investigating intracellular trafficking and degradation of newly synthesized B4GalT1 and MAN2A1.

This work was supported by the NIH grant GM 083144

99. Glycan microarrays shedding light on glycan recognition by viruses

Y. Liu¹; ¹Glycosciences Laboratory, Faculty of Medicine, Imperial College London, London, W12 0NN, UK

Many viruses use host cell surface glycans, as primary or co-receptors, to attach to host cells at initial stages of infection. Glycan microarrays are important tools in elucidation of glycan receptors involved in virus attachment. The neoglycolipid (NGL)-based microarray system is an advanced glycan array platform that is available to the scientific community for recognition studies of diverse glycan binding systems (http://www.imperial.ac.uk/glycosciences). The clustered and flexible presentation of non-covalently immobilized lipid-linked probes printed in a liposomal formulation on a nitrocellulose matrix renders the NGL system powerful in providing information on the molecular basis of the virus-host-glycan interactions.

I will give highlights of the recent contributions of the NGL-based microarrays in collaborative studies with the groups of Niklas Arnberg (Umeå, Sweden) and Thilo Stehle (Tübingen, Germany) on the discoveries of host glycans recognized by human adenoviruses and polyomaviruses. Together with the data from crystallographic studies of virus-glycan interactions at atomic level and cellular biochemical studies, our findings uncover the molecular bases of the host range, tissue tropism, cell entry mechanisms and pathogeneses of the infections. There are implications for therapeutic design, including antiviral agents as well as oncolytic and gene therapies targeting cancer.

100. Possible functions of egcrp2 in cryptococcus neoformans to escape host immune system

T. Watanabe¹, M. Ito¹; ¹Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University

Ergosteryl glucoside (EG), a glycolipid composed of glucose and ergosterol, is synthesized by SGS1, 2 and hydrolyzed by EGCrP2 in Cryptococcus neoformans. Heat treatment of C. neoformans (from environmental temperature 25°C to host body temperature 37°C) increased the SGS1, 2 at mRNA level, by which EG content drastically increased. The disruption of SGS1, 2 genes resulted in the loss of EG and decreased the heat tolerance of C. neoformans at 37°C, suggesting EG synthesis is important for the pathogen to invade mammals. EG content was found to decrease to the control level after several hours with heat shock. In EGCrP2-disrupted mutants of C. neoformans, however, EG content continued to increase and did not return to the control level, indicating that a heat shock-induced increase of EG is normalized with the function of EGCrP2. To investigate the reason why the increased EG content needs to return to the control level, we examined the possibility that EG can activate the innate immune system through the activation of C-type lectin receptors. As a result, we found that EG activates macrophage inducible C-type lectin (Mincle); however, the degree of activation by EG varied depending on the type of Mincle. That is, EG strongly activated human Mincle, and it moderately activated rat Mincle; however, EG hardy activated mouse Mincle. On the other hand, we found that mouse Mincle was strongly activated by acylated EG (AEG), which was actually detected in EGCrP2-disrupted mutants of C. neoformans. However, how AEG is generated from EG in C. neoformans is currently unknown. In contrast to wild-type C. neoformans, EGCrP2-disrupted mutants did not kill mice under the usual conditions of mouse model of cryptococcosis.

These results suggest the possible function of EGCrP2 in the infection process of C. neoformans, i.e., the fungus needs to synthesize EG in order to moderate heat shock when they invade mammals; however, EG and AEG could be a ligand for Mincle, and thus, C. neoformans reduces EG content by EGCrP2 and escapes the host immune system.

This abstract is that of Symposium S6: BACTERIA AND GLYCOCONJUGATES organized by Prof. Sho Yamasaki. Please delete this sentence before printing (or uploading) the program.

101. Glyco-signatures of carcinoembryonic antigen as a fingerprint of body regions and cancer

A. Almeida⁴, F. Jacob³, K. Stavenhagen², K. Alagesan⁴, M. Mischak⁴, M. Wuhrer¹, A. Everest-Dass⁴, C.A. Reis⁵,D. Kolarich⁴; ¹Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands, ²Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands, ³Glyco-Oncology, Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland, ⁴Institute for Glycomics, Griffith University, Southport, QLD, Australia, ⁵Institute for Research and Innovation in Health (i3S), University of Porto, Porto, Portugal

102. Glyco-signatures of carcinoembryonic antigen as a fingerprint of body regions and cancer

Carcinoembryonic Antigen (CEA) is an FDA-approved tumour marker strongly associated with tumour progression and metastasis. Even though 60 % of the CEA molecule are contributed by N-glycans, current knowledge on the CEA specific glycan signatures in health and disease is surprisingly scarce. We used an in-depth glycomics and glycoproteomics workflow to investigate the glycosylation from CEA purified from four different body sources: human colon cancer (cell line and tissue), tissue from liver metastasis of colon cancer and ascites fluid. The analysed CEAs exhibited specific-origin derived N-glycosylation signatures. Distinct glycosylation differences such as N-glycan branching, degree of sialylation and level of bisecting N-glycans were found between the CEA’s from different body sources. Antennae fucosylation, such Lex and Leb/y determinants were found among all CEAs, however with significant abundance differences between the different sources. Our porous graphitized carbon LC- ESI MS/MS approach was also the key to identify a novel glyco-epitope, where a ß1-4 galactosidase resistant hexose is attached to the bisecting GlcNAc. N-glycan structures carrying this epitope was in particular present on colon derived CEA, while almost absent in any other CEAs analysed. Next to confirming that 27 out of the 28 predicted <i>N</i>-glycosylation sites are glycosylated, we also identified a novel, 29^th site of N-glycosylation on Asn⁷⁶ that is located within a non-canonical ⁷¹N-R-Q⁷³ sequence motif in the N-domain. In summary, CEA-specific glycosylation bears a yet unmined potential to improve the specificity of the CEA-marker, but also to understand the role glycosylation plays for its function.

103. The quest for ways to unravel glycan recognition systems

T. Feizi¹; ¹Imperial College London

Glycan arrays are 17 years old and have become essential tools in elucidating roles of glycans in biorecognition systems. Libraries of sequence-defined glycans are expanding as are the diversity of analysis systems including those that are non-slide-based. The glycan sequences thus far assembled are indeed a minuscule proportion of those in mammalian glycomes and those of microbiota and plants. Advances in chemical and chemoenzymatic syntheses are contributing enormously toward populating glycan libraries with bespoke interrelated glycan sequences for close comparisons of their displays of recognition motifs. Concurrently with these developments are Designer and Beam Search array strategies also the Shotgun array approach that are revealing hitherto unsuspected or unknown ligands natural microenvironments. Hand in hand with these are software for glycan array data storage, interpretation and management, also work toward establishment of a public repository under the auspices of GlyGen the NIH-supported Commons Fund bioinformatics initiative. Our symposium will encompass progress in these activities.

104. Two glycomimetic drugs, uproleselan and GMI-1359 in clinical trials for leukemia and breast cancer

W. Fogler¹, J. Peterson¹, J. Magnani¹; ¹GlycoMimetics Inc., Rockville, MD, USA

We have rationally designed two glycomimetic drugs which have successfully passed pre-clinical studies and are now in clinical trials for cancers. Uproleselan (GMI-1271) is a potent and specific antagonist of E-selectin (KD = 450 nM) but not P or L-selectins. In contrast to the normal vasculature, E-selectin is constitutively expressed on the endothelial cells in the microvasculature of the bone marrow (BM). In patients with acute myelogenous leukemia (AML), their AML cells circulate through the BM microvasculature and since express sialyl Lex on the cell surface, they will bind and localize with this protective microdomain of the BM. Upon binding to E-selectin, the NfKb pathway is activated in bound AML cells inducing chemoresistance during cancer therapy. These are the niches from which AML cells will arise during relapse on the disease. In a Phase 2 clinical trial, Uproleselan was administered in combination with standard chemotherapy (MEC; mitoxantrone, etoposide, cytarabine) in relapsed/refracrory patients to mobilize AML blasts and break chemoresistance. This treatment resulted in greater complete response rates in patients and those patients expressing higher levels of sialyl Lex showed the most improvement. Moreover, patients treated with uproleselan that expressed high levels of sialyl Lex on their AML blasts (>10% by flow analysis) demonstrated dramatically longer overall survival (12.7 months vs 5.2 months, P = 0.0056). Based on the Phase 2 results, the FDA granted Uproleselan “breakthrough therapy designation” and uproleselan has now entered Phase 3 clinical studies in relapsed/refractory AML patients. The BM is a protective reservoir for cancer cells metastasizing from solid tumors such as breast and prostate cancer. CXCR4 plays a role in the long term capture of cancer cells. GMI-1359 was designed to inhibit both CXCR4 and E-selectin. In pre-clinical models of metastatic osseous prostate cancer, GMI-1359 showed significant reduction of tumor lesions compared to chemotherapy (doxetaxel) alone (88% vs 38%). GMI-1359 is now entering clinical studies for the treatment of breast cancer.

105. Role of interorganellar membrane contact sites in the neuropathogenesis of GM1-gangliosidosis

A. D’azzo¹; ¹Department of Genetics, St. Jude Children’s Research Hospital, Memphis Tennessee 38105, USA

The monosialylated ganglioside GM1 is a major sialoglycan of neuronal plasma membranes (PMs), present abundantly in membrane microdomains or rafts. Owing to its interaction with Ca2+ binding proteins and Ca2+ channels, GM1 modulates Ca2+ flux across the PM and interorganellar membranes, a process vital for neuronal communication, particularly at the synapses. Catabolism of GM1 is driven by the lysosomal β-galactosidase (β-GAL), a ubiquitous but differentially expressed glycosidase that targets with high affinity this ganglioside, one of its primary natural substrates. β-GAL deficiency in humans results in the progressive accumulation of undegraded GM1 in the nervous system, leading to the generalized neurodegenerative disease, GM1-gangliosidosis. In β-Gal–/– mice, a faithful model of the disease, impaired lysosomal turnover of GM1 leads to its massive buildup at neuronal PMs and other intracellular membranes, particularly those of the ER. We have previously demonstrated that GM1 clusters at specific membrane contact sites between the ER and the mitochondria, known as the mitochondria-associated ER membranes or MAMs. At these contact sites interaction of GM1 with the phosphorylated and active form of the IP3R-1 Ca2+ channel promotes altered Ca2+ flux between the apposing organelles ultimately leading to UPR- and mitochondria-mediated apoptosis. We have now investigated the downstream effects of GM1 accumulation at the PM, focusing on membrane contact sites that tether the ER membranes to the PM, named PM-associated microdomains or PAMs. Our hypothesis is that changes in GM1 levels at the PAMs affect neuronal processes by disrupting Ca2+ homeostasis. Structural analysis of neurites in β-Gal–/– brains showed overt morphological changes, including numerous ectopic dendrites, formation of dendritic beading, and increase in dendritic spines. At the molecular levels, we found that GM1 accumulation at the PAMs induces changes in several Ca2+ binding proteins that govern Ca2+ flux and Ca2+-dependent signaling pathways. These studies emphasize the need to maintain homeostatic levels of GM1 at organellar contact sites in order to ensure proper neuronal functions and prevent the sequence of events leading to neurodegeneration in GM1-gangliosidosis.

106. Inhibiting glyco-immune checkpoints with novel bispecific-like enzyme therapeutics to treat cancer

L. Cao¹, A. Petrone¹, W. Gatlin¹, A. Das¹, J. Che¹, R. Leblanc¹, Z. Siddiquee¹, S. Nerle¹, M. Stanczak³, L. Xu¹, W. Yao¹, K. Normington¹, C. Bertozzi², J. Broderick¹, H. Läubli³, L. Peng¹; ¹Palleon Pharmaceuticals, ²Stanford University, ³University Hospital Basel

Cancer therapy has been revolutionized by the recent developments of immune-checkpoint inhibitors (ICI) to harness the power of the immune system in fighting cancer. However, most patients fail to have durable responses or become resistant to ICI, highlighting the need to identify new mechanisms of immune evasion in cancer. Recently, the glyco-immune checkpoint axis (sialoglycan/Siglec pathway) has emerged as a new mechanism of immune regulation involving both innate and adaptive immunity and an important mechanism of cancer immune escape. However, a therapeutic intervention of this axis remains a great challenge due to the promiscuous receptor-ligand interactions between 15 Siglecs and dense array of various sialoglycans in humans. To overcome this hurdle, we developed a new therapeutic modality named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), which is bispecific-like comprising of a tumor-associated antigen-binding arm and a sialidase moiety, allowing selectively removing terminal sialic acids, the critical binding carbohydrate of Siglecs, from tumor cells. We demonstrated that EAGLE decreased sialic acid levels of tumor cells and enhanced anti-tumor immune responses in multiple human system models in vitro and immunocompetent syngeneic mouse tumor models in vivo. EAGLE treatment released cancer cell-mediated immunosuppression and induced proinflammatory cytokines IFNγ, IL-17A, IL-2, IL-6, and TNFα in human coculture assays of cancer cells with PBMC and primary endothelial cells. Systematic administration of EAGLE increased tumor-infiltrating immune cells and led to robust anti-tumor activities with complete regressions as monotherapy in syngeneic EMT6 mouse tumor models. Re-challenge experiments in EAGLE-cured mice showed that EAGLE induced anti-tumor immunological memory. We further revealed that the mechanism of action of EAGLE involved both innate and adaptive immunity because depleting macrophages or CD8+ T-cells decreased or abolished its efficacy. EAGLE in combination with anti-PD1 mAb treatment achieved ~100% cures in EMT6-Her2 models. In summary, EAGLE is a novel and promising immunomodulatory therapeutics inhibiting the glyco-immune checkpoints and has the potential to overcome resistance to current immunotherapies.

107. Increasing exogenous GM1 ganglioside in vivo improves behavior and attenuates neuropathology in models of neurodegenerative diseases

M.P. Mcdonald², A. Bernardo¹, A. Dhanushkodi², P. Maiti², Y. Akkhawattanangkul², D.F. Delotterie²; ¹Department of Pharmacology, Vanderbilt University, Nashville, TN USA, ²Departments of Neurology and Anatomy & Neurobiology, University of Tennessee Health Science Center, Memphis TN USA

Gangliosides are richly expressed in the brain and are involved in functions as varied as protein transport and cell-to-cell communication. They’re intimately involved in neurodegenerative disorders, but it’s not known exactly how. We have been using genetic and dietary interventions to alter the distribution of gangliosides in vivo, and observe the effects on behavior and neuropathology in mouse models of Alzheimer’s and Parkinson’s diseases. The manipulations include targeted deletion or shRNA knock-down of GD3 synthase, intraventricular infusion of V. cholerae sialidase, AAV-mediated overexpression of Neu3, incorporation of glycomacropeptide (GMP) into chow, or administration of whey protein isolate with or without GMP in the water bottle. The interventions vary with respect to the effect on brain gangliosides, but they all increase GM1 ganglioside, which has long been known to have neuroprotective properties. Although some studies are ongoing, the preponderance of our results show that the interventions improve behavior and reduce neuropathology and neurodegeneration in models of neurodegenerative diseases.

108. Disease-modifying roles of gangliosides in huntington’s disease and beyond

S. Sipione¹, M. Alpaugh¹, D. Galleguillos¹, V. Kadam¹, J. Monyror¹, L.C. Morales¹, Q. Wang¹; ¹Department of Pharmacology and Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada

Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the first exon of the HTT gene. The resulting mutant huntingtin (mHTT) protein acquires toxic conformations and aggregates within the cells, leading to neuronal dysfunction and death.

We have shown that levels of gangliosides, GM1 in particular, are decreased in HD models. Administration of exogenous GM1 reduces levels of soluble and aggregated mutant huntingtin in HD mouse brains, slows down neurodegeneration and corrects motor as well as cognitive and psychiatric-like dysfunctions in HD mice.

I will discuss data suggesting that the disease-modifying effects of GM1 in HD models might be mediated, at least in part, by enhancement of mHTT secretion through extracellular vesicles and by modulation of microglia activity.

109. Understanding and exploiting immune recognition of carbohydrates

J. Gildersleeve¹; ¹National Cancer Institute

Carbohydrate-binding antibodies are abundant in human serum and play a critical role in host defense. At present, we still know very little about how these antibodies arise and what their roles are. In addition to serum antibodies, monoclonal antibodies that bind carbohydrates are valuable research tools and have numerous applications as diagnostics and therapeutics. Unfortunately, there are relatively few high-quality, glycan-binding monoclonal antibodies available for basic research and clinical applications. New insights into the development of antibodies to carbohydrates could aid our understanding of natural immune recognition of glycans as well as improve our ability to generate monoclonal antibodies. Our group is studying the mechanisms by which the mammalian immune system initiates an antibody response to carbohydrates and then evolves antibodies with high affinity and selectivity. These studies are enabled by our carbohydrate antigen microarray, which contains a diverse collection of O-linked glycans, N-linked glycans, glycosaminoglycans, lipid glycans, glycopeptides, and glycoproteins. Although many germline antibodies to proteins and haptens are polyreactive, we find that anti-glycan antibodies to mammalian glycans evolve from highly specific germline precursors. The results have implications for monoclonal antibody development, vaccine development, and autoimmunity.

110. Mechanistic connections between ER stress and mitochondria-associated membranes

T. Simmen¹; ¹University of Alberta, Edmonton, Canada

The mitochondria-associated membrane (MAM) is a biochemical isolate of the endoplasmic reticulum (ER) membrane contact sites with mitochondria. MAMs have been discovered over 6 decades ago, but their functioning became clearer only during the past decade. An important property of MAMs is their functional connection with ER stress. This condition leads to tightening of MAMs and an alteration of their proteome. Within this proteome, ER chaperones constitute an important group of proteins. ER chaperones often interact with calcium-handling proteins, but the significance of this interaction is not yet fully understood. Our results show how the ER folding enzymes calnexin and TMX1 interact with SERCA calcium pumps and how this interaction controls mitochondria metabolism and ER-mitochondria tethering. I will also discuss how other ER folding enzymes form protein complexes that and determine ER-mitochondria signaling and metabolism, dependent on the functioning of other ER-mitochondria tethers.

111. Gentle handling facilitates tandem ms-based structural determinations of glycoproteins and released glycans

C. Costello¹; ¹Boston University School of Medicine

The heterogeneity and fragility of carbohydrate and other co- or post-translational protein modifications present challenges for their analysis by mass spectrometry, but new instrumentation, accessories, and protocols can successfully address these challenges. Electron-Based Dissociation (ExD) methods offer access to a set of dissociation modes that preserve labile modifications to biopolymers and provide both glycosidic and cross-ring cleavage information on glycans, on a time scale that is compatible with online nanoflow ultraperformance liquid chromatographic (nUPLC) separations ± ion mobility (IM) separations, and has potential for use with online capillary electrophoresis (CE). This lecture will present examples from ongoing fundamental and biological studies that show the advantages of soft ionization conditions, stepped collisional dissociation (CID and HCD) including their pairing with electron transfer dissociation (EThcD, CID/ETD), alternative data acquisition modes on Orbitrap instruments, and the implementation of nUPLC-IM-ExD in Fourier transform ion cyclotron resonance (FT-ICR) and quadrupole time-of-flight (QTOF) MS systems.

112. Chemistry and biology of polysialic acid

R. Gerardy-Schahn¹; ¹Institute of Clinical Biochemistry, Hannover Medical School, Hannover

In vertebrates, the α2,8 linked polymer of sialic acid, polysialic acid (polySia), is mainly known as a modification of the neural cell adhesion molecule NCAM. PolySia on NCAM can be produced by two polysialyltransferases, ST8SIA2 and ST8SIA4, with overlapping but distinct expression patterns and enzymatic profiles. As a major determinant of brain development and plasticity, abnormal levels of polySia as well as variants of the human ST8SIA2 gene have been linked to mental disorders like schizophrenia. In a clinical study we contributed to the demonstration that altered polySia-NCAM serum levels in schizophrenic patients are associated with negative symptoms, cognitive impairments and structural changes of brain areas involved in the pathophysiology of the disease. Correspondingly, mice with a conventional knockout of St8sia2 display deficits of cognition associated with impaired brain development resulting in altered long-range connectivity and a loss of interneurons in the prefrontal cortex. Notably, the cognitive deficits of adult St8sia2-/- mice are aggravated by cannabis exposure during adolescence, which in humans is one of the most prominent `second hits’ that render a predisposed person more susceptible to developing schizophrenia. By use of conditional knockout mouse models we now started to dissect, how ST8SIA2-deficiency in different neuron types and brain regions translates into impairments of cognition. In contrast to the neurodevelopmental impact of ST8SIA2, loss of ST8SIA4 leads to reduced polySia levels in adult mice resulting in altered synaptic plasticity and selective impairments of learning and memory. Based on recent studies, deficits of synaptic transmission and learning of St8sia4-/- mice can be rescued by targeting the underlying synaptic mechanisms and, possibly, also by therapeutic application of polySia. Furthermore, impaired memory of adult St8sia4-/- mice can be ameliorated by housing in an enriched environment, known to stimulate hippocampal neurogenesis. This finding raises intriguing questions concerning the role of ST8SIA4 for the age-dependent decline of the polySia-positive fraction of newborn neurons in the human hippocampus and for their further reduction under the condition of hippocampal atrophy in Alzheimer’s disease.

113. Synthetic carbohydrate-based conjugates as promising shigella flexneri vaccine candidates: from concept to first-in-human study

L.A. Mulard¹; ¹Unit Chemistry of Biomolecules, Institut Pasteur, UMR3523 CNRS, Paris, France

Shigellosis, or bacillary dysentery, caused by the enteroinvasive bacteria Shigella, remains one of the top diarrheal diseases in children under five. Species/serotype diversity and geographical distribution strongly support the need for a multivalent S. flexneri vaccine. In the search for a highly immunogenic Shigella vaccine able to generate protective immunity in young children living in low and middle income countries, we have engaged into the development of immunogens consisting of synthetic fragments of selected putative S. flexneri O-antigens (O-Ags) covalently linked via single point attachment to protein carriers.

A multidisciplinary strategy interfacing medicinal chemistry and structure-based vaccinology was implemented. “Protective” epitopes were identified by use of a panel of synthetic O-Ag fragments. Protein conjugates of the most promising oligosaccharides were then evaluated for their immunogenicity in mice. A conjugate encompassing a synthetic hapten corresponding to three repeating units of the O-Ag from S. flexneri 2a, the most prevalent Shigella serotype, was designed accordingly. This vaccine candidate, named SF2a-TT15, has been shown to induce anti-S. flexneri 2a bactericidal antibodies. A GMP batch of SF2a-TT15 was produced and a first-in-human, single-blinded, dose escalation, placebo-controlled study was conducted.

With the first rationally designed synthetic oligosaccharide conjugate vaccine candidate in hand, this presentation primarily provides an overview of our strategy for a broad coverage S. flexneri vaccine. Emphasis is on hapten selection, vaccine design, safety and immunogenicity data following first use in human. In addition, the presentation highlights progress on the way to a S. flexneri bivalent glycovaccine candidate.

114. Gentle handling facilitates tandem MS-based structural determinations of glycoproteins and released glycans

Catherine E. Costello¹; ¹Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA

cecmsms@bu.edu

The heterogeneity and fragility of carbohydrate and other co- or post-translational protein modifications present challenges for their analysis by mass spectrometry, but new instrumentation, accessories, and protocols can successfully address these challenges. Electron-Based Dissociation (ExD) methods offer access to a set of dissociation modes that preserve labile modifications to biopolymers and provide both glycosidic and cross-ring cleavage information on glycans, on a time scale that is compatible with online nanoflow ultraperformance liquid chromatographic (nUPLC) separations ± ion mobility (IM) separations, and has potential for use with online capillary electrophoresis (CE). This lecture will present examples from ongoing fundamental and biological studies that show the advantages of soft ionization conditions, stepped collisional dissociation (CID and HCD) including their pairing with electron transfer dissociation (EThcD, CID/ETD), alternative data acquisition modes on Orbitrap instruments, and the implementation of nUPLC-IM-ExD in Fourier transform ion cyclotron resonance (FT-ICR) and quadrupole time-of-flight (QTOF) MS systems.

Title: The evolution of biological conjugation for vaccine production

Speaker: Jon Cuccui From: The London School of Hygiene and Tropical Medicine

Acknowledgements: Vanessa Terra¹, Sherif Abouelhadid¹, Marta Mauri¹, Emily Kay¹, Ian Passmore¹, Elizabeth Atkins¹, Prerna Vohra², Chintoan-Uta Cosmin², Thippesh Sannasiddappa³, Alex Smith³, Andrew Grant³, Mark Stevens², Brendan Wren¹

1: London School of Hygiene and Tropical Medicine, Infection Biology Department, London, WC1E 7HT. 2: The Roslin Institute & Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, EH25 9RG. 3: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES

Abstract:

Since the discovery of the enzyme PglB from Campylobacter jejuni and its initial characterisation, several groups have sought to learn to harness this oligosaccharyltransferase for the production of glycoconjugates. We and others have found that this is just one enzyme in a plethora of glycosylating machineries that bacteria carry and that are waiting to be exploited. This talk will focus on the development of biological conjugation strategies for the development of vaccines against bacterial pathogens and will discuss some of our latest research to increase vaccine potency and penetrate into new areas of need such as veterinary medicine.

IGO Milano (Glyco25)

Maximal 2300 characters

115. Chemistry and Biology of Polysialic Acid

Rita Gerardy-Schahn¹; ¹Institute of Clinical Biochemistry, Hannover Medical School, 30625 Hannover, Germany

In vertebrates, the α2,8 linked polymer of sialic acid, polysialic acid (polySia), is mainly known as a modification of the neural cell adhesion molecule NCAM. PolySia on NCAM can be produced by two polysialyltransferases, ST8SIA2 and ST8SIA4, with overlapping but distinct expression patterns and enzymatic profiles. As a major determinant of brain development and plasticity, abnormal levels of polySia as well as variants of the human ST8SIA2 gene have been linked to mental disorders like schizophrenia. In a clinical study we contributed to the demonstration that altered polySia-NCAM serum levels in schizophrenic patients are associated with negative symptoms, cognitive impairments and structural changes of brain areas involved in the pathophysiology of the disease. Correspondingly, mice with a conventional knockout of St8sia2 display deficits of cognition associated with impaired brain development resulting in altered long-range connectivity and a loss of interneurons in the prefrontal cortex. Notably, the cognitive deficits of adult St8sia2^-/- mice are aggravated by cannabis exposure during adolescence, which in humans is one of the one most prominent `second hits’ that render a predisposed person more susceptible to developing schizophrenia. By use of conditional knockout mouse models we now started to dissect, how ST8SIA2-deficiency in different neuron types and brain regions translates into impairments of cognition. In contrast to the neurodevelopmental impact of ST8SIA2, loss of ST8SIA4 leads to reduced polySia levels in adult mice resulting in altered synaptic plasticity and selective impairments of learning and memory. Based on recent studies, deficits of synaptic transmission and learning of St8sia4^-/- mice can be rescued by targeting the underlying synaptic mechanisms and, possibly, also by therapeutic application of polySia. Furthermore, impaired memory of adult St8sia4^-/- mice can be ameliorated by housing in an enriched environment, known to stimulate hippocampal neurogenesis. This finding raises intriguing questions concerning the role of ST8SIA4 for the age-dependent decline of the polySia-positive fraction of newborn neurons in the human hippocampus and for their further reduction under the condition of hippocampal atrophy in Alzheimer’s disease.

116. Glycosylated lipids commonly found in microorganisms and human nervous tissues

Yoshio Hirabayashi¹, Hisako Akiyama², Hiroyuki Kamiguchi², Tatsuro Muto³; ¹RIKEN Cluster for Pioneering Research, Saitama, Japan, ²Laboratory for Neural Cell Dynamics, RIKEN Center for Brain Science, Saitama, ³Fujita Health University School of Medicine, Aichi, Japan,

Lipid glycosylation is a highly conserved system in living organisms, indicating its critical role in life. In mammals, the central nervous system tissues contain a large variety of glycosylated lipids including sialic acid-containing complex glycolipids, gangliosides. We have developed a method to isolate isomeric monoglycosylated lipids from brain tissues by using hydrophilic interaction chromatography column (HILIC)-ESI-MS/MS. We successfully detected and isolated cholesteryl beta-glucoside (CholGlc) and beta-galactoside (CholGal) from abundant galactosylceramide (GalCer). Up to now we never detect alpha-linked GalCer in vertebrate brains. Although the bacteria such as Helicobacter pylori synthesized sterylglucoside but its anomeric configuration is alpha but not beta. Moreover, Pyroli ChoGlc as well as plant sterylglucosides is biosynthesized by specific glucosyltransferases. In mammals, however, CholGlc formation is not dependent on UDP-Glc and mediated by retained transglycosylation reaction of glucocerebrosidase (Akiyama et al., 2011,2016). The same lipids were also detectable in human cerebrospinal fluid (CSF) samples and suggested to be a possible maker for neurological diseases (Mutoh, this meeting). We demonstrate that the newly isolated glycolipids are enriched in lipid raft fraction and have immune stimulatory or regulatory potential.

117. The role of sialylation in the life cycle of African Trypanosomes

J S Weber¹; A Gupta¹; S H C Ngomtcho²; S S Shaida³; T T Gbem⁴; G D Chetchet⁵; M Mamman³; A Dotzauer¹; J A Nok⁴; D Achukwi⁶; B. Reinhold-Hurek¹, and S Kelm¹; ¹University Bremen, Centre for Biomolecular Interactions Bremen, Faculty for Biology and Chemistry, Bremen, Germany, ²Departement of Biological Sciences, University of Ngoundéré, Ngoundéré, Cameroon, ³Nigerian Institute for Trypanosomiasis Research (NITR), Kaduna, Nigeria, ⁴Centre for Biotechnology Research and Training, Ahmado Bello University Zaria, Zaria, Nigeria, ⁵Departement of Biochemistry, Ahmado Bello University Zaria, Zaria, Nigeria, ⁶TOZARD Research Laboratory, Bamenda Cameroon

Abstract:

Nagana, the animal form of African trypanosomiasis caused by Trypanosoma species, is a serious livestock production problem in Africa. Glycan biosynthesis machinery, in particular Trans-sialidase (TS) activities play key roles in the pathology of nagana. Active TS genes from T. congolense, TconTS1, TconTS2, TconTS3 and TconTS4 are expressed in both the tsetse vector and the bloodstream of infected animals. Expression levels differ at the various sites of their life cycle. Our research addresses the potential role of these enzymes in the life cycle of the parasite by characterising the enzymatic properties, the potential donor and acceptor substrates as well as the impact of interfering with the trans-sialylation in tsetse flies. The potential of such attempts in controlling the transmission of the disease will be discussed.

118. Tumor angiogenesis activation by hypoxia-dependent glycosylation of tumor suppressors

Claudine Kieda^1,2; ¹Centre de Biophysique Moléculaire, UPR 4301 CNRS, Orléans, 45071, France, ² Laboratory of Molecular Oncology and Innovative Therapies (LOMTI), WIM, Warsaw, 04-141, Poland

claudine.kieda@cnrs-orleans.fr ; ckieda@wim.mil.pl

The hypoxic conditions that appear in any growing tumor contributes to tumor growth, dissemination and participate to select aggressive cancer stem-like cells. The metabolic adaptation of of the microenvironment and tumor cells is ruled by hypoxia which gets to be a key parameter of the tumor evolution. Thus, hypoxia alleviation is the challenge of therapeutic strategies aiming to render the tumor sensitive to chemical and immune response.

In tumor the angiogenesis starts on the basis of endothelial cells (ECs) damage, a key event occurring at the angiogenic switch. It is favored by the particular aerobic glycolytic metabolism of ECs, the latter are to be targets to revert hypoxia through vessel normalization which is the condition to fulfil in order to permit the access to the tumor cells. Tumor suppressor, PTEN is the main control of the PI3K/PDK/AKT/mTOR pathway. Its specifically active glycoforms influence its action. PTEN regulates other tumor suppressors as the von-Hippel-Lindau protein upon PI3K/AKT/mTOR cell growth activating pathway control. This opens a regulatory mean for hypoxia-induced HIF1α and has deep consequences on MDM2 inhibition of the p53 suppressor activity.

Consequently, vasculature normalization obtained by direct O2 delivery increase by red blood cells modified by an allosteric effector of hemoglobin is of major importance as it also allows a stable angiogenesis normalization via PTEN activation and regulating OGlcNAc transferase activity in endothelial cells.

This work was supported by the help of the Kosciuszko grant program. Polish Ministry of Defense (MON) grant “Kosciuszko I”, grant no. 579/2016/DA and NCN “OPUS 12” grant no.357961 to CK

119. Competing interactions between biosynthesis enzymes regulate heparan sulfate structure

Heparan sulfate chains attached to proteoglycans from various tissues and cells show a great variation in structure as well as polysaccharide chain length. However, studies of heparan sulfate from mice clearly show that heparan sulfate from the same cell type or organ of different age-matched mice are indistinguishable. Obviously, structural features of heparan sulfate are under strict control, applied at different levels during and after biosynthesis. In addition to transcriptional and translational regulation of biosynthesis enzyme expression, UDP-sugar and PAPS (sulfate donor) availability, regulatory phosphorylation and sulfation of the glycosaminoglycan linkage region, the formation of functional enzyme complexes appears to be a powerful way to influence biosynthesis. Together with substrate specificity, competing interactions between the enzymes may have a major role in determining the final outcome of biosynthesis.

In collaboration with Sakari Kellokumpu and his group in Oulu we are now investigating interactions between heparan sulfate biosynthesis enzymes, with an emphasis on the N-deacetylase/N-sulfotransferases (NDSTs) which perform the first modification reactions during biosynthesis. By correlating data on heparan sulfate structure in cells over-expressing or lacking a selected enzyme with information about the interaction potential of the enzyme our aim is to evaluate the impact of enzyme-enzyme interactions on heparan sulfate structure.

120. Glycan biomarkers as early predictors of cardiovascular diseases and diabetes

Gordan Lauc^1,2; ¹University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia, ²Genos Glycoscience Research Laboratory, Zagreb, Croatia

glauc@pharma.hr

The majority of proteins that evolved after appearance of multicellular life are glycosylated and glycans significantly affect structure and function of these proteins. However, due to structural complexity of glycans and the absence of a direct genetic template, the analysis of protein glycosylation is much more complicated than the analysis of DNA or proteins. Consequently, the knowledge about the importance of individual variation in glycans for both normal physiological processes and diseases is still limited. In the last few years it is becoming increasingly clear that variations in a DNA sequence are only a beginning of the understanding of complex human diseases. Genetic polymorphisms have to be put in the context of complex biology of life and a more elaborate approach that combines different ‘omics phenotypes is needed to understand disease mechanisms and perform patient stratification that transcends genomics. Glycomics, as by far the most complex posttranslational modification, has an immense potential in this respect, which is only beginning to be investigated. By generating glycomic data for over 80,000 individuals from some of the best characterized clinical and epidemiological cohorts we enabled glycomics to meet other ‘omics. The analysis of this rich gold mind is painting a picture of a very complex genetic and epigenetic regulation of glycosylation that fine tunes protein activity in multiple biological systems and, if altered, contributes to development of different complex diseases. In particular, the evidence is accumulating that in cardiometabolic diseases changes in glycosylation are not only biomarkers, but functional effectors that actively participate in disease development.

121. Glycobiology and Neurodegenerations. The Special role of Gangliosides

Robert Ledeen¹, Samar Alselehdar¹, Gusheng Wu¹; ¹Department of Pharmacology, Physiology, & Neurosciences, New Jersey Medical School, Rutgers University, Newark, NJ, USA

Gangliosides have occupied a prominent role in certain neurodegenerative disorders, both as proposed initiator of neuropathology via subnormal levels of expression and as prospective therapeutic tool. This is particularly the case for Parkinson’s disease (PD) which has been shown to manifest deficiency of a-series gangliosides (GM1, GD1a) in brain as well as a variety of peripheral tissues. A mouse model which recapitulates several pathophysiological aspects of PD occurs due to disruption of one allele of the B4galnt1 gene (GalNAc transferase, GM2 synthase) leading to partial deficiency of a-series similar to that occurring in PD tissues. Such mice experienced resolution of both movement disorders and neuro-pathological lesions following peripheral administration of high doses of GM1 ganglioside or much lower doses of membrane-permeable analogs of GM1 (which enter brain and neuron interior more readily than GM1 itself). GM1 has been administered peripherally to PD patients in clinical trials (Dr. Jay Schneider and associates) resulting in early symptomatic improvement, slower symptom progression, and improved dopamine transporter binding. Both the cause of a-series deficiency and the possible benefit of GM1 analogs in PD therapy remain to be determined. Gangliosides have also received attention in relation to Huntington’s and Alzheimer’s diseases (AD), both of which have been reported to express subnormal levels of ganglioside. AD was studied some years ago by Dr. Lars Svennerholm who administered GM1 directly into brain via osmotic mini-pump in the lateral ventricals of a small group of AD patients. Significant improvement was reported in all cases but unfortunately there was no follow-up to that study. Other gangliosides have also been studied in relation to other neurological disorders, some of which will be described at this symposium.

122. “How sugar metabolism controls protein N-glycosylation - Lessons from CDG”

Dirk J. Lefeber¹; ¹Radboudumc Expertise Centre for Disorders of Glycosylation, Translational Metabolic Laboratory, Department of Neurology, Radboud university medical center, 6525 GA, Nijmegen, The Netherlands

The biological mechanisms that underlie glycan structural abnormalities in human disease are still very poorly understood. Important novel insights into the biochemical mechanisms of protein N-glycosylation have originated from a group of monogenic defects, the Congenital Disorders of Glycosylation (CDG). After initial discovery of many defects in glycosyltransferases, a growing group of additional genetic causes is being identified. These factors cluster in two main groups: defects of Golgi homeostasis and defects in cellular sugar metabolism. These studies provide novel insight into glycosylation mechanisms. For example, SLC35A1, the Golgi transporter of CMP-neu5Ac, was shown to be required for O-mannosylation of alpha-dystroglycan, in a process that is independent from CMP-Neu5Ac.

With a focus on understanding the tissue-specific mechanisms in sugar metabolism, we have established ion-pair LC-QqQ methodology for analysis of sugar metabolites extracted from cells (in 6-well plate format), tissues, and organisms. For example, this allowed to confirm the presence of CDP-ribitol in most human tissues. We are currently applying this technology to unravel the mechanisms of CMP-Neu5Ac synthesis in human. Clinical phenotypes of genetic defects in the sialic acid pathway are contradicting, with an adult-onset myopathy due to GNE mutations, a neurological syndrome due to NANS mutations, the next enzyme in this pathway, and a neuromuscular disease due to mutations in NPL, required for catabolism of Neu5Ac. Biochemical studies are carried out in knock-out and patient-derived cells, including metabolic tracing with ¹³C-labeled sugars and chemically modified sugar derivatives ManNPoc and SiaNPoc. For NANP knock-out cells, we observed normal steady-state levels of CMP-Neu5Ac and decreased de novo synthesis, suggesting the existence of alternative phosphatases. Application of novel N-glycoproteomics technology to plasma samples of sialic acid synthesis defects showed that only subsets of plasma proteins have reduced sialic acid levels. In summary, our preliminary data start to reveal novel mechanisms in sialic acid synthesis that have a protein-specific effect on sialylation.

123. The hot and cold breath of O -GlcNAcylation on cell homeostasis

Stéphan Hardivillé¹, Sadia Raab¹, Maïté Leturcq¹, Ninon Very¹, Gabriela Fuentes-García², Moyira Aquino-Gil¹, Céline Schulz¹, Marlène Mortuaire¹, Angelina Kasprowicz¹, M. Cristina Castañeda-Patlán², Martha Robles-Flores², Ikram El Yazidi¹, Anne-Sophie Vercoutter-Edouart¹, andTony Lefebvre¹; ¹ Univ. Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France, ² Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico.

O-GlcNAcylation is a highly dynamic posttranslational modification (PTM) whose level depends upon nutrient status. Only two enzymes regulate O-GlcNAcylation cycling: the glycosyltransferase OGT (O-GlcNAc transferase) and the glycoside hydrolase OGA (O-GlcNAcase) that add and remove the GlcNAc moiety to and from acceptor proteins respectively. During the last thirty years O-GlcNAcylation has emerged as a master regulator of cell homeostasis. The study of O-GlcNAcylation in various biological systems (bacteria, insects, protists and metazoans) has opened perspectives for understanding its role in many kingdoms of life. O-GlcNAcylation orchestrates many fundamental functions in the cell among which transcription, protein fate, intracellular trafficking and enzyme activity. In the other hand, this glycosylation is implicated in diverse pathologies including neuronal and cardiovascular diseases, and cancers. We will summarize recent findings regarding the roles played by O-GlcNAcylation in cell signaling, cell cycle, resistance to proteasomal degradation and in the stabilization of the MCM complex that is pivotal for DNA replication. Next, we will review the function of this modification in the etiology of colorectal cancer (CRC). We will push forward the molecular dissection of the mechanisms underlying CRC by focusing on the O-GlcNAcylation of TBP. Lastly, we have observed that O-GlcNAcylation regulated the expression of the colon cancer cell markers CD44 and CD133, this point will also be addressed.

124. Organization and dynamics GPI-anchored proteins reveal the cell surface as an active actin membrane composite

Satyajit Mayor¹; ¹National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bellary Road, Bangalore, India

Studying the organization and dynamics of GPI-anchored proteins at the surface of living cells has provided fundamental insights into the structure and function of the plasma membrane¹. The unusual organization of GPI-anchored proteins and other membrane components at the cell surface in the form of small nanoclusters in a sea of monomers has provoked the need for a new understanding of the cell membrane². A detailed understanding of the physico-chemical principles behind the generation of these nanoclusters via the active mechanics of actin filaments and myosin has emerged^3,4. This suggests that the living cell membrane is an active actin-membrane composite⁵. We now find that nanoclusters of GPI-anchored proteins are generated in response to signalling cues from the activation of many membrane receptors. These nanoclusters in turn create specialized membrane microenvironments necessary for the functioning of these receptors. In my talk I will discuss our current understanding of the mechanism of GPI-anchored protein nanoclustering, and how this is relevant for the functioning of integrin receptors.

References:

1.Köster, D. V. & Mayor, S. Cortical actin and the plasma membrane: inextricably intertwined. Curr. Opin. Cell Biol.38, 81–9 (2016).

2.Garcia-parajo, M. F., Cambi, A. & Jacobson, K. Nanoclustering as a dominant motif in plasma membrane organization. J. Cell Sci.127, 4995–5005 (2014).

3.Raghupathy, R. et al. Transbilayer lipid interactions mediate nanoclustering of lipid-anchored proteins. Cell161, 581–94 (2015).

4.Gowrishankar, K. et al. Active remodeling of cortical actin regulates spatiotemporal organization of cell surface molecules. Cell149, 1353–1367 (2012).

5.Rao, M. & Mayor, S. Active organization of membrane constituents in living cells. Current Opinion in Cell Biology29, 126–132 (2014).

ID: 21962

125. Synthetic carbohydrate-based conjugates as promising shigella flexneri vaccine candidates: from concept to first-in-human study

L.A. Mulard¹; ¹Unit Chemistry of Biomolecules, Institut Pasteur, UMR3523 CNRS, Paris, France

Shigellosis, or bacillary dysentery, caused by the enteroinvasive bacteria Shigella, remains one of the top diarrheal diseases in children under five. Species/serotype diversity and geographical distribution strongly support the need for a multivalent S. flexneri vaccine. In the search for a highly immunogenic Shigella vaccine able to generate protective immunity in young children living in low and middle income countries, we have engaged into the development of immunogens consisting of synthetic fragments of selected putative S. flexneri O-antigens (O-Ags) covalently linked via single point attachment to protein carriers.

A multidisciplinary strategy interfacing medicinal chemistry and structure-based vaccinology was implemented. “Protective” epitopes were identified by use of a panel of synthetic O-Ag fragments. Protein conjugates of the most promising oligosaccharides were then evaluated for their immunogenicity in mice. A conjugate encompassing a synthetic hapten corresponding to three repeating units of the O-Ag from S. flexneri 2a, the most prevalent Shigella serotype, was designed accordingly. This vaccine candidate, named SF2a-TT15, has been shown to induce anti-S. flexneri 2a bactericidal antibodies. A GMP batch of SF2a-TT15 was produced and a first-in-human, single-blinded, dose escalation, placebocontrolled study was conducted.

With the first rationally designed synthetic oligosaccharide conjugate vaccine candidate in hand, this presentation primarily provides an overview of our strategy for a broad coverage S. flexneri vaccine. Emphasis is on hapten selection, vaccine design, safety and immunogenicity data following first use in human. In addition, the presentation highlights progress on the way to a S. flexneri bivalent glycovaccine candidate.

126. The Glycobiome: mucin glycosylation and the microbiome

Nicolle H. Packer¹, Arun Everest-Dass¹, David Oliveira¹, Chi-Hung Lin¹, Robyn Peterson¹, Marianne Hesselager¹, Wei Chong¹, Hasinika Gamage¹, Emoke Bendixen¹, Martina Sanderson-Smith¹, Ian Paulsen¹; ¹Department of Molecular Sciences, Macquarie University, and Institute for Glycomics, Griffith University, Australia.

The microbiome was discovered about a decade ago and has become the focus of much research aimed at characterising the effects of a changing microbiota on health and disease. What is clear is that the microbiome is personalised and changes dramatically with numerous diseases and is somehow involved in human metabolism, nutrition, physiology, and immune function. What is unclear are the mechanisms that are regulating and achieving these microbiota-induced changes.

Every surface of the body that is exposed to the environment has a microbiome; including the mouth, gut, vagina and skin. Common to all these exposed surfaces is a mucin layer covering the underlying epithelium. Originally this mucus was thought to only control the surface hydration but it is now clear that the microbiota are using the mucin glycosylation as a landing stage by binding to specific glycoepitopes.

Our work has shown that microbes, both yeast and bacteria, bind to specific glycan substructures in the mouth (Candida albicans), throat (Group A Streptococcus) and sweat (Staphylococcus epidermidis, Corynebacterium spp), and that the strength of this binding can be blood group dependent and be inhibited by competing glycans. We have also shown that the mutation in the fucosyltransferase (FUT1) gene protects pigs from post weaning diarrhoea by preventing the fucose-dependent adhesion of the enterotoxigenic Escherichia coli F18. We now show that there is a correlation between the changes in the gut microbiome and the glycoepitopes displayed on the gut mucins. Both the microbiome and the gut mucin glycosylation profile changes in response to a high fat diet and as a result of dietary fibre intervention. Whether the mucin glycosylation changes observed are controlled by the microbe and/or by the host is a question that needs to be determined if we are to increase our understanding of how to maintain a healthy microbiome.

127. Organization of the Golgi apparatus determines its glycan output

P. Pothukuchi¹, I. Agliarulo¹, M. Pirozzi¹, G. Turacchio¹, R. Rizzo¹, N. Dathan¹, D. Spano¹, L. Capolupo¹, P. Henklein², G. Boncompain³, F. Perez³, L. Obeid⁴, A. Luini¹, G. D'Angelo⁵, S. Parashuraman¹; ¹Institute of Protein Biochemistry IBPCNR, Naples, Italy, ²Charité – Universitätsmedizin Berlin Charitéplatz 1 10117 Berlin, Berlin, Germany, ³Institut Curie, CNRS UMR1 44, Research Centre, Paris, France, Paris, France, ⁴Department of Medicine, Stony Brook University, Stony Brook, NY 11794, New York, United States of America, ⁵Institute of Protein Biochemistry, National Research Council of Italy, Naples, Italy

Glycans play an important role in development and physiology. There are about 150-200 glycosylation enzymes localized to the Golgi that are responsible for most of the cellular glycosylation reactions. Several of them are localized in a restricted manner to one or more cisterna of the Golgi apparatus. How is such a compartmentalized localization achieved? and what are its functional consequences? Our analysis of sphingolipid (SL) biosynthesis system has yielded interesting insights into these questions.

We observed that Grasp55, a medial Golgi localized matrix protein, is important for restricting the localization of two key enzymes of SL biosynthetic pathway – glucosylceramide synthase (GCS) and lactosylceramide synthase (LCS) to trans side of the Golgi stack. In the absence of Grasp55 these enzymes are increasingly localized to the cis-side of the Golgi. Grasp55 binds these enzymes and prevents their entry into retrograde transport carriers and thus their distribution to cis-side of the stack. Generalizing this concept, we propose that Grasp55 and possibly also other matrix proteins act as retainers to bind and restrict the retrograde transport of proteins – (Golgi residents and probably also cargoes) within the Golgi. These retainers act together with adaptors (that promote entry into retrograde carriers) to restrict the localization of the enzymes to specific cisternae of the stack and may also play role in anterograde transport of cargoes.

The mis-localization of GCS and LCS to the cis-side of the Golgi in the absence of Grasp55 correlates with changes in flux across the SL biosynthetic pathway. Using mutant proteins and other means to shift the position of GCS and LCS from the late to the early secretory pathway we observe that cis-localization of these enzymes promotes flux across the glycan pathways initiated by them. We surmise that this is because they encounter their substrates in the absence of competing enzymes. This proposal suggests an interesting concept that the compartmentalized organization allows Golgi apparatus to act as a flux regulator by controlling the localization of its enzymes.

128. Inhibiting Glyco-Immune checkpoints with novel bispecific-like enzyme therapeutics to treat cancer

Lizhi Cao¹, Adam Petrone¹, Wayne Gatlin¹, Jenny Che¹, Abhishek Das¹, Robert LeBlanc¹, Zakir Siddiquee¹, Sujata Nerle¹, Michal Stanczak², Lihui Xu¹, Karl Normington¹, Wei Yao¹, Carolyn Bertozzi³, James Broderick¹, Heinz Läubli², Li Peng¹; ¹Palleon Pharmaceuticals, Waltham, MA, USA, ²Cancer Immunology Laboratory, Department of Biomedicine, University Hospital Basel, Switzerland, ³Department of Chemistry, Stanford University, Stanford, CA, USA

Cancer therapy has been revolutionized by the recent developments of immune-checkpoint inhibitors (ICI) to harness the power of the immune system in fighting cancer. However, most patients fail to have durable responses or become resistant to ICI, highlighting the need to identify new mechanisms of immune evasion in cancer. Recently, the glyco-immune checkpoint axis (sialoglycan/Siglec pathway) has emerged as a new mechanism of immune regulation involving both innate and adaptive immunity and an important mechanism of cancer immune escape. However, a therapeutic intervention of this axis remains a great challenge due to the promiscuous receptor-ligand interactions between 15 Siglecs and dense array of various sialoglycans in humans. To overcome this hurdle, we developed a new therapeutic modality named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), which is bispecific-like comprising of a tumor-associated antigen-binding arm and a sialidase moiety, allowing selectively removing terminal sialic acids, the critical binding carbohydrate of Siglecs, from tumor cells. We demonstrated that EAGLE decreased sialic acid levels of tumor cells and enhanced anti-tumor immune responses in multiple human system models in vitro and immunocompetent syngeneic mouse tumor models in vivo. EAGLE treatment released cancer cell-mediated immunosuppression and induced proinflammatory cytokines IFN gamma, IL-17A, IL-2, IL-6, and TNF alpha in human coculture assays of cancer cells with PBMC and primary endothelial cells. Systematic administration of EAGLE increased tumor-infiltrating immune cells and led to robust anti-tumor activities with complete regressions as monotherapy in syngeneic EMT6 mouse tumor models. Re-challenge experiments in EAGLE-cured mice showed that EAGLE induced anti-tumor immunological memory. We further revealed that the mechanism of action of EAGLE involved both innate and adaptive immunity because depleting macrophages or CD8⁺ T-cells decreased or abolished its efficacy. EAGLE in combination with anti-PD1 mAb treatment achieved ~100% cures in EMT6-Her2 models. In summary, EAGLE is a novel and promising immunomodulatory therapeutics inhibiting the glyco-immune checkpoints and has the potential to overcome resistance to current immunotherapies.

Title: Glyco Abstract Delegates

129 Characterization of new anti-mouse Dectin-1 monoclonal antibody

Y. Adachi¹, N. Maeda¹, J. Tetsui¹, K. Ishibashi¹, D. Yamanaka¹, N. Ohno¹; ¹Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Sciences

Dectin-1 is a small C-type lectin receptor that recognizes fungal cell wall ß-glucan, and is responsible for host defense against fungal infection by producing proinflammatory cytokines. Recent reports suggest that the Dectin-1 contributes the development of diseases such as DSS-induced colitis and type-1 allergy. Controlling dectin-1 function may regulate the inflammatory diseases. To obtain antagonistic monoclonal antibody against mouse Dectin-1, we tried to prepare hybridoma clones by immunizing Dectin-1 KO mice with mouse Dectin-1 soluble protein as an antigen.

The new established 2 clones, 2F3 and 3H8, produced mouse IgG1 and IgG2b kappa chain, respectively. The affinity (K_D) of those mAb determined by BLItz was from 10^-10 to 10^-12 M. The 3H8 significantly inhibited the binding of soluble Dectin-1 to 1,3-ß-D-glucan from Candida albicans. To compare the specificity of two clones, various commercially available Dectin-1 monoclonal antibodies are applied. The binding of 2A11 (Bio-Rad) to mouse Dectin-1 transfectant was inhibited by pretreatment with 2F3, but not with 3H8. The i.p. administration of 3H8 to mice reduced the number of Dectin-1⁺ peritoneal macrophages.

These results suggest that 3H8 is a unique antagonistic mouse monoclonal antibody specific to mouse Dectin-1.

130. Newly identified brain sterol metabolite glycosylated sterols are metabolized by glucocerebrosidase 1 and 2 in vivo

H. Akiyama⁴, M. Ide¹, E. Nakanishi², N. Uemura², Y. Yamaguchi⁵, H. Kamiguchi⁴, R. Takahashi², P. Greimel³, Y. Hirabayashi¹; ¹Cellular Informatics Laboratory, RIKEN, Wako, ²Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, ³Laboratory for Cell Function Dynamics, Center for Brain Science, RIKEN, Wako, ⁴Laboratory for Neural Cell Dynamics, Center for Brain Science, RIKEN, Wako, ⁵Laboratory of Pharmaceutical Physical Chemistry, Tohoku Medical and Pharmaceutical University, Sendai

Sterylglycosides are glycosylated sterols that are found in bacteria, fungi, plants, and animals. In vertebrates, cholesterol is the major sterol component, and its glucosylation forms ß-cholesterylglucoside (ß-GlcChol). Previously, we demonstrated that ß-glucocerebrosidase 1 and 2 (GBA1 and GBA2) possess not only glucosylceramide (GlcCer) hydrolase activity but also transferase activity, catalyzing a transglucosylation reaction to form ß-GlcChol via transfer of the glucose residue from GlcCer to cholesterol [Akiyama and Hirabayashi, Biochim. Biophys. Acta, 2017]. Homozygous mutations in the GBA1 gene cause Gaucher disease, which is the most common lysosomal storage disorder. On the other hand, heterozygous mutations in GBA1 gene are a high-risk factor for Parkinson’s disease. Loss-of-function mutations in the GBA2 gene causes neurological disorders, such as cerebellar ataxia and spastic paraplegia. Although ß-GlcChol metabolism mediated by GBA1 and GBA2 has been studied in several kinds of cells, tissues and plasma, the metabolism of sterylglycosides in the brain is not understood. Since purification of ß-GlcChol from central nervous system tissue has not been reported, and structural analysis based on ß-GlcChol-containing fraction has not been reported, we developed a multi-step chromatographic purification protocol capable of isolating ß-GlcChol from the brain [Akiyama et al., J. Lipid Res., 2016]. Here, we describe the purification and characterization of sterylglycosides from adult rat brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of the isolated brain sterylglycoside revealed the presence of galactosylated sterol, in addition to ß-GlcChol. To better understand the metabolism of ß-GlcChol and the galactosylated sterol in the brain, we analyzed the brains of GBA2-deficient mice and GBA1- and/or GBA2-deficient Oryzias latipes (Japanese medaka). For ß-GlcChol metabolism, GBA1 and GBA2 were responsible for ß-GlcChol degradation and formation, respectively. For galactosylated sterol metabolism, both GBA1 and GBA2 were unexpectedly responsible for galactosylated sterol formation. This is the first report to demonstrate the existence of galactosylated sterol in vertebrates and how in vivo metabolism of sterylglycosides occurs in the brain.

131. A systemic deficiency of ganglioside expression recapitulates parkinsonian symptoms and pathology

S.K. Alselehdar¹, G. Wu¹, Z. Lu¹, R.W. Ledeen¹; ¹Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School at Rutgers University, Newark, NJ, USA

Parkinson’s disease (PD), the second most common neurodegenerative disorder (the majority of which is idiopathic), is considered a disease of aging as the symptoms typically develop around 65. It is now known that the onset of Parkinsonian symptoms begins in the peripheral nervous system (PNS) followed by the emergence of pathology in the central nervous system (CNS), resulting in motor impairment. Here, we present evidence that this systemic dysfunction is related to a widespread reduction in ganglioside expression in the CNS and PNS, primarily of the a-series gangliosides (GM1 and GD1a). We conducted our studies in a mouse model deficit in beta-1,4-N-acetyl-galactosaminyltransferase 1 (GM2 synthase). Using thin-layer chromatography and immunohistochemical studies, we found low GM1 and GD1a levels in the brain (CNS), colon and heart (PNS) of these mice (B4galnt1+/-). Importantly, these mice recapitulate the symptoms and pathology of PD by showing reduced gastrointestinal function, impaired motor coordination, muscle weakness, as well as difficulty in initiating movement, which is successfully reversed using chronic administration of GM1 and GM1 analogs. Additionally, in this study, we identified that a significant number of PD patients also show reduced GM1 and GD1a expression in the brain, colon and heart and exhibit specific PD-like pathology, as evident by less dopaminergic neurons in substantia nigra pars compacta (SNpc) and increased aggregated alpha synuclein in GM1-deficient neurons in the GI tract, heart and brain. This suggests an effective therapy for PD may employ the use of GM1 replacement.

132. Chlorella strains differ from one another by dozens of novel N-glycan structures

R. Mócsai¹, F. Altmann¹; ¹Department of Chemistry, University of Natural Resources and Life Sciences (BOKU), Vienna

Chlorella microalgae are used for various purposes such as fatty acid production, waste water processing or as health-promoting food supplements. MALDI-TOF MS of N-glycans of commercial Chlorella products revealed an unprecedented diversity of N-glycan patterns of which none coincided entirely with that of C. vulgaris type strains. Thirteen glyco-type groups were identified based on differing numbers of methyl groups, pentoses, deoxyhexoses and N-acetylhexosamine. Isobaric N-glycans were shown in several cases to actually adopt different structures. Unexpected features included the occurrence of arabinose, the presence of unusually bound galactose, various types of monosaccharide methylation or the substitution of the second N-acetylglucosamine with arabinose, xylose or galactose. The wealth of as yet undescribed N-glycan structures within a group of organisms regarded as constituting a closely related taxonomic group demands answers as to its evolutionary origin and instigates questions about the function of elaborate glycan diversity on a unicellular organism. Certainly, determination of microalgal glyco-profiles suggests itself as a sharp tool for product characterization and taxonomic classification.

133. ST6GalNAc-IV and ST6GalNAc-VI are responsible for the biosynthesis of glycans recognized by siglec-15 on raw264.7 cells

H.S. Jiang¹, Y.H. Chen¹, P.Y. Low¹, J.Y. Chen¹, K.H. Khoo¹,T. Angata¹; ¹Institute of Biological Chemistry, Academia Sinica

Siglec-15 is a member of Siglec family of sialic acid-recognition lectins of vertebrates. Previous studies have demonstrated that Siglec-15 promotes the fusion of osteoclast precursors by interacting with the sialylated ligands, such as CD44. Siglec-15 preferentially recognizes sialylated glycans containing Sia<2-6HexNAc structural element. However, the sialyltransferase(s) responsible for the biosynthesis of the glycans recognized by Siglec-15 has not been identified. In this study, we sought the sialyltransferase(s) involved in the biosynthesis of the glycans recognized by Siglec-15, using RAW264.7 mouse macrophage cell line as an osteoclast precursor model.

Quantitative PCR analysis of ST6GalNAc isozymes revealed that ST6GalNAc-IV and ST6GalNAc-VI transcripts are present in RAW264.7 cells, and that their expression levels increase by treating the cells with receptor activator of NF│B ligand (RANKL), the inducer of osteoclast differentiation. We prepared ST6GalNAc-IV and ST6GalNAc-VI knockout (KO) RAW264.7 cells by CRISPR/Cas9 technology, and found that the binding of recombinant Siglec-15 to the KO cells is attenuated in both cases. Mass spectrometry-based glycan structural analysis of recombinant CD44 protein produced in a human cell line revealed that co-expression of ST6GalNAc-IV results in an increase in 6-sialyl-T structure (Galß1-3[Neu5Ac<2-6]GalNAc<1-Ser/Thr) and concomitant decrease in core 2-branched O-glycans, whereas co-expression of ST6GalNAc-VI is cytotoxic, greatly diminishing the protein yield and thus preventing further analysis. Taken together, both ST6GalNAc-IV and ST6GalNAc-VI are likely involved in the biosynthesis of glycans recognized by Siglec-15 on RAW264.7 cells.

We thank Academia Sinica Common Mass Spectrometry Facilities (Institute of Biological Chemistry, Academia Sinica) for technical support. This study was supported by the grants from the Ministry of Science and Technology, Taiwan (MOST 104-2311-B-001-017-MY3 and 107-2311-B-001-034).

134. Integration of omics data via the glycosmos portal and the glyspace alliance

M. Shiota², S. Tsuchiya², T. Ono², T. Kuoka², A. Fujita², D. Shinmachi⁵, N. Aoki⁵, Y. Watanabe⁴, S. Okuda⁴, Y. Suzuki¹, N. Fujita¹, K. Angata¹, H. Kaji¹, H. Narimatsu¹, I. Yamada⁶,K. Aoki-Kinoshita³; ¹Biotechnology Research Institute for Drug Discovery, Advanced Industrial Science and Technology, Tsukuba, ²Faculty of Science and Engineering, Soka University, Tokyo, ³Glycan Life Science Integration Center, Faculty of Science and Engineering, Soka University, ⁴Graduate School of Medical and Dental Sciences, Niigata University, Niigata, ⁵SparqLite, Ltd., Tokyo, ⁶The Noguchi Institute, Tokyo

With the advancement of bioinformatics technologies, it is now possible to obtain large amounts of genomics and transcriptomics data. Many advancements in glycomics techniques has also enabled large datasets of glycomics data to be accumulated. It has always been difficult for bioinformatics researchers to fully understand glycomics data, however. Therefore, we have been developing a glycoscience portal called GlyCosmos to enable not only glycoscientists, but also bioinformaticians to more easily access glycan-related data. We have taken advantage of existing data science technologies and bioinformatics tools to incorporate various glycan-related omics information into GlyCosmos such that data can be more readily accessed and analyzed. Currently, information about glycogenes, glycoproteins glycan-related diseases, pathogens, pathways and glycome data can be accessed from GlyCosmos. Moreover, our efforts have allowed us to participate in the GlySpace Alliance in order to easily share our data with other valuable web resources. In this presentation, we will present the GlyCosmos Portal and describe the variety of omics resources available.

135. Possible molecular mechanisms for preventing galectin-2 inactivation under oxidative environment

M. Tamura¹, T. Takeuchi², T. Hatanaka²,Y. Arata¹; ¹Faculty of Pharma-Science, Teikyo University, Tokyo 173-8605, Japan, ²Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, Saitama 350-0295, Japan

Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. A proteomic analysis identified Gal-2 (containing 2 highly conserved Cys residues, Cys57 and Cys75) as a protein that was S-nitrosylated when mouse gastric mucosal lysates were reacted with S-nitrosoglutathione (a physiologically relevant S-nitrosylating agent). The recombinant mouse Gal-2 was S-nitrosylated using nitrosocysteine, following which the sugar-binding specificity and dimerization capacity of the protein was unaffected. On the other hand, Gal-2 oxidation by hydrogen peroxide (H₂O₂) resulted in the loss of sugar-binding ability, whereas S-nitrosylation prevented H₂O₂-induced inactivation. Conversion (mutation) of Cys57 to a Met (C57M) did not result in lectin inactivation following H₂O₂ treatment. When C75S mutant (retains Cys57 residue in the polypeptide chain) was treated with H₂O₂, a typical peak in the CD spectrum, resulting from the rich ß-sheet structure, was gradually lost depending on the H₂O₂ concentration. When C75S protein solution (H₂O₂ treated) was subjected to SDS-PAGE following BS³ (bis(sulfosuccinimidyl)suberate) crosslinker treatment, the cross-linked products with molecular weights higher than the trimers were detected. This suggested that Cys57 residue oxidation resulted in the formation of insoluble higher molecular weight multimers. However, in the presence of lactose, wild type Gal-2 retained its typical ß-sheet peak in the CD spectrum. We have previously revealed that Gal-2 could interact with mucin (an important component of the gastric mucosa) in a ß-galactoside-dependent manner and produce an insoluble precipitate. This suggested that Gal-2 might play a role in the gastric mucosa by strengthening the barrier structure through mucin(s) cross-linking on the mucosal surface. Therefore, the results indicated that the presence of a carbohydrate ligand as well as prevention of Cys57 residue oxidation by S-nitrosylation could contribute towards maintaining Gal-2 activity under oxidative environment and that they may be important for Gal-2 function in the gastric mucosa.

136. High throughput & comprehensive analytical tools to decode the composition of human breast milk

S. Porfirio¹, S. Archer-Hartmann¹, G.B. Moreau², G. Ramakrishnan², W.A. Petri, Jr², R. Haque³, P. Azadi¹; ¹Complex Carbohydrate Research Center, The University of Georgia, Athens, GA, USA, ²Dept. of Medicine/Infectious Diseases, University of Virginia, Charlottesville, VA, USA, ³International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh

Human breast milk is an incredibly rich and complex biofluid composed of many complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides (HMOs). Strikingly, HMOs are not digested by the infant and function as prebiotics for bacterial strains associated with numerous benefits (e.g., protection from enteropathogen infection). Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition (e.g., lactation stage, geographical variation), the analysis of HMO diversity and complexity in human milk samples is of utmost relevance.

Using human milk samples from a cohort of Bangladeshi mothers participating in a study on malnutrition and stunting in children, we have characterized breast milk composition in human samples by means of liquid chromatography and high-resolution tandem mass spectrometry analysis (LC-MS/MS). Although the relationship between HMOs and the growth patterns of infants has not been well characterized, some studies have described a negative correlation between the growth phenotype of severely stunted infants and the abundance of sialylated HMOs in their mother’s milk. Thus, one of our main goals is to use LC-MS/MS techniques to evaluate modification patterns (fucosylation / sialylation) in HMOs. This analytical approach identified over 100 different glycoforms present in human samples, and these results were used to create glycomic composition profiles of each mother’s milk. Our analysis shows a wide diversity of milk composition, with a predominance of fucosylated and sialylated HMOs over neutral HMOs. Although the five most abundant HMOs, comprising up to 60% of the total HMO content of the samples, are typically low molecular weight (MW) compounds, breast milk samples contain a wide variety of glycoforms of much larger MW. Actually, our results show that human breast milk samples tend to contain a total number of 70-80 HMOs, with the highest permethylated masses detected being >5000 mass units. These results demonstrate how current analytical techniques can be used to characterize human milk composition, revealing exciting new details and enhancing our understanding about this fascinating biological matrix.

137. DPMS status in tunicamycin-induced ER stress

J.A. Martinez², Z. Zhang², A. Banerjee², I. Banerjee³, J. Serrano-Negrón², K. Baksi¹, D.K. Banerjee²; ¹Department of Anatomy & Cell Biology, School of Medicine, Universidad Central del Caribe, Bayamón, USA, ²Department of Biochemistry, School of Medicine, University of Puerto Rico, San Juan, PR 00936-5067, USA, ³Department of Chemistry, Fordham University, Bronx, NY 10458, USA

DPMS, a GT-A folded 31kDa endoplasmic reticulum (ER) resident protein essential for the elongation of Man₅GlcNAc₂-PP-Dol to Man₉GlcNAc₂-PP-Dol in asparagine-linked (N-linked) protein glycosylation. Presence of several motifs (DXD, PKA, etc.) makes DPMS subject to regulation following environmental and metabolic challenges. For example, while responding to the cAMP-mediated extracellular signaling, DPMS is phosphorylated. The result is increase in DPMS catalytic activity, which becomes a driving force for enhancing the LLO synthesis and consequently the protein N-glycosylation due to a cross-talk involving DPMS, GlcNAc-1 phosphate transferase and Glc-P-Dol synthase. The outcome is increased cellular proliferation. DPMS overexpression can independently mimics this process. On the other hand, PKA mutation makes cells sensitive to cAMP signaling and the genetic deficiency of DPMS develops congenital disorder of glycosylation (CDG).

Cells treated with the protein N-glycosylation inhibitor tunicamycin exhibits a time-dependent decrease in DPMS activity and correlates with reduced angiogenesis, a hallmark for tumor growth. Accumulation of non- or under-glycosylated proteins beyond a certain threshold develops ER stress interfers with the cell cycle transcriptional machinery and causes the cell cycle arrest. The end point is activation of cellular suicide (i.e., programed cell death/apoptosis) by unfolded protein response (upr). Down regulation of VEGFR I & II phosphorylation fails to reverse the tunicamycin effect of extracellular VEGF₁₆₅. Tunicamycin treatment also causes quantitative inhibition of both non-metastatic and metastatic human breast cancer cells. Testing in pre-clinical models of breast cancer, tunicamycin reduces the progression of ER^-/PR^-/HER2⁺ (double negative) and ER^-/PR^-/HER2^- (triple negative) breast tumors in athymic nude mice in a time- and dose-dependent manner. The reduction was ~55% in three weeks for a double-negative breast cancer given iv at 1mg/Kg once a week whereas it was ~65% for a triple-negative breast cancer given orally at 250⎧g/Kg twice a week.

Tunicamycin gold-nanoparticles are found to be three times more potent in reducing the DPMS expression while inducing ER stress and blocking angiogenesis.

138. New homozygous mutation in ALG12 gene resulted in CDG-I disorder in newborn patient

P. Baráth³, M. Nemčovič³, J. Ziburová³, Z. Pakanová³, S. Šesták³, J. Bellová³, A. Šalingová¹, A. Tekelyová², M. Skokňová², I. Brucknerová², C. Šebová¹, J. Mucha³; ¹National Institute of Children’s Diseases, Department of Laboratory Medicine, Bratislava, Slovakia, ²National Institute of Children’s Diseases, Department of Neonatology and Intensive Medicine, Bratislava, Slovakia, ³Slovak Academy of Sciences, Institute of Chemistry, Department of Glycobiology, Bratislava, Slovakia

ALG12-CDG (CDG Ig; OMIM#607143) inherited rare metabolic disease, caused by defect in the function of alpha-mannosyltransferase 8, which is coded by ALG12 (22q13.33) gene. Up to this date, only 9 patients were reported worldwide. Here we present the eutrophic male, born in 34th gestational week as the second twin. During the gravidity period, polyhydramnion, gestational diabetes, hepatopathy and isthmocervical insufficiency were observed. The child was born with the signs of polystigmatism (low hairline, widely open fontanelles, broad nasal bridge, elongated philtrum, gothic palate, low-set earlobes, pectus excavatum, micropenis, retention of the testes, pedes equinovari) and subcutaneous edema. Due to the clinical and biochemical abnormalities of the newborn patient, isoelectric focusing of transferrin was performed with observed significant hypoglycosylation pattern, typical for CDG-I. Further MALDI TOF/TOF analysis of permethylated serum N-glycans revealed the increased signals of GlcNAc2Man5-7, along with decreased signals of GlcNAc2Man8-9, that suggested the ALG12 defect. Genetic analysis of corresponding ALG12 coding region from patient´s sample found yet unidentified homozygous substitutional variant in the exone 10, while this variant was heterozygous in the samples of both parents and asymptomatic twin. Such comprehensive genomic and glycomic approach led to the discovery of new mutation in ALG12 gene, potentialy responsible for the clinical manifestation of this disorder.

Acknowledgements

This study was supported by the Slovak Research and Development Agency Grant no. APVV-17-0300 and VEGA 2/0130/18. This contribution is the result of the implementation of the project: „Technical infrastructure for biomedical research“, ITMS 26230120008, supported by the Research & Development Operational Programme funded by the ERDF

139. Stereo-specific substrates (ceramide-glycomes) and genomic expression of specific -glycosyltransferases guided-biosynthetic pathways of sphingo-glycolipids

S. Basu¹, A. Agarwal¹, M. Basu¹; ¹University of Notre Dame, Cancer Drug Delivery Research Foundation, Notre Dame, IN 46556, USA

Unlike “Genomic codes” that exist in the nucleus the “Glycomic codes” also exist on the cell surfaces. A glycomic code resides in the stereo specificity of 10 to 15 different carbohydrate units (pentose’s, hexoses, octanoic acid, or nonanoic acids). In this work we investigated the stereo-specificities of acceptors mono- or oligosaccharides that determine the in vivo direction of the pathway (or steps) for the formation of intermediate glycosphingolipids (GSLs) of different families (Ganglio-, Globo-, or Lacto-), which resides on the cell surfaces. Expression of these GSLs varies during embryonic development and disease stages. Besides other functions, “Glycomes” are involved in the intracellular interactions between cell to matrix and cell to extracellular agents such as bacteria, viruses, hormones, toxins, polypeptides, polysaccharides (polyglycomes), polynucleotides, small molecular drugs, and apoptotic agents. In each case interaction between the incoming “glycome” monosaccharide and the molecule attached to the extrinsic molecules and existing oligosaccharides on the cell surfaces is very stereospecific. These stereo-specific glycosphingolipids are biosynthesized in the Golgi-bodies during embryonic development (regulated by genomic expression). By comparing Vmax values of stereospecific glycome oligosaccharides (glycosphingolipids) with same enzyme preparation we proposed the probable pathways of biosynthesis of the three families of GSLs (Ganglio-, Globo-, or Lacto-GSLs) in many species, at different stages of development, or in metastatic stage of cancer cells. The chain growth in any family of ceramide-glycome is strictly guided by the presence of anomeric structure present in the ultimate or penultimate units present in the acceptor or product molecule. Highly purified <-and ß-glycosidases (Exo-or Endo-glycosidases) were prepared from microbial-, plant or sea-animals in novel ways, and were used to assign stereo specificity of many glycome molecules. Expression of these carbohydrate molecules on the cell membranes could be termed as “Glycomic code.”

140. Generation and functional characterization of human cell-based models of leukocyte adhesion deficiency type ii diseases (lad ii).

B. Bazan¹, E. Skurska¹, Y. Shauchuk¹, M. Olczak¹; ¹Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, 14A F. Joliot-Curie St., 50-383 Wroclaw, Poland

Nucleotide sugar transporters are important players in glycosylation process. They deliver activated forms of monosaccharides into the lumen of Golgi apparatus and endoplasmic reticulum (ER). GDP-fucose transporter (C1) is encoded by an SLC35C1 gene. It is localized in the Golgi apparatus. Mutations in this transporter cause defects in the fucosylation of glycoconjugates. In humans lack of fucose in glycoconjugates cause the Leukocyte adhesion deficiency II (LAD II), which is a rare congenital disease leading to immunodeficiency and psychomotor retardation.

We decided to explore five mutation within the SLC35C1 gene which occur in patients with LAD II. Two of them: ⊗G588 and G969A are causing a reading frame shift that leads to a premature termination codon. Others three: C439T, C923T and A1010G cause a point mutations in the gene, which cause amino acid substitution. For generation of the human cell-based model we knocked out the SLC35C1 gene in HepG2 and HEK293T cell lines (KOC1). To achieve it we used CRISPR-Cas9 approach. Using Aleuria aurantia lectin (AAL) specific for fucose we demonstrated that cells with inactivated C1 protein display almost completely lack of fucose in glycoconjugates. C1 variants bearing mutations listed above were stable overproduced in KOC1 cells. We found that C1-C439T, C1-C923T and C1-A1010G protein variants localized properly to the Golgi apparatus whereas C1- ⊗G588 and C1- G969A protein variants delocalized. These mutants mainly colocalise with calnexin, which is the endoplasmic reticulum marker. Inappropriately localized protein would not only influence the fucose transport in to the Golgi apparatus but also on proteins residing in the ER. Those results are identical in both HEK293T-KOC1 and HepG2-KOC1 cells. Our studies will help to better understand the molecular basics of LAD II.

141. Customized glycosylation of therapeutics via a novel production platform

A. Behrens¹, M. Mally¹, A. Arnold¹, A. Harsmann¹, C. Lizak¹, D. Kuhn¹, G. Tontodonati¹, J. Schneider¹, R. Mladenov¹, R. Follador¹, S. Herwig¹, S. Zanon¹, T. Schelbert¹, Y. Turgay¹, A. Faridmoayer¹; ¹LimmaTech Biologics AG

LimmaTech Biologics utilizes its glycosylation expertise to develop a novel glycoengineering platform for better therapeutics.

Scouting of Kinetoplastida led to the identification of a unicellular eukaryote with surprisingly unique N-glycosylation features. We engineered the organism to produce improved therapeutic proteins characterized by custom and homogenous N-glycosylation. Our glycoengineering platform bears the promise of cheaper and faster production. At the same time, it avoids potentially disadvantageous features of mammalian cell production such as glycan microheterogeneity and variable site occupancy.

Several proof of concept studies, including EPO and an anti-CD20 antibody, confirm the potential of this novel glycoengineering platform. Next, we will perform mode of action studies of optimized candidate therapeutics to show advantageous glycoengineering effects.

142. Investigating a putative role for lewis a-structures in plant pathogen-defence

G. Beihammer¹, A. Romero Pérez², E. Van Damme², R. Strasser¹; ¹Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna; ²Department of Biotechnology, Ghent University, Ghent

Terminal sugar residues on complex N-glycans have been quite extensively studied in mammals: they regulate a number of different functions by modulating receptor-ligand interactions and are often sites for pathogen recognition and attachment. On the other hand, surprisingly little is known about the function of complex N-glycans in plants. Here, the repertoire of different N-glycan structures is comparably limited: Lewis A-structures present on complex N-glycans constitute the highest degree of modifications detected so far in plants. These structures are synthesized by the sequential attachment of galactose and fucose to terminal N-Acetylglucosamine, catalyzed by GALT1 and FUT13. Even though Lewis A-modifications are highly conserved throughout the plant kingdom, their biological function remains to be elucidated. Recent studies indicated a possible connection to plant pathogen defense via the stress-inducible lectin F-box-Nictaba. In Arabidopsis thaliana, F-box-Nictaba is upregulated upon infection with Pseudomonas syringae pv. tomato DC3000. Plants overexpressing said lectin show reduced susceptibility to bacterial infection. Furthermore, F-box-Nictaba is co-expressed with GALT1 and FUT13 and interacts with Lewis A-structures in glycan-array studies. Here, we set out to investigate whether Lewis A-structures are involved in the plants response to bacterial challenge. Therefore, we established and characterized various Lewis A-overexpression and knockout lines of A. thaliana. Seedlings of these lines were infected with P. syringae DC3000 using a flood-inoculation assay. The amount of colony-forming units (CFU) within the plants was monitored as well as the disease symptoms on the leaves by quantifying leaf-lesion area. Furthermore, changes in the expression levels of F-box-Nictaba, GALT1 and FUT13 were measured via qPCR and the total N-glycan profile of infected plants analyzed via MALDI-TOF-MS. We detected no difference in respect to bacterial colonization or damaged leaf area between the individual lines. Furthermore, no obvious changes in the abundance of Lewis A-structures in infected plants were observed. Our data indicates that presence or absence of Lewis A-structures does not affect the plants susceptibility to bacterial infection.

143. Avian glycosphingolipid antigens as receptors for shiga toxin

A. Bereznicka³, A. Modlinska¹, M. Duk³, R. Kaczmarek³, K. Szymczak-Kulus³, K. Mikolajczyk³, K. Kapczynska⁴, P. Wittek¹, E.Y. Park², T. Piasecki¹, M. Czerwinski³; ¹Department of Epizootiology and Clinic of Bird and Exotic Animals, Wroclaw University of Environmental and Life Sciences, Wroclaw, ²Laboratory of Biotechnology, Shizuoka University, Shizuoka, ³Laboratory of Glycobiology, Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Science, ⁴Laboratory of Medical Microbiology, Department of Immunology of Infectious Diseases, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Science

Human blood group P^k and P1 antigens are glycosphingolipids synthesized by <1,4-galactosyltransferase, which is encoded by different forms of the A4GALT gene. The P^k and P1 antigens are receptors for pathogens, such as uropathogenic strains of Escherichia coli, as well as toxins, e.g. Shiga toxins (Stx). Stxs are released by Shiga toxin-producing strains of E. coli (STEC) and Shigella dysenteriae of serotype 1; both present a growing threat to human population because of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS), often leading to death.

It has been shown that birds can harbor STEC, but they seem refractory to Stx and could be a spillover hosts. STEC has been isolated from pigeons, starlings and other avian species. However, the avian mechanism of resistance to toxins is unknown. In contrast to humans, the avian PK antigen has been described to be expressed only on glycoproteins.

The neutral glycosphingolipid fraction from RBCs of various avian species were isolated and analyzed by HPTLC and mass spectrometry. In every sample, GlcCer and LacCer were the major neutral glycosphingolipid components. Moreover, RBCs of rock pigeon appeared to contain also Gb3Cer, nLc4Cer and an unidentified glycosphingolipid with molecular weight similar to human P1 antigen. We also identified sugar structures that do not occur in mammals.

We evaluated binding of anti-P1/P^k antibody and Shiga toxin subunit 1B to red blood cells (RBCs) of 40 various species of birds using flow cytofluorometry. In contrast to the RBCs of species from Galliformes order, only RBCs of birds belonging to Neoaves showed binding of anti-P1/P^K antibodies and Stx. We also found that in addition to the previously described A4GALT gene (called “P”), birds have

a homologous gene (called “M”) differing by 55 nucleotides. Specific binding of Shiga toxin subunit 1B were observed on glycoproteins of RBCs from birds belonging to Neoaves and on glycosphingolipids where A4GALT homolog gene appeared. Expression of both genes in human teratocarcinoma 2102Ep cells showed that the enzyme encoded by “P” gene attaches Gal moieties only to glycoproteins, while the “M” gene attaches Gal to glycoproteins and glycosphingolipids.

144. Nuanced interactional diversity between sialic acids and galectins modulates cancer progression

R. Bhat¹; ¹Biological Sciences Division, Indian Institute of Science, Bangalore

Galectins, endogenously expressed lectins, bind to Beta-lactoside-containing conjugates and regulate interactions between cells and extracellular matrices. Sialic acids terminally cap Beta-lactoside-containing N-glycans and modulate the binding of galectins to their cognate glycan partners. Here, I will present two case studies that provide insights into the diversity in possible crosstalk between sialic acids and galectins during cancer progression. In the first case study concerning ovarian cancer metastasis, I show that context-dependent control in expression of sialic acids may in turn regulate non-canonical intracellular functions of Galectin-1 (GAL-1), leading to metastasis-permissive multicellular morphologies. Such regulation has been previously observed in mammary gland morphogenesis and breast cancer and showcases its genericity. In the second study, which deals with cancer of the transitional epithelia of urinary bladder, I show GAL-1 negatively regulates the cell-surface sialic acid levels of cancer urothelia and enhances their interaction with the surrounding matrix microenvironment. Taken together, these case studies reveal that we have only begun to explore the complexity in the dialectic between elements of the glycome and anti-glycome. This complexity holds the key to uncovering the molecular etiologies underlying the debilitative aspects of cancer metastasis.

145. Heparan sulfate structural features in multiple osteochondromas’ disease

N. Veraldi¹, A. Parra², E. Urso¹, C. Cosentino¹, A. Naggi¹, L. Sangiorgi²,A. Bisio¹; ¹Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni, Milan, ²Istituto Ortopedico Rizzoli - IRCCS, Bologna

Multiple Osteochondromas (MO) is the most frequent rare dominant skeletal disorder characterized by the growth of several benign tumors (osteochondromas, OCs) potentially degenerating in malignancy (peripheral chondrosarcomas, PCSs). MO is known to be characterized by the absence or highly reduced amount of heparan sulfate (HS) in the extracellular matrix of growth plate cartilage, resulting in improper signaling networks which causes altered bone growth. Despite the accumulation of HS in the cytoplasm of MO chondrocytes was recently observed, the structural alterations responsible for such an accumulation and the biochemical mechanisms preventing HS from undergoing its physiologic pathway are still unknown.

The present study dealt with the isolation of HS from healthy and pathologic cartilage excisions obtained from fetuses and prepubescent subjects and from patients affected by MO, both benign and malignant tumors. Samples were processed by applying a procedure previously set up to structurally characterize and compare HS from pathologic and physiologic conditions and to investigate the phenotypic differences arising in the presence of exostosin 1 or 2 (EXT1 or EXT2) mutations. The structural characterization of isolated HS was approached by using mono and bi-dimensional NMR and especially using HPLC-MS analysis following exhaustive depolymerization with a cocktail of heparinases I, II and III. The aim of the study was the identification of possible structural alterations of cartilaginous HS in pathologic conditions, such as chain length, oligosaccharide composition, degree of sulfation, and a possible correlation with the original skeletal disorder.

The most important results of the present study were the isolation of HS from human growth plate cartilage and the detection of its relative abundance in MOs and in PCSs compared to healthy conditions. Moreover, the analytical approach applied highlighted two macroscopic aspects of HS structure such as degree of sulfation and chain length, which appeared to be significantly different both between OC and PCS and with respect to healthy cartilage. Interestingly, it appeared that HS structural analysis could be predictive of a possible malignant degeneration of benign tumor.

146. Structure-based design of galectin-specific compounds as potential therapeutics

H. Blanchard¹; ¹Institute for Glycomics, Griffith University

Galectins are a family of galactoside-specific lectins involved in a myriad of metabolic and disease processes. Many galectins demonstrate significant involvement in promoting cancer and inflammation. Some, most notably galectin-3 and galectin-8, have also been identified to have a role in bone-remodelling, leading to osteoporosis (bone loss). Galectins exhibit interactions with cell-surface glycans that can facilitate disease progression. Designing small molecule compounds that target galectins and compete with their interactions toward endogenous carbohydrate ligands is an approach to develop therapeutics, but is challenging due to the characteristics of the galectin carbohydrate-binding site. Our investigations engage structure-based design of compounds to bind within the galectin carbohydrate-binding site and thus have potential to reduce lectin function within biological systems.

This presentation focuses on the design of monosaccharide-based compounds that target galectin-8, a tandem-repeat galectin with two carbohydrate-recognition domains (CRD). X-ray crystallographic structure determination was employed to give insight into structural features important in developing potent and selective inhibitors. Our designed monosaccharide-based compounds successfully mimic the interactions made by the galactose ring, as well as the carboxylic acid part of a natural galectin-8N ligand, 3’-O-sialylated lactose whereby we could promote interaction with a unique arginine located in the galectin-8 N-terminal domain, thus affording selectivity. We have generated novel monosaccharide scaffolds that show good binding and selectivity towards galectin-8.

147. N-glycosylation profile of undifferentiated and differentiated human bone marrow mesenchymal stem cells

H. Hamouda¹, M. Ullah², M. Berger¹, M. Sittinger², R. Tauber¹, J. Ringe²,V. Blanchard¹;¹Institute of Laboratory Medicine, Clinical Chemistry, and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany, ²Tissue Engineering Laboratory & Berlin-Brandenburg Center for Regenerative Therapies, Dept. of Rheumatology and Clinical Immunology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

Mesenchymal stem cells (MSC) are multipotent cells that are easy to isolate and culture, develop into several tissues including fat, migrate to diseased organs, have immunosuppressive properties and secrete regenerative factors. The aim of this work was to characterize the cell surface N-glycome of MSC and of their differentiated progenies. In the present study, human MSC were isolated from the bone marrow of donors, differentiated into adipocytes, chondrocytes and osteocytes. The differentiation state was verified with specific markers. Cell surface glycopeptides were released from living cells using our well-established protocol and N-glycans were subsequently analysed after permethylation using MALDI-TOF mass spectrometry. We were able to detect over then 100 different N-glycans including high-mannose, hybrid, complex N-glycans as well as poly-N-acetyllactosamine chains.

Differentiation of MSC into adipogenic direction was accompanied by an increased amount of biantennary fucosylated structures, a decreased amount of fucosylated as well as afucosylated tri- and tetraantennary structures, and an increased sialylation, which was corroborated by an upregulation of ST3GAL1 and a downregulation of FUT8 gene. The N-glycosylation profile of chondrogenically differentiated MSCs was investigated before and after isolation from their extracellular matrix (ECM) and compared to the profile of undifferentiated MSCs. There was more high-mannose and less complex N-glycans when their ECM was present. Differentiation was accompanied with an increased amount of biantennary N-glycans when the ECM was absent. Cell surface N-glycosylation of chondrogenically differentiated MSCs was generally less branched than the one of undifferentiated MSCs. N-Glycans had higher amounts of core-fucosylation as well as Lewis^X epitopes. Osteogenic differentiation of MSCs was accompanied by an increased amount of biantennary structures. In addition, cell surface N-glycosylation was less branched in osteogenically differentiated MSCs. Furthermore, the amount of fucosylated structures increased with differentiation and contained mainly core-fucose residues.

To conclude, the N-glycome of human MSC and their differentiated counterparts, namely adipocyte-, chondrocyte- and osteocyte-like cells, possess characteristic cell-type specific glycosylation features.

148. Enzymatic generation of dynamic combinatorial libraries of functionalised cyclodextrins

P. Both¹, M. Deabreu¹, C. Gray¹, J. Van Munster¹, D. Shirion¹, R. Castangia¹, A. Green¹, S. Flitsch¹; ¹Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, United Kingdom.

Cyclodextrins are a family of macrocyclic oligosaccharides most commonly composed of 6, 7 or 8 glucosidic units. These supramolecular structures have found broad application in the pharmaceutical, cosmetic, food and textile industries, largely due to their ability to bind guest molecules within their hydrophobic cavity. Functionalisation of cyclodextrins is often required to enhance their properties for a desired application or to broaden their use in other research fields such as molecular recognition and the development of enzyme mimics. The key challenge in developing novel cyclodextrin applications is the identification and production of cyclodextrins with a ring size and arrangement of functional group(s) that confer the required properties. Here we generate dynamic combinatorial libraries of cyclodextrins with single and multiple as well as homo- and hetero- functionalizations, with a range of ring sizes and functional group configurations. We demonstrate that these libraries, generated via exploiting the broad substrate specificity of a cyclodextrin glycosyltransferase, allow guest-molecule complexing resulting in selective amplification of functionalised host cyclodextrins. The work presented is expected to greatly facilitate the identification and synthesis of novel cyclodextrin derivatives with diverse applications.

149. Rapid synthesis of heparan sulfate mimicking oligomers through peptide coupling

A. Brackovic¹, A.M. Daines¹, S.F. Hinkley¹; ¹Ferrier Research Institute, Victoria University of Wellington

Heparan sulfate (HS) is glycosaminoglycan (GAG) polymer intrinsic to life. Comprised of repeating glucosamine - glucuronic acid disaccharide units, it is hugely information rich due to varying chain length and a complex sulfation pattern: O-sulfation may be observed at the C-2 position of the uronic acids and C-6 and C-3 positions of the glucosamine. Furthermore, the glucosamine can also be N-sulfated, N-acetylated or, rarely, a free amine. These modifications give rise to a varied negative charge distribution pattern along the polymer which effects protein interactions and in part explains the biological promiscuity of HS.

The optimum toolkit to probe biological processes would be a range of monodisperse HS oligomers, but many congeners are required to quantify structure activity relationships (SAR), and although multistep synthesis methodology is improving, this approach is tedious. We present the synthesis of building blocks suitable of rapid oligomer synthesis peptide-linked modified saccharides. This strategy permits resin-assisted peptide coupling giving access to carbohydrate amide-linked polymers. Our protecting group strategy was devised to allow for controlled sulfation. Ultimately, we will create a library of polymers with well defined chain-length and sulfation patterns for evaluation of bioactivity in bone repair processes.

The synthesis of the glucuronic acid mimicking monomer unit starts from the commercially available ®-D-galactose pentaacetate and the C-1 methyl ester is installed using a cyanation/methanolysis sequence. The protecting group strategy allowed for orthogonal protection of the hydroxyl groups at C-2 (silyl ether or acetate), C-3 (silyl ether) and C-6 (para-methoxy benzyl ether), and the stereochemistry at C-4 was subsequently inverted to obtain the C-4 amine which was protected as a fluorenylmethyl carbamate. Next, the C-6 hydroxyl was converted into a tert-butyl ester. The presence of another ester functionality and a carbamate made the deprotection of the methyl ester challenging but successful selective deprotection was achieved through an SN2 reaction with lithium iodide under neutral conditions. This way, the synthesis of the glucuronic acid mimicking unit was completed in 13 steps.

150. A cell free enzyme-based protocol for the tailored synthesis of nature-identical actinobacillus pleuropneumoniae serotype 1 capsular oligosaccharides

I. Budde², C. Litschko², R. Gerardy-Schahn², M. Schubert¹, T. Fiebig²; ¹Department of Biosciences, University of Salzburg, Salzburg, Austria, ²Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. Based on its capsule polymer composition, App is classified into 18 different serotypes. The capsule polymer of App serotype 1 (App1), a serotype often involved in outbreaks, consist of [4)-GlcNAc-®(1,6)-Gal-<-1-(PO4-] repeating units that are O-acetylated at O-6 of the galactose. It is a major virulence factor and was used in previous studies for the successful generation of an experimental glycoconjugate vaccine. However, the distribution of glycoconjugate vaccines in the animal health sector is limited by the high cost of production, which involves mass cultivation of the pathogen.

We therefore aim to exploit the capsule polymerase Cps1B of App1 for the development of potentially more affordable in vitro synthesis protocols for the polymer. Besides the two catalytic domains (a glycosyltransferase and hexose-phosphate transferase domain), Cps1B contains a domain rich in tetratrico peptide repeats (TPR) that was shown to be involved in dimerization. We analyzed the elongation mechanism of wildtype Cps1B in comparison to a ⊗TPR truncation (Cps1B-⊗TPR). Interestingly, the product profiles displayed by Cps1B suggest processive elongation of the nascent polymer, whereas Cps1B-⊗TPR elongates in a distributive manner. The latter is favored in biotechnological applications, because it allows control over the product length and thus the generation of tailored oligosaccharides. With the aim to further decrease the heterogeneity of the synthesized products, single-domain mutants were generated and immobilized on individual columns, separating the two catalytic activities and allowing the generation of a pool of oligosaccharides with even lower dispersity. Since the natural capsule polymer harvested from App1 is modified by O-acetylation, we also identified and purified the putative O-acetyltransferase Cps1D from the capsule gene cluster of App1 and used it to modify the polymers produced by Cps1B. 2D NMR analyses of the products revealed O-acetylation levels identical to the natural polymer. Taken together, we established the pathogen-free in vitro synthesis of nature-identical App1 capsule polymers with defined length.

151. Chemoenzymatic synthesis of sulfated ganglioside glycans

M. Bunyatov², G. Boons¹; ¹1. Utrecht University, Chemical Biology and Drug Discovery 2. Complex Carbohydrate Research Center, ²Utrecht University, Chemical Biology and Drug Discovery

Gangliosides are one of the most abundant class of glycolipids that reside mainly on the outer surface of cells of the nervous system. These sialic acid-containing gangliosides represent acidic glycosphingolipid family due to their carboxylic acid group of sialic acid. The structural diversity of acidic glycosphingolipids further increases by the presence of sulfate groups on several positions of ganglioside backbone, for some gangliosides in the place of sialic acid. The presence of those sulfate esters on the glycans of gangliosides have been attributed to several carcinomas.

Methodologies describing the synthesis of sulfated glycolipids mostly focus on the synthesis of either sulfated or sialylated forms of gangliosides. The preparation of glycans of sulfated ganglioside is impeded by the unavailability of several sulfo- and sialyltransferases. To get a deeper insight about the sulfated gangliosides we develop a chemoenzymatic route which comprises the chemical synthesis of core ganglioside glycan which contains sulfate esters at various positions and enzymatic modification of this core by several bacterial or mammalian glycosyltransferases.

The core ganglioside backbone (GM1) structure, was successfully synthesized containing sulfate groups at several positions. The use of different sialyltransferases allowed us to further diversify this glycan into more complex ganglioside forms such as GD1a, GT1a, GT1alfa and their sulfated analogues.

152. Lipid-associated chitin transglycosylases of saccharomyces cerevisiae yeast cell surface - intermediate form in processing are revealed in the cell wall.

A. Bykova¹, V. Rekstina¹, R. Ziganshin², T. Kalebina¹; ¹Lomonosov Moscow State University, ²Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry

Introduction

Yeast cell wall (CW) includes GPI-proteins covalently anchored to glucan. Some of them are transglycosylases. Strict regulation of their activity is vital for the cell, but it could be hampered as a consequence of immobile attachment. We assumed that there is a pool of non-covalently bound GPI-proteins in the CW.

Considering that vesicles were revealed in yeast CW, we suggested that there are some GPI-proteins that are non-covalently bound to glucan, but incorporate in CW in the form of microcompartments associated with lipids. Additionally, we suppose glucantransferase Bgl2 could influence these GPI-proteins incorporation. This research is devoted to the verification of the proposed assumptions.

Methods

Isolation yeast CW, protein biotinylation, differential CW-protein extraction, Western blotting, LC-MS/MS –analysis.

Results and discussion

In delipidated CW two non-covalently bound chitin transglycosylases Crh1 and Crh2 were revealed. It is assumed that the detected proteins have already lost the lipid part of the GPI-anchor, but are non-covalently attached to the glucan yet. We believe that the identified chitin transglycosylases in the CW are associated with lipids and form a microcompartment.

Crh1 and Crh2 are found also in the CW of mutant strain devoid of Bgl2p. Apparently, their incorporation into the cell wall is independent of the functions of Bgl2p glucantransferase.

Conclusion

Non-covalently bound to high-molecular-weight glucan chitin transglycosylases Crh1 and Crh2 were revealed in the yeast CW. It is supposed to be associated with lipids and incorporated in the CW independently of Bgl2p.

This work was supported by Russian Foundation for Basic Research grant N°18-34-00915mol_a.

153. Identification of the antigen recognized in vitro by rHIgM22, a remyelination-promoting human monoclonal antibody

L. Cabitta¹, S. Grassi¹, S. Prioni¹, L. Mauri¹, M.G. Ciampa¹, Y. Zorina², S. Sonnino¹, A. Prinetti¹; ¹Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy, ²Acorda Therapeutics, Inc, Ardsley, NY, USA

Recombinant human IgM22 (rHIgM22) binds to myelin and oligodendrocytes (OLs), and promotes remyelination in models of multiple sclerosis. rHIgM22 preferentially reacts with sulfatide-positive (O4+) OLs, and its binding is abolished in brain slices from cerebroside sulfotransferase (CST) ko mice, suggesting its binding requires the presence of a product of cerebroside sulfotransferase. However, literature suggests that cell populations lacking sulfatide expression, such as microglia and oligodendrocyte precursor cells, are responsive to rHIgM22, thus the identity of the antigen recognized by this antibody remains to be elucidated.

We tested the binding of rHIgM22 to purified lipids and lipid extracts from various sources using TLC immunostaining and surface plasmon resonance (SPR) with lipid monolayers. Our results show that IgM22 binds to sulfatide and lysosulfatide in vitro, while it does not bind to other myelin sphingolipids. In addition, rHIgM22 also reacts with phosphatidylinositol, phosphatidylserine and phosphatidic acid, present in lipid extracts from various sources, including CST ko mice brains, mixed glial cultures, isolated astrocytes and microglia.

These data suggest that sulfatide at the OLs surface might be important for the binding of rHIgM22 to these cells. On the other hand, its ability to bind some glycerophospholipids could explain the biological responses elicited by rHIgM22 in cells lacking sulfatide expression. The in vitro reactivity of rHIgM22 suggests that binding of rHIgM22 to intact cells might require a complex molecular arrangement, and, in particular, sulfatide and other membrane lipids might be part of the functional rHIgM22 antigen localized at the cell surface.

154. The impact and mechanism of abnormal metabolised sulfatide on the m6a methylation modification in tumor cells

Q. Cai¹, X. Wu¹; ¹Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Key Lab of Glycoconjugate Research, Ministry of Public Health, Shanghai, 200032

Our previous study found that sphingolipid metabolism was abnormal in either hepatocellular carcinoma(HCC) or renal clear cell carcinoma(KIRC). We had further analyzed the pathways of abnormal sphingolipid metabolism in HCC and KIRC by lipidomics and found that the level of sulfatide was abnormally elevated, but ceramide, sphingomyelin and sphingosine levels were decreased. Our preliminary study revealed that sulfatide promoted the expression of integrin <V subunit and enhanced the metastasis ability of HCC cells. Meanwhile, we found that sulfatide down-regulated the mRNA adenine 6 methylation level of some genes (MTF1, ZMYND8) in HCC cells by MeRIP-seq, and had proved the sulfatide effect on the expression levels of these genes by RNA-seq and RT-PCR. To elucidate the mechanism of sulfatide affecting the methylation and expression of MTF1 and ZMYND8 mRNA, we used the m6A methylation inhibitor 3-Deazaadenosine as a control to detect the methylation level of MTF1 and ZMYND8 mRNA m6A and the stability of mRNA in HCC cells after sulfatide treatment. We also examined the effects of sulfatide on the expression levels and enzyme activities of the enzymes METTL3/14, WTAP, FTO, ALKBH5, YTHDF1-3, YTHDC1-3, which are involved in the methylation modification of RNA m6A. We also examined the effect of sulfatide on the formation of RNA m6A methylase complex by Co-IP. In conclusion, we proved that the abnormal metabolism of sulfatide in tumor cells may play an important role in m6A methylation modification and expression regulation of the mRNA, and elucidated the significance of abnormal lipid metabolites in tumor development.

155. Triple incorporation of an unnatural amino acid to create a glycoconjugate vaccine against streptoccocus pneumoniae

T. Violo², R.B. Quast¹, A. Allot², F. Benhadda², C. Dussouy², C. Tellier², C. Grandjean², E. Camberlein²; ¹LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, 31077 Toulouse CEDEX 04, France, ²Université de Nantes, CNRS, UFIP, UMR 6286, F-44000 Nantes, France

Streptoccocus pneumoniae is a bacteria responsible for serious diseases such as pneumonia or meningitis and leads to the death of approximately one million of children each year. Efficient anti-pneumococcal glycoconjugate vaccines made of bacterial capsular polysaccharides have been launched. However, these vaccines confer protection to seven or thirteen out of the approximately 90s pneumoccocus serotypes identified so far. Therefore finding an efficient vaccine against all of them is still a challenge. Moreover, the current conjugation techniques used to prepare the glycoconjugate vaccines do not allow to finely controlling the amount and position of conjugated carbohydrate haptens, a feature which strongly impacts the fate of the immune response. In this context, we plan to generate a vaccine that consists in a conjugate of a well-conserved surface protein of S. Pneumoniae (PsaA), for the targeting of a wider range of serotypes, with a capsular tetrasaccharide (“Gal-Glc-(Gal-)GlcNAc”) that induces effective immunity against one of the most common serotype of S. Pneumoniae (serotype 14). To finely control the site of the capsule tetrasaccharide conjugation on the protein we used the incorporation of propargyl-Lysine, an unnatural amino acid, during the translation of PsaA. The propargyl-Lysine introduced at three distinct positions, individually or at the same time, brings click chemistry functions necessary to perform homogenous and site-specific glycoconjugation with the synthetic tetrasaccharide equipped with a bioorthogonal azide-functionalized spacer.

156. Deciphering casd1 role in gd2 o-acetylation in breast cancer cells

S. Cavdarli⁴, N. Yamakawa², Y. Guerardel³, M. Terme¹, J. Le Doussal¹, P. Delannoy², S. Groux-Degroote²; ¹OGD2 pharma, Nantes, ²UGSF, UMR 8576, Lille University, ³UGSF, UMR 8576, Lille University- OGD2 pharma SA Nantes, ⁴UGSF, UMR 8576, Lille University- OGD2 pharma, Nantes.

Mainly restricted to the nervous system in healthy adults, disialoganglioside GD2 has been shown to be involved in aggressiveness and metastasis of neuro-ectoderm derived tumors such as melanoma and neuroblastoma. However, the use of the ch14.18 anti-GD2 therapeutic antibody in neuroblastoma patients induces toxicity due to GD2 expression in nerves. Interestingly, the O-acetylated form of GD2, OAcGD2, is not expressed in human peripheral fibers but highly expressed in GD2 positive tumor cells. Very little information is known regarding the expression and roles of O-acetylated disialogangliosides in breast cancer (BC) cell lines. Recently, we reported OAcGD2 expression in BC cell lines, whereas OAcGD3 was not detected. Our results suggest a direct synthesis of OAcGD2 from GD2. However, the acetylation mechanisms are still poorly understood. The biosynthesis of O-acetylated sialic acids involves sialate O-acetyltransferases that transfer an acetyl group from acetyl-coenzyme A onto the C4/C7/C8/C9 positions of sialic acids at the terminal position of sialylated glycoconjugates. The only sialyl-O-acetyltransferase known to date is encoded by CASD1 and is involved in GD3 O-acetylation. In order to get insights into the mechanisms of OAcGD2 biosynthesis, we tried to decipher the potential role of CASD1 in GD2 O-acetylation by modulation of CASD1 expression in SUM 159PT BC cell line. Our results show that the depletion of CASD1 using siRNA reduced OAcGD2 expression in SUM 159PT BC cell line by immunofluorescence and confocal microscopy. In contrast, CASD1 overexpression using transient transfection increased OAcGD2 expression, whereas stable transfection did not have any impact on OAcGD2 expression. These preliminary results predict a possible role of CASD1 in GD2 O-acetylation in BC cells.

157. Downregulation of <1-2 fucosyltransferase induces human pluripotent stem cell differentiation

S. Chen¹, S. Cohen², R. G. Lichtenstein²; ¹ Faculty of Engineering, Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel, ²Faculty of Engineering, Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

The composition of cell surface glycoconjugates is determined by glycosyltransferases whose function in the context of cell fate decisions is poorly understood. One of the glycosyltransferases which function in human pluripotent stem cells (hPSCs) is the <1-2 fucosyltransferase (FUT1) that catalyzes the synthesis of fucosylated glycoconjugates in an <1-2 linkage. Using a model of hPSCs-derived germ layer cells and cardiomyocytes, we uncover an imperative function of FUT1 and its consequent <1-2 fucosylated glycoconjugates in preserving the self-renewal of hPSC or inducing differentiation, depends on FUT1 expression level.

Down-regulation of FUT1 is observed at an early stage of differentiation toward mesoderm, endoderm, and ectoderm, in 5 various hPSC lines using different differentiation protocols. Specifically, hPSCs-derived intermediate cardiac progenitors transcribe the Hop Homeobox (Hopx), while poorly transcribe FUT1 and total <-fucosyl glycoconjugates.

Downregulation of FUT1 using small interfering RNA induces expression of lateral mesoderm transcription factors, mainly of Hopx, whereas FUT1 overexpression damages proper differentiation towards cardiomyocytes, definitive endoderm lineage, and neural progenitor cells. These findings demonstrate an early effect of FUT1 in the hPSC-fate decision, opening a new approach to control stem cell fate.

158. In-depth glycomic analysis targeting specific glycotopes in cancer cells using anti-carbohydrate antibody

Y. Chen², C. Hsiao³, K. Khoo³, M. Yang¹, T. Chang¹; ¹GlycoNex Inc., Taiwan, ²Institute of Biochemical Sciences, National Taiwan University, Taiwan, ³Institute of Biological Chemistry, Academia Sinica, Taiwan

Different combination and relative expression level of assorted terminal sialylated, fucosylated and/or sulfated glyco-epitopes (glycotopes) collectively define the glycomic characteristics of a cell or tissue type at a specific patho-physiological state. For instance, the aberrant glycosylation of cancer cells is characterized by overexpression of specific glycotopes, which has been recognized as a hallmark of tumorigenesis and metastasis in multiple cancer types. Several well-known tumor-associated carbohydrate antigens such as Lewis y, Globo H, Sialyl Tn, and GD2 are being actively pursued as targets of rapidly developing cancer immunotherapies. Other novel and/or critical glycotopes implicated by immunohistochemistry are, however, often not detected by conventional glycomic analytical workflow due to their low abundance. Their exact chemical settings and individual glycan carriers therefore remain largely unknown. We have previously established a one-fraction glycan preparation approach in combination with a one-shot nanoLC-MS2-product dependent-MS3 analytical workflow that would shorten analytical time without compromising the attainable glycomic resolution and precision. We now further developed and incorporated an immuno-capture strategy utilizing anti-carbohydrate antibodies to enrich the glycans carrying glycotopes of interest from the glycomes of cancer cells. It was demonstrated in this work that a therapeutic anti-carbohydrate antibody in preclinical development would enrich a distinctive population of heavily fucosylated glycans from gastric and colorectal cancer cells. Further MS2/MS3 sequencing revealed a full capping of terminal Type I or Type II chain units with Fuc on branched structures, representing glycan entities with multivalent Lewis b/y units. This single or multiple immuno-capture driven analytical platform proves to be capable of enhancing the depth of targeted glycomic analysis and is particularly suited for identifying glycotopes of clinical significance.

159. Sialylation of D2 dopamine receptor and A2A adenosine receptor by St8sia3 modulates their receptor heteromerization and pharmacological responses in mice

Y. Chern⁴, C. Lin⁴, C. Wu², C. Sato¹, K. Khoo³; ¹Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japen, ²Genomics Research Center, Academia Sinica, Taipei, Taiwan, ³Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ⁴Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

Sialic acids are commonly observed at the end of glycoconjugates and have been implicated in neuronal plasticity and inflammation. The functions and regulations of sialylation in the brain remain largely unknown. The ST8 <-N-acetyl-neuraminide <-2,8-sialyltransferase 3 (ST8SIA3) exists in many brain areas of the brain and is highly enriched in the striatum. We showed in the present study that ST8SIA3 is expressed mainly in neurons but not in astrocytes or microglia in the striatum. In addition, the lack of St8sia3 (St8sia3-KO) reduced the levels of disialylated and trisialylated terminal glycotopes in the striatum of mice. Interestingly, the apparent sizes of several striatum-enriched G-protein-coupled receptors (GPCRs) (including the A_2A adenosine receptor (A_2AR), and dopamine D1 and D2 receptors (D1R and D2R)) were smaller in the absence of St8sia3. Treatments with sialidases eliminated the differences in the sizes of these molecules between the striatum of St8sia3-KO and WT mice. Exogenous expression of ST8SIA3 in vivo using adeno-associated viruses normalized the abnormal sizes of these proteins. Collectively, these findings suggest that several GPCRs are substrates of ST8SIA3 in the striatum. The function of disialylated/trisialylated terminal glycotopes in the regulation of these striatal GPCRs is worth further investigation. Specifically, we reported here that the lack of sialylation enhanced the distribution of D2R and A_2AR in lipid rafts and the interaction between them. The locomotor activity assay revealed that a larger population of D2R appeared to form heteromers with A2AR in the striatum of St8sia3-KO mice than in WT mice. Since the A_2AR-D2R heteromer is an important drug target for several basal ganglia diseases (such as schizophrenia and Parkinson’s disease), our findings provide new insights into the mechanisms regulating the striatum and may pave the way for the development of novel therapeutic strategies for basal ganglia diseases.

160. Dissecting the site-specific n- and o-glycosylation of the ectodomains of receptor protein tyrosine phosphatases by concerted mass spectrometry analyses

Y.C. Chien¹, C.W. Kuo¹, T. Angata¹, S.T.D. Hsu¹, T.C. Meng¹, K.H. Khoo¹; ¹Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan

Glycosylation is a well-known post-translational modification on the extracellular domains of transmembrane receptor-like protein tyrosine phosphatases (RPTPs). Among the RPTPs, the R4 subtype members, RPTP alpha (PTPRA) and RPTP epsilon (PTPRE), carry the shortest ectodomains of only 120 and 25 amino acid residues, respectively. Yet in the case of PTPRA, there are 7 consensus N-glycosylation sequons distributed over a full stretch of essentially Ser/Thr/Pro rich mucin domain containing more than 40 potential O-glycosylation sites. While the heavily N-glycosylated nature of PTPRA was appreciated, the density and nature of O-glycosylation was not previously investigated. PTPRA/E were implicated in many diseases but the functional relevance of their heavily glycosylated extracellular domain is not known. Interestingly, both endogenous PTPRA and PTPRE were separated into two distinct bands instead of a continuous smear on SDS-PAGE indicative of 2 discrete sub-populations of glycoforms. To understand how the glycosylation may affect its physicochemical properties and modulate its intracellular phosphatase activities, a detailed mass spectrometry-based bottom-up analysis was undertaken. In gel digestion followed by separate mapping of its released glycans and glycopeptides revealed that incomplete O-glycosylation appears to be the main determinant for the smaller size glycoforms, which failed to be expressed on surface. The lack of tryptic cleavage sites largely prevented efficient sequencing of long stretches of heavily glycosylated tryptic peptides with convoluted patterns of site-specific O- and N-glycosylation patterns. Our strategy therefore relied on deploying different combination of proteases and subject the variably cleaved shorter glycopeptides to nanoLC-MS/MS analysis using a HCD-pd-EThcD analytical workflow. A higher Byonic cut-off score was employed to reduce false positives in glycopeptide spectrum matches, whereby second steps of manual inspection/interpretation coupled with fishing out of the glycoforms related by accurate glycosyl residue mass at LC-MS1 level would decrease false negative. We report in this work the first mapping of the glycosylation heterogeneity of PTPRA and its physical impact on the conformation of the ectodomains.

161. Mucosal absorption of therapeutic peptides by harnessing the endogenous sorting of glycosphingolipids

D. Chinnapen¹, M.D. Garcia-Castillo¹, W. Lencer¹; ¹Department of Pediatrics - Division of Gastroenterology, Harvard Medical School - Boston Children’s Hospital, Boston MA USA

We aim to harness glycosphingolipid trafficking across barrier epithelial cells for clinical applications: in this case, for treatment of type 2 diabetes.

Mucosal surfaces represent vast areas where host tissues are separated from the environment only by a delicate but highly effective single layer of columnar epithelial cells, joined by tight junctions that are impermeable to proteins and even small peptides. So far, the lack of rational and efficient methods to circumvent this barrier has prevented the application of most therapeutic proteins and peptides for mucosal (oral/nasal) drug delivery.

The pathway for biologics (i.e. therapeutic proteins and peptides) to cross the barrier of the intestine is by transcytosis – a process of transcellular endocytic membrane trafficking that connects one surface of polarized cells with the other. We have found that certain glycosphingolipids cross epithelial barriers by transcytosis and that the structure of its ceramide domain dictates transport through this pathway. Recent structure-function studies show that ganglioside GM containing non-native “short chain” fatty acids in the ceramide domain allow for more efficient transport across simple epithelial barriers by transcytosis, and some species allow for easy release from the cell back into solution after transcytosis. Conjugation of therapeutic peptides (GLP-1) to the extra-cellular oligosaccharide domain of these short-chain species enables the lipid to then act as a trafficking molecule, allowing for systemic absorption of the fusion molecule after gastric gavage and nasal administration. In mice we find absorption of the fusion molecules surpassing the best currently achieved for oral delivery of peptides and biologics. Our interpretations and challenges for developing the technology will be discussed.

162. Arginine glycosylation mechanism and structural dynamics of SseK2 from salmonella enterica

J. Park¹, Y. Yoo¹, J. Kim¹, M. Choi¹; ¹Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea

The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-│B-dependent responses to infection. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like GTs, although they differ in protein substrate specificity. We reported (Park. et al, Nature Communications, 2018) that these enzymes are retaining glycosyltransferases composed of a helix-loop-helix (HLH) domain, a lid domain, and a catalytic domain. A conserved HEN motif (His-Glu-Asn) in the active site is important for enzyme catalysis and bacterial virulence. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are critical for enzyme recognition. Long Molecular Dynamics simulations suggest that the HLH domain determines substrate specificity and the lid-domain regulates the opening of the active site. Overall, our data suggest a front-face SNi mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens.

163. Sialic acid in the communication between the gut microbiota and the brain

E.S.R.E.D.E.A.G.M.N. Coletto, Tribolo, Griffith, Crost, Latousakis, Santamarina, Telatin, Savva, Hajihosseini, Juge¹; ¹Quadram Institute Bioscience, Norwich Research Park, Norwich, UK School of Biological Science, University of East Anglia, Norwich, UK(Santamarina, Hajihosseini)

A bidirectional communication between the gastrointestinal (GI) tract and the brain has long been acknowledged. Several studies showed that the onset of several brain diseases, such as neurodegenerative disease, stress and anxiety are linked with an altered composition of the gut microbiota. However, the molecular mechanisms underpinning the communication between gut microbes and the brain remain to be elucidated.

Here we explored the importance of mucin derived sialic acid metabolites in gut-brain communication. In the vertebrates nervous system, gangliosides and polysialic acid chain are enriched with sialic acid residues, which are critical at the early stage of brain development, but also for neuroplasticity and cell signalling.

In the GI tract, sialic acids are abundant in the colon where they are found in terminal location of mucin glycan chains and can be released by gut microbes inhabiting the mucus layer. In this work, we first characterised sialic acid derivatives in the caecal content, brain and serum of mice fed diets rich or low in complex carbohydrates, by HPLC. A range of sialic acid derivatives were identified. Statistical analyses showed positive associations between mice fed a diet low in complex carbohydrates and the serum levels of Neu5,8Ac2 and Neu5,7Ac2, caecal content levels of Neu5,7Ac2 and Neu5,Gc9Ac and brain levels of Neu5,8Ac2 and Neu5,9Ac2. Interestingly, microbial profile of the gut microbiota of these mice, by 16S sequencing analysis, showed association of specific gut bacterial species with the serum level of Neu5,8Ac2 and Neu5,7Ac2 derivatives.

Preliminary data of mouse model with an alteration in mucin glycosylation profile and gut microbiota composition showed differences in the pattern of distribution and expression of non-neuronal cells (microglia, astrocytes and oligodendrocytes) involved in neuroinflammation state, as compared to wild-type mice.

Together these data provide a basis to further explore the role of sialic acid derivatives and metabolites in the communication between the gut and the brain.

164. Stabilized N-oxyamide anionic glycoglycerolipids targeting protein kinase B (Akt)

D. Colombo¹, G. Orsini¹, M. Zuccolo¹, P. Perego², C. Corno²; ¹Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Via Saldini 50, 20133 Milano, Italy, ²Molecular Pharmacology Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42/via Venezian 1, 20133 Milan, Italy

Protein kinase are enzymes involved in the regulation of many crucial cellular processes like proliferation, differentiation and apoptosis. Among them, the serine/threonine protein kinase B, also known as Akt, plays a key role as a component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis, which is implicated in aberrant tumor cell signaling. Inappropriate activation of the Akt kinase is a common event in human tumors making Akt a critical player in cell survival and, consequently, inhibitors that target Akt are potentially relevant for cancer therapy.

The structure of the phosphatidylinositol 3,4,5 triphosphate (PIP3), the natural ligand of Akt PH domain, is composed by an inositol, with a phosphate group in position 3 and a glycerol moiety in position 1 carrying long acyl chains. Recently, two classes of anionic glycoglycerolipids based on 2-O-β-D-glucosylglycerol mimicking PIP3 bearing a carboxylate or sulfonate group on the sugar moiety were synthesized. The antiproliferative activity of the compounds was examined in the human ovarian carcinoma IGROV-1 cell line, in which we observed a more marked growth inhibitory effect when drug incubation occurred in a serum-free medium as compared to complete medium, thereby suggesting that serum affects the drug bioavailability. Conversion into N-oxyamide is an eligible modification to improve metabolic stability of carboxylic derivatives. Therefore, in this study N-oxyamide-linked anionic glycoglycerolipids analogues of the previously studied Akt inhibitors were prepared and tested for their antiproliferative activity in the human ovarian carcinoma IGROV-1 cell line.

165. Glycocalixarenes decorated with streptococcus pneumoniae 19F capsular polysaccharide fragments bind to anti-19F antibodies

F. Faroldi¹, L. Morelli², M. Giuliani¹, E. Torre³, S. Fallarini³, F. Sansone¹,F. Compostella²; ¹Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, ²Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, ³Department of Pharmaceutical Sciences, University of “Piemonte Orientale”, Novara

Capsular polysaccharides (CPS) of encapsulated bacteria have been recognized as critical determinants of bacterial virulence and found to be able to stimulate protective immunity against infection disease, laying the bases for the development of current antibacterial vaccines. CPSs are cell-surface polymers consisting of oligosaccharide repeating units. High molecular mass repetitive polysaccharide structures are able to display simultaneously a greater number of densely displayed epitopes capable to effectively interact in a clustered form with specific antibodies. The study of the chemical determinants necessary for an effective interaction still requires detailed molecular insights. Nanomaterials or multivalent scaffolds loaded with carbohydrate antigens allow repetitive antigen display, and are emerging as promising synthetic vaccine candidates, alternative to classic polysaccharide/protein conjugate vaccines. In this framework, we have decided to explore the potential of calixarenes as scaffolds for the multipresentation of bacterial CPS fragments related to Streptococcus pneumoniae serotype 19F (SP19F). The hypothesis was to assess if such scaffolds, presenting a limited number of copies of short SP19F fragments, displayed in a multivalent form, are able to gain affinities and potencies towards the natural antibodies similar to those observed for the natural polysaccharide. Herein, we will report the preparation and biological evaluation of a family of calixarenes functionalized with saccharide fragments related to the trisaccharide repeating unit of SP19F. In particular, a calix[6]arene, functionalized with six copies of the trisaccharide repeating unit of SP19F, resulted very effective in competing with natural 19F polysaccharide in the binding to specific anti-19F antibody. This compound shows efficacies and affinities higher than those exhibited by the single trisaccharide, thus evidencing the effect of multivalency in increasing the ability of the single saccharide unit to compete with natural 19F polysaccharide in the binding to specific anti-19F antibody.

166. Acetal pH-sensitive linkers for glyco-protein conjugation

L. Confalonieri², D. Imperio², L. Panza², F. Compostella¹; ¹Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milano, Italy, ²Dipartimento di Scienze del Farmaco, Università degli Studi del Piemonte Orientale, Novara, Italy

The environment of tumor tissue has a slightly lower pH compared to that of normal tissue. Cancer cells have high rates of converting glucose to lactate. Their intracellular pH is near to 5.0, while in the extracellular system the pH value is around 7.4. This difference can be exploited to design cleavable linker for drug conjugation to protein that are stable during circulation in the blood, while are activated to release the drug at more acidic pH. These drug delivery systems are at the base of a targeted therapy, since they are able to selectively distinguish between healthy and cancer cells by the use of cellular tags. In this way it is possible to reduce the unwanted, dose-limiting, and debilitating side effects of some chemotherapy agents.

Acetals are acid labile functional groups commonly employed as protecting groups for alcohols in organic synthesis. They have not been widely used in the design of pH-responsive linkers, even if they are appropriate for the conjugation of a drug containing free hydroxyl groups. So, acetals can be helpful in the design of bifunctional linkers, that are involved in the conjugation of a drug to a protein, since they are cleavable in biocompatible condition.

In this context, we have developed bifunctional linkers with a carboxylic function at one end and a dimethyl acetal at the other. The carboxylic group allows the conjugation to the protein through amide bonds with the lysine residues, while a transacetylation reaction on the dimethyl acetal is required to link the drug through an hydroxyl group. Herein, we will report the preparation of two linkers differing in the chain length, starting from ©-valerolactone. Furthermore, we will describe our results in the optimization of the transacetylation reaction for drug conjugation and on linker stability/solubility.

167. Molecular basis for the interaction between haemophilus influenzae type b capsular polysaccharide and a protective monoclonal antibody

F. Nonne¹, A. Corrado¹, R. De Ricco¹, L. Dello Iacono¹, D. Proietti¹, N. Norais¹, M. Taddei², I. Margarit Y Ros¹, R. Adamo¹, F. Carboni¹, M.R. Romano¹; ¹GSK Vaccines, ²Università di Siena, Dipartimento di Biotecnologie, Chimica e Farmacia

Haemophilus influenzae infections are a major cause of bacterial respiratory tract morbidity and can lead to severe diseases such as pneumonia, sepsis, and meningitis.1 H. Influenzae type b (Hib) is coated with a capsular polysaccharide (CPS) made up of polyribosyl-ribitol-phosphate (PRP) repeating units (RUs) that has been conjugated to carrier proteins for efficacious vaccination.2 Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. It has been demonstrated that an octasaccharide antigen (containing 4 RU) resembles PRP polysaccharide in terms of immunogenicity and recognition by anti-Hib antibodies.2 Identifying the minimal epitope directly involved in the antibody binding can facilitate vaccine design. For this purpose short Hib oligosaccharide (OS) fragments (2 to 5 RU), obtained from acid hydrolysis of natural polysaccharide,3 were used to characterize the interaction with the human protective anti-Hib monoclonal antibody (hmAb)4 by STD-NMR (Saturation Transfer Difference NMR). A Fab fragment from the hmAb digestion was obtained and its interaction with the Hib OS fragments were characterized by SPR (Surface Plasmon Resonance). Attempts to achieve X-ray crystallography of the Fab complex with Hib OS are underway.

References

1. J. R. Gilsdorf, J. Infect., 2015, 71, S10.

2. J. Y. Baek et al. Chem. Sci., 2018, 9, 1279

3. N. Ravenscroft et al. Vaccine, 1999, 17, 2802

4. A.H.Lucas et al. Infection and Immunity, 1994, 62, 3873

168. Unravelling the carbohydrate-specificity of a novel lysm domain from clostridium thermocellum

R. L. Costa³, D. O. Ribeiro³, B. A. Pinheiro³, J. L. A. M. Brás², L. Silva¹, Y. Zhang¹, Y. Liu¹, W. Chai¹, M.J. Romão³, T. Feizi¹, C. M. G. A. Fontes², A.L. Carvalho³, A. S. Palma³; ¹Glycosciences Laboratory, Department of Medicine, Imperial College London, London W12 0NN, UK, ²NZYTech – genes & enzymes, Estrada do Paço do Lumiar, Campus do Lumiar, Edif. E, 1649-038 Lisboa, Portugal, ³UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal

LysM (lysin motif) domains are widespread protein domains found in prokaryotes and eukaryotes that are known to bind to chitin or peptidoglycan. These domains are present in proteins involved in many biological functions, such as in bacterial cell wall degradation, signalling in plant-bacteria/fungi pathogenesis and symbiosis, and in bacterial spores morphogenesis. LysM domains are classified as family 50 carbohydrate-binding modules (CBM50) in the CAZy database (www.cazy.org). Several CBM50 modules have been identified in the genome of Clostridium thermocellum (Ct), a highly efficient cellulolytic bacterium that vastly contributes for the degradation of plant cell-wall polysaccharides. However, the molecular details of carbohydrate recognition, the 3D structure and the function of these CBMs are yet to be elucidated.

In this study, we performed carbohydrate microarray screening analysis of family 50 CtCBMs against representative plant and fungal cell-wall oligosaccharide sequences to identify the carbohydrate ligands. The results revealed a high specificity towards ®-1,4-linked-N-acetylglucosamine (GlcNAc) chitin oligosaccharides with a minimum chain length requirement of 3 residues. The characterization of the carbohydrate-binding interaction was pursued for a CtCBM50 and its oligosaccharide ligands by X-ray crystallography, site-directed mutagenesis and ITC. These studies showed that the CBM has a typical ®<<® LysM domain fold and identified the key residues for the specificity of recognition and chain-length dependency. In particular, a complex intermolecular interaction of two CBM molecules with a ®1,4-GlcNAc trisaccharide sequence was observed, mediated by □-CH (Trp11, Tyr38) and hydrogen bonding (Asn35) interactions. The information derived allowed to understand mechanisms of carbohydrate-recognition to chitin and peptidoglycan by family 50 CtCBMs, which contribute to elucidating their role in C. thermocellum.

Financial support: FCT-MCTES through grants UID/Multi/04378/2013, POCI-01-0145-FEDER-007728, PTDC/BBB-BEP/0869/2014, PTDC/BIA-MIC/5947/2014; SFRH/BD/100569/2014; SFRH/BPD/68563/2010; PD/BD/135517/2018.

169. N-glycosylation of plasma proteins and immunoglobulin G in multiple sclerosis

A. Cvetko¹, D. Kifer¹, J. Wilson³, G. Lauc², T. Pavić¹; ¹Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia; ²Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia; Genos Glycoscience Research Laboratory, 10000 Zagreb, Croatia; ³MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom; Usher Institute for Population Health Sciences and Informatics, University of Edinburgh, Edinburgh, United Kingdom

Multiple sclerosis (MS), a disease affecting the central nervous system, is characterized by demyelination and inflammation. Mechanism underlying the development of MS is not yet fully understood, but it is considered to be influenced by both genetic and environmental factors. N-glycosylation is one of the most complex co- and post-translational modifications and a highly regulated process. N-glycans do not only increase protein structural diversity, but can modify their function as well. Multiple studies have shown the importance of N-glycan changes in different autoimmune and inflammatory diseases, which highlights N-glycans as potential diagnostic and/or prognostic biomarkers. Even though plasma protein and immunoglobulin G (IgG) N-glycosylation changes are well investigated in many diseases, the studies on N-glycan changes in multiple sclerosis are scarce. Hence, we aimed to investigate N-glycosylation patterns of plasma proteins and IgG in multiple sclerosis. We have analysed plasma and IgG N-glycosylation profiles in 83 multiple sclerosis cases, 88 age- and sex-matched controls and 85 sex-matched controls, but who passed the majority of lifetime risk for MS development (>70 years of age). Core fucosylation showed to be the most prominently altered IgG glycosylation trait, as it was significantly decreased in MS subjects compared to the healthy controls (adjusted p=0.0069). Among all generated plasma glycosylation traits, the most significant changes were observed in antennary fucosylated and high branched N-glycans, which were both increased in the MS group (adjusted p=0.0067 and 0.0219, respectively). On the other hand, low branched N-glycans were significantly decreased in MS subjects (adjusted p=0.0167). Using logistic regression that incorporated all directly measured N-glycans for plasma and IgG, we were able to distinguish MS patients from controls (plasma p=2.6851x10^-11, IgG p=1.1314x10^-7, respectively). Receiver operator characteristic curves showed better results for plasma N-glycan model (Area under the curve [AUC]=0.852) over the IgG N-glycan model (AUC=0.798). Our results demonstrated that plasma protein and IgG N-glycosylation markedly changes in multiple sclerosis. However, additional studies are needed to determine the background of these changes, their role in multiple sclerosis development and their potential use as diagnostic biomarkers.

170. Novel muscular sialic acid related disorder caused by defects in the N-acetylneuraminate pyruvate lyase (NPL) gene

A. Da Silva⁴, X. Pan⁴, G. Muscarnera⁴, J. Dort⁴, Z. Orfi⁴, N. Dumont⁴, P. Van Vliet⁴, G. Andelfinger⁴, S. Jamet², Y. Yamanaka³, X. Wen⁶, C.D. Van Karnebeek¹, D.J. Lefeber⁵, C. Crist², A.V. Pshezhetsky⁴; ¹Department of Pediatrics, Emma Children’s Hospital, University of Amsterdam, The Netherlands, ²Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, Canada, ³McGill University Transgenic core facility, McGill University, Montreal, QC, Canada, ⁴Ste-Justine University Hospital Center, University of Montreal, QC, Canada, ⁵Translational Metabolic Laboratory, Radboud University Medical Center, Nijmegen, The Netherlands, ⁶Zebrafish Centre for Advanced Drug Discovery, St. Michael’s Hospital, Toronto, ON, Canada

Sialic acids are important components of glycoproteins, glycolipids and are essential for cellular communication, infection and tumor metastasis. Despite the known importance of sialic acids in human physiology and the existence of several genetic disorders caused by defects of sialic acid metabolism, the biological roles of enzymes involved in sialic acid degradation remain unclear.

Neuraminic acid pyruvate-lyase (NPL), cleaves sialic acid to produce N-acetylmannosamine and pyruvate. Based on homology with bacterial enzymes, it has been proposed to be involved in the regulation of cellular free sialic acid levels. Recently, we identified disease-causing mutations in the NPL gene in patients presenting with cardiomyopathy, skeletal myopathy, sensorineural hearing loss symptoms and urinary excretion of free sialic acid (Wen, X-Y et al., JCI Insight, 2018). Using CRISPR/cas 9 technology, we developed the first mouse model of NPL deficiency carrying the human missense mutation p.Arg63Cys affecting enzyme stability and activity (NplR63C). Mice showed 6-fold increased sialic acid levels in the urine thus mimicking human metabolic phenotype. Importantly they also demonstrated early-onset muscle weakness in a number of neuromuscular tests. The maximum tetanic force of their extensor digitorum longus (EDL) muscle was 2-fold reduced as compared to the WT littermates whereas analysis of the muscle tissue by immunofluorescent microscopy revealed abnormal and disarrayed muscle fibers. Levels of several metabolites related to sialic acid catabolism revealed drastic changes in the number of tissues of NplR63C mice including the brain and suggested that the disease caused by NPL deficiency has a systemic manifestation. The majority of other metabolites showing drastic reduction in the tissues of NplR63C mice are important components of the glycogenolysis pathway including Glucose-1-phosphate suggesting NPL deficiency affects glycogenolysis, an important pathway for energy generation during hypoglycemia.

Altogether, our results provide new insights into the roles of the sialic acid catabolism in humans, suggest an important role of NPL in glycogenolysis and should potentially help finding new treatments for patients with genetic glycosylation-related myopathies.

171. Development of tools to study 3-o sulfated heparan sulfate involved in (patho-)physiological processes

L. Damen², T.B. Phuong¹, E. Van De Westerlo², A. Oosterhof², D. Fernig¹, W. Daamen², T. Van Kuppevelt²; ¹Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, UK; ²Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud university medical center, The Netherlands.

Heparan sulfate (HS) is a linear polysaccharide with high functional diversity, resulting from a number of different sulfation motifs. The sulfation of the C3-hydroxyl group of the glucosamine moiety modulates many protein interactions and is associated with several pathologies. This modification is generated by seven HS glucosaminyl 3-O-sulfotransferases resulting in motifs that can be classified either as AT-type (GlcA-GlcNS3S) or gD-type (IdoA2S-GlcNS3S). However, since the number of isoenzymes exceeds the number of motifs, larger, more complex motifs are anticipated. Previously, we obtained the single chain variable fragment antibody (scFv) HS4C3 from a phage-display library, reactive with 3-O-sulfate of both motifs. To target different 3-O sulfate motifs more specifically, characterization of the interaction between HS4C3 and the 3-O sulfated HS is required. Here we characterize the interaction between HS4C3 and heparin, using the “protect & label” methodology. Lysine and arginine residues not binding to heparin were chemically modified with respectively N-hydroxysuccinimide (NHS) acetate and phenylglyoxal, whereas interacting residues were labeled with NHS-biotin and hydroxy-phenylglyoxal . After digestion, labeled residues were identified using mass spectrometry. Results indicate that most labeled arginines and lysines were concentrated in and surrounding the complementarity determining region 3 (CDR3) of the antibody, but were also present in other regions. Using 3D models of heparin (1HPN) and HS4C3, we predict that the arginine residue preceding the CDR3 of the heavy chain is essential for binding to the 3-O sulfate epitope, while arginines and lysines of the CDR3 bind surrounding sulfate groups. Labeled residues located in other regions provided a binding site for flanking saccharides. Comparison of these data with those obtained for antibodies that recognize different HS epitopes (e.g. antibodies LKiv69 and HS3A8) will give us insight in specific mutations that need to be introduced in the HS4C3 antibody to change its reactivity towards specific 3-O sulfate containing motifs. Antibodies targeting different 3-O sulfation patterns will be used to investigate the function of 3-O sulfation in (patho)physiological processes.

172. Disruption of the glycocalyx of canine melanoma cells underpins a promising new cancer vaccination strategy

A. Dell¹, D. Lu¹, A. Antonopoulos¹, S. Haslam¹, J. Bryan³, G. Clark²; ¹Department of Life Sciences, Imperial College London, Exhibition Road, South Kensington, London, UK SW7 2AZ, ²Department of Obstetrics, Gynecology and Women’s Health, School of Medicine, University of Missouri, Columbia, MO, USA 65211, ³Department of Veterinary Medicine & Surgery, University of Missouri, Columbia, MO, USA 65211

Aberrant glycosylation is a classical hallmark of human cancer. It is well established that the structures and expression levels of polylactosamine-containing glycans are greatly altered during malignant transformation and tumour progression. Such glycans have been implicated in the trafficking of tumour cells and the suppression of both the innate and adaptive immune response. Canine cancers are excellent models for analogous cancers in humans. In the current study, we have employed MALDI-TOF and GC-MS glycomic methodologies to show that the N-glycome of canine melanoma cells is broadly similar to that associated with human melanoma cells, except that, as expected, some of the glycans are capped with N-glycolylneuraminic acid and Gal<1-3Gal sequences instead of N-acetylneuraminic acid. Importantly, both express elevated levels of linear polylactosamine sequences which have been implicated in the suppression of both the adaptive and innate immune responses. Moreover the ganglioside content of canine melanoma mirrors human melanomas.

We hypothesised that the global inactivation of immunosuppressive carbohydrate sequences from tumour cells would result in a greatly enhanced anti-tumour immune response. A pilot clinical trial involving canines, following up on this hypothesis, is in progress at the University of Missouri. Remarkably we have observed complete remissions in 35% of canine oral melanoma patients vaccinated with tumour cell lysates treated to inactivate their carbohydrate sequences. We conclude that glycans act as immune checkpoint molecules that interfere with the induction of adaptive immune responses directed against tumour cells.

173. Effect of change in glycosylation of human serum transferrin on iron release to competing chelator EDTA

H. Deris¹, T. Weitner², M. Gabricevic², D. Sakic²; ¹Genos Ltd, Glycoscience Research Laboratory, Zagreb, Croatia, ²University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia

Glycans may occupy a greater part of a glycoprotein structure and are directly involved in their communication with the environment. Therefore, changes in glycosylation may alter glycoprotein - ligand interaction. Transferrins (Tf) are homologous glycoproteins with the main role in maintaining iron homeostasis. The purpose of this study was to demonstrate the influence of the glycan part of the Tf structure on its function, specifically on the iron binding strength. The most abundant Tf glycoform is composed of two biantennary oligosaccharide chains with four terminal sialic acids. Terminal sialic acids were cleaved from the holo-Tf by neuraminidase and asialo-Tf was obtained. All experiments were carried out by using ethylenediaminetetraacetic acid (EDTA) as a competitive chelator and UV-Vis spectroscopy in PIPES buffer (pH = 7.4 at 25° C). Concentrations of apo-Tf, holo-Tf and free iron were obtained from the absorbance data at 450 nm and the added EDTA concentration, from which corresponding equilibrium constants were calculated. The obtained equilibrium constants are an order of magnitude higher for the iron binding to the native Tf when compared to asialo-Tf. Additionally, the iron release kinetics is altered by the neuraminidase treatment of the native transferrin. Thus, these results indicate that glycosylation changes of transferrin under certain conditions may impact normal iron homeostasis.

174. Biosensor-based determination of protein-glycosphingolipid interaction: binding of shiga toxins to membrane microdomains of human brain endothelial cells

J. Detzner¹, D. Steil¹, G. Pohlentz¹, N. Legros¹, H. Humpf², A. Mellmann¹, H. Karch¹, J. Müthing¹; ¹Institute for Hygiene, University of Münster, Münster, ²Institute of Food Chemistry, University of Münster, Münster

Beyond intestinal disturbances, enterohemorrhagic Escherichia coli (EHEC) may cause severe extraintestinal complications such as damage of the brain. After translocation of EHEC-released Shiga toxins (Stxs) from the gut into the circulation, Stxs target human brain microvascular endothelial cells (HBMECs) resulting in serious cerebral dysfunction. The B pentamer of the AB5 toxins binds to the glycosphingolipid (GSL) receptors globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), which are compounds of the plasma membrane of HBMECs. GSLs are deemed to cluster in cholesterol- and sphingomyelin (SM)-rich microdomains termed lipid rafts. Detergent-resistant membranes (DRMs) are used as lipid raft-equivalents resembling the liquid-ordered membrane phase and nonDRMs as the counterpart for the liquid-disordered phase being useful tools in membrane research.

The goal of this study was to elucidate the DRM- and nonDRM-specific lipoforms of Gb3Cer and Gb4Cer and to determine the binding characteristics of label-free Stxs in real time towards DRMs and nonDRMs.

DRM and nonDRM fractions were obtained from sucrose density gradients, which were prepared from HBMECs by ultracentrifugation. Gb3Cer and Gb4Cer were detected immunochemically using a thin-layer chromatography (TLC) overlay assay. The various lipoforms of Gb3Cer and Gb4Cer were determined by means of electrospray ionization mass spectrometry (ESI MS). Interaction analyses of Stx with DRMs and nonDRMs were recorded with a surface acoustic wave (SAW) biosensor in real time.

The immunochemical detection of the TLC-separated GSLs revealed accumulation of Gb3Cer and Gb4Cer in the DRM versus the nonDRM fractions of HBMECs indicating their association with lipid rafts. Gb3Cer and Gb4Cer lipoforms with saturated fatty acids dominated in the DRMs, whereas GSLs with unsaturated fatty acids prevailed in the nonDRM fractions. Real-time interaction analysis of affinity-purified Stxs with DRMs and nonDRMs evidenced adherence of Stxs to DRMs and failure in binding towards nonDRMs. SAW binding kinetics allowed for calculation of the binding strength of Stxs towards GSL-carrying DRMs.

The specific interaction of Stxs with DRMs suggests lipid raft association of the Stx GSL receptors in the plasma membrane of HBMECs.

175. Neurotrophic properties of gm1 oligosaccharide: evidence on the development of primary neurons in culture

E. Di Biase¹, G. Lunghi¹, M. Fazzari¹, S. Prioni¹, E. Chiricozzi¹, S. Sonnino¹; ¹University of Milano

Gangliosides are sialic-acid containing glycosphingolipids, mostly abundant in the nervous tissues. Among these, the monosialo-ganglioside GM1 plays a pivotal role during neuronal development and its neuroprotective and neurotrophic properties have been largely reported both in vitro and in vivo. In cultured neurons, the GM1 enrichment in plasma membrane (PM) microdomains crucially contributes to the activation of neurotrophins receptors belonging to Trk family. This event triggers a specific signaling cascade resulting in actin depolymerization, axon protrusion and elongation. Despite this evidence, the mechanism of action of GM1 is still unknown. In neuroblastoma cells we demonstrated that GM1 oligosaccharide (OligoGM1) was able to induce cell differentiation and also protect cells against neurotoxins. This effect was due to a direct OligoGM1 binding to NGF receptor TrkA, resulting in the TrkA-MAPK pathway activation.

Here, we characterize OligoGM1 effect on the developmental process of mouse primary neurons. Time-lapse recordings of plated neurons showed that exogenously administered OligoGM1 enhances neuron clustering, arborization and networking. Accordingly, in the presence of OligoGM1 neurons shows a higher phosphorylation rate of FAK and Src proteins, the intracellular key regulators of neuronal motility. Moreover, treated cells express increased level of specific neuronal markers, suggesting an advanced stage of maturation compared to controls. Moreover, in the presence of OligoGM1, neurons anticipate the expression of more complex ganglioside and reduce the level of simpler ones, displaying the typical ganglioside pathway of mature neurons. Concerning its mechanism of action, OligoGM1 interacts with cell surface without entering the cells, suggesting the presence of a biological target on neuronal PM. Interestingly, we observed the TrkA-MAPK pathway activation as an early event underlying OligoGM1 effects in neurons.

Our data reveal that the specific role of GM1 in neuronal differentiation and maturation, described in the past, is determined by its oligosaccharide portion which, by interacting with the cell surface, triggers the activation of intracellular biochemical pathways responsible for neuronal migration, dendrites emission and axon growth.

176. Lipopolysaccharides from plants: the importance of the structure in the plant-microbe cross-talk

F. Di Lorenzo¹, A. Silipo¹, A. Molinaro¹; ¹Department of Chemical Sciences, University of Naples Federico II

Lipopolysaccharides (LPS), the major components of the outer membrane of Gram-negative bacteria, are crucial cell wall glycoconjugates acting as MAMPs (Microbe-Associated Molecular Patterns) in plant/bacteria interactions. They exert a central role in the mechanisms of bacterial invasion and adaptation to the host environment. In addition, in some cases, the LPS is a fundamental actor throughout the symbiotic process becoming necessary for the host plant.

The LPS fine structure is at the basis of this dynamic host-guest recognition that, in the case of phytopathogens is followed by the innate response whereas in the case of symbiosis is followed by its “suppression”. Herein, the structural determination of LPS is an important step toward for the comprehension of its structure to function relationships, either in the case of phytopathogens or in symbiotic bacteria.

In this communication I will present two bright examples on how structural modifications of pathogenic and symbiotic LPSs have repercussions on the plant-microbe cross-talk.

On the “symbiotic” side, I will show how the absence of a single sugar, in the unprecedented structure of the soybean symbiont Sinorhizobium fredii HH103 LPS, results in a severe impairment of the symbiotic process itself. In parallel, I will present how the LPS of the phytopathogen Xanthomonas campestris pv. campestris chemically changes during the infection process to escape the plant innate immune surveillance.

177. Function of glucuronyl c5-epimerase in tumor and the mechanism underlying action of polysaccharide by targeting the enzyme

F. He¹, S. Zhang¹, K. Ding¹; ¹Glycochmistry & Glycobiology Lab, Shanghai Institute of Materia Medica, Chinese Academy of Sciences

According to recent database of Integrity, there are 461 carbohydrate based new drug on the clinical trial, including carbohydrate, glycosides, nucleosides, vaccine, and carbohydrate mimetics. So far, there are about more than 500 clinical carbohydrate-based drugs available. However, this field still lags far behind small molecule and biopharmaceutical field due to the complex and heterogeneity structural feature of glycans, and there is no sequencer and high-efficiency automatic synthesis equipment. Tumorigenesis is orchestrated by a series of growth factor-HS (heparan sulfate) interactions which are involved in cell growth, inflammation, and blood coagulation. GLCE (glucuronic acid epimerase) is a critical enzyme involved in HS synthesis, which converts GlcA (D-glucuronic acid) into IdoA (L-iduronic acid). However, the function of GLCE in tumorigenesis is largely unknown. In the present study we showed that GLCE has different expressions in different tumors. GLCE silencing caused arrested lung cancer A549 cell growth and cervical cancer HeLa cell growth but promoted pancreatic cancer Bxpc-3 cell growth. We showed further that GLCE silencing worked on the expressions of PPAR and PRDM16. Interestingly, we found that the polysaccharides LRP1-s2 could inhibit pancreatic cell growth via targeting to GLCE. LRP1-s2 activates AMPK< and inhibits Smad1/5/9, AKT, GSK-3®, NF-│B and p38 phosophorylation, up-regulates p21 and attenuates Ras expression. The above results suggested that LRP1-s2 might be a new drug candidate for the anti-pancreatic cancer therapy.

178. N-glycan signatures of type 2 diabetes, its complications and medication use

V. Dotz¹, E. Memarian², R. Lemmers⁹, E. Schoep⁴, A. Naber⁷, S. Singh⁷, A. Lieverse⁸, F. Rutters⁵, A. Van Der Heijden⁶, P. Elders⁶, J. Beulens⁵, R. Slieker⁴, E. Sijbrands⁹, L. `t Hart³, M. Van Hoek⁹, M. Wuhrer¹; ¹Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, ²Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden; Genos Glycoscience Research Laboratory, Zagreb, Croatia, ³Department of Cell and Chemical Biology & Department of Biomedical data Sciences, Leiden University Medical Center, Leiden; Department of Epidemiology and Biostatistics, Amsterdam University Medical Center, location VUmc, Amsterdam, ⁴Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, ⁵Department of Epidemiology and Biostatistics, Amsterdam University Medical Center, location VUmc University Medical Center, Amsterdam, ⁶Department of General Practice and Elderly Care, Amsterdam University Medical Center, location VUmc University Medical Center, Amsterdam, ⁷Department of Internal Medicine, Erasmus University Medical Center, Rotterdam; Department of Internal Medicine, Maxima Medical Center, Eindhoven, ⁸Department of Internal Medicine, Maxima Medical Center, Eindhoven, ⁹Erasmus University Medical Center, Department of Internal Medicine, Rotterdam

The total plasma N-glycome (TPNG) reflects the levels and glycosylation of the major plasma glycoproteins, among which are immunoglobulins, acute-phase proteins and apolipoproteins. Type 2 diabetes mellitus (T2D) affects millions of people worldwide and causes considerable morbidity and mortality due to its long term vascular complications. Although associations of TPNG with T2D have been recently reported by us and others, data on the association between TPNG and T2D complications or medication use are scarce. Using automated sample preparation prior to relative quantitation by MALDI mass spectrometry, we determined the TPNG in two large, independent clinical cohorts (DiaGene: n = 1583 vs. 728; Hoorn Diabetes Care System (DCS) cohort: n = 1519 vs. 192 T2D cases vs. nondiabetic controls, respectively). By applying multiple logistic or linear regression models, we tested for associations of glycan features with case-control status, medication use of metformin, statin and ACE-inhibitors/Angiotensin receptor blockers, and micro- and macrovascular complications, with adjustment for age, sex, and T2D risk factors. T2D complications were also assessed prospectively by Cox regression. We found cross-sectional associations of all major glycosylation traits, i.e. sialylation, fucosylation, galactosylation, bisection, and glycan complexity with T2D, which were replicated in the DCS cohort. Some of the associations with disease seem to be related to medication, since we found overlapping features associating with both T2D and metformin or statin use. Strikingly, the strong negative association between <2,3-sialylation and T2D was not affected by medication use, but was instead significant in our analyses regarding T2D complications. Similarly, the bisection and galactosylation of immunoglobulin-derived glycans was significant in several analyses of macro- and microvascular complications both cross-sectionally and prospectively. In conclusion, our data provide great insights into the association between TPNG signatures and T2D, T2D medication and complications. These results will be taken forward to advance our knowledge on T2D pathophysiology and to improve personalized approaches in T2D and related complications.

179. < 2,6-linked sialic acid regulates ErbB2 biology and gastric cancer cell response to trastuzumab in a glycosite-specific manner

H.O. Duarte², J. Rodrigues², C. Gomes², S. Mereiter², P.J. Hensbergen¹, A.L.H. Ederveen¹, P. Van Veelen¹, A. Polónia², M. Wuhrer¹, L.L. Santos³, J. Gomes², C.A. Reis²; ¹CPM/LUMC (Center for Proteomics and Metabolomics of the Leiden University Medical Center), ²Ipatimup/i3S (Institute of Molecular Pathology and Immunology of the University of Porto), ³IPO-Porto (Portuguese Institute of Oncology - Porto)

The overexpression and hyperactivation of the Human Epidermal Growth Factor Receptor 2 (ErbB2) actively drive the malignant transformation of the human gastric mucosa. Although the anti-ErbB2 monoclonal antibody (mAb) trastuzumab (TTZ) is currently in use for the treatment of ErbB2-positive gastric cancer (GC) patients, both intrinsic and acquired resistance to TTZ are commonly observed. Despite this transmembrane receptor tyrosine kinase (RTK) being a target for extensive glycosylation, its detailed glycosylation profile and the molecular mechanisms through which it actively tunes the malignant traits of GC cells, including resistance to TTZ, remain elusive.

Previously, we have identified ErbB2 as a carrier of the tumor-associated <2,6-linked sialic acid (<2,6NeuAc) glycan antigen. To assess the biological relevance of <2,6 sialylation within the ErbB2 glycosylation landscape, we used CRISPR/Cas9 technology to generate an ErbB2-positive <2,6NeuAc-null GC cell line (ST6GAL1-/-). ErbB2 immunoprecipitation from wild-type (WT), ST6GAL1-/- cells and GC clinical specimens was performed, followed by mass spectrometry-based analysis of the receptor’s glycosylation repertoire. Proximity ligation assay (PLA) between ErbB2 and sialylated glycan epitopes was performed in GC clinical samples. Moreover, the phenotypic response of both WT and ST6GAL1-/- cells to trastuzumab treatment was evaluated.

Lectin labeling of ST6GAL1-/- cells confirmed the abrogation of the <2,6NeuAc, and further unveiled a drastic upregulation of multi-fucosylated glycans at the cell surface. Furthermore, glycoproteomic and glycomic profiling of <2,6NeuAc-null ErbB2 disclosed the replacement of the <2,6NeuAc glycan motif by multi-fucosylated species, in a glycosite-specific manner. PLA further validated ErbB2 as an in situ carrier of tumor-associated sialylated glycan epitopes. Finally, ST6GAL1-/- cells exhibited enhanced sensitivity to trastuzumab treatment through a reduced capacity to sustain oncogenic signaling.

Herein, we have identified a novel molecular mechanism through which ErbB2 glycosylation actively tunes the differential response of GC cells to trastuzumab. The disclosed glycosylation signatures represent a promising molecular target in the clinical management of GC patients.

180. User-friendly extraction and multistage tandem mass spectrometry based analysis of lipid-linked oligosaccharides in microalgae

R. Dumontier¹, P. Lucas¹, C. Loutelier-Bourhis³, A. Mareck¹, C. Afonso³, P. Lerouge¹, N. Mati-Baouche¹, M. Bardor²; ¹UNIROUEN, Laboratoire Glyco-MEV EA4358, Normandie Univ, 76000 Rouen, France, ²UNIROUEN, Laboratoire Glyco-MEV EA4358, Normandie Univ, 76000 Rouen, France / Institut Universitaire de France (IUF), 75000 Paris, France, ³UNIROUEN, Rouen INSA, CNRS, COBRA, Normandie Univ, 76000 Rouen, France

The protein N-glycosylation is initiated within the endoplasmic reticulum through the synthesis of a lipid-linked oligosaccharide (LLO) precursor. This precursor is then transferred en bloc on neo-synthesized proteins through the action of the oligosaccharyltransferase (OST) leading to the formation of glycoproteins. The N-linked glycans bore by the glycoproteins are then processed into oligomannosides prior to the exit of the glycoproteins from the endoplasmic reticulum and its entrance into the Golgi apparatus. In this compartment, the N-linked glycans are further maturated into complex type N-glycans, step which are specific to each species. This process has been well studied in a lot of eukaryotes including higher plants. In contrast, little information regarding the LLO precursor and synthesis of N-linked glycans in general is available in microalgae. In this work, a user-friendly extraction method combining microsomal enrichment and solvent extractions followed by purification steps is described. This strategy is useful to extract LLO precursor from microalgae which are then pure enough to be analyzed by mass spectrometry. Then, the oligosaccharide moiety released from the extracted LLO were analyzed by multistage tandem mass spectrometry in two microalgae models namely the green microalgae, Chlamydomonas reinhardtii and the diatom, Phaeodactylum tricornutum. This method allowed to highlight the LLO structure and to improve understanding of the N-glycosylation pathway in these two species.

181. The role of heparan sulfate in thymus organogenesis and T cell development

H. Hsu¹, Y. Chen¹, P. Chen¹, W. Chen¹, Y. Chen¹, S.A. Herrera-Heredia¹, I. Dzhagalov¹; ¹National Yang-Ming University

T cell development in the thymus is a well-organized, dynamic and compartmentalized process that requires carefully choreographed interactions between different cell types. The glycosaminoglycan Heparan sulfate (HS) can bind to many secreted signaling molecules and can participate in the communication between cells. We found out that HS was expressed at much higher level on the fibroblasts than on any other cell type in the thymus. Selective deletion in fibroblasts of the gene encoding the glycosyltransferase Ext1 that is crucial for the synthesis of HS resulted in non-viable embryos, underscoring the importance of HS for development in utero. Fetal thymic organ cultures and thymus transplantations revealed that absence of HS led to significantly smaller size and cellularity of the organ, but did not affect the progression of T cell development. To understand the mechanism through which HS affected the size of the thymus, we tested the hypothesis that HS is important for the formation of immobilized gradients of soluble signaling molecules. The chemokines CCL21, CXCL12 and CCL19 all bound to thymic fibroblasts in HS-dependent manner. Moreover, the migration of mature bone marrow-derived dendritic cells into thymic slices was impaired after treatment of the slices with Heparinase or Heparin. Thus, we conclude that HS plays a very important role during thymus organogenesis at least in part due to its ability to bind to and form immobilized gradients of chemokines that are essential for the directed migration of cells in the thymus.

182. Novel biomarkers for detection of circulating tumor cells – a model using the combination of glycan-targeting fluorescent molecularly imprinted polymers and non-invasive volume measurements

Z. El-Schich¹, Y. Zhang¹, T. Göransson¹, S. Shinde¹, N. Dizeyi³, K. Von Wachenfeldt⁵, K. Alm⁴, B. Janicke⁴, J. L. Persson², B. Sellergren¹, A. Gjörloff Wingren¹; ¹Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, Malmö, Sweden, ²Department of Molecular Biology, Umeå University, Sweden, ³Department of Translational Medicine, Lund University, Malmö, Sweden, ⁴Phase holographic imaging AB, Lund, Sweden, ⁵Truly Translational Sweden AB, Lund, Sweden

Tumor cells express high levels of glycans and is often associated with the increased invasive potential in clinical tumors correlating with poor prognosis. To improve the detection of the rare circulating tumour cells (CTC), we have used a combination of sialic acid (SA)-targeting molecularly imprinted polymers (MIPs), or “plastic antibodies”, and digital holographic cytometry (DHC). Expression of the epithelial cell adhesion molecule (EpCAM) was analyzed by flow cytometry, together with fluorescent labelled SA-targeted lectins and SA-imprinted MIPs. Here, we show the result of EpCAM and SA expression when using the SA-MIPs on a collection of breast cancer cell lines. The lectins Maackia Amurensis I (MALI) and Sambucus Nigra (SNA) specifically bind to the 2,3-SA and 2,6-SA variants, respectively, and was used for analysis of SA using both confocal microscopy and flow cytometry. We also demonstrate the SA-MIP binding to the breast cancer cell lines using confocal microscopy. Since CTCs generally are larger, we have measured physical and optical properties of both CTC and white blood cells with DHC. We show that CD45- CTC and CD45+ white blood cells can be analyzed and distinguished from each other by differences in cell area, cell volume and cell thickness. We confirm with DHC that CD45- CTCs are larger compared to CD45+ white blood cells. In conclusion, we show a combination of biomarkers that can be a new powerful tool in the diagnosis of CTCs.

183. Hilic-MS based glycomics in the diagnosis of congenital disorders of glycosylation (CDG)

F. Esposito², A. Palmigiano², A. Messina², R. Barone¹, L. Sturiale², D. Garozzo²; ¹Child Neuropsychiatry Unit, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy, ²CNR, Institute for Polymers, Composites and Biomaterials, Catania, Italy

N-glycans are often studied to discover disease-associated biomarkers, as well as to identify changing in glycomic profiles supporting diagnosis of those disorders directly connected to impaired glycan biosynthesis of glycoconjugates, such as congenital disorders of glycosylation (CDG). Strategies based on liquid chromatography (LC) coupled preferentially to electrospray ionization (ESI) - mass spectrometry (MS) have emerged as powerful analytical methods for N-glycan identification and characterization.

To enhance detection sensitivity, glycans are commonly functionalized with a tag prior to LC-MS analysis. Since the majority of the techniques used for glycan derivatization are notoriously time-consuming, some commercial analytical kits were developed to perform a rapid deglycosylation and labelling of N-glycans linked specifically to monoclonal antibodies (mAbs).

Adopting one of these commercial kits, RapiFluor-MS (RFMS), through a slightly modified protocol, we performed the enzymatic release and the N-glycan labelling of total serum and single serum glycoproteins from selected CDG patients (MAN1B1-CDG, ALG12-CDG, MOGS-CDG, TMEM199-CDG). Hydrophilic Interaction Chromatography (HILIC)-UPLC-ESI-MS separation of derivatized N-glycans allowed us to differentiate either structural isomers and isomers differing for the linkage type, as hybrid glycan structures accumulating in N-glycan profile of MAN1B1-CDG and ALG12-CDG.

According to patient immunological phenotype, serum IgG analysis of the Glucosidase 1-deficient patient (MOGS-CDG) showed significant N-glycosylation differences compared to control as the occurrence of the accumulating Glc3-Man7-GlcNAc2 glycoform (in accordance with the molecular defect) and, moreover, a generalized increase of sialylation.

Serum N-glycan profiling of TMEM199-CDG showed, aside an increased amount of undersialylated biantennary structures, also an overall increase of the triantennary N-glycan component.

All these applications demonstrated that RFMS method coupled to HILIC-UPLC-ESI-MS, represents a sensitive high throughput approach for serum N-glycome analysis and a valuable option for glycan detection and separation particularly for isomeric species.

184. Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of fc-fused lectin and antibody

C.Y. Fan¹; ¹National Tsing Hua University

The utilization of immuno-magnetic nanoparticles (MNPs) for the selective capture, enrichment, and separation of specific glycoproteins from complicated biological samples is appealing for the discovery of disease biomarkers. Herein, MNPs were designed and anchored with abundant boronic acid (BA) and photoreactive alkyl diazirine (Diaz) functional groups to obtain permanently tethered Fc-fused Siglec-2 (a mammalian cell surface lectin also known as CD22) and antiserum amyloid A (SAA) mAb with the assistance of reversible boronate affinity and UV light activation in an orientation-controlled manner. The Siglec-2-Fc-functionalized MNPs showed excellent stability in fetal bovine serum (FBS) and excellent efficiency in the extraction of cell membrane glycoproteins. The anti-SAA mAb-functionalized MNPs maintained active Ab orientation and preserved antigen recognition capability in biological samples. Thus, the BA-Diaz-based strategy holds promise for the immobilization of glycoproteins, such as antibodies, with the original protein binding activity maintained, which can provide better enrichment for the sensitive detection of target proteins.

185. MItochondrial modulation: a novel role for gm1 oligosaccharide

M. Fazzari¹, G. Lunghi¹, E. Di Biase¹, M. Audano¹, E. Maffioli¹, F. Grassi Scalvini¹, G. Tedeschi¹, L. Mauri¹, N. Mitro¹, E. Chiricozzi¹, S. Sonnino¹, ¹University of Milano

Functional data and clinical studies suggest the existence of a positive loop between the age-dependent GM1 deficiency and alpha-synuclein (<S) accumulation determining the neurodegeneration onset of sporadic Parkinson Disease (PD). This loop is triggered by the plasma membrane GM1 deficiency, which leads to a failure of trophic signaling and to the <S accumulation, increasing the susceptibility to neuronal death.

Recently we shed new light on the molecular basis underlying GM1 effects highlighting that GM1 oligosaccharide (OligoGM1) directly binds TrkA receptor, triggering TrkA-MAPK pathway activation which leads to neuronal differentiation and protection. Following its administration to PD mouse model, OligoGM1 was found to completely rescue the physical symptoms, reduce <S aggregates and restore dopaminergic neurons.

Since the mitochondrial dysfunction plays a central role in the exacerbation of nigrostriatal degeneration in PD, we decide to evaluate the putative OligoGM1 mitochondrial modulation in murine neuroblastoma cells, N2a.

Following its exogenous administration, proteomic analysis revealed an increased expression of proteins involved in mitochondrial bioenergetics and in oxidative stress protection. By biochemical studies we found that OligoGM1 protects N2a cells from MPTP toxic effect as well as from mitochondrial oxidative stress. Moreover, by immunoblotting we identified an increased expression of Tom20/HtrA2 mitochondrial proteins, whose reduced expression has been associated with PD. At functional level, we found increased basal and uncoupled mitochondrial respiration following OligoGM1 administration.

Collectively our data indicate a possible role of OligoGM1 as mitochondrial regulator that by inducing mitochondriogenesis and enhancing mitochondrial activity could determine mitochondrial restoration in PD neurons.

186. Structural and mechanistic basis of capsule O-acetylation in neisseria meningitidis serogroup A

T. Fiebig⁴, J.T. Cramer³, M. Schubert¹, A. Bethe⁴, F.F.R. Büttner⁴, R. Fedorov², M. Mühlenhoff²; ¹Department of Biosciences, University of Salzburg, Salzburg, Austria, ²Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany, ³Institute for Virology, Hannover Medical School, Hannover, Germany, ⁴Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany

Neisseria meningitidis serogroup A has been the major cause of bacterial meningitis in the African meningitis belt for decades. A major breakthrough in combatting the disease worldwide has been the development of glycoconjugate vaccines consisting of the capsule polymer of the pathogen, coupled to a carrier protein. The capsule of serogoup A consists of O-acetylated [→6)-<-D-ManNAc-(1→OPO3^-→]n repeating units, and O-acetylation of the ManNAc residues at O-3 and to a minor extent at O-4 was found to be mandatory to induce a sufficient immune response after vaccination. The enzyme catalyzing this modification, CsaC, belongs to a structurally uncharacterized family of O-acetyltransferases and represents an important synthetic tool for vaccine developmental studies.

Here, we report the structures of CsaC in its unbound form as well as bound to the acetyl-donor acetyl-CoA, and to a tetramer of the capsule polymer. Supported by a biochemical characterization of the enzyme and mass spectrometry analyses of wildtype and mutant constructs, the structural data demonstrates that CsaC transfers the O-acetyl group onto O-3 of the ManNAc ring, using a double displacement mechanism via a covalent intermediate with Ser114. We show that O-acetylation at O-4 results from spontaneous migration of the acetyl-group, which leads to the formation of nine different epitopes that could be identified and characterized in detail using 2D NMR spectroscopy. A comparative stability assay of non-O-acetylated vs. O-acetylated polymer demonstrated that O-acetylation considerably stabilizes the polymer and that hydrolysis only takes place between non-O-acetylated residues. Our findings are of crucial relevance for future vaccine design.

187. Study of pamp - binding activity of the lectin from the mussel mytilus trossulus

A. Filshtein¹, I. Chikalovets¹, V. Molchanova¹, O. Chernikov¹; ¹G.B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS, Vladivostok, Russia

Interest in lectins is due to their high selectivity of binding to the carbohydrate regions of the cell membrane. Because of this, lectins take part in the most subtle processes at the cellular, subcellular and organ levels in living organisms. Lectins are components involved in innate immunity of invertebrates, which are deprived of acquired immunity. The system of innate immunity is not specific, because it responds to certain molecular structures inherent in all pathogenic microorganisms. These structures are called pathogen-associated molecular patterns (PAMP). Such PAMP are the molecules that make up the bacterial cell membrane. Lectins interacting with PAMP are combined into group of pattern recognition receptors (PRRs). Early, the Gal/GalNAc-specific lectin was identified and characterized from the sea mussel Mytilus trossulus (MTL). To determine whether MTL belongs to PRRs, enzyme-linked immunosorbent assay (ELISA) was performed to detect the direct binding of MTL to LPS, peptidoglycan and beta-1,3-glucan.

It was revealed that MTL exhibits concentration-dependent binding to the PAMP in the following relationship: peptidoglycan> beta-1,3-glucan> LPS. In order to determine the biological activity of MTL, experiments on lectin binding to microorganisms (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Vibrio proteolyticus, Pichia pastoris), inhibiting the growth of microorganisms and the effect of lectin on the formation of biofilms were conducted.

As a result, it was found that MTL binds to all the studied microorganisms, agglutinates them, tightly binding them into aggregates. Further, using turbidimetry, it was found that MTL inhibits the growth of P. pastoris, B. subtilis and E. coli cells by 46%, 20% and 79.2%, respectively. It is also shown that MTL affects the formation of biofilms, reducing their biomass by 52.7% in the case of E. coli, by 23% - S. aureus, 3.6% - V. proteolyticus.

As a result, it can be assumed that MTL can be included in PRRs group and is a component of the immune system of the mussel, participating in the protection of the body of an invertebrate from the effects of external pathogens.

This work was supported by RFBR debts No. 18-34-00210.

188. The interplay of nmr spectroscopy and molecular modeling revealed novel insights on the recognition of sialylated glycans by hn protein of mumps virus

R.E. Forgione¹, A. Molinaro¹, R. Marchetti¹, A. Silipo¹; ¹Department of Chemical Sciences, University of Naples “Federico II”, Naples, Italy

Protein– glycans interactions set the basis of several molecular recognition processes and are implicated in events like cell–cell interactions, signal transduction, inflammation, viral entry and host bacteria recognition, thereby participating in disease, defence and symbiosis. Of particular interest is the role of sialylated glycans in mediating bacterial and viral infections, acting as receptors for many pathogens, including paramyxoviruses as mumps virus (MuV). MuV represents the main cause of mumps disease, a systemic viral illness characterized by painful swelling the salivary glands, typically the parotid glands. The pathogenesis of the virus in humans is a long-standing question. Although several vaccines have been developed, unexpectedly, MuV still causes outbreaks, even in highly vaccinated populations worldwide. Within this frame, the present work is focused on the study of the molecular recognition between the MuV attachment protein hemagglutinin-neuraminidase (MuV-HN) and sialylated ligands. The MuV-HN protein displays several functions essential for virus entry and infection, since is located on the surface of the MuV and mediates the viral attachment to the host cell surface. Actually, MuV-HN recognizes sialic acid-containing glycoconjugates on cell surfaces, thus assisting the fusion activity of the viral cell surface F protein, leading to the penetration of MuV into the host cell. In combination to that, the HN neuraminidase activity removes the sialic acid from progeny virus particles thereby avoiding viral self-agglutination. Nuclear magnetic resonance (NMR) techniques combined with computational studies were applied to dissect the interaction between HN protein and sialylated ligands. Novel insights on the kinetic of the hydrolysis reaction at the basis of the neuraminidase activity of HN have been provided; moreover, the definition of the bioactive conformation and of the binding epitope of the sialylated ligands allowed a deeper comprehension of the biological processes at the basis of MuV- HN infectivity. Our results represent a first step toward the development of more effective antiviral vaccines to prevent MuV infection and reinfection globally.

189. Antimicrobial potential of lectins isolated from cayaponia martiana and penicillium polonicum

F. Clavijo², N. Kiedanski¹, S. Alborés², L. Franco Fraguas¹; ¹Área de Bioquímica, DepBio, Facultad de Química, UdelaR., ²Área de Microbiología, DepBio, Facultad de Química, UdelaR.

The isolation of molecules from natural sources with antimicrobial properties increases continuously, since microorganisms frequently develop new resistance mechanisms to escape to antimicrobial actions. Lectins are carbohydrate-binding proteins ubiquitously present in organisms, and many of them have been found to exhibit antimicrobial properties. Our group is presently evaluating the antimicrobial potential of lectins including plants and fungal sources. From previous screenings we have selected two lectin sources to evaluate this activity: Cayaponia martiana, a plant native species from Uruguay belonging to the Cucurbitaceae family, and a fungus isolated from Antarctica (King George Island) belonging to the Penicillium genus. The lectin from C. martiana (CML) fruits extract was purified for the group, by affinity chromatography on a mannosyl-Sepharose column. For the identification of the fungus, the analysis was performed by sequencing the ITS1/ITS2 regions and the beta-tubuline gen and comparing them to the GenBank sequences database. From these results, the fungal strain was identified as Penicillium polonicum. An aqueous extract from mycelia in 50mM acetate buffer pH 5.5 was applied to a DEAE-Sepharose FF column and the lectin was eluted with the buffer supplemented with 0.5 M NaCl. The P. polonicum lectin (PPL) showed specificity towards mannose and methyl-mannoside, as determined by hemagglutination (HAG) inhibition assays using rat red cells. The HAG was also inhibited by the glycoprotein bovine sialo- and asialolactoferrin. The antimicrobial activity of these two lectins was evaluated by the minimal inhibitory concentration (MIC), against different microorganisms: fungi (Penicillium expansum, Fusarium graminearum, Aspergillus flavus, Candida albicans) and bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Xantomonas vesicatoria, Klebsiella pneumoniae, Acinetobacter baumanii, Enterobacter cloacae, Enterococcus faecalis). The CML inhibited the growth of F. graminearum and P. aeruginosa, while the PPL inhibited the growth of A. niger. The biological and biochemical characterization of these two lectins with antimicrobial properties, is under progress.

190. A dynamic view on IgG Fc/Fc©r complexes – insights from microsecond md simulations

M. Frank¹; ¹Biognos AB, Gothenburg, Sweden

The increasing popularity of antibody-based treatment demands a better understanding of antibody functions and in particular their interaction with cellular receptors. It has recently been found that IgG Fc / Fc(gamma)R interaction is sensitive to N-glycosylation on both interacting partners. Also the correct presentation of the receptor on the cell membrane is essential for proper immune cell function. With the advance of computer technology it has become feasible to simulate such large molecular systems on the microsecond timescale. Here also first results from high-level MD simulations of membrane-embedded CD16A and CD64 interacting with IgG1 Fc with different N-glycans are presented. The simulations provide insight into the dynamics and specificity of the molecular interactions at atomistic resolution. This helps to interpret existing experimental results better and can give valuable new ideas for planning the next round of experiments or for the design of improved antibodies.

191. Insulinotropic activity of sialidase inhibitor 2,3-dehydro-2-deoxy-n-acetylneuraminic acid with a glucose-dependent manner

Y. Fujita¹, A. Minami¹, Y. Kaneko¹, T. Sawatani¹, T. Otsubo², K. Ikeda², Y. Kurebayashi¹, T. Takahashi¹, T. Miyagi³, T. Ishikawa¹, T. Suzuki¹; ¹Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuka, ²Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University, ³Miyagi Cancer Center Research Institute, Natori, japan

Sialidase cleaves sialic acids from sialoglycoconjugates. Sialidase inhibitor 2,3-dehydro-2-deoxy-N- acetylneuraminic acid(DANA)remarkably enhances glutamate release from hippocampal neurons. Because synaptic vesicle exocytosis has many common processes with secretory granule exocytosis, in the present study, we demonstrated the effect of DANA on insulin release from ®-cells. We first demonstrated the distribution of sialidase activity in the pancreas. Histochemical imaging of mouse pancreas using a benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac), a highly sensitive histochemical imaging probe for sialidase activity, showed that pancreatic islets have intense sialidase activity. Insulin release from INS-1D clonal rat ®-like cells induced by 8.3 mM glucose was enhanced by DANA. Although hypoglycemia is the most common and serious side effect of glucose-lowering drugs, DANA did not cause insulin release from INS-1D cells under the non-stimulated conditions with the 2.8 mM glucose. In a mouse intraperitoneal glucose tolerance test, increase in serum insulin level was enhanced by intravenous injection of DANA. Under the fasting condition, insulin release, however, was not enhanced by DANA. Additionally, blood insulin level in sialidase isozyme Neu3-deficient mice was significantly higher than that in WT mice under the ad libitum-fed condition but not under a fasting condition. The lectin staining with Maackia amurensis agglutinin which is a sialic acid-binding lectin that mainly recognizes <2-3-linked sialic acid [Neu5Ac<2-3Gal-®(1-3)-GalNAc] showed that the binding of MAA to pancreatic islets was strengthened by DANA. Calcium oscillations induced by 8.3 mM glucose in INS-1D cells was not affected by DANA. These results indicate that DANA is a potentiator of insulin secretion in a glucose-dependent manner. Sialidase inhibitor is expected to be useful for antidiabetic treatment with low risk of hypoglycemia.

192. Hydroxylation of ceramide modifies sensitivity of GD3-positive cancer cells to the killing of natural killer cells

K. Furukawa², N. Hashimoto³, A. Yamamoto³, P. Crocker⁴, Y. Ohmi¹, R. Bhuiyan¹, K. Furukawa¹;¹College of Life and Health Sciences, Chubu University, ²Department of Biomedical Sciences, College of Life and Health Sciences,Chubu University, ³Graduate School of Medical Pharmaceutical and Dental Sciences, Tokushima University, ⁴School of Life Sciences, Dundee University

Many of sialic acid-recognizing lectins, Siglecs, have an inhibitory motif “Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)” in cytoplasm, and recognition of sialylated carbohydrates by Siglecs results in the suppression of Siglec-expressing cells. Natural immune cells such as natural killer (NK) and monocytes express Siglec-7 and Siglec-9, respectively, and their interaction with sialyl carbohydrates triggers inhibitory signals. In turn, malignant transformation of cells often induces novel expression of sialylated carbohydrates. Here, we studied recognition specificity of sialylated glycolipids on cancer cells by Siglec-7 on NK cells, and investigated their phenotypic changes based on the interaction.

To analyze Siglec-7 binding, we established GD3-expressing colon cancer cell line, DLD-1, by GD3 synthase cDNA. Using this DLD-1GD3S, binding of Siglec-7-Fc was analyzed by flow cytometry. Chemical structures of glycolipids were analyzed by QTRAP6500.

Although DLD-1GD3S cells showed high GD3 expression by an anti-GD3 antibody, no binding of Siglec-7 was found. On the other hand, DLD-1 cells cultured with milk-derived GD3 (milk-GD3) showed definite binding of Siglec-7. In the comparison of molecular species of GD3 expressed in DLD-1GD3S and DLD-1/milk-GD3+ cells revealed that milk-GD3 contained mainly regular ceramides, while DLD-1GD3S-derived GD3 contained mainly hydroxylated ceramides, probably phytoceramides, in which C4 of long chain base (LCB) was hydroxylated. Consequently, GD3 with different ceramide structures differentially reacted with Siglec-7 on the cell surface.

As ceramide-modification enzymes, DES2 is involved in the synthesis of phytoceramides. In addition to LCB, fatty acids in cearmides are modified by FA2H, which is involved in C2 hydroxylation. To clarify effects of these hydroxylations in Siglec-7 binding, knockout of DES2 and/or FA2H was done using DLD-1GD3S cells based on CRISPR/Cas9 system. Consequently, Siglec-7 binding was detected in DES2-KO cells and also in FA2H-KO cells, and double KO cells showed stronger binding. Effects of the structure alteration of sialyl-glycolipids on the sensitivity to NK killing were examined, resulting in the reduction of the killing in all GD3-expressing cells with dehydroxy-ceramides.

193. Core 1—derived O-glycan is required the maintain of glomerular function in glomerular podocyte

S. Fuseya⁴, R. Suzuki⁵, R. Okada¹, T. Usui³, N. Morito³, K. Hagiwara², T. Sato², H. Narimatsu², S. Takahashi¹, T. Kudo¹; ¹Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, ²Department of Life Science and Biotechnology, Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, ³Department of Nephrology, Faculty of Medicine, University of Tsukuba, ⁴Doctor’s Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, ⁵Ph.D. program in Human Biology, School of Integrative and Global Majors, University of Tsukuba

The glomerular filtration barrier is composed of the podocyte, glomerular basement membrane (GBM) and fenestrated endothelial cells. The disruptions of these structures cause several glomerular injuries such as focal segmental glomerular sclerosis (FSGS) in some cases. In 2006, mice with a mutation of core 1 beta-1,3-galactosyltransferase (C1galt1) which elongated core 1-derived O-glycan in conjunction with C1galt1 specific molecular chaperone (Cosmc) showed kidney disease (Alexander W.S. et al., PNAS., 2006). Moreover, C1galt1 systemically conditional knockout mouse in adult showed albuminuria and some glomerular sclerosis (Song K. et al., JBC., 2017). On the other hand, the loss of proteoglycans in GBM didn’t cause the proteinuria (Aoki S. et al., Nephrol. Dial. Transplant., 2018). Therefore, we hypothesized that core 1-derived O-glycan in podocytes is required for preventing the leakage of albumin from blood vessels. Then, we exploited the tamoxifen-inducible NPHS2-CreER^T2 transgene to ablate a conditional mutant Cosmc allele (cKO).

cKO mice exhibit transient proteinuria and the foot process effacements in podocyte at 20 days after the final tamoxifen injection. Subsequently, cKO showed the pathogenesis of FSGS. Immunohistochemical staining of MALII lectin which recognizes alpha2,3-linked sialic acids revealed that the MALII signal was decreased in cKO podocytes. It is well-known that Podocalyxin sialoglycoprotein is expressed in podocytes. To clarify the key molecules of the FSGS-like phenotype, we focused on this sialoglycoprotein. As a result, western blot analysis of the isolated glomeruli revealed that the mature podocalyxin protein was significantly reduced in cKO compared to WT mice. Then, some signals of degraded podocalyxin appeared in cKO mice. Our results suggest that core 1-derived O-glycan is required for normal glomerular filtration in podocyte and a decrease of podocalyxin by the absence of core 1-derived O-glycan might be due to proteolytic attack.

194. First x-ray crystal structure of human clec10a (MGL) crd: binding mode elucidation, thermodynamics and affinity for multivalent ligands

A. Gabba⁴, U. Westerlind³, S.J. Van Vliet², G. Birrane¹, P.V. Murphy⁴; ¹Beth Israel Deaconess Medical Center, Division of Experimental Medicine, Harvard Medical School,Boston,US, ²Department of Molecular Cell Biology and Immunology, Amsterdam UMC, Vrije Universiteit Amsterdam, Netherlands, ³Leibniz Institut fur Analytische Wissenschaften, ISAS, Dortmund,Germany; Department of Chemistry, Umea University, Umea, Sweden, ⁴School of Chemistry, National University of Ireland Galway, Galway, Ireland

Lectins are the main class of protein which recognize glycans. A member of the C-type lectin family is CLEC10A (h-MGL, CD301), an endocytic receptor located on the surface of some immune system cells, mainly dendritic cells (DCs) and macrophages. This protein displays exceptional selectivity for the truncated glycans bearing GalNAc present on tumour mucins. The binding event is believed to trigger the antigen internalization/presentation, such as in the case of aberrant MUC1 overexpressed in several tumours histotype, but there are also strong evidence suggesting CLEC10A involvement in Ebola virus entry into DCs. The role of h-MGL in DCs function is not fully understood and therefore the development of potent inhibitors of h-MGL could be significant in gaining understanding of the purpose of this receptor in DCs biology and in improving DC-based vaccine development and immunotherapy.

A series of CLEC10A inhibitors and more than fifty MUC1 glycopeptides decorated with h-MGL ligands were synthesized. Their ability to be recognized and internalized by h-MGL and their ability to create immunological memory in a rabbit model is currently under evaluation.

We obtained the first crystal structure of CLEC10A carbohydrate recognition domain (CRD) and have elucidated the GalNAc binding mode in the solid state. Our new data are complementary to previous STD NMR study and provide structural information relevant to the glycobiology and immunology community. Furthermore, an extensive study of the thermodynamic binding parameters of the full length CLEC10A and CRD of CLEC10A with multivalent glycoclusters revealed they may have different binding modes.

195. Chemo-enzymatic approach for production of various N-glycans

X. Gao¹; ¹Jiangnan University

Asparagine (N)-linked glycosylation is the most common type of glycosylation in eukaryotic cells. N-glycans, which can be divided into high-mannose type, complex type and hybrid type, play important roles in maintaining the stability of glycoproteins, extending their half-life period and enhancing the targeting ability. Understanding how the glycan structures contribute to these functions has been stymied by the inability to produce the N-glycans and their biosynthetic intermediates as homogenous structures in sufficient quantities. Thus, an efficient in vitro strategy to prepare various types of N-glycan structures is absolutely desired.

Recently, our lab described the high yield reconstruction of eukaryotic lipid-linked oligosaccharide (LLO) pathway to synthesis high-mannose type M9 and various high-mannose type N-glycans in vitro, providing an effective tool for investigating the features and mechanisms in the correct folding and secretion for N-linked glycoprotein. The chemo-enzymatic synthesis was accomplished by the successive glycosylation catalyzed by recombinant mannosyltransferases expressed in prokaryotic system, leading to the milligram scale of the final product. Meanwhile, we are aiming to produce other types of N-glycans with significant amount by elongating the glycan chain with a series of recombinant glycosyltransferases (GTases) selected from the eukaryotic N-glycosylation pathway. So far, various Golgi GTases, such as GnTs, GalT, Fut8 and SiaT, have been successfully expressed in E. coli as their soluble form in our lab. It is supposed that various complex type N-glycans could be efficiently assembled in a well-designed shortened pathway by shifting the GTases.

196. Application of immobilized neuraminidase from clostridium perfringens to the enzymatic desialylation of glycoproteins

L. Bidondo¹, F. Festari², T. Freire², C. Giacomini¹; ¹Biochemistry Laboratory, Bioscience Department, Chemistry Faculty,Universidad de la República, ²Immunobiology Department, Medical Faculty, Universidad de la República

Biological glycoconjugates have several structural and functional roles. Changes in glycan profile of cell glycoproteins are associated to pathological processes such as cancer. In fact, breast cancer cells secrete mucins carrying carbohydrate antigens such as sialyl-Tn antigen into cancer tissues and/or the bloodstream, in which mucins may interact with Siglec-2 (sialic acid-binding, immunoglobulin like lectin 2). Siglec-2 binds to sialyl-Tn antigen present on tumor-derived mucins down regulating splenic marginal zone B-cells in tumor-bearing mice. Immobilized neuraminidases can selectively remove sialic acid from glycoproteins without altering the protein backbone, being easily removed from the reaction mixture by a simple filtration step. This allows their reuse and avoids further purification steps in order to separate the desialylated protein from the neuraminidase. Thus, immobilized neuraminidase is a useful glycomic tool for the study of the biological role of sialic acid, which in turn enables the development of diagnostic methods and disease treatments. Costridium perfringens neuraminidase is specific for the cleavage of <-2,3- <-2,6- or <-2,8 terminal sialic acid from several glycoproteins. Here we report the immobilization of C. perfringens neuraminidase and its application to the selective desialylation of glycoproteins. Neuraminidase was successfully immobilized onto agarose activated with cyanate ester groups with high yield (90%). The immobilized enzyme expressed 40% of its initial activity and was stable at 4°C for at least four months. It was re-used for 7 desialylation cycles without loss of activity. Immobilized neuraminidase was effective in the selective desialylation of bovine fetuine and ovine submaxilar mucine. A decrease in the recognition of Sambucus Nigrans lectin of 84% was evidenced after enzymatic desialylation of fetuin both at pH 4.6 and 7.4. Desialylation of mucin was evaluated using anti Neuraminic 5 acid-2, 6-N-acetyl galactosamine antibody, showing a decrease in antibody recognition of 72% due to enzymatic desialylation. The success in the desialylation of model glycoproteins encouraged us to study the performance of this tool in the desialylation of ascitc lysates and ascitc cells, which is currently under evaluation.

197. Methylglyoxal increases sialic acid concentration and alters the adhesion and invasion properties of the neuroblastoma.

V.S. Gnanapragassam¹, M. Scheer¹, M. Nagasundaram¹, R. Horstkorte¹; ¹Institute for Physiological Chemistry, Martin Luther University Halle-Wittenberg, Germany.

Neuroblastoma is the second most frequent extracranial tumor, affecting young children worldwide. Oncogene MYCN is associated with 25% of high-risk neuroblastoma subtype. Methylglyoxal (MGO) is a highly reactive carbonyl compound elevated in cancer due to increased aerobic glycolysis. We investigated the effect of methylglyoxal in the MYCN-amplified (Kelly) and MYCN-non-amplified neuroblastoma cell lines (SHSY5Y). We have observed increased sialic acids (Sia), and altered tumor-associated glycan epitopes after treatment with MGO (0-100 micromolar).

Since we have found altered expression of glycans and tumor-associated glycan epitopes, cancer cell properties such as adhesion, migration, invasion, and colony formation were examined. We identified decreased adhesion of the cells towards a variety of ECM proteins, but display increased migration. Furthermore, enhanced invasion and colony forming ability of the Kelly and SHSY5Y cells were confirmed. Overall, treatment of neuroblastoma cells with MGO induces metastatic potential in MYCN amplified and non-MYCN amplified cells potentially through altered glycans associated with changes in cell signaling.

198. Carbohydrate-derived small molecule galectin inhibitors: design, screening and application as tools

C. Grandjean¹; ¹Université de Nantes, CNRS, Unité Fonctionnalité et Ingénierie des Protéines (UFIP), UMR 6286, F-44000 Nantes, France

Galectins form a ubiquitous family of proteins which exert their functions upon interacting with either glycan or protein ligands. Galectins take part in numerous physiological processes during development and later to maintain homeostasis and to set up the immune response.

Succeeding in designing highly potent and specific sugar-based small molecule inhibitors of the galectins is an important challenge in order to develop 1) tools to study the modes of action of galectins which remain largely unknown at the cellular levels and 2) drugs for the treatment of diseases such as pulmonary fibrosis, arthritic diseases, hepatitis steatosis or cancer… which are directly or indirectly linked to the deregulation of the expression or the function of the galectins.

We will first report the design and the synthesis of oligo-galactosides based on an iterative click-chemistry strategy as well as that of unprecedented type I lactosamine derivatives obtained according to a chemo-enzymatic approach making use of engineered glycoside hydrolases.

Interactions of these derivatives with galectins have next been characterized by combining fluorescence anisotropy and X-ray crystallography.

Finally some selective and potent galectin-3 inhibitors have been selected and used to question some biological function involving this galectin such as collective cell migration.

Observed differences among inhibitors in term of binding and in cellulo activity have been tentatively ascribed to differences in inhibitor respective structures, highlighting some guidelines to be followed for designing tools in the future.

199. Effects of the remyelination-promoting antibody rHIgM22 on glycosphingolipid metabolism in primary cultured glial cells

S. Grassi¹, S. Prioni¹, L. Cabitta¹, S. Sonnino¹, A. Prinetti¹; ¹Department of Medical Biotechnology and Translational Medicine, University of Milano

Recombinant human IgM22 (rHIgM22) binds to myelin and oligodendrocytes (OLs) and promotes remyelination in mouse models of multiple sclerosis. Literature suggests that rHIgM22 recruits a multimolecular complex formed by Lyn, integrin <v®3 and PDGFR<, triggering Lyn activation and promoting oligodendrocyte precursor cells (OPCs) survival and proliferation. However, its exact mechanism of action remains to be elucidated.

We have shown the involvement of different glycosphingolipids in rHIgM22 binding at the cell surface, suggesting that reorganization of lipid membrane microenvironment might be relevant in its biological activity.

Thus, we assessed the effect of a 24 hours, single dose treatment with rHIgM22 on glycosphingolipid metabolism in cultured rat mixed glial cells (MGC), OPCs and OLs. The treatment had no significant effects on the lipid pattern of MGC. However, in OPCs and OLs it determined an increase in the levels of gangliosides GD3 and GM3, both known for their ability to interact with and modulate the activity of different growth factor receptors. The treatment also determined an increase in the levels of PDGFR<, integrin <V, Lyn and in Lyn activation.

In addition, rHIgM22 determined a reduced activity of the acid sphingomyelinase (ASMase), with a consequent reduction of ceramide (Cer) generation. Ceramide generated by the action of ASMase represents an important pro-apoptotic signal, but also potent regulator for the organization of sphingolipid-rich signaling platforms. Remarkably, genetic deficiency or pharmacological inhibition of ASMase effectively protect against demyelination and other detrimental effects in MS models. Moreover, previous studies in OLs linked this reduced activity to the anti-apoptotic effect of Lyn activation, suggesting that rHIgM22-mediated increased Lyn expression and activation could result in a decrease in ASMase activity and in Cer generation thus inhibiting pro-apoptotic signaling and/or the organization of sphingolipid-dependent signaling platforms.

Altogether, our results support the notion that rHIgM22 protective effects might be mediated by alterations of lipid-dependent membrane organization and/or signalling in different cell types present in the nice of MS lesions.

200. B4GALNT2 gene expression controls the biosynthesis of sda and sialyl lewisx antigens in healthy and cancer human gastrointestinal tract

S. Groux-Degroote¹, V. Cogez¹, C. Wavelet¹, C. Schulz¹, A. Mihalache¹, P. Delannoy¹, F. Dall’olio², A. Harduin-Lepers¹; ¹Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France, ²University of Bologna, Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Italy.

The histo blood group carbohydrate Sd^a antigen and its cognate biosynthetic enzyme B4GALNT2 show the highest level of expression in healthy colon. Their dramatic downregulation previously observed in colon cancer tissues could play a role in the concomitant elevation of the selectin ligand sLe^x, involved in metastasis. However, the mechanisms involved in B4GALNT2 down-regulation in colon cancer are not clear. The human B4GALNT2 gene specifies at least two transcripts, diverging in the first exon. The long form contains a 253 nt exon 1L and encodes the long form B4GALNT2 protein (LF-B4GALNT2); the short form contains a 38 nt exon 1S and encodes the short form B4GALNT2 protein (SF-B4GALNT2).

Using qPCR, we show that normal and cancer colon tissues express mainly the short form transcripts, while the long form is primarily found in embryonic colon cell lines. Using an antibody that recognizes the stem region common to all B4GALNT2 proteins, we observed several isoforms in human normal and cancer colon tissues: the expression of SF-B4GALNT2 protein was major in healthy colon, and down-regulated in cancer tissues. Other isoforms were sometimes observed, such as low levels of the LF-B4GALNT2 in most tissue samples, but also a shorter (possibly soluble) isoform. This was observed in samples from different parts of the colon, notably with a higher expression in descending colon compared to ascending colon. SF-B4GALNT2 expression in gastro-intestinal tissues usually correlated with Sd^a expression and low sLe^x expression, both of which are expressed mainly on PNGase F-insensitive carbohydrate chains.

The mechanisms of B4GALNT2 down-regulation in colon cancer involve transcriptional regulation of SF-B4GALNT2: we have identified the minimal promoter region and general transcription factors controlling the expression of the short form B4GALNT2 in colon cells. Other mechanisms could be involved in decreased Sd^a synthesis in colon cancer, such as different functions of the SF-B4GALNT2 vs LF-B4GALNT2, which exhibit different cellular trafficking and localization.

201. Semi-synthetic O2A-CRM197 conjugate as a potential vaccine candidate for klebsiella pneumoniae

S. Guddehalli Parameswarappa², B. Monnanda², J. Przygodda², D. Knopp², S. Oestriech², M. Lisboa², A. Naini², A. Von Bonin¹, C.L. Pereira²; ¹Vaxxilon AG, Christoph-Merian Ring 11, 4153 Reinach, Switzerland, ²Vaxxilon Deutschland GmbH, Magnuss Strasse 11, 12489 Berlin, Germany

Infections caused by multi-drug resistant bacteria e.g., carbapenem-resistant Klebsiella pneumoniae (KP) are difficult to treat and results in high mortality rate in many regions of the world. Some pathogens are now controlled by last resort of antibiotics thereby making new forms of treatments inevitable. Vaccines play a significant role in governing these diseases and are also an effective way to cope with multi-drug resistance. KP is one such pathogen for which currently no vaccines are available. Highly diversified surface-exposed polysaccharides such as K-antigens and O-antigens are major virulence factors in KP. O1, O2 and O3 are most prevalent of O-antigens and are potential vaccine targets. The O2a antigen consists of a disaccharide repeating unit called D-galactan I and is also present in O1 as a low molecular domain. Currently we are developing an O-antigen based semi-synthetic conjugate vaccine for KP-O2a. The well defined O2a synthetic oligosaccharide varying in length are conjugated to CRM197 and immunologically evaluated as potential vaccine candidates.

202. Galabiose-binding streptococcal adhesin P (SADP) is inhibited with rationally designed tetravalent phenylurea-gb3 glycodendrimer at picomolar level

S. Haataja⁴, P. Verma¹, O. Fu², A.C. Papageorgiou⁵, S. Pöysti⁴, R.J. Pieters², U.J. Nilsson¹, J. Finne³; ¹Centre for Analysis and Synthesis, Department of Chemistry, Lund University, Lund, Sweden, ²Department of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands, ³Department ofeBiosciences, Division of Biochemistry and Biotechnology , University of Helsinki, Helsinki, Finland, ⁴Institute of Biomedicine, University of Turku, Turku, Finland, ⁵Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland

In the era of increasing antibiotic resistance of bacterial pathogens, new effective antimicrobial therapies are needed. Host cell surface carbohydrate receptors are an important target in the design of novel anti-adhesion compounds. However, the poor affinity of carbohydrates to bacterial adhesins is hampering the design of effective inhibitors useful in therapy. Streptococcal adhesin P (SadP) binds galabiose (Gal-alpha-1–4Gal), mediates bacterial binding to host cell surface glycolipids such as trihexosyl ceramide (Gb3) and plays a role in the pathogenesis of meningitis. We have designed a tetravalent dendrimer composed of phenylurea-galabiose glycomimetic and tested it as an inhibitor of the binding of SadP adhesin to its receptor. Using a homogenous AlphaScreen-based competitive inhibition assay, the phenylurea-modified galabiose-containing dendrimer inhibited SadP receptor binding at low picomolar level with an inhibitory power of 26 500 compared to the Gb3 trisaccharide. The results are encouraging in terms of using glycomimetics in antimicrobial therapies.

203. A sweet glimpse of rat immunoglobulin g glycosylation: towards comprehensive rodent animal model glyco(proteo)mics

S. Habazin², M. Novokmet², J. Štambuk², G. Razdorov², T. Keser¹, G. Lauc²; ¹Department of biochemistry and molecular biology, Faculty of pharmacy and biochemistry, University of Zagreb, Ulica Ante Kovačića 1, Zagreb, Croatia, ²Glycobiology laboratory, Genos Ltd., Borongajska cesta 83h, Zagreb, Croatia

Lab rats have traditionally been favoured as experimental animals because, compared to mice now holding primacy as a vertebrate disease model, their physiology and metabolism resembles more closely that of the humans. Since no -omics studies can be considered complete anymore without introducing glycomics dataset, mouse IgG N-glycosylation is already well explored. The fact that rat serum glycoprofiling has been conducted only recently calls for a thorough examination of normal and pathological rat IgG N-glycosylation patterns, encompassing both glycomic and glycoproteomic approaches.

An array of protocols for efficient rat IgG isolation using protein L affinity chromatography, PNGase F deglycosylation, fluorescent labelling of free glycans, purification and PGC enrichment as well as tryptic glycopeptides preparation combined with HILIC- or RP-SPE for subclass-specific IgG analysis was developed. Suitable UPLC-FLR and nano-LC-ESI-QqTOF methods were also established. Specific-pathogen-free Wistar rat serum was used as a test sample and preliminary results indicate an excellent recovery of IgG. Rat IgG glycome is dominated by mono- and digalactosylated glycans bearing core fucose followed by bisected structures. Interestingly, sialylated glycans carry almost exclusively N-glycolylneuraminic acid although N-acetylneuraminic acid is also expressed in rat. These findings suggest that glycosylation patterns share some similarities with those of mice and humans, yet full elucidation of how glycans alter rat IgG effector functions remains to be done.

A high-throughput rat IgG glycoproteomic analysis workflow suitable for IgG2a, IgG2b and IgG2c analysis in larger cohorts has recently been introduced in our lab. Thorough understanding of rat antibodies glycosylation will contribute to immunobiology of this popular animal model and hopefully open new fields of research such as nutriglycomics or pharmacoglycomics.

204. Glucosamine restores cognitive deficit induced by sleep deprivation in adult zebrafish

Y. Lee¹, J. Park¹, S. Kim¹, W. Lim², Y. Song³, I. Han¹; ¹Department of Physiology and Biophysics, College of Medicine, Inha University, Incheon, ²Department of Psychiatry, Medical School, Ewha Woman’s University, ³Ilsong Institute of Life Science, Hanllym University, Kyeonggi-Do

Sleep is an evolutionarily conserved physiological process implicated in the consolidation of learning and memory (L/M). In our experiments, sleep deprivation (SD) modulated the gene expression of neurotransmitters and immediate early genes implicated in L/M whereas expression of the circadian clock proteins, Bmal-1 and Per-2, was not significantly altered. Moreover, defects in motor neuron function and L/M ability in zebrafish were induced under conditions of SD. At the molecular level, SD triggered downregulation of O-GlcNAcylation and protein/mRNA levels of O-GlcNAc transferase (OGT) along with increased O-GlcNAcase (OGA) expression. Enhancement of HBP/O-GlcNAcylation by glucosamine or the OGA inhibitor, Thiamet-G, significantly restored cognitive deficits and increased PKA/CREB signaling in the SD group of zebrafish. To our knowledge, the current study has provided valuable insights into the molecular and biochemical changes associated with SD-induced cognitive dysfunction at the whole-brain level using the zebrafish system for the first time. Our findings highlight the role of the glucosamine for cognitive function and provide potential therapeutic agents for SD-induced cognitive defects.

205. Vertebrate <2,8-Sialyltransferases : a teleost perspective

M. Decloquement⁵, M. Tindara Venuto⁴, M. Noel², V. Cogez², S. Galuska¹, D. Petit³,A. Harduin-Lepers⁵; ¹ Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany, ² Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS 8576, Université de Lille, Faculté des Sciences et Technologies, 59655 Villeneuve d’Ascq cedex, France, ³Glycosylation et différenciation cellulaire, EA 7500, Laboratoire PEIRENE, Université de Limoges, 123 avenue Albert Thomas, 87060 Limoges cedex, France, ⁴Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany, ⁵Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS 8576, Université de Lille, Faculté des Sciences et Technologies, 59655 Villeneuve d’Ascq cedex, France

Sialic acids found at the outermost position of glycolipids and glycoproteins in Deuterostomes tissues control numerous biological functions like embryonic development or host-pathogen interactions. Sialic acids, such as Neu5Ac, Neu5Gc and Kdn, their glycosidic linkage and their degree of polymerization (DP) have critical roles in these biological processes. Interestingly, the structural diversity of sialic acid polymers (polySia) is remarkable on ray-finned fish fertilized eggs and is prone to changes over embryonic development. Biosynthesis of these diverse polySia conjugates is mediated by <2,8-sialyltransferases (ST8Sia), which catalyze the transfer of sialic acid residues from activated sugar donors (CMP-sialic acids) to a variety of acceptor substrates. In an effort to bring functional basis to the exquisite distribution and diversity of <2,8-sialoconjugates described in teleosts, we have studied the evolutionary relationships of the vertebrate ST8Sia. Towards this end, more than two hundred of fish ST8Sia sequences were identified through BLAST screening in the NCBI database suggesting an expansion of this family in fish. Our data indicated that while the mammalian ST8Sia family is comprised of 6 subfamilies forming di-, oligo- or polymers of <2,8-linked sialic acids, the fish <2,8-sialyltransferase family appears to be much more diverse and heterogenously distributed among fish species. Our molecular phylogenetic and phylogenomic analyses underscore the loss of several teleost fish st8sia loci and also the presence of several new st8sia paralogues, e.g. for st8sia7 and st8sia8 in fish genomes, which might be a source of phenotypic novelty. Furthermore, sequence-based analyses unveiled potential changes of function after gene duplication in fish genomes as for the polysialyltransferases . Functional genomics studies using several fish recombinant enzymes were undertaken to characterize these novel fish ST8Sia activities.

206. Characterization of in vitro and in vivo anti-herpes simplex virus activity of monogalactosyl diacylglyceride isolated from a green microalga, coccomyxa sp. KJ

K. Hayashi³, J. Lee⁴, S. Komatsu², M. Kanazashi², H. Kuno², K. Atsumi², T. Hayashi¹, T. Kawahara¹; ¹College of Life and Health Sciences, Chubu University, Japan, ²DENSO CORPORATION, Japan, ³Graduate School of Engineering, Chubu University, Japan, ⁴Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Japan

Herpes simplex virus type 2 (HSV-2) causes genital herpes, a sexually transmitted lifelong infection in populations worldwide. Some immunocompromised individuals such as elderly people, transplant patients, and acquired immune deficiency syndrome patients, experience severe primary diseases and frequent symptomatic recurrences. Recently, we have isolated a monogalactosyl diacylglyceride (MGDG) as an anti-HSV-2 component from the ethanol extract of Coccomyxa sp. KJ via a bioassay-guided fractionation. In the present study, the mechanism of action of the compound was elucidated by in vitro and in vivo assays.

<-Linolenic acid (C18:3) and 7,10,13-hexadecatrienoic acid (C16:3) accounted for approximately 72% and 23%, respectively, of total fatty acids of the MGDG. When the virus-compound mixture was assessed for the virus infectivity via a plaque assay, the MGDG was found to show potent virucidal activity against HSV-2. Possible mechanisms underlying the virucidal activity of MGDG were elucidated by the binding ability of virus to host cell membrane. As the results, MGDG-treated HSV-2 could not propagate in the cells. In accordance with the findings that virus particles lost their ability of binding to host cells, physical changes in HSV-2 shape were observed after treatment with MGDG, including a decrease in particle size, and possible damage to the viral envelope and capsids, as assessed using electron microscopy.

When we investigated whether exposing mice to MGDG-treated HSV-2 would protect the animals from genital herpes, the inoculum showed no pathogenicity in the animal model, indicating that MGDG exhibits irreversible virucidal activity against HSV-2 particles. In the animal model of HSV-2-induced genital herpes, intravaginally administered MGDG exerted a prophylactic effect by suppressing viral yields in the genital cavity and formation of herpetic lesions, resulting in a higher survival rate in treated mice than control mice administered solvent.

Thus, the current data suggest that the MGDG has potential as a novel tropical therapeutics for HSV infections, including cold scores, viral keratitis, and anogenital herpes.

207. Assembly of a multivalent neo-glycoprotein glycan library to determine efficient clostridium difficile toxin a binders

V. Heine³, S. Boesveld¹, H. Pelantová², V. Křen², C. Trautwein¹, G. Sellge¹, P. Strnad¹, L. Elling³; ¹Department of Internal Medicine III, University Hospital, RWTH Aachen University, Aachen, Germany, ²Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, Prague, Czech Republic, ³Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany

Clostridium difficile infections are the leading cause of nosocomial diarrhea and pseudomembranous colitis and trigger the necessity for new therapies. In the initial step of infection, C. difficile secretes the virulence factors toxin A (TcdA) and B (TcdB) that bind to the surface glycans of epithelial colon cells. To inhibit further development of the disease, scavenging of the toxins is crucial. The Galili epitope (Gal<3Gal®4GlcNAc) is considered a potent binder for TcdA but only scavenges its receptor domain (TcdA-R). Furthermore, multivalent approaches have been hardly elaborated but are likely to present a solution for proper toxin binding. For this, we assembled a multivalent neo-glycoprotein glycan ligand library of 40 different epitopes, based on N-Acetyllactosamine (LacNAc) backbone structures. Sequential application of different glycosyltransferases facilitated the buildup of natural and artificial structures; specific lectins detected the newly added glycosyl residues and thus verified the synthesized glycans. The structures were tested in binding assays with TcdA-R to find ligands with higher binding affinities than the Galili epitope. A selection of structures comprising fucosylated and sialylated epitopes (Lewis^y, Lewis^x, H-Antigen, LacNAc, Sia<6LacNAc) was chosen for binding assays with the holotoxin. Fucosylation was observed to strongly enhance binding of TcdA. The best performing structure, Lewis^y-Lewis^x, was selected for de novo synthesis. It was found that high occupation of BSA with the glycan facilitates proper scavenging of TcdA and effectively protects human colon cells in presence of TcdA. In conclusion, Lewis^y-Lewis^x neo-glycoproteins are novel multivalent glycoconjugates for toxin detection and scavenging in biomedical applications.

208. Polysialic acid and siglec-e orchestrate negative feedback regulation of microglia activation

H. Hildebrandt¹, H. Thiesler¹; ¹Clinical Biochemistry, Hannover Medical School, Hannover, Germany

Polysialic acid (polySia) emerges as a novel regulator of microglia and macrophage reactivity. We recently identified a pool of polySia in murine microglia and human macrophages residing on neuropilin-2 (NRP2) and the E-selectin ligand-1 (ESL-1). This pool of polySia is confined to the Golgi compartment and released in response to pro-inflammatory stimulation. Since exogenously added polySia is able to attenuate the inflammatory response we proposed that the release of polysialylated proteins constitutes a mechanism for negative feedback regulation of microglia and macrophage activation. Here, we demonstrate that translocation of polySia from the Golgi to the cell surface can be induced by calcium depletion of the ER and Golgi compartment and show a continuous shedding of polysialylated proteins over 24 hours after LPS stimulation. The latter was unexpected, because polySia signals detected by immunocytochemistry are rapidly depleted. However, it indicates that the amount of released polySia is much higher than anticipated based on immunostaining. This may be crucial for microglial responses during traumatic brain injury (TBI), as we detected a pattern of transient polySia signals in the Golgi of activated microglia around a stab wound in the adult mouse brain. Using CRISPR/Cas9-mediated knockout in BV2 microglia we demonstrate that the loss of the putative polySia receptor Siglec-E not only prevents the inhibition of pro-inflammatory activation by exogenously added polySia but also leads to a strong increase of LPS-induced activation. A comparable increase of LPS-induced activation is observed in microglia with abolished polySia synthesis. Together, these results provide strong evidence that the release of the microglia-intrinsic polySia pool, as observed in TBI, inhibits the inflammatory response by acting as a trans-activating ligand of Siglec-E.

Funding: German Research Foundation (DFG), grant Hi 678/9-1

209. Effects of antiviral treatment on the modulation of igg production and glycosylation in hepatitis b virus-related liver cirrhosis

C. Ho², T. Chang¹, R. Chien³; ¹Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ²Department of Medical Laboratory Science, College of Medicine, I-Shou University, Kaohsiung, Taiwan, ³Liver Research Unit, Linkou Chang Gung Memorial Hospital and University College of Medicine, Taoyuan, Taiwan

As debate rumbles on about whether anti-hepatitis B virus (HBV) nucleos(t)ide analogue treatments modulate host immune system during end-stage liver diseases, we studied effects of two potent anti-HBV agents, telbivudine or entecavir, on IgG-Fc glycosylation, which has a profound effect on the modulation of inflammatory responses, as well as various humoral immune activities including cytokine secretion, immunoglobulin production, in liver cirrhosis. Serum IgG-Fc N-glycan structures in patients with HBV-related liver cirrhosis, who had received either telbivudine treatment or entecavir treatment for at least 48 weeks, were analyzed using liquid chromatography tandem-mass spectrometry. Levels of cytokines and each immunoglobulin isotype were measured using enzyme-linked immunosorbent assays. Results showed that 48 weeks of entecavir treatment caused HBV DNA loss, alanine aminotransferase normalization, and an amelioration of hypergammaglobulinemia in cirrhotic patients; however, telbivudine treatment, though possessing similar efficacies on HBV suppression and an improvement in liver inflammation to entecavir treatment, did not mitigate IgG-related hypergammaglobulinemia. Moreover, telbivudine treatment enhanced the proportion of serum IgG-Fc agalactosylation, which was associated with enhanced levels of transforming growth factor (TGF)-®1 and IgG, in cirrhotic patients. Levels of IgG and TGF-®1 in sera of the cirrhotic patients before and during treatment were positively correlated. In vitro assays revealed that telbivudine treatment induced TGF-®1 expression in human macrophagic cells. Moreover, recombinant TGF-®1 treatment stimulated cell proliferation, IgG overproduction, and an increase in IgG agalactosylation in human IgG-producing B cell lines. In conclusion, telbivudine therapy was associated with TGF-®1 hyperactivity, IgG-related hypergammaglobulinemia, and IgG-Fc agalactosylation in HBV-related liver cirrhosis.

210. Molokhia (corchorus olitorius) leaf polysaccharides as potential prebiotics with their immunomodulating activity

H. Park², H. Lee², M. Oh³, K. Shin¹; ¹Department of Food Science and Biotechnology, Kyonggi University, Gyeonggi, ²Research Division of Food Functionality, Korea Food Research Institute, Jeollabuk-do, ³Technical Assistance Center, Korea Food Research Institute, Jeollabuk-do

This research reports on the extraction, partial characterization, and the potential application of molokhia leaf crude polysaccharides (MCP) as a prebiotic. MCP was a heteropolysaccharide mainly contained 61.8% (w/w) neutral sugar and 15.9% (w/w) uronic acid which predominantly composed of xylose, glucuronic acid, glucose, arabinose, and galactose with a molar ratio of 1.0:6.7:9.6:18.0:33.5. Moreover, methylation analysis indicated that MCP may be a polysaccharide belonging to rhamnogalacturonan-I. FT-IR and gel GPC analysis also confirmed the results obtained by HPLC and characterized preliminary structure features of the MCP. Furthermore, the MCP remained 93.7% undigested in the simulated digestion process and stimulated the growth of four commercial probiotics such as Bifidobacteria longum, B. Bifidum, Lactobacillus plantarum, and L. paracasei and increased production of the short chain fatty acids (SCFAs) using normal mouse model. In addition, MCP enhanced the production of immunoglobulin A from Peyer’s patch cells in vitro. Totally, the MCP showed a considerable prebiotic capability in vitro and high SCFA properties in vivo suggesting that the MCP is a potent functional ingredient which can be used in food and drug industries.

211. Development of a database of glycan-binding analysis results of glycan array experimental data

M. Hosoda⁴, Y. Takahashi¹, M. Shiota², D. Shinmachi⁵, E. Amagasa³, K.F. Aoki-Kinoshita⁴; ¹Department of Bioinformatics, Graduate school of Engineering, SOKA University, Tokyo, ²Faculty of Science and Engineering, SOKA University, Tokyo 4SparqLite LLC., Tokyo, ³Faculty of Science and Engineering, SOKA University, Tokyo, ⁴Glycan & Life System Integration Center (GalSIC), Faculty of Science and Engineering, SOKA University, Tokyo, ⁵SparqLite LLC., Tokyo.

We have developed the MCAW (Multiple Carbohydrate Alignment with Weights) tool that can compare multiple glycan structures and visualize common patterns among the glycans. The MCAW tool can visualize the aligned glycans by aligning the common glycan substructures similar to multiple amino acid sequence alignment, which is used to find common amino acid sequence motifs. Therefore, we aimed to elucidate glycan recognizing mechanisms of glycan binding proteins (GBPs) by analyzing glycan array data using this tool. We investigated the glycan array data from the Consortium for Functional Glycomics (CFG).

More than 5,000 glycan array experiments including animal lectins, plant lectins and viruses that have been published in CFG were downloaded. For each experiment, the glycans towards which GBPs showed higher affinity were used as input to the MCAW tool.

Experimental data with low binding affinity or in which the data did not meet the conditions for executing the tool were excluded from the analysis. As a result, 1,081 analyses were performed by the MCAW tool. Afterward, we constructed the MCAW-DB database (https://mcawdb.glycoinfo.org) and released it as a database containing our analysis results.

In MCAW-DB, by clicking the sample name on the top page, the analysis results of MCAW tool will be displayed. Common monosaccharides and binding information are displayed as ratios. For example, if a site is shown to contain lactose 100%, this indicates that lactose is a common motif across all the high-affinity glycan structures that bound to the GBP. In addition, it is possible to view the high-affinity glycan structures that were used in the analysis. The analysis data of MCAW-DB is linked with the data in the Lectin resource of the glycoscience portal GlyCosmos (https://glycosmos.org).

212. Bullfrog sialic acid-binding lectin reduces the expression of egf receptors in cancer cells

T. Tatsuta², S. Sato¹, S. Sugawara², M. Hosono²; ¹Department of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, ²Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University

cSBL isolated from bullfrog (Rana catesbeiana) eggs is a multifunctional protein having properties of a sialic acid-binding lectin and a ribonuclease (RNase). cSBL induces apoptosis in malignant tumor cells due to its RNase activity after binding through its lectin activity followed by incorporation to the cells. cSBL also exerts potent cytotoxicity in various tumor cell types, but low cytotoxicity in normal cells. Recently we found that cSBL significantly inhibited tumor growth in human xenograft mouse model of malignant mesothelioma, without any adverse effects.

In the previous study, it is reported that cSBL induced cell death with decreasing the expression level of estrogen receptor (ER). In this study, we assessed the expressions of several key molecules after cSBL treatment including ER, progesterone receptor (PgR), and human epidermal growth factor receptor (HER) family, which are associated with the breast cancer classification and with the decision of therapeutic strategy. The expressions of ER, PgR, and HER2 in ZR-75-1 and MCF-7 breast cancer cell lines were significantly decreased by the treatment of cSBL (10 ⎧M) for 72 hrs. cSBL also suppressed every HER family molecules (HER1~4) that expressed in seven types of breast cancer cells. Interestingly, it has a tendency to decrease any HER family molecules regardless of cancer cell types. The suppressive effect of EGFR/HER1 was also observed in malignant mesothelioma, lung and bladder cancer cells. However EGFR would not always necessary for the effect of cSBL because the ERK potential, a growth-accelerating factor being in the downstream of EGFR pathway, were not affected by decreasing of EGFR. Therefore cSBL triggers apoptosis induction without affecting EGFR signaling, but the downregulation of HER could be accompanied with cSBL-induced cell death under this experimental conditions. There are some chemotherapeutic problems; e.g. mutation of HER family receptors in cancer cells can cause resistance to the molecular targeted drugs, and some inhibitors of AKT and PI3K activate HER1 and HER3. Therefore the combinatorial approach using these antitumor agents and cSBL might contribute a new chemotherapeutic strategy.

213. Extracellular vesicles from clec2-activated platelets enhance dengue virus-induced lethality via CLEC5A/TLR2

P. Sung Pei-Shan², T. Huang¹, S. Hsieh²; ¹Department of Medicine, Mackay Medical College, New Taipei City, Taiwan, ²Genomics Research Center, Academia Sinica, Taipei, Taiwan

Platelet-leukocyte interactions amplify inflammatory reactions, but the underlying mechanism is still unclear. CLEC5A and CLEC2 are spleen tyrosine kinase (Syk)-coupled C-type lectin receptors, abundantly expressed by leukocytes and platelets, respectively. Whereas CLEC5A is a pattern recognition receptor (PRR) to flaviviruses and bacteria, CLEC2 is the receptor for platelet-activating snake venom aggretin. Here we show that dengue virus (DV) activates platelets via CLEC2 to release extracellular vesicles (EVs), including exosomes (EXOs) and microvesicles (MVs). DV-induced EXOs (DV-EXOs) and MVs (DV-MVs) further activate CLEC5A and TLR2 on neutrophils and macrophages, thereby induc neutrophil extracellular trap (NET) formation and proinflammatory cytokine release. Compared toIn stat1-/- mice, simultaneous blockade of CLEC5A and TLR2 effectively attenuates DV-induced inflammatory response and increases survival rate from 30% to 90%. The identification of critical roles of for CLEC2 and CLEC5A/TLR2 in platelet-leukocyte interactions will support the development of novel strategies to treat acute viral infection in the future.

214. De novo structural determination of oligosaccharides with logically derived sequence tandem mass spectrometry and discovery of new oligosaccharides in bovine milk

H.C. Hsu¹, S. Tsai¹, W. Weng¹, C.Y. Liew¹, S. Huang¹, Y. Kuo¹, C. Ni¹; ¹Institute of Atomic and Molecular Sciences, Academia Sinica

Structural determination of glycans remains an obstacle and constitutes a bottleneck in carbohydrate research. Glycan structures were conventionally determined by chemical methods or nuclear magnetic resonance spectroscopy which require a large amount of sample and are not readily applicable for glycans extracted from biological samples. Although it has a high sensitivity and widely used for structural determination of molecules, mass spectrometry can only reveal parts of the glycan structure. Here we demonstrated a new mass spectrometry method that the full structures of glycans, including diastereomers and anomericity of each monosaccharide and linkage position of each glycosidic bond, can be determined using collision induced dissociation tandem mass spectrometry guided by a logically derived sequence (LODES). This new method, which was validated by blind test of various oligosaccharides, provided de novo structural identifications with high sensitivity and was applied to in situ structural determination of the oligosaccharides eluted by high performance liquid chromatography. Bovine milk oligosaccharides that have not been observed in previous studies were discovered.

215. N-glycosylation on il-6 modulates its biological activity in lung cancer cells

C. Hung², H. Yeh¹, H. Wang³, C. Lin², W. Su²; ¹Center of Applied Nanomedicine, National Cheng Kung University, Tainan, Taiwan, ²Department of Internal Medicine, National Cheng Kung University Hospital and College of Medicine, Tainan, Taiwan, ³Department of Surgery, National Cheng Kung University Hospital and College of Medicine, Tainan, Taiwan

Interleukin-6 (IL-6) is overexpressed in various cancer cells, intriguingly contributing to both tumor growth and anti-tumor immunity. Cell-type-specific glycosylations on IL-6 have been mentioned in early studies. However, the glycosylation pattern and biological function of cancer cell-secreted IL-6 remains unclear. Here, we describe the glycosylation pattern of lung cancer cell-secreted IL-6 and its impact on the activation of JAK/STAT pathway. IL-6 molecules with different molecular weights were detected in the conditional media of IL-6-overexpressed lung cancer cell lines by immunoblot. Because in NetNGlyc 1.0 Server, one possible N-glycosylation site has been predicted at N73 on IL-6, we used treatment of N-glycosylation-specific inhibitors and site-directed mutagenesis on N73 to demonstrate that lung cancer cell-secreted IL-6 is modified by N-glycosylation. To examine which glycotransferases may participate in the modification, we screened the expression of glycosyltransferases by qPCR. Among them, the expression of fucosyltransferase 8 (FUT8), responsible for core fucosylation on N-glycosylated proteins, was higher in lung cancer cells as compared to normal bronchial cells. In paired normal (N) and tumor (T) tissues from lung cancer patients, higher FUT8 mRNA was detected in tumor part than normal part. Besides, we found similar glycosylation pattern in the secreted IL-6 of short-term cultured lung cancer cells derived from malignant pleural effusions. To uncover the potential influences on cellular signaling from glycosylation on IL-6, we generated core fucosylation-reduced IL-6 by silencing FUT8 with shRNA transduction in lung cancer cells. Subsequently, acceptor cells were treated with conditional media containing fully-glycosylated or core fucose-reduced IL-6. The fully-gycosylated IL-6 induced prolonged STAT3Y705 phosphorylation and distinct gene population compared to core fucose-depleted IL-6. Together, we report the presence of specific IL-6 glycoforms secreted from lung cancer cell lines and lung cancer cells from clinical samples. Moreover, the glycosylation on IL-6 changes its activity on JAK/STAT pathway.

216. Characterization of specific cell-surface heparan sulfate-protein interactions

S. Hung¹; ¹Genomics Research Center, Academia Sinica, Taipei, Taiwan

Heparan sulfate (HS) is a linear polysaccharide that is widely distributed on the cell surface, where it exists as proteoglycan component. The HS chain is initially assembled as a simple 1→4-linked copolymer of N-acetyl-<-D-glucosamine and ®-D-glucuronic acid (GlcA), but the seemingly regulated but non-template driven modifications cause extensive microheterogeneity along the sugar backbone comprised of around 50 to 200 disaccharide units. These modifications, which include N-deacetylation, N-sulfonation, GlcA 5-C epimerization forming <-L-iduronic acid, and multiple O-sulfonations, are implemented by several enzyme isoforms of varying specificities and are always incomplete, accounting to theoretically 48 disaccharide variations. The myriad of functional group patterns decorating the sugar backbone allowed HS to encode a high density of structural information. Such array of modifications is responsible for mediating or modulating protein activity. Keen interests are focused in deciphering the molecular level details of the HS–protein interactions because they may present therapeutic opportunities. Here, chemical synthesis of HS oligosaccharides in conducting structure–activity relationship studies will be presented.

217. New insights into the n-glycome of the honeybee superfood royal jelly by off-line LC-MS analysis

A. Hykollari², D. Malzl¹, P. Scheidl¹, J. Vanbeselaere¹, B. Eckmair¹, I. Wilson¹, K. Paschinger¹; ¹Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria, ²Proteomics Unit, Veterinärmedizinische Universität, 1210 Wien, Austria

Royal jelly is produced by the worker honeybees, primarily for lifelong feeding of the queen bee and has a number of applications in the food and cosmetic industry, as it is widely believed to have health benefits such as antibiotic and anti-inflammatory effects. A number of proteins in royal jelly are glycosylated; in this study, we detected unexpected, potentially bioactive N-glycan modifications capable of binding pentraxins, which may modulate the human immune system.

The N-glycans were enzymatically released and, after sequential purification steps including solid phase extraction on non-porous graphitized carbon, labelled with 2-aminopyridine for further analysis. In order to resolve isomers or low abundant structures, N-glycans were subject to RP-HPLC followed by HIAX chromatography. All the collected fractions were analysed with MALDI TOF MS and MS/MS (Bruker Autoflex Speed) in positive and negative ion modes. In addition to the “off-line” N-glycan analysis, protein-specific glycopeptide and N-glycan analyses were also performed. The affinity of specific residues towards pentraxins and specific antibodies was tested by probing N-glycan pools in a microarray format.

The neutral overall N-glycome displayed a range of oligomannosidic, hybrid and complex N-glycans. Chemical and exoglycosidase treatment of single isomers confirmed the presence of residues such as core <-fucose, core ß-mannose and capping of multiantennary glycans with galactose and N-acetylgalactosamine. The anionic N-glycan structures, enriched on non-porous graphitized carbon, were modified with sulphate and glucuronic acid, as found also in insect cell lines (Hi5), as well as with the zwitterion phosphoethanolamine, observed also in T. vaginalis and fungal Penicillium. Significant amounts of glucuronylated and sulphated structures were also identified in combination with antennal and double fucosylated core residues. Western blot and microarray analysis showed binding of royal jelly N-glycan epitopes to a human pentraxin (serum amyloid P) and anti-HNK-1 as well as to selected lectins.

In conclusion, our data demonstrate that fractionation and MS fragmentation, in both positive and negative modes, are necessary to reveal the complexity of N-glycan structures in an invertebrate species.

218. Synthesis and evaluation of 1,5-anhydro-d-fructose derivatives as inflammasome inhibitors

H. Ideo², K. Goto², A. Tsuchida², Y. Hirose², I. Maruyama¹, S. Noma¹, T. Shirai², J. Amano², M. Mizuno², A. Matsuda²;¹Grad. sch. of Medical and Dental Sci., Kagoshima Univ., Kagoshima, ²The Noguchi Institute, Tokyo

Inflammasomes are protein complexes consisting of particular pattern recognition receptors (PRRs), ASC, and caspase-1 which are formed intracellularly in response to various stimulating factors. First, a PPR such as NLRP3, AIM2, or NLRC4 recognizes a particular stimulating factor, then caspase-1 is activated, and the active caspase-1 induces inflammation by promoting the maturation and secretion of inflammatory cytokines such as IL-1® and IL-18 in inflammasomes. Inflammatory responses based on the formation of inflammasomes are often effective for defense against infections. This mechanism is implicated in the development of various inflammatory responses such as seen in infection, diabetes mellitus, arteriosclerosis, and autoimmune diseases. As the mechanisms of inflammasome formation have been gradually revealed, many studies into inflammasome inhibitors have been carried out with the aim of developing diagnostic and therapeutic medicines.

1,5-Anhydro-D-fructose (1,5-AF) is a functional monosaccharide that can be formed directly from starch and glycogen in vivo by <-1,4-glucan lyase. It occurs in edible morels, red seaweeds, and certain mammalian tissues, and has been revealed to exhibit strong anti-oxidant activity and antibacterial activity. Therefore, 1,5-AF is expected to find use in various fields such as health foods and medicines. Recently, 1,5-AF was reported to suppress the inflammasome, although with only low activity. Herein, we show the synthesis and the evaluation of inflammasome inhibitors of which 1,5-AF is a lead compound. We focused on the hydration of 2-keto form of 1,5-AF and speculated that this hydration was the cause of low activity. Therefore, we synthesized some 1,5-AF derivatives that would not be able to form the dimer conformation and can be expected to have high activity against inflammasome, and then evaluated their inhibitory activities with respect to the NLRP3 inflammasome by using mouse bone marrow–derived macrophages and human THP-1 cells. As a result, some synthesized 2-keto form compounds had much higher inhibitory activities with respect to the NLRP3 inflammasome than did 1,5-AF.

219. Exploring new strategies for drug delivery

D. Imperio¹, F. Campo¹, F. Mazzola², L. Confalonieri¹, L. Varani², L. Panza¹; ¹Dip. di Scienze del Farmaco, Università del Piemonte Orientale, Novara (IT), ²Istituto di Ricerca in Biomedicina, Università della Svizzera Italiana, Bellinzona (CH)

A major problem in cancer chemotherapy is represented by the unsatisfactory selectivity of anticancer drugs. Several approaches are under study to improve the selectivity of the therapy, mainly exploiting overexpressed tumor cell surface structures through targeted delivery. Within this frame, we present here two approaches for drug delivery and targeting exploiting cyclodextrins and liposomes.

1. Cyclodextrins are used as complexing agents to increase aqueous solubility of poorly soluble drugs, as well as their bioavailability, safety, and stability. Studies in both humans and animals have shown that cyclodextrins can be used to improve drug delivery from many types of drug formulation.

We will describe here the synthesis of a modified ®-cyclodextrin bearing a well-established ligand for prostate specific membrane antigen (PSMA) to target prostate cancer. PSMA is a type II transmembrane glycoprotein, an established prostate cancer marker and has been considered as a promising biological target for anti-PC drug delivery and diagnostics.

The PSMA we chose ligand is a urea dimer of (S)-glutamic acid. It was modified at the side chain carboxylic groups of one of the glutamic acid, to allow the conjugation with per-6-azido-®-cyclodextrin through a new spacer.

2. Liposomes-based drug delivery systems have significant potential due to high carrier capacity and possibility of incorporation of both hydrophilic and hydrophobic substances. Liposomes can also be designed to allow controlled (sustained) drug release and to target drugs to the tissue of interest. These properties enable improvement of drug bioavailability and reduction of the dosing frequency.

We will present here the preparation of acid-sensitive liposomes decorated with specific antibodies light chains directed against two tumor markers expressed on blast cells in acute myeloid leukemia (AML), namely T-cell immunoglobulin and mucin domain-containing molecule3 (TIM3) and the alpha-chain of the interleukin-3 receptor (CD123). Within the liposomes, for preliminary experiments, was encapsulated daunorubicine, one of the drugs used in the standard care of AML.

Acknowledgements: we gratefully thank Programma di cooperazione Interreg V-A Italia-Svizzera 2014-2020 – Progetto LEUCITI for financial support.

220. Alteration of glycans localization with cellular senescence and human aging

Y. Itakura¹, N. Sasaki¹, M. Toyoda¹; ¹Research Team for Geriatric Medicine, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan

Glycans play essential roles in biological functions such as differentiation and immune response. However, the details of the specific glycans involved in aging are limited. Moreover, there is only a little information for intracellular glycans. Here, we investigated cellular senescence- and human aging-dependent cell surface and intracellular glycan alterations in human diploid fibroblasts derived from differently aged skin using the lectin microarray.

We found that sialylated glycans on the cell surface decreased with cellular senescence and human aging. The intracellular glycans of cells derived from aged fetus and from elderly subjects showed similar localized patterns while repeating unsteady changes. The localization of sialic acids of cells shown by lectin staining were in good agreement with the ratio of abundance observed by lectin microarray analysis. Moreover, the ratio of increasing sialylated intracellular glycoproteins in the fetus-derived cells showing cellular senescence was similar to that in cells derived from the different ages with human aging.

We consider that the glycan changes during cellular senescence are correlated with human aging. Therefore, glycan profiling provides mechanistic insight into the molecular changes during aging and might be useful for the characterization of aging biomarkers.

221. Catfish (silurus asotus) lectin enhances the cytotoxic effects of sunitinib on renal cell carcinoma

J. Ito¹, S. Shigeki¹, M. Hosono¹, M. Sato¹; ¹Tohoku Medical and Pharmaceutical University, Sendai, Japan

(Introduction and Objective) Renal cell carcinoma (RCC) is highly malignant. Although most patients with RCC can be surgically cured, there are several patients who experience recurrence. In the last few years, the number of therapeutic agents for patients with RCC recurrence and/or metastases has increased, including targeted agents against VEGF, TKI, and mTOR. We have been conducting researches on glycans as a new target of RCC therapy, which we call “glycotherapy”. Previous experiments by our group showed that Catfish (Silurus asotus) egg lectin (SAL) bound to globotriaosylceramide (Gb3) expressed on the surface of HeLa cells, and that SAL increased intracellular uptake of sunitinib. This study sought to analyze the combined effects of SAL and sunitinib against RCC cells.(Methods) SAL was isolated by sequential chromatography. RCC cell lines, TOS1, TOS3, and TOS3LN, were established in our institution. We performed flowcytometric analysis of Gb3 expression on RCC cells, binding of SAL to Gb3+ RCC cells, and binding of annexin-V to and propidium iodide (PI) incorporated into Gb3+ RCC cells. We also assessed the single and combined effects of SAL and molecular targeted drugs against RCC cells. (Results) Gb3 expression levels were higher in TOS1 and TOS3 cells than in TOS3LN cells. SAL bound abundantly to TOS1 and TOS3 cells, and the binding was dependent on the amount of Gb3 expressed on the cell’s surface. SAL increased annexin-V binding to, and PI incorporation into, TOS1 and TOS3 cells. In the reaction between TOS1 cells and single molecular targeted drugs, such as sunitinib, axitinib, pazopanib, and everolimus, we observed that cell viability decreased in a concentration-dependent manner for each of the drugs. However, when SAL and molecular targeted drugs were used in combination there was only a significant decrease in cell survival rate with sunitinib. Moreover, there was an increase in cell apoptosis when SAL and sunitinib were used in combination than when sunitinib was used alone. Furthermore, SAL increased incorporation of sunitinib into TOS1 cells and consequently enhanced sunitinib’s cytotoxic effects.(Conclusion) The effects of SAL varied depending on the amount of Gb3 expressed on RCC cells. A combination effect was observed when TOS1 cells were treated with a combination of SAL and sunitinib. Further studies are needed in order to better understand the clinical applications of these drug combinations; however, glycans and lectins may be new targets for cancer therapy.

222. The effects of cu(ii) and zn(ii) ions on amyloid beta aggregation induced by gm1-containing lipid bilayer

K. Ito¹, T. Matsubara¹, T. Sato¹; ¹Department of Sci. and Tech., Keio Univ., Kanagawa

The Alzheimer’s disease (AD) is one of the most common and serious neurodegenerative disease. In AD patient’s brain, amyloid beta protein (Aß) is deposited and its Aß deposition leads to AD progression. The mechanism of Aß aggregation is still unclear, but Aß is known to bind to the monosialoganglioside GM1 in neuronal cell membranes and form toxic aggregates and fibrils. Moreover, higher concentrations of metal ions such as Cu²⁺, Zn²⁺, and Fe³⁺ in AD brains than that in healthy brains have also been found. It has also been reported that metal ions form complexes with Aß, promote, and/or inhibit Aß aggregation. However, few studies have been reported for the effects of metal ions on the ganglioside-induced Aß fibril formation. Therefore, we have investigated the aggregations of Aß₄₂ on lipid bilayer of 10% GM1 (GM1/sphingomyelin/cholesterol=10:45:45) monolayer and phospholipid-coated mica. In the present study, Cu²⁺ and Zn²⁺ were interacted with Aß₄₂ on the lipid bilayer. After incubation, the morphology of Aß aggregations was observed by atomic force microscopy (AFM). AFM observation indicates that formation of Aß₄₂ aggregates were promoted by both metal ions. But the morphology of Aß₄₂ aggregations was different depending on species of metal ions interacted. In the presence of Cu²⁺, Aß₄₂ non-fibrillar aggregates formation was different depending on the concentration of Cu²⁺. In the presence of Zn²⁺, Aß₄₂ fibril formation was promoted. These results indicate that each metal ion, Cu²⁺ or Zn²⁺ has different effect on the ganglioside-induced Aß₄₂ fibril formation. Moreover, in order to investigate the mechanism on Aß₄₂ aggregates interacted with Cu²⁺, the fluorescence of Tyr10 in Aß₄₂ was observed in the presence of Cu²⁺. The results showed the complexes of Aß₄₂ and Cu²⁺ were formed and they interacted with 10% GM1 membrane. By understanding the mechanism of Aß aggregation with metal ions and ganglioside, the new approaches to therapy of AD are expected.

223. Molecular pathogenesis and innovative therapy for lysosomal neuraminidase 1 (neu1) deficiencies (sialidosis and galactosialidosis)

K. Itoh¹, J. Tsukimoto¹, D. Tsuji¹, Y. Horii¹, T. Iniwa¹, Y. Fukushi¹, H. Ando¹, P. Simona², L. Cabitta², S. Grassi², A. Prinetti², S. Sonnino²; ¹Department of Medicinal Biotechnology, Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima; ²Department of Medical Biotechnology and Translational Medicine, University of Milano, Milan

Neuraminidase 1 (NEU1) is a lysosomal sialidase, which is activated through binding with cathepsin A (CTSA) and transported to lysosomes to cleave the terminal <2,3- or <2,6-sialic acid residues from sialylglycoconjugates at acidic pH. The CTSA itself has acid carboxypeptidase activity as well as the protective functions for the NEU1 and ß-galactosidase 1 (GLB1) to form multienzymatic complex. However, intracellular crystallization of NEU1 has been observed when singly overexpressed in mammalian cells. Sialidosis (SD) and Galactosialidosis (GS) are autosomal recessive NEU1 deficiencies caused by the heterogeneous gene mutations of NEU1 and CTSA, respectively. These incurable metabolic disorders associate with excessive accumulation of sialyloligosaccharides in the organs and urinal secretion and neurovisceral symptoms including myoclonus, cherry-red spot, hepatosplenomegaly and so on.

To develop the effective therapy for these NEU1 deficiencies, an innovative technology is crucial for simultaneous and stoichiometric expression of NEU1 and CTSA as activator and chaperone in the same cells.

We constructed a novel modified NEU1 gene (modNEU1) and succeeded in producing the modified NEU1 which does not form in cellulo crystals itself but can associate with CTSA to exhibit NEU1 activity. We demonstrated that the gene transfer with an adeno-associated viral (AAV) vector encoding both modHEU1 and CTSA under the control of dual promoters increased both NEU1 and CTSA activities in the fibroblasts derived from SD and GS patients to reduce the accumulated sialyloligosaccharides, suggesting the therapeutic potential of the bicistronic AAV-modNEU1/CTSA vector for both SD and GS.

We also established a novel GS mouse model with homozygous single base substitution in murine Ctsa causing splicing abnormality, associated with the combined deficiencies of Neu1 and Ctsa, excessive accumulation of sialyloligosaccharides in a wide range of tissues and neurovisceral manifestations including myoclonus. Significant decreases in galactosylceramide (GalCer) and sulfatides in the brains of the GS mice, suggesting the abnormality in oligodendrocytes should be responsible for the neurological symptoms similar to those of SD and GS patients.

224. Shigella flexneri lipopolysaccharide conformations: effects of glucosylation and o-acetylation in serogroup 2

J. Hlozek¹, M. Kuttel², N. Ravenscroft¹; ¹Department of Chemistry, University of Cape Town, ²Department of Computer Science, University of Cape Town

The Shigella genus is one of the main bacterial causes of intestinal disease worldwide. Shigella bacteria have a high morbidity and claim more than 800 000 fatalities annually – predominantly young children in low income areas. Shigella flexneri is the dominant species causing Shigella infection, with up to 60% of shigellosis cases. S. flexneri strains are serotyped by the O-antigen lipopolysaccharides (LPS), which are a primary vaccine target. Except for serogroup 6, S. flexneri serotypes share a common tetrasaccharide repeating unit backbone (serotype Y): →2)-<-L-RhapIII-(1→2)-<-L-RhapII-(1→3)-<-L-RhapI-(1→3)-®-D-GlcpNAc-(1→. The various other serotypes arise from differing glucosylation and/or O-acetylation patterns on the common backbone. Due to the similarity in S. flexneri serotypes, it has been suggested that broad protection against shigella can be achieved from a vaccine including just four serotypes.

Serotype 2a is the most prevalent S. flexneri serotype worldwide and is one of the four proposed vaccine serotypes. Serotype 2a is characterized by glucosylation at O4 of Rhal with partial O-acetylation predominantly at O-3 of Rhalll and O-6 of GlcNAc. Serotype 2b has the same glucosylated structure of 2a with an additional glucose at O-3 of Rhalll and no O-acetylation. A previous study of serogroup 5 indicated that glucosylation alters the linear backbone conformation to significantly more compact structure. Another study reported the individual linkage conformations of S. flexneri O-antigens from relatively short molecular dynamics simulations. Here we explore the conformations of serogroup 2 LPS antigens through microsecond-scale molecular dynamics simulations of six repeating units of serotype Y, Y with glucosylation (2a and 2b), and Y with glucosylation and O-acetylation (2a with Rhalll3Ac). Preliminary results indicate that glycosylation and O-acetylation of the backbone bring about significant restriction in the motion of the adjacent linkages. This in turn causes significant changes in the conformational preferences of the flexible chains.

225. Synthetic arabinomannan glycolipids inhibit mycobacterial growth and biofilm

G.C. Samanta¹, A. Mahapa¹, D. Chatterji¹, N. Jayaraman¹; ¹Indian Institute of Science

Mycobacterial outer cell-wall envelop constitutes a rich glycoconjugate constellation, primarily of arabinogalactans, lipoarabinomannans, phosphoglycans, phosphatidyl mannoside and mycolic acids. Among several factors, the presence of capping glycans at the non-reducing end of lipoarabinomannan component is one of the detriments to the virulence characteristics of the mycobacterium. Synthetic versions of the glycolipids possess an interesting functional role in the metabolic activities of the mycobacterium. Studies conducted over a period more than a decade in our laboratories demonstrate that arabinomannan glycolipids interfere in the mycobacterial growth and, more importantly, also interfere the altered metabolic processes pertaining to the mycobacterium at a quorum sensing phase, namely, the biofilm phase. Mycobacterial growth and biofilm inhibitions by the synthetic glycolipids show a distinct trend, wherein higher arabinan component in the glycolipids inhibits the planktonic mycobacterial growth, whereas higher mannan component exhibits a profound mycobacterial biofilm disruption. Branching pattern constituting the arabinomannan glycolipids also exhibits a trend in the interference of the mycobacterial metabolic processes. A series of lipodomics profiling studies show that the synthetic glycolipids could down-regulate biosynthesis of a number of components of the mycobacterial biofilm growth. Among the components, phosphatidylinositol mannosides, alpha-, alphaprime- and keto-mycolic acids and phosphoglyceroethanolamines are more heavily down-regulated. Following a cue on these results, the relative mole fractions of mannan and arabinans were varied, leading to further observations of the preferential inhibition activities to mycobacterial growth or biofilm growth. Front-line drug isoniazid is also found to undergo a synergism when combined with the glycolipids. The drug, which is a cell-wall biosynthesis inhibitor, along with the synthetic glycolipids exhibits a synergistic effect and a 3- to 4-fold increase in the inhibition potency to the biofilm grown mycobacterium. The presentation will cover important developments in advancing glycolipids as mycobacterial cell-wall inhibitors.

References: (a) Mahapa, A.; Samanta, G.C.; Maiti, K.G.; Chatterji, D.; Jayaraman, N. ChemBioChem., 2019, In print. (b) Maiti, K.; Syal, K.; Chatterji, D.; Jayaraman, N. ChemBioChem., 2017, 18, 1959 – 1970. (c) Syal, K.; Maiti, K.; Kottari, N.; Avaji, P. G.; Chatterji, D.; Jayaraman, N. Glycoconjugate J., 2016, 33, 763 – 777.

226. Arrays of mammalian glycan-binding receptors for screening of microbial ligands

S.A.F. Jégouzo¹, M.E. Taylor¹, K. Drickamer¹; ¹Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom

A novel lectin array format is being developed to characterize the roles of glycan-binding receptors in innate immunity by probing their interactions with pathogenic micro-organisms. The array is constructed with carbohydrate-recognition domains (CRDs) from mammalian C-type lectins modified with single-site biotin tags, thus avoiding potential confounding effects of appended Fc domains or chemical immobilization. Biotin-tagged CRDs are created by encoding a biotinylation sequence, based on the natural target for biotin ligase in pyruvate oxidase, in bacterial expression systems for the CRDs. The tag is biotinylated during expression in the bacteria by co-expression of biotin ligase. Structural analysis shows that the C-terminal biotin tag projects from the opposite site of the CRD compared to the sugar-binding site. The tag is accessible to streptavidin binding and the CRDs have been extensively characterized in binding assays with glycans and glycoproteins. The tag allows immobilization on streptavidin in a 96-well format. Key advantages of this approach are that the CRDs can be abundantly expressed, they are stable, and purification is by affinity purification on sugar ligands, which ensures that the proteins are fully active. This feature gives confidence in negative as well as positive results. A model array containing all of the sugar-binding C-type lectins from cow has been generated. Some key results have emerged from screening of the array: (1) Yeast, Gram positive bacteria, and Gram negative bacteria bind to distinct but overlapping sets of receptors; (2) in all cases, multiple receptors interact with each type of micro-organism; (3) binding to each class of micro-organism is targeted to common sugar structures rather than to strain-specific oligosaccharides. These results provide a unique overview of the interactions between mammalian glycan-binding proteins in the innate immune system and different classes of potential pathogens.

227. O-galnacylation modification of tgf-beta type ii receptor by ppgalnac-t4 supresses migration and invasion of breast cancer cells via met process

Q. Wu¹, Y. Liu¹, B. Niang², X. Chen¹, T. Huang¹, W. Li¹, S. Wang², J. Zhang¹; ¹School of Life Science and Medicine, Dalian University of Technology, Panjin, China, ²Department of Biochemistry, Dalian Medical Uiversity, Dalian, China

GalNAc-type O-glycosylation, which is initially catalyzed by polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) isoenzymes, is the most abundant and complex protein post-translational modification. Accumulating evidences have proven that ppGalNAc-Ts are involved in pathological disorders and malignant transformation by modifying their target proteins. However, the exact molecular functions and mechanisms of ppGalNAc-Ts are still not well understood. In this study, the role of ppGalNAc-T4, T4 in breast cancer was investigated. The results showed that the expression of T4 reduced significantly in invasive lobular type breast cancer tissues than that in invasive ductal type. T4 expression is negatively associated with migration and invasion ability in breast carcinoma cell lines. Over-expression and CRISPR/Cas9-mediated loss-off function studies revealed that T4 expression attenuated the migration and invasion capability of breast cancer cells through MET process. Furthermore, TGF-beta signaling-induced EMT is impaired by up-regulation of T4 expression and is enhanced by knocking out T4 expression in breast cancer cells. Importantly, in T4 knocking-out MCF cells, O-GalNAc modifications of TGF-beta type II receptor decreased, more stable TGF-beta type I and II dimer complex formed, and TGF-beta signaling activity was markedly elevated. This was further confirmed by identification of Ser31 corresponding to the extracellular domain of TGF -beta R II as the putative O-GalNAcylation site via T4 directly enzyme-catalytic glycosylation in vitro. In addition, S31A mutagenesis led to reducing the O-GalNAcylation on TGF-beta R II and raising TGF-beta signaling activity in breast cancer cells.

Together, these results unravel a novel mechanism of ppGalNAc-T4-catalysing TGF-beta receptors O-GalNAc modification that suppresses breast carcinoma cells migration and invasion via MET process. Targeting ppGalNAc-T4 may be a potential therapeutic strategy for breast cancer treatment.

This study was supported by grant from the National Natural Science Foundation of China (No. 31570802).

228. A beta-glucan from grifola frondosa effectively delivers oligonucleotides to antigen presenting cells

H. Cui², X. Zhu², Z. Huo², J. Fang¹; ¹GlycoNovo Technologies Co., Ltd., Shanghai, 201318, China, ²National Research Center for Carbohydrate Synthesis, Jiangxi Normal University, Nanchang 330022, China

Grifola frondosa is an edible and medicinal mushroom with great nutritional value and bioactivity. In the present study, A soluble homogeneous βß-glucan, GFPS, with a molecular mass of 5.42x106 Da was purified from the fruit bodies of Grifola frondosa using 5% cold NaOH. The structure of GFPS was determined with infrared spectroscopy, NMR, and monosaccharide composition analysis, and was identified to be a βß-D-(1-3)-linked glucan backbone with a singleβß-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. Our results indicated that GFPS had a triple helical structure and could form a complex with polydeoxyadenylic acid [poly(A)]. Further studies demonstrated that GFPS could interact with poly(dA) moiety of the designed TNF-<αantisense oligonucleotide (ASO). The complex could incorporate the ASO into the macrophage cells via dectin-1 receptor and attenuate lipopolysaccharide (LPS)-induced secretion of TNF-<.αOur results suggested that GFPS could be employed to deliver therapeutic oligonucleotides for the treatment of inflammatory diseases and cancers such as lung cancer.

229. Towards the generation of comprehensive human kidney glycome array

Y. Jin¹, C. Farrar³, C. Nauser³, M. Howard³, R. Wallis⁴, P. Garred², W. Chai¹, S. Sacks³, T. Feizi¹, Y. Liu¹; ¹Glycosciences Laboratory, Faculty of Medicine, Imperial College London, UK, ²Laboratory of Molecular Medicine, University of Copenhagen, UK, ³MRC Centre for Transplantation, King’s College London, UK, ⁴School of Biological Sciences, University of Leicester, UK

Glycosylation is a post-translational modification crucial to molecular mechanisms in health and disease, involved in recognition by antibodies, endogens and exogenous lectins and adhesion proteins of microbiota. Exploring the total glycome of cells and tissues to identify the ligands involved in specific recognition events is often laborious and time consuming, due to the heterogeneity of the glycan populations. The recently developed Beam Search array strategy is to handle complex glycan using a workflow for iterative arraying, probing and further resolving the ligand positive fractions. In conjunction with MS, NMR and immuno-sequencing methods, this approach has shown considerable potential in natural ligand discovery and led to elucidation of novel glycan sequences in gastric mucin recognized by rotaviruses.

Here we present the first application of the Beam Search strategy to human tissue samples. A primary human kidney Beam Search array is being generated integrating glycan elements from human renal cortex tissue obtained from radical nephrectomy. This includes glycolipids, N- and O-glycans and glycosaminoglycans using sequential separation steps monitored by MS: organic solvent extractions for glycolipids, endoglycosidase-H followed by PNGase F for N-glycans and reductive elimination for O-glycans. The isolated N- and O-glycans are converted into neoglycolipids (NGLs) for arraying side-by-side with the kidney glycolipid fractions and various sequence-defined glycan standards. We will discuss validation of this primary array with a range of glycan-binding reagents (lectins and antibodies) with known specificities, and initial screening with glycan-binding proteins known to be associated with kidney pathology.

230. Effect of estrogen deficiency on human serum and IGG N-glycosylation

J. Jurić⁴, W. Kohrt¹, D. Kifer³, M. Pezer⁴, P. Nigrovic², G. Lauc⁴; ¹Division of Geriatric Medicine, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA, ²Division of Rheumatology, Immunology and Allergy, Brigham and Women´s Hospital, Boston, Massachusetts, USA, ³Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia, ⁴Genos Glycoscience Research Laboratory, Zagreb, Croatia

Most proteins in human serum are glycosylated by diverse glycan structures that fine-tune their function. Regulatory role of glycans has been most extensively explored in immunoglobulin G (IgG). Different glycoforms of this antibody initiate versatile immune responses. It is known that diseases and aging alter N-glycosylation of IgG, but recently, sex hormones also emerged as a significant regulatory factor. Major differences in IgG N-glycosylation were reported in females during pregnancy, late puberty, and with entering the menopause. However, current knowledge of the associations between female sex hormones and IgG N-glycosylation is mostly based on epidemiological studies. Here we present an experimental study aiming to investigate the effect of menopause-like estrogen levels on N-glycosylation of IgG and total human serum proteins. Participants were healthy premenopausal women with normal menstrual cycle function, deprived of estrogen with monthly injections of gonadotropin-releasing hormone agonist (GnRH_AG). During GnRH_AG therapy, one group was given estrogen supplementation in the form of estradiol (E2) patches and the other group received a placebo. The study terminated with the spontaneous recovery of normal menstrual cycle function. Each participant was tested 3 times during the study. Each time, 5 ml of peripheral blood was drawn and serum was obtained. Additionally, IgG was isolated from sera by affinity chromatography on a protein G monolithic plate. Serum and IgG N-glycans were released by PNGase F, labeled with 2-AB and analyzed by hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC). Our study indicates that circulating estrogen levels associated with serum and IgG N-glycosylation. Changes of N-glycosylation detected in serum most likely originate from IgG. Our results show that estrogen-deficient hormonal milieu causes higher bisection, and lower galactosylation and sialylation level that is usually involved in the proinflammatory background.

231. Breakthrough of O-glycan liberation and its application to analysis of mucins separated by supported molecular matrix electrophoresis

A. Kameyama¹, W.W. Thet Tin¹, M. Toyoda², M. Sakaguchi²; ¹Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), JAPAN, ²Sumitomo Bakelite Co., Ltd., Japan

Rapid and easy preparation of glycans from glycoproteins is a crucial step to glycomics. Preparation of N-glycans has been well-established, while O-glycan preparations have yet to satisfy the recent needs for glycan analysis in glycobiology research and biopharmaceutical industry. Here, we introduce a novel method of O-glycan liberation that can be executed in aqueous solution and completed within 20 min. O-Glycans could be released from glycoproteins by using 50% hydroxylamine and an organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The reaction condition was optimized by using bovine fetuin as a model glycoprotein. The optimized method afforded comparable recovery rates to those of gas-phase hydrazinolysis, but with less degraded products. Unlike hydrazinolysis, the glycans containing NeuGc could also be released without any degradation. Fluorescent tag such as anthranilic acid (2-AA) and 2-aminobenzamide (2-AB) was successfully introduced to label the released glycans by a reductive amination. To demonstrate the feasibility of the method, we analyzed O-glycans of mucins separated by supported molecular matrix electrophoresis (SMME) which is previously developed to characterize mucins. Mucins are hardly characterized by proteomics because of highly glycosylation and their inability to enter a polyacrylamide gel due to their large molecular size. SMME is a membrane electrophoresis using hydrophilic polymer soaking into porous membrane of polyvinylidene difluoride as the separation medium. Mucins can be separated as narrow bands by SMME. The bands are excised and subjected to the O-glycan liberation. We analyzed commercially available bovine submaxillary mucin and mucins extracted from porcine submaxillary gland using a combination of SMME and the developed O-glycan liberation as described above. 2-AA Labeled O-glycans obtained from each band generated upon SMME were successfully analyzed by LC-MS. Thus, the O-glycan liberation developed in this study demonstrated its rapidness and easiness, besides overcoming defects of gas-phase hydrazinolysis without sacrificing recovery rates. We believe that the method would be quite useful as a general technique of O-glycan liberation for glycomics, glycobiology research, and biopharmaceutical industry.

232. Increase in sialidase activity during cerebral infarction and its role on glutamate release in rat hippocampus

T. Kano¹, A. Minami¹, T. Otsubo², Y. Kurebayashi¹, T. Takahashi¹, K. Ikeda², T. Suzuki¹; ¹Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, ²Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University

Sialidase removes sialic acid residues in sialoglycoconjugates. We previously found that sialidase is essential for hippocampal memory and synaptic plasticity. Enzyme activity of sialidase is rapidly increased in response to neural excitation. Because sialic acid bound to gangliosides such as the tetra-sialoganglioside GQ1b is crucial for calcium signaling and neurotransmitter release, neural activity-dependent removal of sialic acid may affect hippocampal neurotransmission. In the present study, we investigated the desialylation by neural excitation during cerebral infarction and it’s role on glutamate release.

For in vivo monitoring of desialylation on the cell surface, the amount of free sialic acid collected from the hippocampal extracellular space was measured using in vivo microdialysis. The hippocampus was perfused with artificial cerebrospinal fluid (ACSF) and then injected with exogenous sialidase from Arthrobacter ureafaciens (AUSA). Neu5Ac, the main molecular species of sialic acid, in the perfusate was increased by AUSA injection. In contrast, Neu5Ac in the perfusate was decreased by novel sialidase inhibitor 2-deoxy-2,3-didehydro-D-N-propionylneuraminic acid. We next evaluated desialylation during cerebral infarction. The Neu5Ac level as well as glutamate level in the hippocampal extracellular space was significantly increased by was photochemically induced cortical infarction with the photosensitizing dye, rose bengal. In this study, we also investigated the role of endogenous sialidase in glutamate release in the rat hippocampus. In the hippocampal CA3 region, the extracellular glutamate level measured by in vivo microdialysis was significantly increased by perfusion with sialidase inhibitors, 2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid and 2-deoxy-2,3-didehydro-D-N-glycolylneuraminic acid. These results indicate that sialidase activity is increased by neural excitation during cerebral infarction, and that sialidase down-regulates glutamate release from hippocampal neurons. Neural activity dependent desialylation by sialidase may be a negativefeedback factor against presynaptic activity.

233. Determination of serum proteins glycosylation in cancer before and after the treatment: lectin-based protein microarray and maldi-tof ms approaches

J. Katrlík¹, L. Pažitná¹, M. Križáková¹, Z. Pakanová¹, P. Baráth¹, S. Kim², B. Bystrický³; ¹Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia, ²Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup, Republic of Korea, ³Oncology Department, Faculty Hospital in Trenčín, Slovakia

Glycosylation status of glycoconjugates is one of the cancer-related markers which can significantly increase the informative value of protein biomarkers. We determined the differences in glycan composition in serum samples obtained from patients with various types of cancer (colorectal, breast, ovarian and others) before chemotherapy and 3 months after the end of the treatment. We used two glycoprofiling techniques: lectin-based protein microarray platform and MALDI-TOF MS. First of the platforms enables high-throughput glycoprofiling of biomarkers and recognition of glycan – lectin interactions. The serum samples were first depleted to remove abundant proteins (albumin and IgG) and then the samples were spotted into arrays on microarray slide and incubated with a panel of 16 chosen biotinylated lectins. The detection was performed after incubation with fluorescent conjugate of streptavidin using microarray scanner. MALDI-TOF MS was used for the direct glycoprofiling of serum apolipoprotein C-III without protein isolation or glycan release. The same approaches were used for the measurement of samples from control group of healthy individuals taken three months apart. Determined differences in signal intensities measured by lectin-based protein microarray showed statistically significant changes in glycosylation for the patients’ samples. Although analytical assay based on lectin-glycan interactions does not allow identification of glycan structures, this platform is suitable for rapid screening and detecting glycosylation changes or abnormalities for a large number of samples and is complementary to MS methods enabling identification of glycan structures. It makes it very useful in biomarker research and diagnostics. We use this method also in another applications as e.g. glycoprofiling Congenital Disorders of Glycosylation (CDG) patients’ samples, monitoring glycosylation changes related to the aging or determination of glycostructure of therapeutic proteins. Acknowledgement: this work was supported by the projects SK-KR-18-0004, SK-SRB-18-0028, APVV-17-0300, VEGA 2/0137/18, COST CA16113, COST CA18132, Centre for materials, layers and systems for applications and chemical processes under extreme conditions - Stage II (ITMS No.: 26240120021, R&D OP funded by ERDF).

234. Profiling of n-linked glycans from ultra-small samples by capillary electrophoresis with a large-volume dual preconcentration technique

T. Kawai¹; ¹RIKEN Center for Biosystems Dynamics Research

Structure of N-linked glycans is changed in response with pathogenesis like cancer. Glycomic profiling from limited number of cells in an early-stage tissue and/or progressed tumor micro-environment is essential for discovering effective drugs. For analyzing tiny biological samples, we developed sensitive, high-resolution method for analyzing trace level of N-linked glycans by coupling capillary electrophoresis (CE), laser-induced fluorescence (LIF) detection, and an online sample preconcentration (OSP) method named “large-volume dual preconcentration by isotachophoresis and stacking (LDIS)”, which is composed of two OSP methods, large-volume sample stacking and transient isotachophoresis [1]. A typical LDIS-CE-LIF protocol was simple: a short-plug of leading electrolyte (LE) and large-volume sample solution were introduced to a capillary, followed by application of constant voltage. In the analysis of glucose ladder labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) with 10 mM sodium chloride as LE, up to 2300-fold sensitivity increase was achieved with higher resolution than those in normal CE. N-glycans from approximately 100 cells (HeLa, MCF7, and HepG2) were then analyzed as the model of localized tumor cells. N-glycans were released from cellular lysate with peptide-N-glycosidase F, labeled with APTS dye, purified with magnetic beads, and analyzed by LDIS-CE-LIF. Up to 40 glycans were successfully detected with almost same profile of large-scale sample. N-glycan structure could be predicted by searching glucose-unit value via Glycostore database. It suggests the potential of LDIS-CE-LIF for discovery of disease-specific N-linked glycans in microscale environment.

[1] T. Kawai et al. J. Chromatogr. A, 2018, 1565, 138.

235. Characterization of glycan structures of human induced pluripotent stem (hips) cells by marker antibodies

T. Kawasaki¹, H. Nakao², T. Yamaguchi⁵, K. Kawabata⁵, Y. Ohta³, N. Kawasaki³, H. Toyoda⁴, N. Kawasaki¹; ¹Glycobiotech. Lab., Ritsumeikan Univ., Shiga, Japan, ²Glycobiotech. Lab., Ritsumeikan Univ., Shiga, Japan, Lab. Stem Cell Reg., NIBIOHN, Osaka, Japan, ³Grad. Sch, Med. LifeSci., Yokohama City Univ., Kanagawa, Japan, ⁴Lab. Bio-anal. Chem., Col. Pharm. Sci., Ritsumeikan Univ., Shiga, Japan, ⁵Lab. Stem Cell Reg., NIBIOHN, Osaka, Japan

Glycomics on human embryonic stem (ES) cells and induced pleuripotent stem (iPS) cells indicated that structures of glycans of all classes, N-glycans, O-glycans, Glycosaminoglycans (GAGs) and glyco-sphingolipid (GSL) glycans tended to be immature and rich in polylactosamine motif.

To investigate stem cell specific glycoforms in more detail, we have developed antibodies against hiPS cells, R-10G, R-17F, R-6C, R-13E for recent several years. The epitopes of these antibodies appear to be present mostly on O-glycans except for R-17F, which is expressed predominantly on GSL as well as on O-glycans and N-glycans. The minimum epitope of R-10G is Gal®1-4GlcNAc(6S)®1-3Gal®1-4GlcNAc(6S)®1, (low sulfate keratan sulfate), that of R-17F is Fuc<1-2Gal®1-3GlcNAc ®1-3Gal®1-4Glc, (lacto-N-fucopentaose I (LNFP I)) and that of R-6C is Sia<2-3Gal®1-3GlcNAc(6S)®1-3Gal®1-4GlcNAc(6S)®1. Binding specificity of tumor rejection antigen (TRA) -1-60, which was generated by Andrews et al., in 1984, was re-investigated on ELISA using newly synthesized oligosaccharides, because currently two different TRA-1-60 antibodies are commercially available. One TRA-1-60 reagent recognized Gal®1-3GlcNAc®1-3Gal®1-4GlcNAc®1 as reported by Natunen et al., in 2011, but the other TRA-1-60 reagent recognized not only Gal®1-3GlcNAc®1-3Gal®1-4GlcNAc®1 but also Sia<2-3Gal®1-3 GlcNAc®1-3Gal®1-4GlcNAc ®1 and Gal®1-3GlcNAc(6S)®1-3Gal®1-4GlcNAc(6S)®1, the latter can be an immediate biosynthetic-precursor of the R-6C epitope. These results together with identification of sialyl-lactotetra GSL as a new hES marker (by Barone et al., in 2014) suggested that polylactosamine motif such as Type 1-Type 2 and Type 2-Type 2, with or without sulfate on GlcNAc, with or without sialic acid or fucose on Gal and with different numbers of polylactosamine unit may bring about a unique diversity of glycoforms on hiPS/hES cells. Interestingly, one of the hiPS/hES membrane proteins, podocalyxin, is identified to be a common core protein for these polylactosamine-based epitopes. In addition, R-17F antibody (IgG1) was revealed to have a strong direct cytotoxic activity to hiPS/ES cells, which may be beneficial in regenerative medicine to remove undifferentiated hiPS/ES cells from preparations of differentiated cells.

236. Anion exchanger 2 (slc4a2a)-mediated bicarbonate import facilitates proton leakage across golgi membranes

E. Khosrowabadi¹, A. Rivinoja², S. Kellokumpu¹; ¹Glycan Biosynthesis Research Unit,Faculty of Biochemistry and Molecular Medicine,University of Oulu,Finland; ²Northern Finland Laboratory Centre, Oulu, Finland

Most secretory and endocytic compartments have each their own uniquely acidic pH environment that is essential for their proper functioning. In each case, the resting pH of an acidic organelle is thought to reflect changes either in the rate proton pumping by the vacuolar H+-ATPase (V-ATPase) or its leakage through organellar membranes via a “H+ channel” whose identity still remains obscure. Here we show instead, that proton leakage across Golgi membranes does not involve a H+ channel, but rather, relies on AE2a (SLC4A2a)-mediated bicarbonate import. Specifically, we show that proton leak is dependent on bicarbonate-chloride exchange, and markedly increased by overexpression of AE2a anion exchanger or decreased by shRNA-mediated knockdown of the AE2a. These findings suggest that proton leakage from the Golgi lumen is facilitated by a common chemical buffering reaction in which imported HCO3- and luminal protons combine to form CO2 and water, i.e. products that can readily cross Golgi membranes either via diffusion or via aquaporin channels. The findings also provide a rational explanation for the flattened morphology of the Golgi cisternae, as their shape is optimal for expelling CO2 and H2O from the Golgi lumen.

237. Macroevolutionary reconstruction of glycosylation from the perspective of homo sapiens genome

D. Kifer³, M. Ledinski¹, M. Domazet-Lošo², G. Lauc⁴, G. Klobučar¹, T. Domazet-Lošo⁵; ¹Department of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia, ²Faculty of Electrical Engineering and Computing, University of Zagreb, Zagreb, Croatia, ³Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia, ⁴Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia; Genos Glycoscience Research Laboratory, Zagreb, Croatia, ⁵Ruđer Bošković Institute, Zagreb, Croatia; Catholic University of Croatia, Zagreb, Croatia

Glycosylation is present in all living organisms, ranging from bacteria and archaea all the way to humans. Glycan structures attached to lipids and proteins are not coded into genome but are precisely added by enzymes involved in glycosylation process. Observed increase in glycan structure complexity through the evolution suggests that the glycosylation processes are under continuous evolutionary change. To reconstruct the evolutionary origin of the genes involved in glycosylation and their target proteins in H. sapiens we applied phylostratigraphic approach. Results showed that homologs of most human genes involved in glycosylation could be traced back to the last universal common ancestor (LUCA) suggesting that the protein glycosylation is an essential to all cellular life. By focusing on the evolutionary origin of genes involved in N-glycosylation pathway, we observed significant enrichment in the last eukaryotic common ancestor (LECA), probably linked to the emergence of endomembrane system which plays important role in eukaryotic glycosylation. Similarly, in the last common ancestor of all eumetazoans we found enrichment of genes related to glycosaminoglycans (GAGs) suggesting importance of GAGs for development of true tissues in the eumetazoan clade. Comparable to genes involved in glycosylation we found that significant number of glycoproteins, genes which are glycosylated, could be traced back to LUCA. The phylostrata (phylogenetic internodes) that mark origins of metazoans, eumetazoans, deuterostomes and vertebrates are also periods loaded with glycoproteins. Functional enrichment analyses on the organ level revealed that high proportion of genes related to immune, nervous and circulatory system processes were coding for glycoproteins. On the cellular level, cell-cell signaling, cell differentiation and cell maturation are functions enriched in glycoproteins. These results suggest the ancient origin of the glycosylation machinery, but also point to its fine tuning in metazoans and vertebrates.

238. Possible physiological function of neu-medullocytes that are sialidase and cd5 positive b-1 cells, expressing autoimmune regulator (aire) in the mouse thymus

S. Kijimoto-Ochiai¹; ¹Life Space COSMOS

Neu-medullocytes, which are sialidase positive and express immunoglobulin and Mac-1 (CD11b/CD18) in the mouse thymus, are found histochemically using 5-bromo-4-chloro-3-indolyl-<-D-N-acetylneuraminic acid salt (X-NANA) in 2004 and named in 2008 (S. Kijimoto-Ochiai et.al.). Very recently (2019, Scientific reports 9:858), we showed that almost of Neu-medullocytes were CD5 positive B cells, i.e. B-1 cells, and a few of them expressed the transcription factor autoimmune regulator (AIRE). The function of B-1 cells is known to contribute to the antibody production against pathogenic bacterial glycan but not against peptides. Thus, I would like to propose the physiological function of Neu-medullocytes in the mouse thymus as antigen-presenting cells (APCs) for mainly pathogenic glycan during negative selection step. For the physiological function of AIRE-positive Neu-medullocytes, I would like to propose as APCs for self-glycan antigens such as blood types to eliminate the auto-reactive T cells against self-glycan. (In a human thymus, cells similar to Neu-medullocytes may act as APCs at the negative selection step to eliminate the auto-reactive T cells responding to self-ABO blood antigens or other self-glycan antigens to keep self-tolerance.)

On the other hand, we have shown that existence of NEU1 sialidase on mouse thymocytes whose natural substrate is CD5 (S. Kijimoto-Ochiai et al. 2018 Glycobiology 28, 306-317.). However, as I mentioned above, very recently we showed that Neu-medullocytes have CD5. It is also well know that single positive T (SPT) cells in the mouse thymus express CD5 (Azzam HS. et al. (1998) J Exp Med. 188, 2301-2311). Because the thymocyte-preparation used in the report (2018, Glycobiology) probably contained a part of B-1 cells, it is unclear that NEU1 exists on T cells or on B-1 cells and that which CD5, on SPT cell or on Neu-medullocyte, is used as a substrate for NEU1. Although these should be clarified, at present possible hypothetical, physiological functions of Neu-medullocytes and the fate of T cells will be shown with the addition and removal of sialic acid during the development of T cells in the mouse thymus.

239. Stress-activated mir-204 governs senescent phenotypes of chondrocytes to promote osteoarthritis development

J. Kim¹; ¹Department of Biological Sciences, College of Natural Sciences, Seoul National University

A progressive loss of cartilage matrix leads to the development of osteoarthritis (OA). Matrix homeostasis is disturbed in OA cartilage as the result of reduced production of cartilage-specific matrix and increased secretion of catabolic mediators by chondrocytes. Chondrocyte senescence is a crucial cellular event contributing to such imbalance in matrix metabolism during OA development. Here, we identify miR-204 as a senescence-associated microRNA (miRNA) which is markedly upregulated in OA cartilage. The upregulated miR-204 simultaneously targets multiple components of the sulfated proteoglycan (PG) biosynthesis pathway, effectively shutting down PG anabolism. Ectopic expression of the miR-204 in joints triggers spontaneous cartilage loss and OA development, whereas inhibition of miR-204 ameliorates experimental OA, with concomitant recovery of PG synthesis and suppression of inflammatory senescence-associated secretory phenotype (SASP) factors in cartilage. Collectively, we unravel a stress-activated senescence pathway that underlies disrupted matrix homeostasis in OA cartilage.

240. Hericium erinaceus and sterem hirsutum ricin b-like lectin orthologs recognize fucosylated n-glycans in a human colorectal carcinoma cell line, SW1116

S. Kim¹; ¹Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology, 181 Ipsin-gil, Jeongeup 56212, Korea

The fruiting body of the edible mushroom Hericium erinaceus has isolectins with the ricin B-like lectin, HEL1, and the core 1 O-glycan-binding lectin, HEL2. In previous works, the glycan binding specificity of the recombinant HEL2 was confirmed, but that of the recombinant HEL1 (rHEL1) could not be characterized. To evaluate further the glycan binding activities for rHEL1, a mushroom Stereum hirsutum lectin (SHL1), which is highly homologous protein to HEL1 containing a typical ricin-B like motif (QxW)₃, was selected and characterized together. Interestingly, these recombinant lectins showed different multiple complex forms under non-denaturation: rSHL1 was dimer form with agglutination activity, but rHEL1 was still a monomer with non-activity toward animal erythrocytes. Glycan array analysis revealed these recombinant ricin-B like lectins exhibited relatively different binding strength toward fucosylated N-glycans harboring Fuc <(1,2)Gal or Fuc <(1,4)GlcNAc. Isothermal calorimetry showed rSHL1 interacted strongly Fuc <(1,2)Gal rather than other fucosylated GlcNAc glycoconjugates comparing to that of rHEL1. These glycan binding activity as well as physicochemical property of rSHL1 were comparable to that of rHEL1in their detection ability for highly fucosylated N-glycans in the cell surface glycoproteins present on the SW1116 human colorectal carcinoma cell. These results are remarkable that the SHL1 might be a novel lectin to detect highly fucosylated glycoepitopes in carcinoma cell lines for the diagnosis application. [Acknowledgments: This work was supported by the bilateral collaboration program (NRF-2018K1A3A1A39071061) between Korea and Slovakia through the National Research Foundation of Korea (NRF) and partially by KRIBB Research Initiative Program grant.]

241. Structural features of high-mannose type free n-glycans (FNGS) in a. thaliana mutant lines lacking in erad related proteins

K. Furuta², M. Maeda², R. Strasser¹, Y. Kimura²; ¹Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Austria; ²Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Japan

In the ERAD system working in eucaryotic cells, misfolded glycoproteins are retro-transported from the endoplasmic reticulum (ER) to the cytosol and then degraded by the proteasome complex after deglycosylation by the cytosolic PNGase. In plant cells, it has been believed that the high-mannose type N-glycan, Man9GlcNAc2, on the misfolded glycoproteins are trimmed by <1,2-Man’ase (MNS4/5) to be Man7GlcNAc2 structure with an exposed <1,6-Man residues at the non-reducing terminal. This glycan on the misfolded glycoproteins is recognized by a lectin-like receptor (OS9), and then the misfolded glycoproteins are retro-transported to the cytosol thorough the retrotranslocon composed of several proteins such as SEL1L and ubiquitin ligases (HRD1A/B). Prior to the proteasomal degradation, the misfolded glycoproteins are deglycosylated by the cytosolic PNGase to release high mannose type free N-glycans (GN2-HMT-FNGs). The resulting FNGs are further processed by ENGase to be GN1-HMT-FNGs carrying one GlcNAc residue. As a part of study to elucidate the ERAD system working in plant cells, in this study, we analyzed the structural features of GN1-HMT-FNGs produced in four mutant-lines of A. thaliana (MNS4/5, OS9, SEL1, and HRD1A/B).

FNGs prepared from wild type and the four mutant-lines of A. thaliana were pyridylaminated, and the fluorescence-labeled HMT-FNGs were purified by Con A affinity chromatography, and then GN1-FNGs and GN2-FNGs were separated by RP-HPLC. The structural features of GN1-HMT-FNGs were analyzed by SF-HPLC, <-Man’ase digestion, partial acetolysis, and ESI-MS analysis.

The structural features and amount of GN1-HMT-FNGs in MNS4/5 mutant, a growth defect line, were almost the same as those in wild type plant, suggesting that misfolded glycoproteins were retro-transported from the ER to cytosol by a glycan-independent mechanism. The structural features and amount of GN1-HMT-FNGs in HRD1A/B mutant line were almost the same as those in wild type plant, suggesting that another ubiquitin ligase other than HRD1A/B should be involved in the ubiquitination of misfolded glycoproteins. On the other hands, the structural features and amount of GN1-HMT-FNGs the OS9 mutant and the SEL1 mutant were quite different from those in wild type plant. The amount of FNGs in both mutant lines increased more than two folds, and trimmed small size HTMs, Man5-4GlcNAc1, were accumulated. Furthermore, a glucosylated FNG, Glc1Man7GlcNAc, was specifically found in the SEL1 mutant line, suggesting that Glc’ase II may require an interaction with SEL1 for the full-activity.

242. Bisecting glcnac is a general suppressor of terminal modifications of N-glycans

M. Nakano³, N. Taniguchi², Y. Kizuka¹; ¹Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu, Japan, ²Department of Glyco-Oncology and Medical Biochemistry, Osaka International Cancer Institute, Osaka, Japan, ³Graduate School of Advanced Sciences of Matter, Hiroshima University, Hiroshima, Japan

Bisecting GlcNAc, a branched GlcNAc in N-glycan synthesized by GnT-III (encoded by MGAT3 gene), is most highly expressed in brain. Our previous studies using Mgat3-deficient mice showed that bisecting GlcNAc promotes Alzheimer’s pathology by positively regulating the functions of amyloid beta-producing enzyme BACE1. However, physiological functions of bisecting GlcNAc remain largely unclear.

Several in vitro biochemical studies already showed that introduction of bisecting GlcNAc suppresses further actions of other GlcNAc-branching enzymes, such as GnT-IV and -V, suggesting that bisecting GlcNAc regulates overall N-glycan profiles in vivo. To clarify this issue, we analyzed N-glycan structures of Mgat3-deficient brain and found that various terminal modifications were upregulated in Mgat3-knockout, such as Lewis-fucose, sialic acid and HNK-1 epitopes. The levels of glycosyltransferase mRNAs and their donor substrates were unchanged between WT and Mgat3-knockout. In contrast, our enzyme assays using bisected and non-bisected acceptor substrates revealed that most enzymes working on N-glycan terminals prefer the non-bisected glycan as a substrate, indicating that the upregulation of terminal N-glycan epitopes were attributed tosubstrate specificity of glycosyltransferases. Molecular dynamics simulation of glycosyltransferase-glycan complexes showed that the presence of bisecting GlcNAc changed overall N-glycan conformation from extended type to back-fold type where alpha1,6-arm loses interaction with the enzymes, suggesting that the bisected glycan is not preferred by various glycosyltransferases for N-glycan terminals due to alteration of N-glycan conformation. These results highlight the roles of bisecting GlcNAc as a general suppressor of terminal modifications of N-glycans, providing new insight into how protein N-glycosylation is regulated in cells.

243. The effect of salinomycin on N–glycome in a cancer stem cell model

F. Klicek¹, M. Novokmet¹, M. Marjanovic², M. Kralj², G. Lauc¹; ¹Genos Glycoscience Research Laboratory, 10000 Zagreb, Croatia, ²Rudjer Boskovic Institute, Division of Molecular Medicine Laboratory for Experimental Therapy, 10000 Zagreb, Croatia

N-glycosilation is a post-translational modification of proteins which has an important role in their function and structure. Almost all secreted and membrane proteins are glycosylated. The N-glycan precursors are synthesized in endoplasmatic reticulum and further modified to final form in the Golgi apparatus. Modifications of N-glycans in the Golgi apparatus are pH dependent and a disruption of pH homeostasis in Golgi is visible in change of N-glycan structures. We investigated the effect of salinomycin, a cancer stem cell (CSC) targeting compound, on N-glyco profile of a CSC model (HMLE-pBp and HMLE-Twist cell lines). To determine the N-glycome profile of secreted proteins glycans were released by PNGaseF digestion and fluorescently labeled with procainamide. We also determined the composition of released N-glycans in the profile with MS and MS/MS experiments. N-glycome profile of secreted proteins changed in both cell lines after the treatment with salinomycin. The most evident difference in profiles after treatment is the loss of highly branched complex glycan structures and the relative increase of simpler glycans, indicating that salinomycin disrupts the Golgi apparatus homeostasis while the endoplasmatic reticulum remains intact. Moreover, we noticed that the effect of is more prominent on HMLE-Twist than on HMLE-pBp cell line.

244. Preferential expression of sialyl 6’-sulfo n-acetyllactosamine-capped o-glycans on high endothelial venules in human peripheral lymph nodes

M. Kobayashi², M. Tsutsumiuchi², H. Hoshino², A. Kogami², T. Tsutsumiuchi², O. Yokoyama², T. Akama¹; ¹Kansai Medical University, ²University of Fukui

Lymphocyte “homing”, the physiologic trafficking of lymphocytes from the circulation to secondary lymphoid organs, is regulated by sequential adhesive interactions between lymphocytes and endothelial cells that constitute high endothelial venules (HEVs). Initial lymphocyte “rolling” is mediated by relatively weak, transient adhesive interactions between L-selectin expressed on lymphocytes and sulfated mucin-type O-glycans expressed on HEVs. Keratan sulfate galactose (Gal)-6-O-sulfotransferase (KSGal6ST) catalyzes 6-O-sulfation of Gal in keratan sulfate glycosaminoglycan chains but also transfers sulfate to Gal in much shorter glycan chains, such as sialylated N-acetyllactosamine (LacNAc)-capped O-glycans. In mice, KSGal6ST is reportedly expressed in HEVs and functions in synthesizing 6-sulfo Gal-containing O-glycans on HEVs. However, in humans, the presence of 6-sulfo Gal-containing O-glycans on HEVs is not reported. Employing the newly developed monoclonal antibody 297-11A, which recognizes non-sialylated terminal 6’-sulfo LacNAc, we demonstrate that sialyl 6’-sulfo (and/or 6,6’-disulfo) LacNAc-capped O-glycans are preferentially displayed on HEVs in human peripheral lymph nodes (PLNs) and to a lesser extent in mesenteric LNs (MLNs) but not in Peyer’s patches (PPs). We also found that the scaffold protein mucosal addressin cell adhesion molecule 1 (MAdCAM-1), which is expressed on HEVs in PPs and MLNs but not PLNs, was modified by 297-11A-positive sulfated glycans less efficiently than was CD34. Moreover, 297-11A-positive sulfated glycans were also displayed on HEV-like vessels induced in tumor-infiltrating lymphocyte (TIL) aggregates formed in various cancers. These findings collectively indicate that 297-11A-positive sulfated glycans potentially play a role in physiologic lymphocyte homing as well as in lymphocyte recruitment under pathologic conditions.

245. Advancements in glycoanalytical strategies for quantitative n- and o-glycan analysis from in vitro established cell-lines.

M. Kotsias², R.P. Kozak², R.A. Gardner², G.C.M. Vreeker¹, K. Madunic¹, S. Nicolardi¹, J. Hendel², M. Wuhrer¹, D.I.R. Spencer²; ¹Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands, ²Ludger Ltd, Culham Science Centre, Abingdon, Oxfordshire, UK

Protein glycosylation has shown to be critical for a wide range of biological processes, resulting in an increased interest in studying its alterations as disease biomarkers as well as for patient stratification and personalized medicine. However, glycosylation analysis of biological samples such as in vitro cell lines is often demanding due to the high complexity of the glycan structures found in these samples. Advancements in the current technologies is necessary to reduce the time required to prepare and measure the glycans in these samples as well as provide a system that is easy to operate, reliable, repeatable and accurate.

Notably, glycan changes can be complex and subtle, often requiring the analysis of a large number of samples in order to obtain conclusive results. Automated platforms can offer a solution to replace many of the time-consuming and labour-intensive steps, providing similar performance to expert manual labour and even outperforming manual sample preparation when large cohorts are measured.

With regard to rapid glycan characterization, MALDI-MS is commonly performed on a time-of-flight-mass analyser. However, in the case of N-glycans, peak overlapping has been observed for large tri- and tetra-antennary glycans, while high resolving power (RP) is needed in order to distinguish small O-glycan structures from matrix signals. In this context, higher RP and mass accuracy can be obtained by using Fourier Transform Ion Cyclotron Resonance Mass Spectrometers (FTICR-MS) for detailed glycosylation analysis of complex samples.

Various approaches are currently employed for the relative or absolute quantitation of glycans. Hence, we will introduce glycan analysis strategies that we routinely use for structural N- and O-glycan analysis, describing the information that each provides and presenting recent advances in terms of throughput (automation vs. manual), resolution (FTICR vs. TOF) and precision in an effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines.

246. Stepwise triple-click functionalization of synthetic peptides

A. Kovalová¹, R. Pohl¹, M. Vrábel¹; ¹Institute of Organic Chemistry and Biochemistry of the CAS, Prague

Interactions between saccharides and proteins take place in all forms of life and play a fundamental role in numerous biological processes. These include signaling, recognition events, growth, host-pathogen interactions, immune response, and others. Despite the crucial role of sugars in these biological events we still do not have adequate tools to decipher their complexity and to elucidate their biological role. The sugar-protein interactions are usually highly specific and generally very efficient. However, one individual sugar-protein interaction can be surprisingly weak. In most cases, multiple binding events contribute to the necessary affinity. This means that not only the type of saccharide units, but also their number, distance, and orientation in space are of importance for the overall interaction with the active protein or enzyme.

In order to simulate this multiplicity which occurs in Nature, we focus on the development of a dynamic, hetero-glycosylated system that we use to study binding preferences of biologically relevant proteins. Our approach is based on the construction of glycopeptides containing up to three different saccharide units. We prepare those hetero-glycopeptides by combining two robust methods – solid phase peptide synthesis (SPPS) and copper-catalyzed alkyne-azide cycloadditions (CuAAC). In the first instance, unnatural amino acids containing orthogonally protected terminal alkyne groups are incorporated into the peptide sequence. Subsequent selective deprotection and sequential CuAAC reactions using various azide-containing sugar building blocks enable decoration of the peptide sequence by up to three different sugar units. This LEGO^®-like approach allows us to synthesize single modified sequences, but also to prepare whole glycopeptide libraries, which can be subsequently used for studies of the sugar-protein interactions.

To demonstrate the utility of the same approach in a different context, we show a simple assay where we used our methodology to prepare a fluorogenic substrate for model protease. The developed methodology thus enables the construction of multi-functionalized peptides bearing various useful moieties and represents a straightforward approach for post-synthetic modification of synthetic peptides and peptide libraries.

247. Synthesis of glycoclusters containing thiodisaccharides related to heparan sulfate

D. Koffi Teki¹, V. Moreau¹, V. Chagnault¹, J. Kovensky¹; ¹Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources LG2A CNRS UMR 7378, Université de Picardie Jules Verne, Amiens, France

Glycosaminoglycans (GAGs) are involved in the regulation of a large number of biological processes such as inflammation, cell signalling, angiogenesis, viral infection and coagulation. Heparin and heparan sulfate (HS) share the same basic structure, IdoA/GlcA-(1-4)-GlcNAc/GlcNS. Sulfate groups can be located at O-2 of uronic acid residues, at O-6 and/or O-3 of glucosamine units. Heparin is more sulfated than HS and its main uronic acid is IdoA, whereas it is GlcA for HS. Unlike molecules isolated from tissues, pure molecules, derived from organic synthesis, can prevent side effects and are very useful tools for understanding the structure-activity relationships of many biological and pharmacological activities. In our research group, we focus particularly on the synthesis of multivalent thioglycoside analogs. We report herein the synthesis of new glycoclusters with thiodisaccharide units, S-analogs of heparan sulfate.

First, the synthesis of a new thiodisaccharide analog of the HS sequence was performed. Two different strategies were assayed: glycosylation of an activated donor with a thiol functionalized acceptor, and bimolecular substitution of an electrophile equipped with a triflate leaving group with an anomeric thiol. The thiodisaccharide analog was obtained by nucleophilic displacement of a 4-triflate galactoside derivative, by an anomeric thiol of a glucuronic acid precursor. After modifying the aglycone part to introduce an azide, the thiodisaccharide was coupled to maltotriose scaffolds carrying one, two or three propargyl groups by CuAAC, affording one monovalent, two different divalent and one trivalent derivatives. These new compounds are powerful tools for the study of HS-protein interactions.

248. Variability and longitudinal stability of glycosylation profile of igg produced by different hybridoma clones

J. Krištić², O.O. Zaytseva², T. Lenac-Roviš¹, S. Jonjić¹, G. Lauc²; ¹Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, ²Genos Glycoscience Research Laboratory, Zagreb

Glycans attached to immunoglobulin G (IgG) have significant impact on structural and functional properties of IgG antibody. Glycosylation of IgG appears to be determined in an antigen-specific manner which probably enables IgG to exhibit appropriate mode of action. Here we analysed glycosylation of monoclonal IgGs produced by 27 different hybridoma clones and determined stability of IgG glycosylation profile within the two week culture period and also in different lots of individual monoclonal IgGs (i.e. IgGs produced in independent cultures of the same hybridoma clone). Hybridoma clones were grown in serum free medium and produced IgG were purified from cell culture supernatants using Protein G columns/disks. IgG Fc glycosylation was analysed by ESI-LC-MS. We observed some clear differences between glycosylation profiles of IgGs produced by different clones but the level of dissimilarity/similarity varied across different clones. We also observed that glycosylation profile of IgGs produced in the culture of individual hybridoma clones remained relatively stable across three time points spaced throughout the two week culture period. Furthermore, although comparison of different lots of IgGs produced in separate cultures of the same hybridoma clone showed some notable differences in IgG glycosylation profile, some characteristic features of profile were retained. Mechanisms that dictate the glycosylation of IgG produced by individual clones and enable persistence of glycosylation pattern remain to be identified.

249. Developing non-carbohydrate glycomimetics targeted to bacterial lectins

S. Kuhaudomlarp¹, P. Da Silva⁵, E. Siebs², E. Shanina⁴, E. Gillon¹, A. Titz³, C. Rademacher⁴, D. Rognan⁵, A. Imberty¹; ¹CERMAV, 601 Rue de la Chimie, 38041, Grenoble cedex 9, France, ²Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), 66123, Saarbrücken, Germany, ³Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), 66123, Saarbrücken, Germany. Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover-Braunschweig, Germany, ⁴Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14424 Potsdam, Germany; Institute of Chemistry and Biochemistry, Department of Biology, Chemistry, and Pharmacy, Freie Universität Berlin, Germany, ⁵Laboratoire d’Innovation Thérapeutique, UMR 7200 CNRSUniversité de Strasbourg, 74 route du Rhin, 67400 Illkirch, France

Pathogenic bacteria utilise glycan epitopes on host tissue for specific recognition and host infection, which are mediated by the interaction between host glycan epitopes and the pathogenic carbohydrate binding protein called lectins. Several lectin inhibitors have previously been designed to interfere with the glycan-lectin interaction, but these inhibitors often exhibit poor pharmacokinetic and pharmacodynamics parameters. Moreover, the structural diversity of current lectin inhibitors is restricted to the derivatives of the native glycan, leaving a vast underexplored chemical space of compound myriads. To address these challenges, we aim to develop non-carbohydrate drug-like lectin inhibitors, which could overcome limitations of the current glycan-based therapeutics inhibitors. We utilised multidisciplinary approaches including virtual and in vitro functional screening of chemical libraries. Lead compounds from the initial screens were further validated by biophysical assay (SPR), and X-ray crystallographic study of the lead compound in complex with lectin target. To demonstrate the validity of our approaches, we utilised our workflow to identify non-glycan inhibitor candidates for a galactose-binding lectin from Pseudomonas aeruginosa strain PAO1 (LecA) through virtual and functional screening by NMR, followed by SPR analysis. These combinatorial approaches identified promising candidates that could be followed up by X-ray crystallography. The outcome of our work provides a robust workflow for identification of novel lectin inhibitors, that could potentially be applied to other lectin targets. Most importantly, we have identified novel, non-carbohydrate chemical leads that could be further developed into new therapeutics for treatment of bacterial infection.

250. Strategic applications of complementary data dependent multistage fragmentation in negative ion mode to expedite mass spectrometry-based sequencing of glycopeptides

C. Kuo¹, K. Khoo¹; ¹Institute of Biological Chemistry, Academia Sinica, Taiwan

While glycomics aims at mapping the entire range of glycan entities expressed at any one patho-physiological stage of a particular cell type, tissue or organism, glycoproteomics ventures a step further in locating their protein site-specific distribution. Although recent advances in mass spectrometry (MS) have enabled meaningful glycoproteomic undertakings, many technical limitations remain unsolved. Among these, ability to sequence the peptide backbone for de novo identification, delineating multiple N- and O-glycosylation sites on single glycopeptides and deriving more glycan structure information to discriminate isomeric glycoforms, are well acknowledged practical problems to be urgently tackled. We have been exploring all different kinds of experimental workflow for a most productive LC-MS/MS-based glycopeptide analysis, focusing particularly on the multiple O-glycosylated peptides and those carrying sulfated glycans. We have examined the complementarity of various fragmentation modes and most recently the judicious applications of negative ion mode sequencing to supplement a robust positive mode LC-MS²/MS³ workflow. We found that sulfation and sialylation drastically alter the fragmentation pattern in negative ion mode and the characteristic features identified can be utilized to program the most informative MS³. Moreover, facile elimination of the O-glycan moieties under MS² affords an easy way to discover additional O-glycosylation on a glycopeptide that would otherwise not obvious. By high sensitivity detection of the characteristic glycan fragmentation ions produced by N- or O-glycans in combination with readily distinguishable elimination of the entire O-glycans in negative ion mode, we demonstrated in this work that unambiguous MS/MS sequencing of intact glycopeptides could be extended to those bearing both N- and O-glycans, or multiple O-glycans. Ability to advance from definitive identification of single to multiple site-specific glycosylation pattern on the same peptide backbones is anticipated to have a significant impact on the level of structural and biological insights one can gain in glycoproteomics applications.

251. A synthetic glycosylated small molecule as a novel autophagy inducer that inhibits the nlrp3 inflammasome

K. Hua¹, L. Lo², W. Wong¹; ¹Department of Biotechnology and Animal Science, National Ilan University, Ilan, Taiwan, ²Department of Chemistry, National Taiwan University, Taipei, Taiwan

The NLRP3 inflammasome promotes the pathogenesis of metabolic, neurodegenerative and infectious diseases, making it is a promising therapeutic target in these human diseases. A synthetic glycosylated small molecule (G-01) was evaluated for its inhibitory potential towards the NLRP3 inflammasome and the possible underlying molecular mechanisms were studied. We found that G-01 attenuated NLRP3 inflammasome activation in mouse J774A.1 macrophages and bone-marrow derived macrophages. Mechanistic analysis revealed that G-01 induced sirt1-dependent autophagy induction, which prevented the mitochondrial damage and reduced ASC oligomerization and ASC/NLRP3 complex formation. In addition, oral administration of G-01 attenuated inflammatory responses by suppressing NLRP3 inflammasome pathways in DSS-induced acute colitis and uric acid crystal-induced peritonitis in mice. Our results indicated that G-01 is a novel autophagy inducer that inhibits the NLRP3 inflammasome and can be used for ameliorating NLRP3-associated complications in the future.

252. In-silico simulation of o-linked glycan biosynthesis in cho cells

T. Kuoka¹, S. Akase¹, C. Jin², N.G. Karlsson², K.F. Aoki-Kinoshita³; ¹Department of Bioinformatics, Graduate School of Engineering, Soka University, Tokyo, ²Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, Gothenburg, ³Glycan & Life System Integration Center (GaLSIC), Faculty of Science and Engineering, Soka University, Tokyo

We have been developing a Web tool to perform systems biology analysis of glycan biosynthesis. This tool, called GlycoSim, allows users to select glycosyltransferases and an initial glycan substrate, it then computes all possible glycan structures that could be synthesized. After the input of enzyme kinetic parameters, GlycoSim simulates the biosynthesis of all the glycans. We have been aiming to provide a user-friendly interface so that non-experts can perform these simulations. After the successful simulation of N-linked glycans in Drosophila embryo using GlycoSim, we utilized the same tool to simulate the biosynthesis pathways of the O-linked glycans in Chinese Hamster Ovary (CHO) cell lines.

In our model, we collected the enzymes involved in the biosynthesis of O-glycans in CHO from over 25 glycogenes stored in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Each enzyme’s substrate specificity, product, and reaction constraints were collected from literature, and using GlycoSim, we predicted all possible glycans and the reaction network from the initial glycan to final products. We then used COPASI to simulate the dynamics of the synthesizing process by combining the previously predicted reaction network with the kinetics of each reaction.

To validate our model, we used mass spectrometry data of O-glycans found on P-selectin glycoprotein ligand-1 (PSGL-1) in CHO cells while transiently expressing different glycosyltransferase(s). The wild-type profile consisted of three glycans and it required five enzymes to synthesize. Using COPASI’s tools, the parameter values were estimated for the wild type and used to estimate the parameters for the other cell lines. In this presentation, we will present our progress in our analyses.

253. Capsular polysaccharide antigens: unravelling the helical hypothesis.

M. Kuttel², N. Ravenscroft¹; ¹Department of Chemistry, University of Cape Town, ²Department of Computer Science, University of Cape Town

Extended helical conformations have been proposed for a wide range of bacterial capsular polysaccharide antigens, from homopolymers, such Group B meningococcal polysaccharide, to heteropolymers with five- or six-residue repeating units, such as Group B Streptococcus. In some cases, helices are assumed to comprise a conformational epitope that is necessary for antibody binding. However, compelling experimental evident to support the helical hypothesis is often ambiguous or absent. Indeed, our recent molecular modelling studies indicate that helices are not as common as is often assumed in capsular polysaccharides: small changes in structure (such as O-acetylation) can disrupt the formation of a helix. Here we compare the primary structure, conformation and dynamic motion in key meningococcal, pneumococcal and streptococcal capsular polysaccharides to establish general heuristics for when extended helices do, and do not, form in carbohydrate antigens. Such rules of thumb can prove useful in vaccine development for estimates of cross-protection between carbohydrate antigens.

254. Atheromatous plaque injuries in coronary arteries elevate local serum sulfatide levels, which might contribute to the pathogenesis of atherothrombosis

A. Kudo², S. Nakahara², K. Sako¹, T. Inoue³, I. Taguchi²,M. Kyogashima⁴; ¹Clinical Pharmacological Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama, Japan, ²Department of Cardiology, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan, ³Department of Cardiovascular Medicine, Dokkyo Medical University, Mibu-machi, Tochigi, Japan, ⁴Division of Microbiology and Molecular Cell Biology, Nihon Pharmaceutical University, Ina-machi, Saitama, Japan

Sulfatides, are a representative group of sulfated glycosphingolipids. From early 1960s, there were many reports that sulfatides concern thrombosis and haemostasis, which often contradicts each other. We previously demonstrated that sulfatides play crucial roles in neointimal thickening after bare-metal stent implantation in patients with coronary atherosclerosis. Recently Li et al. reported high peripheral serum sulfatide levels in patients with ST-segment elevation myocardial infarction. These observations suggest that sulfatides in sera and atheromatous plaques might contribute to the pathogenesis of acute coronary syndrome. We determined whether plaque injuries influence local sulfatide levels by directly measuring them using microcather in 41 patients, who were treated with percutaneous coronary intervention (PCI). Blood was sampled at four locations in each patient. Points one and two, proximal and distal to a stenotic lesion, respectively, were sampled before PCI procedure. Points three and four, proximal and distal to the stenotic lesion, respectively, were sampled 15 minutes after the procedure. Patients were principally treated by either drug-eluting stent or drug-coated balloon. All patients were divided into two groups according to the prerequisite of preballoon dilation (pre-BD) before sampling blood at point two because of the inability to pass a microcatheter through the lesion. In those patients without pre-BD, mean serum sulfatide levels at point two (4.60 ± 4.04 nmol/ml), point three (4.35 ± 3.76 nmol/ml), and point four (4.53 ± 3.26 nmol/ml) were significantly higher than that at point one (2.49 ± 1.11 nmol/ml, vs point two, p < 0.02; vs point three, p < 0.03; vs point four, p < 0.03, respectively). Patients who underwent pre-BD required additional time (about 15–20 minutes) for sampling at points two to four compared with those who did not undergo pre-BD; there was no significant difference between the groups, probably because of dilution of sulfatides in the circulation. These results suggest that plaque injuries elevate local serum sulfatide level which could induce atherothrombosis in coronary arteries.

255. Investigation of bacterial cell surface glycoconjugates by solid-state nmr

C. Laguri¹, C. Bougault¹, P. Schanda¹, A. Molinaro², J. Simorre¹; ¹IBS Grenoble, ²University Napoli Federico II

Since their introduction in the middle of the last century, antibiotics have become essential for the treatment of bacterial infections and in virtually all modern medicine. However, the emergence and spread of multi-drug resistant pathogens in the last two decades has seriously reduced the drug arsenal to combat infectious diseases. The cell envelope is of critical importance in antimicrobial chemotherapy, since it possesses validated targets for clinically useful antibiotics, it plays a central role in acquired and intrinsic resistance to existing and new antibiotics via efflux and impermeability and it is more readily accessible to antimicrobial agents than cytoplasmic proteins. In this context we focus here on two glycoconjugate targets of importance. The LipoPolySaccharides (LPS) decorating the cell surface of Gram-negative bacteria serve as a physical barrier providing the bacteria protection from its surroundings. They are also recognized by the immune system as a marker for the detection of bacterial pathogen invasion, responsible for the development of inflammatory response, and in extreme cases for endotoxic shock. The PeptidoGlycan (PG) determines cell shape and provides resistance to the turgor pressure. These high molecular weight systems are highly heterogeneous and flexible and are difficult to characterize alone or in the presence of their assembly proteins to retrieve structural information at atomic scale. We present here the potency of NMR towards this goal.

256. Prospects of glycoconjugates recognizing system (C4В + C4A)

M. Lakhtin¹, V. Lakhtin¹; ¹Department of Medical Biotechnology, G.N. Gabrichevsky Research Institute for Epidemiology and Microbiology. Moscow.

The aim. On the basis of own data, to summarize potential of functional directed assembly analysis of serum complement C4 components - isotypes/ subisotypes of C4В and C4A which preferentially bind carbohydrate moiety of glycoconjugates (GC) or protein moiety, accordingly (in addition to usual immunodetection of isotypes as antigens) for establishment of healthy donor status and in prognostics of risks of diseases as well as in diagnostics of limited spectrum of diseases. Methods. Own developed procedures of functional ELISA in microplate (FELISAM) of the patient serum complement C4 isotypes C4В and C4A (C4BFELISAM, C4AFELISAM, C4AC4BFELISAM) and on the blot were used. Peroxidase chemiluminescent blot ELISA (PCBELISA) bioimagination variants in regime of a real time using BioChemi System, UVP were advanced. Proteins were registered using fluorescent SYPRO Protein Blot Stain. Results. 1. Variants of microplate ELISA of isotypes (in separate sensibilized preliminary prepared microplates for C4BFELISAM and C4AFELISAM or, for the first time, in a hybrid microplates for simultaneous detection of functional C4B and C4A using C4AC4BFELISAM) were developed. The proposed algorhythms of evaluation of deficits of functionally active complement components were effective in prognostics and diagnostics of such diseases as gastric ulcer, duodenal ulcer, systemic lupus erythematosus (SLE), phospholipid syndrome (PLS), SLP and PLS, rheumatoid arthritis. Interaction of isotypes to the microplate well bottom GC (aggregated human IgG/ IgG3 or Salmonella LPS) involved lectin—GC-type manner of recognition. Functional similarity of isotypes to WGA and PHA (phytolectins possessing complex systems and revealing activities against tumor cells) was observed. 2. For the first time variants of PCBELISA of isotypes and their subisotypes (up to 5-7 forms in isotype) in the new diagnostic-prognostic assembly region (within pI 3,5-5) created during isoelectric focusing of sera (at least 0,3 mkl was enough) in plate of polyacrylamide gel in gradient of pН 3-5 were developed. Visual qualitatively bioimagination and quantitatively controlled patient sera desialylation with Clostridium perfringens sialidase-proteinase cocktail in conditions of termotreatments increasing diagnostic potential of “universal blood” is described. Upon bioimagination registration of isotypes we used additional acetate anion binding site of the radish peroxidase (in addition to its catalytic site) to improve blot background and increase final resolution of procedure. FELISAM and PCBELISA established reliable ranging patient sera according to absolute contents of functionally complexed C4, C4В, C4A; revealed supported bioimagination of complete deficits of C4 (C4A+C4В), C4В or C4A; allowed reliable grouping sera characterized with small deficits of C4 when C4В> C4A or C4B< C4A. The latter grouping is of importance for establishment of disease risk tendencies. 3. Results of FELISAM and PCBELISA of complete or partial deficits of C4В or C4A, partial deficit of C4В/ C4A or C4A/ C4B ratios, complete absence of both isotypes supported each other. Conclusions. 1. Results indicate extended prospects of serum isotypes C4В, C4A and their subisotypes in prognostics and diagnostics of autoimmune, other system and infectious diseases. 2. Consideration of (sub)isotypes complexed to selected natural and artificial GC (of interest, initiators of further directed solid phased assembling, modulators of intermolecular and intercellular communications, as drugs) will open new way for construction of drugs and drug networks (their modeling, control, monitoring, choice and action) supported with PCBELISA in combination with FELISAM. 3. Prospects of further typing and standardization of both isotypes and subisotypes using water-soluble artificial polymeric multifunctional polyvalent mucin like conformationally adaptive GC (www.lectinity.com) in combination of antibodies (including sequential treatments of blots with antibodies and then GC) will open possibilities for further increasing sensitivity of prognostics and diagnostics of diseases.

257. Prospects of glycopatterns recognizing erythropoietins

V. Lakhtin¹, M. Lakhtin¹; ¹Department of Medical Biotechnology, G.N. Gabrichevsky Research Institute for Epidemiology and Microbiology. Moscow.

The aim was to summarize own data on lectin systems (LS recognizing and reversibly binding patterns of glycoconjugates [GC]) of human erythropoietins (EPO: erytrostim, epocrin, recormon, other recombinant variants; endogenic glycosylated highly acidic forms). Methods. Isoelectric focusing in polyacrylamide plate, electroblotting, display with biotin-streptavidin system using immune sandwich or/and polymeric multivalent watersoluble conformationally adaptive synthetic mucins-imitating GC (www.lectinity.com) were used. Fluorescence of proteins (labeled with SYPRO Protein Blot Stain) and peroxidase chemiluminescence of LS in a real time were registered in BioChemi System (UVP). Capability of EPO to recognize and bind targets is compared to hormone extended possible biological activities (literature). Results and discussion. 1. The network of EPOLS—GC functions in variants of such recognizing systems (major and minor ones) as “Lectin—Set of available/ exposed GC ranging on affinity” and “GC—Ranging set of components of EPOLS”. Expression of EPOLS—GC is changed depending on mosaic of solid phased protein forms distribution and types of GC. EPOLS are characterized with increased affinity to panels of selected GC (for example, LacNAc-GC, L-Fuc-GC)> Man-GC (combinations of GC can be considered as tuning/ superlinear modulators of solid phased [like cell surface] complexed mosaics coupled to EPO final variations of spectrum of activities). 2. Compared to epocrin system (glycosylated products expressed in CНO cells), erytrostim system (non-glycosylated products expressed in E. coli) was characterized with increased visualization by, affinity to and distribution of GC in target forms within appropriate sites of binding. 2-3 types of GC can be enough to discriminate different recombinant EPO preparations. 3. Upon assembly or aggregation, EPO complexes form megapattern sites of recognition of GC (intersubunit, intermolecular, cryptic) that result in increasing expression of GC binding (as in cases of Sia-GC or SO3-GC binding). 4. The synergism between antibodies and GC binding as well as between types of GC is registered. 5. Compared to original systems of EPO (some EPO forms can be originally complexed), EPOLS—GC are visualized as asymmetric mosaics accompanied with appearance of new signal forms (in endogenic and recombinant pI-regions). 6. EPOLS as cascade (EPO—GC followed by EPO—Immune sandwich—GC systems) was observed. Conclusions. 1. It was suggested that cascades of 2-3 EPOLS observed are depended on and modulated by selected panels of GC. Being forming on cell surfaces, such EPOLS sequentially and progressively influence other second events involving redistribution of EPO coupled tuning systems of cell receptors in direction of choice of EPO activity type to be realized. 2. Obtained results were compared to data of non-traditional/ non-ordinary EPO activities. The possible on duty participation of GC patterns in initiation and modulation of “exciting levels of readiness” of receptor mosaics coupled to further development of EPO final activities corresponds and does not contradict to the knowledge concerning mechanisms of innate immunity cells communications. 3. Conception of the role of recognizing glycopatterns cascade systems of therapeutic (glyco)proteins as the basis, accessory, preliminary, initiating events, for fine tuning of intercellular ligand-receptor communications is formulated on example of protein hormones. According to conception the glycosylation of protein may serve a key to extend EPO spectrum of communication activities and to choice the way in direction of EPO final activity expected and needed.

258. The role of siglec-7 in the interaction of colorectal cancer-associated bacteria f. nucleatum ssp. with the innate immune system

D. Lamprinaki⁷, C. Hellmich⁶, P. Garcia-Vello², K. M Bowles⁵, K. Drickamer³, C. De Castro¹, P. Crocker⁴, N. Juge⁷; ¹Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy, ²Department of Chemical Sciences, University of Naples Federico II, Portici, Italy, ³Department of Life Sciences, Imperial College London, UK, ⁴Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, UK, ⁵Norfolk and Norwich University Hospitals, NHS foundation trust and Norwich Medical School, University of East Anglia, Norwich, UK, ⁶Norfolk and Norwich University Hospitals, NHS foundation trust, Norwich, UK, ⁷Quadram Institute Bioscience, Norwich Research Park, Norwich, UK

The gut microbiota plays a major role in human health. An alteration of the gut microbiota composition or dysbiosis, leads to the prevalence of certain bacterial species which can contribute to disease development such as in colorectal cancer (CRC). Fusobacterium nucleatum associated with CRC tumors has been shown to contribute to myeloid-derived suppressor cell expansion. However, the molecular mechanisms underpinning F. nucleatum interactions with immune cells are not fully understood.

Here we test the hypothesis that F. nucleatum interaction with immune cells is mediated by the interaction between glycoconjugates decorating the bacterial cell surface and lectins expressed on antigen presenting cells.

We first tested the binding of F. nucleatum strains (ATCC 25586, 10953 and 51191) to a panel of recombinant human lectins (Dectin-2, Galectin-3 and Siglecs) by flow cytometry and showed that F. nucleatum specifically bound to Siglec-7 and to a lesser degree to the other lectins tested. O-antigen characterization of the purified lipopolysaccharide of F. nucleatum ATCC 51191 showed a trisaccharide repeating unit made of two aminuronic monosaccharides and one 6-deoxy-2,4-diamino unit with a heterogenous 4N-acetylation pattern. Binding assays are investigating the O-antigen role in the interaction with Siglec-7.

Stimulation of human monocyte-derived dendritic cells (moDCs) with F. nucleatum ssp. showed induction of a pro-inflammatory profile with the production of TNF< and IL-8 cytokines and the induction of CD80, CD86, PD-L1 cell surface markers. Further, human monocyte-derived macrophages (moMacs) showed a tumor associated profile characterized by the induction of IL-10, IL-6, IL-8 cytokines and PD-L1 marker and a downregulation of CD86 cell surface marker expression. Experiments are on-going using Siglec-7 RNA-silenced moDCs and moMacs to confirm Siglec-7 involvement in the innate immune response mediated by F. nucleatum.

259. Matricellular- and matrix- proteins constitute a morphogenetic program for the epithelial ovarian cancer metastatic niche

J. Langthasa¹, S. Kumar. S², S. Swamy², R. Gawari², R. Bhat¹; ¹Indian Institute of Science, ²Kidwai Cancer Institute

Epithelial ovarian cancer (EOC) is one of the most common and aggressive of gynaecological malignancies with a very poor five-year survival rate. The mortality associated with EOC correlates with the micrometastasis of ‘spheroids’: multicellular aggregates of cancer cells disseminated within the peritoneal cavity of EOC patients. We hypothesized that cell-cell adhesion is crucial to the formation of spheroids that helps these metastatic cells evade the biological (immune) and physical (shear) stresses associated with the hostile fluid milieu of the peritoneal cavity. We investigated whether the ®-galactoside-binding matricellular adhesion protein Galectin-1 (GAL-1), known to be upregulated in several cancers and involved epithelial-mesenchymal transitions could be involved in ovarian cancer spheroidogenesis. In cell line- and patient ascites-derived primary spheroids, GAL-1 repartitioned from the nucleus to an intercellular locale as cancer cells constituted spheroids. Pharmacological inhibition of GAL-1 resulted in an impaired spheroidal formation; overexpression of GAL-1 increased spheroid size. Disintegration of early spheroids was observed with GAL-1 inhibition, whereas mature spheroids remain intact, revealing additional mechanisms of morphogenesis were required to stabilize the cell adhesive role of GAL-1. Further investigation with mature spheroids led us to the discovery of a basement membrane-like extracellular matrix that stabilizes the structure of mature spheroids. Our in vitro and ex vivo results reveal a novel bi-step mechanism of ovarian cancer spheroid formation revealing new potential targets for therapy.

260. Glutamine fructose 6-phosphate aminotransferase (gfat) from plasmodium berghei: a clue for novel therapeutic perspectives?

M. Laugieri¹, M. Tonetti¹; ¹Department of Experimental Medicine, University of Genova, Italy

GFAT (GlmS in Bacteria) catalyzes the first step in the hexosamine pathway, both in Eukaryotes and Prokaryotes. It displays a N-terminal glutaminase domain and a C-terminal isomerase one. The free N-terminal amino group of Cys1 is essential for catalysis; in fact, it is required to activate the –SH group to act as a nucleophile, to attack the amide carbon of the glutamine substrate, a mechanisms typical the N-terminal nucleophile hydrolases. Indeed, all GFATs (and bacterial GlmS) so far characterized display an N-terminal Cys and modifications of the free amino group impair the catalytic activity.

Sequence and phylogenetic analyses of GFAT available in the databases revealed that a group of protozoan putative GFAT displays long N-terminal extensions, thus challenging the proposed catalytic mechanism. These extensions do not have apparently a clear biological significance in the analyzed organisms: they are highly variable and they are not typical signal sequences for targeting in sub-cellular compartments. Moreover, protozoan GFAT lack a 40-70 aminoacid insertion, typical of eukaryotic enzymes, but absent in the bacterial GlmS, which is involved in feed-back regulation of enzyme activity by UDP-GlcNAc.

To understand the role of the protozoan N-terminal extensions, we have expressed Plasmodium berghei GFAT, as wild type form and as a truncated form exposing the N-terminal free Cys1. Enzymatic characterization has revealed that wt and truncated GFAT display comparable glutaminase activity, indicating a different catalytic mechanism for this group of enzymes.

Studies are in progress to identify the functional role of GFAT N-terminal extension and the catalytic mechanism in comparison to human GFAT. Since the N-terminal extension is present in important human protozoan pathogens, belonging to Apicomplexa (i.e. Plasmodium, Toxoplasma), Heterokonta (i.e. Blastocystis) and Amoebozoa (i.e. Entamoeba, Acanthamoeba), these studies could represent a starting point for the development of more specific inhibitors for therapeutic applications.

261. Investigating the functional link between TMEM165 and SPCA1

E. Lebredonchel³, M. Houdou⁴, H. Hoffmann¹, M. Krzewinski⁴, D. Vicogne⁴, C.M. Rice¹, A. Klein², F. Foulquier⁴; ¹Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA, ²UGSF (Unité de Glycobiologie Structurale et Fonctionnelle) Villeneuve d’Ascq and Lille Medical Center France, ³UGSF (Unité de Glycobiologie Structurale et Fonctionnelle) Villeneuve d’Ascq and Lille Medical Center, France, ⁴UGSF (Unité de Glycobiologie Structurale et Fonctionnelle), Villeneuve d’Ascq (Lille), France

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases named TMEM165-CDG (Congenital Disorders of Glycosylation). Although the precise role of TMEM165 in glycosylation is still controversial, our results highly suggest that TMEM165 would act as a Golgi Ca2+/ Mn2+ antiport transporter regulating both Ca2+ and Mn2+ Golgi homeostasis, the latter is required as a major cofactor of many Golgi glycosylation enzymes. Strikingly, we recently demonstrated that besides its role in regulating Golgi Mn2+ homeostasis and consequently Golgi glycosylation, TMEM165 was sensitive to high manganese exposure.

On the other hand, another Golgi protein is known to import manganese from the cytosol to the Golgi lumen: SPCA1, which is the Golgi Ca2+ / Mn2+ P-type ATPase pump. Studying a potential functional link between TMEM165 and SPCA1, we first noticed a nearly complete loss of TMEM165 in SPCA1 deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1’s function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the the mutant that blocks Ca2+ but not Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Finally, the expression of TMEM165 was shown to be linked to the function of SPCA1.

262. Glycodelin-a sialylation stimulates the conversion of decidual nk cell via l-selectin binding

C. Lee¹, M. Vijayan¹, P.C. Chiu²; ¹Department of Obstetrics and Gynaecology, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, ²Shenzhen Key Laboratory of Fertility Regulation, Department of Obstetrics and Gynecology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China

Glycodelin-A (GdA) is an abundant sialylated secretory N-glycoprotein of the human endometrial glands. It plays important role in early placental development by interacting with immune cells and trophoblasts at the maternal-fetal interface. This interaction is largely sialylation dependent which involves the recognition of defined sialylated N-glycan on GdA by sialic-acid-binding proteins located on the target cell surface. Decidual natural killer (dNK) cells are the most abundant leukocyte in the decidua during early human pregnancy. The primary role of dNK cells is to promote placental vascular remodeling via both direct interaction with the endothelial cells and indirect interaction with trophoblasts. In this study, we hypothesized that GdA stimulates the generation of dNK cells and enhances the vascular remodeling activities of the transformed cells via the sialic acid dependent L-selectin receptor.

GdA treatment enhanced the expression of dNK cell specific markers CD9 and CD49a in the dNK cell precursor CD16−CD56bright NK cells. It also up-regulated the stimulatory activities of CD16-CD56bright NK cells on endothelial cell angiogenesis and invasiveness of extravillous trophoblasts. Immuno-precipitation confirmed the interaction of GdA with L-selectin on the cell membrane of NK cells. L-selectin is highly expressed in CD16-CD56bright NK cells, but not in CD16+CD56dim NK cells, and thus GdA had a stronger binding affinity to the former than the latter NK cells. GdA de-sialylation or anti-L-selectin functional blocking antibody treatment suppressed the biological activities and binding of GdA on the NK cells. To conclude, GdA stimulates the partial conversion of dNK cells and enhances their vascular remodelling activities via L-selectin binding. Further investigation on the glycosylation and functions of GdA will enhance our understanding on human placentation and placenta-associated complications with altered NK cell biology.

263. Characterization of glycosyltransferases involved in benzoxazinoid biosynthesis in scoparia dulcis

J. Lee², Y. Kasai¹, T. Kuraoka¹, Y. Yamamura²; ¹Faculty of Pharmacy and Pharmaceutical Sciences, University of Toyama, Japan, ²Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Japan.

Plants produce numerous secondary metabolites which no role in growth, photosynthesis, reproduction, or other primary functions. These chemicals are extremely diverse; many thousands have been identifiled in several major classes. Benzoxazinoids (BXs) include benzoxazinones and benzoxazilinones, and they are major ones playing an important role in disease resistance and allelopathy. In addition, they also have many other unique properties including anti-bacterial and anti-fungal activity. It is well-known that monocots could synthesize BXs, whereas a little dicots could produce them. Biosynthetic machineries of BXs have been well examined in Zea mays, and glycosylation steps are important for their storage and prevention of self-toxicity. The important glycosyl transferases have been named to be BX8 and BX9. Scoparia dulcis is a perennial herb widely distributed in the torrid zone, and it has been reported to synthesize BXs. Here, we would show the characterization of UDP glycosyltransferases (UGTs) involved in BX metabolism in S. dulcis.

Our screening based on Pfam search of UDPGT domain (PF00216) revealed that 196 contigs were found from in-house transcriptomic database of S. dulcis. Among these candidates, we selected gene candidates involved BX metabolism based on the homology search of known enzymes, BX8s. As a result, we could obtain 19 full-length clones, and phylogenetic analysis indicated that SdUGT1 closely related to UGT76 family enzymes. Nine candidates were placed into the clade of UGT85 family. Because a dicot BX8 enzyme from Consolida orientalis was UGT85N1, therefore, these nine enzymes were suggested to be potential candidates. To clarify their function, they were transformed into pET-44b vector for heterologous expression in Escherichia coli. Recombinant proteins were assayed for their enzymatic activity using DIMBOA or DIBOA as substrates. Among our cloned genes, three gene products might be involved in BX metabolism. recombinant SdGT1 was found to possess glycosyl transferase activity against DIMBOA, whereas recombinant SdGT3 and SdGT9 did against DIBOA. The present results are the first report to be characterize BX8/9 candidates from S. dulcis.

264. Nano-mechanical interaction measurement between lectin and saccharide to understand immune response

S. Lee¹, D.S. Hwang¹; ¹Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Korea

Saccharides are Gram-negative bacteria, Gram-positive bacteria, and fungi cell wall components for giving rigidity of the structure and for being recognized by host immune cells. Normally the saccharides are known as a pathogen-associated molecular pattern(PAMP) or microbe-associated molecular pattern(MAMP) which are recognized by pattern recognition receptors(PRRs) in the immune cell surface. When PRRs recognize and interact with PAMP in bacteria, virus or other infectious molecules the immune signaling is activated to protect the host. PRRs and PAMP, the receptor-ligand interaction especially the protein and saccharide interaction is important to understand the mechanism of the immune signaling and immune response. According to receptor-ligand immune studies conducted so far, most of them focus on the binding structure, signaling molecules, and secreting cytokines. However, there is not enough information about when receptor and ligand are binding on how strongly they interact from the interaction force point of view. Due to that, our research group measures the interaction force between lectin and saccharide binding to understand the mechanism of immune activity in a biophysical way. In this study, the interaction force of various lectins and saccharides were measured with nano-mechanical interaction measuring tool which is surface forces apparatus(SFA). SFA can measure molecular interaction between two surfaces with a force sensitivity of <10nN and distance resolution of ~0.1nm. SFA can mimic physiological condition such as pH, temperature, and ion concentration and when the interaction measured, two surfaces are meet in one spot so sample damage is low. Due to these reasons, it has been extensively used for various types of biological molecules including specific or non-specific interaction. Receptor-ligand interaction force measuring can provide a reference for related studies and give in-depth insight into immune activity by interdisciplinary research combining immunology and biophysics.

265. Biodistribution of metabolic glycan labeled extracelluar vesicles of metastatic breast cancer

T.S. Lee¹, J.I. Shin¹; ¹Division of RI Application, Korea Institute of Radiological and Medical Sciences, Seoul

Extracellular vesicles (EVs) are nano-sized vesicles that play critical roles in various pathophysiological processes. Tracking of EVs became important issue for their diagnostic and therapeutic use in cancer.

Here, we report a EVs labeling strategy through naturally metabolic incorporation of azido-sugar into glycan of cells and EVs, and strain-promoted azide-alkyne click (SPAAC) reaction. Tetra-acetylated N-azidoacetyl-D-mannosamine (Ac₄ManNAz) greatly incorporated into glycan in breast cancer cells and their EVs, and subsequently isolated azido containing EVs were labeled with azadibenzyl cyclooctyne (ADIBO)-fluorescent dyes by bioorthogonal click reaction. Cellular uptake and In vivo tracking of fluorescent labeled EVs were evaluated in breast cancer cells and tumor bearing and normal mice.

Metabolic glycan exosome labeling strategy with azido sugar and clickable fluorescent dyes by click chemistry was successfully applied in breast cancer cells and cancer derived EVs. Metabolic glycan-fluorescent dye labeled EVs showed similar uptake patterns with non-metabolic labeled EVs. Cellular uptake of metabolic glycan-fluorescent dye labeled cancer derived EVs showed dosage and temperature dependent pattern. Metastatic breast cancer-derived EVs showed specific organ targeting effect, compared to non-metastatic breast cancer’s EVs and its targeting was modulated by integrin alpha 6 blocking antibody and extracelluar matrix binding blocker, HYD-1 peptide.

Our metabolic EVs labeling strategy can be highly useful in studying the interaction and communication between cancer cells and EVs and it maybe a promising tool in studying biodistribution of EVs, and optimizing EVs based therapeutic approaches.

266. Galectin-4 secreted by intestinal epithelial cells modulates the composition of microbiota and protects the host against pathogens

C. Li¹, T. Tu¹, F. Liu¹; ¹ Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

Galectins are a member of animal lectins with conserved carbohydrate-recognition domains (CRDs) that recognize galactosides. They exist in the cytosol and nucleus, but also in extracellular space, although they do not have a classical signal peptide. Recent studies have identified the ability of galectin-4 (Gal4) and -8 to bind to and kill bacteria. We showed that a number of different galectins, especially Gal4, were present in the secretions from freshly isolated intestines of mice. In addition, a small population of intestinal commensal bacteria was found to be coated by Gal4. To investigate the effect of Gal4 on the microbiota composition, Gal4-associated fraction of commensal recovered from the intestines were enriched by anti-Gal4 antibody labeling and magnetic separation. 16S rDNA sequencing analysis revealed that several bacterial taxa were enriched in this fraction, including Bifidobacterium and Lactobacillus murinus. Interestingly, the relative abundance of Bifidobacterium was significantly higher in wild-type mice compared to Gal4-deficient littermates. To examine the role of Gal4 in host-defense against pathogens in the intestine, we studied its interaction with four strains of human bacteria: Escherichia (E.) coli O19ab, E. coli O86, Salmonella (S.) enterica Cubana and S. enterica Worthington. We found that recombinant human Gal4 bound to these strains through its CRDs. Gal4 did not affect bacterial survival, but induced chaining and clustering. The formation of chains was from the divided cells failing to separate into individual daughter cells. Gal4 was enriched at the cell-cell junctions and bridged the daughter cells through binding to lipopolysaccharides. Moreover, Gal4 restricted bacterial motility significantly. When mice were subjected to oral challenge with S. enterica Worthington after dextran sulfate sodium treatment, the number of S. enterica Worthington was lower in mesenteric lymph nodes and colon of human Gal4 transgenic mice compared to wild-type littermates. Our data suggest that Gal4 protects the host by inducing bacterial chaining and restricting bacterial motility.

267. Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies

H.L. Tytgat³, C. Lin², M.D. Levasseur⁴, J. Mock³, M.F. Bachmann¹, M. Aebi³, T.G. Keys³; ¹Department of Immunology, Inselspital, University of Bern, 3010 Bern, Switzerland, ²Functional Genomic Center Zurich, ETH Zurich, 8057 Zurich, Switzerland, ³Institute of Microbiology, ETH Zurich, 8093 Zurich, Switzerland, ⁴Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland

Glycosylation of proteins has a profound impact on their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. To circumvent these bottlenecks, we have pioneered a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling glycoprotein nanomaterials bearing hundreds of copies of the glycan epitope.

268. Chemoenzymatic synthesis of sialylated globosides and their derivatives

C. Lin², P. Li², S. Huang², P. Chiang², C. Fan², T. Angata¹; ¹Academia Sinica, ²National Tsing Hua University

Globosides, composed by ceramide and a series of glycans featuring with a Gal-<1,4-Gal linkage, have been found on various cancer cell surfaces and related to cancer development. Although many methods have been developed for the globosides, the synthesis of disialosylglobopentaose (DSGb5) has not been reported yet. We successfully synthesized DSGb5 glycan with azide-modified linker from Gb5 (SSEA-3) with total 25% yield by one-pot multienzyme (OPME) method and 12%~26% by three different chemoenzymatic methods. Moreover, Gb3, Gb4, and Gb5 were catalyzed by several kinds of sialyltransferases to afford mono-sialylated (<2,3- and <2,6-), linear di-sialylated (<2,8-<2,3- and <2,8-<2,6-), and branched di-sialylated (<2,6- and <2,3-) globo-series glycans.

Although the glycan moiety is considered to be the key of globoside’s bioactivity, the ceramide of globosides was also indicated to be critical for signal transduction in recent studies. Therefore, it is demanded to develop an efficient method to synthesize globo-ceramides and their derivatives for investigation of the bioactivities toward cancer cell. Herein, OPME method was applied to synthesize Gb3, Gb4, Gb5, SSEA-4, and Globo-H sphingolipids by using chemically synthesized lactose (Lac) sphingolipid as starting material, which shows better water solubility than that of Lac-ceramide. The fatty acid was introduced to the above glycosphingolipids at the late stage to yield globosides.

269. Development of potent and selective inhibitors for gut bacterial ®-glucuronidases

C.H. Lin¹; ¹Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

Gut bacterial ®-D-glucuronidases (GUSs) catalyze the removal of glucuronic acid from liver-produced ®-D-glucuronides. These reactions can have deleterious consequences when they reverse the metabolism of xenobiotics. The human gut contains hundreds of GUSs of variable sequences and structures. To understand how any particular bacterial GUS(s) contributes to global GUS activity and affects human health, the individual substrate preference(s) must be known. Herein we report that representative GUSs vary in their ability to produce various xenobiotics from their respective glucuronides. To attempt to explain the distinct substrate preference, we solved the structure of a bacterial GUS complexed with coumarin-3-®-D-glucuronide. Comparisons of this structure with other GUS structures identified differences in loop 3 (or the <2-helix loop) and loop 5 at the aglycone-binding site, where differences in their conformations, hydrophobicities and flexibilities appear to underlie the distinct substrate preference(s) of the GUSs. Additional sequence, structural and functional analysis indicated that several groups of functionally related gut bacterial GUSs exist. Our results pinpoint opportunistic gut bacterial GUSs as those that cause xenobiotic-induced toxicity. We propose a structure-activity relationship that should allow the prediction of the functional roles of GUSs.

Additionally we will present the development of potent and selective inhibitors for desirable bacterial GUSs. By using the mechanism-based design, several iminocyclitols and analogues were synthesized and evaluated to understand how the charge and conformation play a role in determining the inhibition potency. We will also discuss how to enhance the selectivity for undesirable group(s) of gut bacterial GUSs.

270. Mice lacking st8sia vi specifically in b cells mount higher igm responses

H. Tsai¹, Y. Yu¹, K. Khoo², T. Angata², K. Lin¹; ¹Genomics Research Center, Academia Sinica, Taipei, ²Institute of Biological Chemistry, Academia Sinica, Taipei

How glycosylation affects the function of immune cells is largely unknown. We previously characterized the glycome of a mouse B-cell lymphoma cell line and revealed the presence of Neu5Gc<2-8Neu5Gc diSialyl motif that occurs not only on the simple core 1 O-glycans, but also on the poly-N-acetyllactosamine (polyLacNAc) and non-extended LacNAc of the N-glycans. We also demonstrated that the expression of <-N-acetyl-neuraminide <-2,8-sialyltransferase 6 (ST8Sia VI) was increased during primary mouse splenic B cell differentiation into antibody-secreting plasma cells and contributed to the presence of diSialyl motif. Whether up-regulation of ST8Sia VI during B cell activation and plasma cell differentiation plays any regulatory roles in immune responses is still unexplored. Here, we investigated into the physiological roles of ST8Sia VI and diSialyl motif in B cells. We created St8sia6^f/f mice that carry two lox-P sites franking the exon 6 of St8sia6 gene, resulting in a genetic deletion of St8sia6 specifically in B cell lineage upon DNA recombination by Cre recombinase controlled by the B cell specific CD19 promoter. We found that general B cell development was not severely disturbed, but higher IgM responses were observed following immunization with T cell-dependent or T cell-independent antigens in mice lacking St8sia6 in B cells, as compared with littermate controls. Consistently, more antigen-specific plasma cells were found in the spleen and bone marrow of B cell specific St8sia6 knockout mice. B cells lacking St8sia6 activate better than the control B cells when B cell receptor is cross-linked with anti-IgM. We are attempting to identify the diSialyl carrier proteins on B cells and investigating into the molecular impact of diSialyl motif on B cell activation and IgM production. Results from this study may contribute to the better understanding of the significance of diSialyl motif in IgM responses, which may be relevant to the control of some autoimmune and infectious diseases.

271. Intracellular galectin-1 involves in erythropoiesis via regulating stats and SMAD2/3 signaling

W. Lin¹, C. Cheng¹; ¹Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan

Erythropoiesis is a tightly regulated process of differentiation from erythroid progenitor cells to mature erythrocytes. Several growth factors are involved in erythropoiesis including erythropoietin (EPO) and GM-CSF. In our previous study, we have demonstrated non-receptor tyrosine kinase Syk as a novel upstream signaling molecule of EPOR and GM-CSF receptors in TF-1 erythroleukemia cells to mediate downstream signaling pathways like ERK, STAT5 and Akt for erythropoiesis. In this study, we like to address the functions of galectin-1 (Gal-1) in erythropoiesis, which is a soluble beta-galactoside binding animal lectin, and can exert cellular functions through intracellular and extracellular pathway. We found Gal-1 is expressed in plasma membrane, cytosol and nucleus in TF-1 and K562 cells. Using hemin-induced erythrocyte differentiation in K562 cells as a cell model, we found hemin treatment increases intracellular Gal-1 protein expression but not its mRNA level. Moreover, treatment of MG132 or Bafilomycin A1 further increases Gal-1 protein level in hemin-treated K562 cells, suggesting that Gal-1 stability is controlled by proteasome and lysosome. By using Gal-1 shRNA to suppress Gal-1 expression in K562 cells, our results showed that Gal-1 has no effect on cell proliferation and survival. However, Gal-1 shRNA significantly inhibits hemin-induced hemoglobin-© (Hb-©) expression, and this inhibition cannot be reversed by exogenous Gal-1. Further study showed the influence of Gal-1 on signaling pathways which are involved in erythropoiesis. Induction of the phosphorylation level of STAT3, STAT5 and ERK is observed in hemin-treated K562 cells, whereas Gal-1 shRNA not only counteracts hemin-induced phosphorylation of STAT5 and ERK, but also suppresses Syk, STAT3, and Smad2/3 protein expression, therefore leading to inhibition of hemin-induced nuclear translocation of STAT3, ERK, and Smad2/3. Inhibitors of Syk (GS9973), ERK (U0126), Smad2/3 (SB431542), STAT5 (573108), and STAT3 (Stattic) suppress hemin-induced Hb-© expression as well as Gal-1 shRNA. Besides modulating the transduction of signaling pathways, Gal-1 can bind to ERK, STAT3, and Smad2/3, but not STAT5 under hemin stimulation. By comparing colony formation assay in hematopoietic progenitor cells obtained from bone marrows of WT and Gal-1−/− mice, significant inhibitions of both BFU-E and CFU-E formation in Gal-1-deficient cells as compared to WT cells were detected. Taken together, our results indicate that Gal-1 is beneficial for erythrocyte differentiation through regulating Syk-STATs, ERK and Smad2/3 signaling pathways, interacting with and controlling nuclear translocation of these signaling molecules.

272. Opening up prospects for bioinformatics-aided glycoscience

F. Lisacek³, J. Mariethoz³, F. Bonnardel³, A. Imberty¹, N. Packer²; ¹CNRS- Université Grenoble, France, ²Macquarie University, Sydney, Australia, ³SIB Swiss Institute of Bioinformatics, Geneva, Switzerland

It is commonly recognised that bioinformatics has boosted the fields of genomics and proteomics and as such was instrumental in generating new knowledge. With increasing throughput in glycomics and glycoproteomics, we expect glycoinformatics to play a similar role and have launched the Glycomics@ExPASy initiative (www.expasy.org/glycomics) in that spirit in 2016. Our implementation promotes easy navigation correlating glycan structures to binders or glycoproteins to diseases, etc. In the past three years, we have released a tool set that enables glycome profiling, glyco-epitope mapping, protein glyco-site characterisation and lectin classification.

The overall purpose of Glycomics@ExPASy is to provide web-based resources, which put together, reflect a consistent picture of the structural and functional aspects of glycosylation. Two recent inclusions in the collection, i.e., GlyConnect and UniLectin exemplify this purpose. To feed information into each of these two platforms, we process and curate large datasets. This larger scale approach imposes grouping and classifying and raises new issues relative to defining appropriate yet not too constraining comparison criteria. We discuss this thin balance through examples of profiling glycomes, glycoproteins and lectin families.

273. Resistance to bortezomib in breast cancer cells that downregulate bim through foxa1 o-glcnacylation

Y. Liu¹, X. Wang¹, J. Zhang¹; ¹School of life science & medicine, Dalian University of Technology

Bortezomib (BTZ), a well-established proteasome inhibitor used in the clinical therapy, leads the modulation of several biological alterations and in turn induces apoptosis. Although clinical trials with BTZ have shown promising results for some types of cancers, but not for some others, including those of the breast. The molecular basis of BTZ resistance in breast cancer remains elusive. In the present study, we found that cellular O-GlcNAc modification was dramatically elevated by BTZ treatment in intrinsic resistant MCF-7 and T47D cells, but not in sensitive MDA-MB-231 cells. A progressive increase in O-GlcNAcylation characterized the increased acquired resistance of MDA-MB-231-derived cells. We showed that elevated O-GlcNAc subsequently modified breast cancer related pioneer factor FOXA1 and reduced its protein stability. Further, we demonstrated that FOXA1 attenuation was involved in transcriptional downregulation of proapoptotic Bim and thus suppressed breast cancer cell apoptosis. Finally, the combination of O-GlcNAc inhibitor L01 to BTZ sensitized resistant cells. Our results have revealed a new regulatory mechanism that involves O-GlcNAc elevation mediated Bim deficiency, which plays a key role in the apoptotic dysregulation and BTZ resistance in breast cancer cells.

274. Ganglioside gm1 interacts and stabilizes the cystic fibrosis transmembrane conductance regulator at the plasma membrane

N. Loberto¹, G. Mancini³, C. Trabucchi², M.C. Dechecchi², L. Mauri¹, D. Olioso², S. Sonnino¹, N. Pedemonte⁴, V. Tomati⁴, G. Cabrini², A. Tamanini², M. Aureli¹; ¹Department of Medical Biotechnology and Translational Medicine, University of Milan, ²Department of Pathology and Diagnostics, University Hospital of Verona, Verona, Italy, ³Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Hospital, Milano, Italy, ⁴U.O.C. Genetica Medica, Istituto Gianna Gaslini, Genova, Italy

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel expressed at the apical surface of epithelial cells. Mutations in CFTR gene cause Cystic Fibrosis (CF), an autosomal recessive disease characterized by severe lung impairment.

The new therapeutic strategies used for CF therapy are based on the use of pharmacological agents able to increase the surface level of mutated CFTR (correctors), as well as its plasma membrane (PM) activity (potentiators). Unfortunately, for the most common CF-causing mutation F508del, their efficacy seems to be time-limited due to a reduced stability of corrected protein at PM.

During the years an increasing number of proteins involved in the CFTR PM stabilization and function were described, but scant information is available regarding the lipid counterpart. Interestingly, the lack of CFTR expression at the PM of bronchial epithelial cells correlates with a reduction of the ganglioside GM1 cellular levels. By the use of a radioactive and photoactivable GM1, we established that the ganglioside directly interacts with CFTR at the PM level.

Since this lipid has been described to play an important role in the activity of some PM proteins, we investigated the effects of GM1 on the PM stability of F508del CFTR rescued by the use of Lumacaftor (VX-809) and Ivacaftor (VX-770).

As result, we found that when GM1 is exogenously administered to epithelial bronchial cells expressing F508del CFTR, the amount of the mutated protein that remains associated with the PM is high if compared with the drugs treatment alone. In addition, we demonstrated that the rescued F508del CFTR directly interacts with the exogenously administered GM1. Consequently, we found that the treatment with GM1 induces an increase of the CFTR scaffolding proteins NHERF1 and ezrin.

Taken together, our results indicate GM1 as a new interactor of CFTR at PM and, due to its fundamental role in the mutated CFTR stabilization, this ganglioside could be considered as adjuvant for the CF therapeutic strategy based on the use of corrector and potentiators.

Supported by the Italian Cystic Fibrosis Research Foundation grant FFC # 09/2015 to AT and #02/2018 to MA

275. Large-scale preparation and characterization of recombinant human golgi glycosyltransferaseS GNT-I, GNT-II AND FUT8

T. Lu¹, N. Wang¹, X. Gao¹; ¹Jiangnan University

Asparagine (N)-linked glycoproteins are assembled in the endoplasmic reticulum (ER) and transported to Golgi apparatus, where the N-glycan is trimmed to the mature form. During this process, most of the glycans on the secretory proteins in eukaryotic cell are shifted from the high-mannose type to the complex type, which is known to be critical for their biological functions. Several glycosyltransferases are involved in the N-glycan trimming in the Golgi apparatus, while three of them are considered as the key enzymes to produce complex type N-glycans, namely GnT-I, GnT-II and Fut8. GnT-I adds a b-1,2-GlcNAc to the A arm a-1,3 mannose, while GnT-II and Fut8 work after GnT-I, adding a b-1,2-GlcNAc to the a-1,6 mannose and Fut8 add an a-1,6-fucose to the reducing terminal GlcNAc.

To develop a better understanding of these three enzymes, in this study, we establish the overexpression and purification of recombinant human GnT-I, GnT-II and Fut8 using prokaryotic expression system. All the three N-terminal transmembrane domain truncated proteins were successfully obtained in their soluble form from E.coli ROSETTA strain. The expressed enzymes were purified and characterized. It was proved that the acceptor specificity of GnT-I is rather board, while GnT-II and Fut8 are strict with the substrates having an a-1,3-mannose-b-1,4-GlcNAc branch. On the other hand, both GnT-I and GnT-II need a cation cofactor such as Mn2+, which is not critical for Fut8. Kinetic analysis showed the Km value of GnT-II was 55 μM against its natural glycan substrate. These results may be applicable to the in vitro enzymatic-synthesis of complex type N-glycans.

276. GM1 oligosaccharide modulation of calcium signaling in neuronal functions

G. Lunghi¹, M. Fazzari¹, E. Di Biase¹, L. Mauri¹, E. Maffioli¹, F. Grassi Scalvini¹, G. Tedeschi¹, E. Chiricozzi¹, S. Sonnino¹; ¹University of Milano

Recently, we demonstrated that the oligosaccharide portion of ganglioside GM1 (OligoGM1) is responsible, via the direct activation of the TrkA-MAPK pathway, for the ability of GM1 to induce neuritogenesis and to confer neuroprotection, in a mouse neuroblastoma cell line N2a. This suggests that the specific role of ganglioside GM1 in neuronal functions described in the past is determined by a direct interaction of its oligosaccharide portion with specific proteins expressed on the plasma membrane (PM). Among them, an important GM1 property would regard the activation of PM ion channels that permit the regulation of cytosolic calcium concentration necessary for neuronal functions. Thus, we investigated if GM1 oligosaccharide could be involved in the regulation of calcium signaling using murine neuroblastoma cell line, N2a. First, we observed by calcium imaging that OligoGM1 administration to undifferentiated N2a cells resulted in an increased calcium influx, partly mediated by the activation of TrkA receptor. As demonstrated by biochemical analysis, PLC© activation follows the TrkA stimulation by OligoGM1, leading to the opening of calcium channels on the PM and on intracellular storages. Subsequently, we found that neurite induction in N2a cells was blocked by subtoxic administration of extracellular and intracellular calcium chelators, suggesting that the increase of intracellular calcium is responsible of OligoGM1-mediated differentiation. Moreover, proteomic analysis identified 324 proteins expressed only in OligoGM1 treated cells and, interestingly, many of them are involved in the regulation of calcium homeostasis and in calcium-dependent differentiative and neuroprotective pathways. These results suggest that GM1 oligosaccharide is responsible for the regulation of calcium signaling and homeostasis at the base of the neuronal functions mediated by PM ganglioside GM1.

277. Deciphering the recognition patterns of lectin interaction with bacterial lipopolysaccharides using nuclear magnetic resonance (NMR) spectroscopy

M. Maalej², R. Marchetti³, C. Laguri¹, A. Molinaro³, J. Simorre¹; ¹Institut de Biologie Structurale (IBS de Grenoble), ²Institut de Biologie Structurale (IBS de Grenoble) / Universita degli studi di Napoli (Federico II), ³Universita degli studi di Napoli (Federico II)

The interaction between C-type lectin immune cell receptors (CLRs) and glycolipid variants from Gram-negative bacteria outer membrane (i.e. lipopolysaccharide LPS and Lipooligosaccharide LOS) is one of the first molecular events required for the activation of mammalian immune system and inflammation in septic shock patients. The specific recognition of the surface exposed glycans expressed by pathogens is a result of C-type lectin’s different affinities toward complex sugar arrangements, which makes its investigation very challenging. Besides, the role and molecular mechanisms of these interactions are not fully understood. Within the frame of this PhD project we are focusing on obtaining atomic-scale information on the interaction between several eukaryotic lectins (i.e. MGL, DC-SIGN and Langerin) and bacterial LPS/LOS scaffolds. A first study on the binding of the core oligosaccharides from E. coli R1 and R3 LOSs with the human Macrophage Galactose-type Lectin receptor, h-MGL, was performed by the means of Saturation-Transfer Difference (STD)-NMR spectroscopy and computational methods. Our results showed that the de-acetylated LOS from E. coli R1 is recognized by h-MGL through the terminal disaccharide Gal<(1,2)Gal. Moreover, the recognition and behaviour of LPS/LOS from several strains by human C-type lectins will be subjected to further investigations using Electron Microscopy, Surface Plasmon Resonance and solid-state NMR spectroscopy.

278. Analysis of suppressive function of dendritic cells by glycopolymers carrying multivalent plant antigenic n-glycans bearing lewis a antigens

M. Maeda¹, C. Tanabe¹, M. Tani¹, Y. Kimura¹; ¹Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan

[Introduction] Most pollen allergens are glycoproteins carrying plant antigenic N-glycans. We reported, in a previous paper, that a free plant antigenic N-glycans, Man3Xyl1Fuc1GlcNAc2 (M3FX), suppressed Th2 immune response caused by a Japanese cedar pollen allergen, Cry j1, suggesting that the core structure of plant specific N-glycan, M3FX, is a useful candidate as a therapeutic agent for pollinosis. Recently, a novel Japanese cypress (Chamaecyparis obtuse) pollen allergen was identified and named Cha o3, which is a glycoprotein containing a cellulase-like domain. The structural analysis showed that plant antigenic N-glycans bearing Lewis a antigen(s) accounts for 50% of the N-glycans of Cha o3, suggesting that this structural feature is very similar to that of Cry j1. In this study, we have synthesized glycopolymers carrying multivalent plant antigenic N-glycans containing Lewis a antigens, and analyzed the suppressive function of the glycopolymers on dendritic cells (DC) differentiation and maturation. [Methods and Results] Two types of Asn-glycopeptides (plant antigenic type containing Lewis a antigen, and high mannose type) were prepared from water plants (Egeria densa), and royal jelly glycoproteins. Asn-glycopeptides were coupled to poly-©-L-glutamic acid (©PGA). The resulting glycopolymers were purified by a combination of gel-permeation and RP-HPLC. The incorporation of N-glycans into ©PGA (mol%) was estimated by amino acid composition analysis. The coupling efficiency of plant antigenic type, and high mannose type N-glycans to ©PGA were 3.8%, and 3.6%, indicating that nearly 200 molecules of N-glycans were incorporated into ©PGA. DC like cells were induced from THP-1 cell lines with culturing in the presence of PMA and IL-4 for four days. The induction was confirmed with expression level of low CD14 (monocyte maker) and high CD11c (DC maker). DC like cells were cultured in the presence or absence of the two kinds of glycopolymers for three days. The differentiation and maturation were assessed by the expression level of CD86 (co-stimulatory protein) and HLA-DR (MHC class2 protein) by FACS analysis. The expression of HLA-DR was significantly increased in 10 ⎧g and decreased in 100 ⎧g of plant antigenic type glycopolymers, although there was no significant difference in CD86 expression. [Discussion] These results suggested a possibility that the plant antigenic type glycopolymers may inhibit Th2 immune response via suppression of antigen presentation of DC.

279. Cancer cell derived extracellular vesicles carry tumour associated glycan epitopes

A. Magalhães¹, D. Freitas¹, M. Balmaña¹, J. Poças¹, D. Campos¹, H. Osório¹, A. Konstantinidi², S.Y. Vakhrushev², C.A. Reis¹, ¹i3S/IPATIMUP - Institute of Molecular Pathology and Immunology of University of Porto, Portugal, ²University of Copenhagen, Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Faculty of Health Sciences, Copenhagen, Denmark.

Aberrant cell surface glycosylation is a hallmark of cancer. Glycoconjugates have been shown to be important players in cancer cell interaction with the extracellular matrix and to modulate the tumour microenvironment and the disease progression. Paramount evidence shows that extracellular vesicles (EVs) are important signalling particles which play critical roles in intercellular communication. EVs are largely produced by cancer cells and have been demonstrated to impact cancer spread namely by defining the pre-metastatic niches. Importantly, glycoconjugates were shown to play key roles in EVs biogenesis and to act as receptors for capture and internalization of cancer cell-derived EVs. Although several seminal studies have addressed the lipid, protein and nucleic acid cargo of EVs, its glycan content remains poorly characterised.

In the present study, we have isolated EVs using different state-of-the-art methodologies, namely differential ultracentrifugation, total exosome isolation kit, OptiPrepTM density gradient and size exclusion chromatography, and assessed its impact in the subpopulation of EVs obtained. The EVs were characterized regarding the protein cargo and the glycosylation profile. Using a genetically engineered cancer cell line displaying homogeneous truncated O-glycans we were able to isolate, for the first time, EVs carrying proteins modified with the tumour-associated glycan STn. Further isolation of specific subsets of EVs showed that different subpopulations can harbour distinct glycan profiles.

The analysis of the glycosylation features of the proteins carried by EVs isolated from cancer cells demonstrated that the different isolation approaches result in distinct EV subpopulations, highlighting the need of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. The identification of unique tumour-associated glycosylation alterations in cancer-derived EVs holds great potential as a source of non-invasive biomarkers for early diagnosis of cancer.

280. Metabolism of polysialic acid (polysia) is defective in multiple pre-clinical models of huntington’s disease

G. Pepe², E. Amico², L. Capocci², A. Sönmez¹, T.J. Boltje³, R. Parlato¹, A. Di Pardo², V. Maglione²; ¹Institute of Applied Physiology, University of Ulm, Ulm, Germany, ²IRCCS Neuromed, Pozzilli, Italy, ³Radboud University Nijmegen, The Netherlands

Defective metabolism of sialic acid-containing glycosphingolipids (gangliosides) has been described as important determinant of disease pathogenesis in Huntington’s disease (HD), a rare hereditary neurodegenerative condition with complex pathogenic profile and with no cure available. Reduced levels of gangliosides in HD mice and in human cells is associated with perturbed expression of the biosynthetic enzymes, sialyltransferases, which are commonly involved also in the synthesis of glycoproteins.

Thus, the hypothesis is that alteration of ganglioside metabolism in HD may represent a phenomenon of a more general global defective metabolism of sialoconjugates which may likely interfere also with glycoprotein homeostasis.

In this study, we explored for the first time whether or not the metabolism of sialic acid-containing glycoproteins is actually compromised in HD. Particular attention was paid to the analysis of the Polysialic acid (polySia), the sugar polymer commonly linked to the neural cell adhesion molecule (NCAM) that is well known to be essential for brain development and plasticity.

Interestingly, our findings demonstrate that metabolism of polySia becomes precociously perturbed in HD, even at the early stages of mouse embryonic development. Although only speculative by now, the early aberrant levels of polySia in HD may provide evidence to elaborate a different concept of the disease and eventually investigate still unexplored aspect of any associated neurodevelopmental impairment.

281. Dynamic characterisation of the interactions between complex glycans and host immune proteins

R. Marchetti¹, A. Molinaro¹, A. Silipo¹; ¹Department of Chemical Sciences, University of Naples Federico II

Glycans display incredible structural diversity (the Glycome) and are involved in fundamental molecular and biological processes, including protein folding, cell-adhesion, molecular trafficking, signal transduction, modulation of receptor activity, as well as many critical functions in immunoregulation and pathological processes (autoimmunity, cancer).

The ability of glycans to regulate various aspects of the immune response is mediated by their recognition by various host receptor proteins, including lectins, such as C-type lectins (as Dectin-1) and Siglecs (Sialic Acid Binding Immunoglobulin Like Lectins). Among them, Siglecs are transmembrane receptors that function to recognize ubiquitous sialic acid epitopes on cell surface glycoconjugates, predominantly expressed by innate immune cells.

Siglecs are increasingly recognized for their role in helping immune cells to distinguish between “self” and “non-self”, dampening autoimmune responses and controlling inflammation in response to various pathogens. Conversely, many feared pathogens, including membrane-enveloped viruses and bacteria, have the ability to cloak themselves with sialylated glycans that mimic “self” allowing them to elude or subvert the host immune responses.

Given that Sialic acid - Siglec interactions have been associated with a broad spectrum of diseases, ranging from autoimmunity to neurodegeneration and cancer, strategies to tune these interactions could have great therapeutic potential.

Within this frame, a multidisciplinary approach, which combines cutting-edge methodologies including advanced ligand-based NMR (Nuclear Magnetic Resonance) techniques and computational studies, was applied with the aim to improve our knowledge on the molecular basis of the interaction between functional glycan epitopes and their protein receptors.

Our results provide crucial information on different binding mechanisms and represent a key step in the rational design of glycomimetics able to modulate the function of host immune receptor proteins.

282. Glycan-based molecular interactions and their role in bacterial surface adhesion

K. Dunker¹, S.G.d.l.T. Canny¹, I. Bakke¹, C.T. Nordgård¹,M. Sletmoen¹; ¹Norwegian university of science and technology

The increased awareness of the numerous and important functions of glycans in biological systems, including but not limited to bacterial adhesion, recently motivated researches to conclude that glycoconjugates, and glycoproteins in particular, are an underappreciated and potentially crucial factor in understanding bacteria–host interactions. In a recent review, we provided an overview of the role of glycans in bacterial adhesion to mucosal surfaces, and also argued that the single-molecule techniques are powerful tools to advance our understanding of the mechanisms underlying bacterial adhesion. In the talk, I will present a study in which we characterize the mechanical properties of mucosal surfaces covering the skin of Atlantic salmon fry as well as the adhesion of microbes to this mucosal surface using atomic-force microscopy methods. We have quantified the interactions between five chosen bacteria that are found in fish skin and the skin mucus on germ-free (GF) and conventionally raised (CVR) yolk-sac fry. The use of intact fish skin circumvents the need for mucin extraction, purification and surface immobilization, i.e. processes that might influence on the molecular structure and availability, and thus interfere with the interaction abilities. Irrespective of the type of bacteria investigated, the average percentage of force curves containing signatures of interaction was lower for the CVR fish than the GF fish. The vast majority of binding strengths recorded were weak (<300 pN), and in the same range as previously documented for single protein-glycan interactions. The Young’s modulus was found to be lower for the mucosal surface of GF fry compared to the CVR fry, reflecting that the colonization by bacteria introduces structural changes in the mucosal layer.

283. Production and structural characterization of a fucose binding lectin from the emerging microfungi scedosporium apiospermum; a target for the development of antiadhesive glycodrugs.

D. Martinez Alarcon², R. Pieters³, A. Varrot¹; ¹CERMAV, CNRS, Univ. Grenoble Alpes, Grenoble, France, ²CERMAV, Univ. Grenoble Alpes, CNRS, Grenoble, France, ³Utrecht University, Utrecht, The Netherlands.

Scedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised patients and notably bronchopulmonary infections in cystic fibrosis patients. It represents a therapeutic challenge due to its intrinsic resistance towards traditional antifungals and to its high recurrence. Since lectins are involved on host-pathogen interactions, several approaches have been suggested to inhibit their binding to host cells through carbohydrates mimics. However, in the case of Scedosporium, no such lectin has been identified to date, hindering the development of anti-adhesive molecules as antifungals against this pathogen. We present the identification of a lectin from S. apiospermum (SapL1), its recombinant production, an analysis of its fine specificity and its structural characterization by X–ray crystallography. SapL1 was identified by data mining using the sequence of FleA (AFL), a conidial surface lectin from Aspergillus fumigatus, as bait on Scedosporium genome. Expression in soluble form in Escherichia coli necessitates unexpectedly the addition of rhamnose to the culture media. SapL1 is purified with a final yield of ~4 mg·L-1. It forms homodimers and display strong hemagglutination activity on blood cells. Glycan array data demonstrated that SapL1 recognized fucosylated oligosaccharides from ABO and Lewis histo-blood group antigens (<1-2/3/4 linked) better than core fucose (<1-6) with a clear preference for H type II antigen. Affinity was measured by Isothermal Titration Calorimetry (ITC). SapL1 was co-crystalized with <-methyl-fucoside and its structure was solved at 2.3 Å by molecular replacement. Refined model exhibits a dimer of six-bladed ®-propellers with the ligand bound to every binding sites. The latest were found at the interface between blades and all present a conserved arginine, glutamate and tryptophan residues involved in essential hydrogen bonds with the O4 and O3 hydroxyls. This information contributes to the understanding of glycosylated surfaces recognition by Scedosporium and can now be used for the development of glycodrugs for antiadhesive therapies targeting SapL1 inhibition.

284. Inhibition of amyloid assemblies generated on ganglioside-containing lipid bilayer by a gm1 cluster-binding peptide

T. Matsubara¹, M. Nakai¹, M. Nishihara¹, T. Sato¹; ¹Keio University

The onset of Alzheimer’s disease (AD) is involved in amyloid ß-protein (Aß) assemblies on ganglioside-enriched microdomains in presynaptic neuronal membranes. We have investigated the Aß assembly on neuronal membranes by atomic force microscopic (AFM) imaging of the surface topography of lipid membranes, resulted in that ganglioside cluster has a potential as a major therapeutic target against AD. In the present study, GM1 cluster-binding peptide identified by a phage display technology was chosen to inhibit Aß assemblies. Ganglioside-containing lipid membrane composed of GM1, sphingomyelin, and cholesterol was prepared and the interaction between Aß and GM1 on the membrane was investigated by AFM. We found that the number of Aß fibrils generated on the ganglioside-containing lipid membrane decreased in the presence of the peptide. This results indicated that the binding of the peptide to GM1 cluster in the membrane inhibits the interaction between Aß and GM1. Furthermore, peptide dendrimers with high affinity for GM1 showed effective inhibition of Aß assemblies. Our results suggest that the GM1 cluster-binding peptide has a potential as a novel prophylactic and therapeutic agent for AD.

285. Comparative analysis of exosomal glycomes with lectin microarray

A. Matsuda², M. Yoshida¹, T. Nakagawa³, T. Wagatsuma¹, M. Suematsu³, H. Narimatsu¹, Y. Kabe³, A. Kuno¹; ¹Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, ²Department of Biochemistry, Keio University School of Medicine, Tokyo, ³Department of Biochemistry, Keio University School of Medicine, Tokyo.

Exosomes are generally covered with glycocalyx, which are controlled by a host cell glyco-synthetic machinery as similar to secreted and plasma membrane glycoproteins. Several exosomal subpopulations classified by tetraspanins have been investigated in relationship to diseases. However, their comparative analysis has never been attempted in terms of glycomics. In this study, we demonstrate comparative glycomic analysis of exosome subpopulations derived from pancreatic cancer cells using lectin microarray system. 8 pancreatic cancer culture cells derived secreted, plasma membrane glycoproteins, and exosomes were prepared, respectively. Exosomes were isolated and purified using commercially available exosome isolation kits. Further each CD antigens-positive exosomes were fractionated from total exosomes using specific antibodies against tetraspanin (CD9, CD63, CD81) and were subjected to the comparative glycan profiling with lectin microarray. As the result it was found that the total exosome-derived glycoproteins contain abundant glycoproteins with specific glycan structures compared with secreted and membrane glycoproteins. Moreover, the multivariable analysis of the glycan profiling of the antigen positive exosomes indicated that each exosome fraction showed specific lectin signal patterns. The results suggest that the surface glycan structures vary depending on the exosome subpopulations. In conclusion, in this study, we succeeded in establishing the protocol for rapid glycan profiling on the surface of small particles of about 100 nm diameter (exosome). This method is expected to be useful for clarification of biological significance of glycan structures on exosome.

286. Oligomannose-coated liposomes induce activation and maturation of human mononuclear phagocytes in vitro

Y. Matsuoka², Y. Kawauchi², K. Kawauchi¹, Y. Kuroda², N. Kojima²; ¹ Medical Center East, Department of Medicine, Tokyo Women’s Medical University, Tokyo; ²Department of Applied Biochemistry, Tokai University, Hiratsuka

We have shown that administration of liposomes coated with a neoglycolipid containing <1-3,<1-6-mannotriose residues (Man3-DPPE) can induce antigen-specific cellular immunity in mice by delivering both activating/maturation signals and liposome-encased antigens to antigen presenting cells (APCs) in mice. The aim of this study was to assess the potential of the oligomannose-coated lipsomes for in vitro activation/maturation of human APCs. We first evaluated the effect of the liposomes coated with various types of oligomannose-containing neoglycolipids on PMA-stimulated human monocytic THP-1 cells as a model for human APCs. In response to treatment with Man3-DPPE-coated liposomes (Man3-OML), PMA-stimulated THP-1 cells showed enhanced expression of CD40, CD80 and HLA-DR and secreted significant levels of IL-12p40. Among various linkages of Man2-DPPE-coated liposomes, only <1,6-Man2-DPPE-coated liposomes induced these cellular responses. On the other hand, Man5-DPPE-coated liposomes failed to activate the cells. These results suggest that unsubstituted <1-6Man branch in Man3-DPPE is required for in vitro activation of human APCs. Man3-OML-induced IL-12p40 production was not inhibited by BAY11-7082, an inhibitor of the MyD88-dependent signaling network, suggesting that Toll like receptors (TLRs) are not involved in activation of human mononuclear phagocytes by Man3-OMLs.

Then the effect of Man3-OML on human monocyte-derived dendritic cells (moDCs) was assessed. Stimulation of inflammatory monocytes or moDCs with Man3-OMLs also induced enhanced expression of co-stimulatory molecules, HLA-DR, and CCR7, and IL-12p40 production from both types of cells. In response to Man3-OML treatment, moDCs but not inflammatory monocytes produced bioactive IL-12p70, which was enhanced by CD40 ligation. Thus, Man3-OMLs can activate naïve human mononuclear phagocytes and lead human moDCs to a fully matured status in vitro to elicit CTLs and a Th1 response without addition of inflammatory cytokines or TLR agonists.

287. Structure-activity relationship study of tlr4/md-2 ligands that synergistically regulate receptor function

Y. Matsuura², A. Shimoyama¹, K. Fukase¹; ¹1) Grad. Sch. Sci., Osaka Univ. Toyonaka. 2) PRC,. Grad. Sch. Sci., Osaka Univ. Toyonaka, ²Grad. Sch. Sci., Osaka Univ. Toyonaka

LPS (Lipopolysaccharide), one of cell surface component of Gram negative bacteria, is known as a representative innate immune stimulator. The active principal of LPS, lipid A which is a glycolipid present at the terminal of LPS, is recognized by TLR4/MD-2 receptor.

Lipid A is composed by hydrophilic phosphorylated-glucosamine moiety and hydrophobic fatty acid moiety. The balance of these moieties strongly affects to the immune activity of lipid A. For example, hexa-acylated Escherichia coli lipid A is known as strong immune stimulator (potent TLR4 agonist), but its biosynthetic precursor (lipid IVa), composed by less fatty acid moiety, is known as a TLR4 antagonist. Interestingly, we found that lipid IVa shows synergistic effect at lower concentration of lipid IVa to enhance immune stimulation activity of E. coli lipid A. These result suggested the composition of lipid A mixtures deeply affects their function. Therefore, in this study, we prepared a mixture of chemically synthesized pure lipid As and evaluated their biological activities to investigate the effect of compositional heterogeneity of lipid A.

We mixed lipid IVa and its analogues with E. coli lipid A and evaluated their NF-│B activation. We used two kinds of mono-phosphorylated lipid IVa analogs, one is 1-dephosphorylated, the other is 4’-dephosphorylated in addition to 1,4’-diphosphorylated E. coli lipid A and lipid IVa. Lipid IVa and both mono-phosphorylated lipid IVa analogs switched its activity from antagonist to synergist under the lower concentration. In addition, different concentration dependency in synergistic effects was observed amaong lipid IVa (di-phosphorylated) and its mono-phosphorylated analogs. Mono-phosphorylated analogs showed synergistic effects at higher concentration than lipid IVa.

288. Glycated hemoglobin versus fructosamine in the control of type 2 diabetes with iron deficiency anemia

S. Meherhera², S. Abdi¹; ¹Biochemistery laboratory, Department of Medecine, SAAD DAHLAB University of Blida, Blida, ²Biochemistery laboratory, Department of Pharmacy, SAAD DAHLAB University of Blida, Blida

Purpose

Glycated hemoglobin (HbA1C) is the standard biomarker for controlling glycemic levels and managing the risk of long-term complications in diabetic patients. In spite of its advantages, its rate is affected by numerous physiological and pathological variations as seen during renal failure, hemoglobinosis, thalassemia and iron deficiency anemia during which concentrations are falsely higher.

Particular interest was given to fructosamine, a control of the average glycemic level of two to three weeks preceding the dosage and independent of the fluctuations incurred by hemoglobin.

Our present study aims to:

• To determine the impact of iron deficiency anemia on glycated hemoglobin determination;

• Correlation study between glycated hemoglobin and fructosamine in cases and controls and determine the value of fructosamine determination in anemic patients.

Materiel and methods

We conducted a case-control study at the central laboratory of medical analysis, CHU FRANTZ FANON Blida, Algeria between January and May, 2019. We realize different biochemical parameters in cases and controls such as: glycaemia, HbA1C, Fructosamine, iron, ferritin, FNS the statistical study done with excel and SPSS Software

Results and discussion

Our study focused on a series of 100 type 2 diabetic patients, 50 of whom were anemic (cases) with a sex ratio of 0.21 (M/F) and 50 non anemic (controls), with a sex ratio of 0.72 (M/F).

The average age of cases is 59.56 years (62.2 years in controls) with a standard deviation of 11.954 years (9.293years in controls) with no significant difference

The average hemoglobin in cases is 10.80g/dl (SD=0.81) and 13.66g/dl (1.04) in controls with significant difference (p <0.05)

The mean serum ferritin is 27.34μg/l (SD=27.79) in cases which is significantly different from that of controls 104,35μg/l (SD=65.60)

The average glycated hemoglobin in cases is 8.05% (SD=2.11) and 7.77%(SD=1. in controls. The average fructosamine in cases is 432.82 umol/dl (SD=126,21) and 449.11 umol/dl (SD=146,43) in controls. The student test demonstrated the lack of significant difference in these two parameters in the cases and controls with p <0.05.

In anemic diabetics we found no correlation between glycated hemoglobin and fructosamine, with a Bravais-Pearson r equal to 0.226 and a coefficient of determination r² equal to 0. 051; the mean of significance is 0.115.In non-anemic diabetics there is a positive correlation between the two variables “glycated hemoglobin” and “fructosamine” in the controls with a Bravais-Pearson r equal to 0.529 and a coefficient of determination r² equal to 0.277. This correlation is therefore significant with a p less than 0.0001.

Conclusion

Our results confirm that fructosamine values evolve in parallel with those of glycated hemoglobin, but are not subject to variations in hemoglobin hence the usefulness of this parameter in these situations

289. Accumulation of 14c-n-glycolylneuraminic acid in the rat brain after tail vein injection

Y. Mikami¹, A. Minami¹, T. Otsubo², Y. Kurebayashi¹, T. Takahashi¹, K. Ikeda², T. Suzuki¹; ¹Department of Biochemistry, School of Pharmaceutical Science, University of Shizuka, ²Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University

The two major molecular species of sialic acid existing in nature are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Neu5Ac is abundant in mammalian brains and plays crucial roles in many neural functions. In contrast, Neu5Gc is present only at a trace level in vertebrate brains. The brain-specific suppression of Neu5Gc synthesis, which is a common feature in mammals, suggests that Neu5Gc has toxicity against brain functions. However, in vivo kinetics of Neu5Gc in the whole body, especially in the brain, has not been studied in sufficient detail. To determine the in vivo kinetics of Neu5Gc, ¹⁴C-Neu5Gc was enzymatically synthesized and injected into rat tail veins. Although most of ¹⁴C-Neu5Gc was excreted in urine, a small amount of ¹⁴C-Neu5Gc was detected in the brain. Brain autoradiography indicated that ¹⁴C-Neu5Gc was accumulated predominantly in the hippocampus. Most of the ¹⁴C-Neu5Gc transferred into the brain was incorporated into gangliosides including GM1, GD1a, GD1b, GT1b and GQ1b. Reduction of ¹⁴C-Neu5Gc after intracerebroventricular infusion was slower than that of ¹⁴C-Neu5Ac in the brain and hippocampus. These results suggest that Neu5Gc is transferred from blood into the brain across the blood brain barrier and accumulates in the brain.

290. Human <1,4-galactosyltrasferase: influence of n-glycans on enzyme activity

K. Mikolajczyk¹, A. Bereznicka¹, K. Szymczak-Kulus¹, K. Kapczynska², A. Urbaniak¹, R. Kaczmarek¹, M. Czerwinski¹; ¹Laboratory of Glycobiology, Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Science, ²Laboratory of Medical Microbiology, Department of Immunology of Infectious Diseases, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Science

Human <1,4-galactosyltrasferase (Gb3/CD77 or P1/P^k synthase), encoded by the A4GALT gene, transfers galactose from UDP-Gal to glycosphingolipid and glycoprotein acceptors, synthesizing carbohydrate antigens of the human P1PK blood group system. The enzyme with Q at position 211 of polypeptide chain attaches Gal to Gal only, creating Gal<1,4→Gal disaccharide which is a fragment of P^k and P1 antigens. <1,4-galactosyltrasferase with p.Q211E substitution reveals additional acceptor specificity, attaching Gal not only to Gal but also to GalNAc and creating NOR antigen (which terminates with Gal<1,4→GalNAc). The P^k antigen is the main receptor for Shiga toxins, which are secreted by Shigella dysenteriae serotype 1 and enterohaemorrhagic Escherichia coli (EHEC), causing hemorrhagic colitis and haemolytic-uremic syndrome (HUS) in humans. In addition, P^k serves as a receptor for uropathogenic E. coli responsible for pyelonephritis.

Human <1,4-galactosyltrasferase contains two N-glycosylation sites at N121 and N203 (Uniprot Q9NPC4). We demonstrated before that the enzyme expressed in insect cells was inactive after PNGase F treatment, thus N-glycans seem to be necessary for its activity.

In this study, we evaluate <1,4-galactosyltrasferase variants with destroyed N-glycosylation sites (p.S123A, p.T205A and p.S123A/p.T205A) expressed in CHO Lec2 and HEK293E cells. The variants differ by molecular mass, indicating that the wild-type enzyme is N-glycosylated at both sites, with probably more complex N-glycan structure at N121. In addition, the p.S123A variant shows increased activity in comparison to the wild-type control, while p.T205A and p.S123A/p.T205A are less active. These data are corroborated by the treatment of CHO Lec2 cells expressing <1,4-galactosyltrasferase variants with tunicamycin, which caused a decrease of the enzyme activity and its molecular mass. All these findings are the first report about the essential role of N-glycans in maintaining the activity of human <1,4-galactosyltrasferase.

291. Role of sialidase in hippocampal memory processing

A. Minami¹, T. Otsubo², Y. Kurebayashi¹, T. Takahashi¹, K. Ikeda², T. Suzuki¹; ¹Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, ²Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University

Sialic acid, an acidic monosaccharide, is expressed most frequently at the ends of glycans and creates a negative charge on the cell surface. Sialic acid residues in sialylglycoconjugates are removed by sialidase on the extracellular cell surface as well as inside the cell. Here, we showed that sialidase is necessary for normal long-term potentiation (LTP) at hippocampal mossy fiber-CA3 pyramidal cell synapses and for hippocampus-dependent spatial memory. Thus, we investigated in detail the role of sialidase in memory processing. To visualize sialidase activities on the rat hippocampus, we developed a novel benzothiazolylphenol-based sialic acid derivative (BTP3-Neu5Ac) as a highly sensitive histochemical imaging probe for sialidase activity. Sialidase activity detected with BTP3-Neu5Ac was immediately increased in response to LTP-inducible high-frequency stimulation on a time scale of seconds at the CA3 stratum lucidum in acute rat brain slices. Sialidase activity was also increased by neural excitation by high K⁺-induced membrane depolarization. The increase in sialidase activity by neural excitation appears to be caused not by secreted sialidase or by increase in sialidase expression, but by a change in the subcellular localization of sialidase. To obtain direct evidence for sialic acid removal on the extracellular cell surface during neural excitation, extracellular free sialic acid level in the hippocampus was monitored using in vivo microdialysis. The free sialic acid level was increased during hippocampus-dependent memory formation in a contextual fear-conditioning paradigm. Our results show that neural activity-dependent desialylation by sialidase may be involved in hippocampal memory processing.

292. A novel synthetic pathway for alternating glycopolymers as prototypes of glycosaminoglycan mimics

M. Minoda¹; ¹Faculty of Molecular Chemistry and Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology

Glycosaminoglycans (GAGs) are physiologically active polysaccharides consisting of alternating copolymer structure. As for glycopolymers so far synthesized as GAG mimics by controlled radical polymerization, sequence control of repeating units, even alternating structure, is still beyond our reach. This study developed a novel synthetic pathway to attain alternating glycopolymers as prototypes of GAG mimics. It has been recognized that copolymerization of an electron-rich and electron-deficient vinyl monomers can afford alternating copolymers by radical copolymerization. By developing this method, the author has succeeded in the synthesis of well-defined alternating copolymers by RAFT (Reversible Addition Fragmentation chain Transfer) copolymerization of a functionalized vinyl ether (VE) and maleimide (MI). In this study, VEs and MIs having pendant unprotected saccharide moieties were newly synthesized by Huisgen click reaction between alkyne-substituted VE or MI and glycosyl azides. The RAFT copolymerization of maltose-substituted VE (Mal-VE) and ethyl MI (EtMI) was firstly carried out by employing 2-benzyl-sulfanyl thiocarbonyl sulfonyl ethanol as a chain transfer agent and commercial azo initiator VA-044 in water/CH₃CN (4/3, v/v) at 60^oC ([Mal-VE]₀/[EtMI]₀ = 50/50). GPC analysis indicated the formation of copolymers with unimodal GPC traces and MWDs < 1.5. Moreover, M_n of the copolymers proportionally increased as the conversion increased, suggesting the copolymerization proceeded in a controlled manner. Due to the homopolymerizability of EtMI, the copolymers contained a small excess amount of EtMI units. MALDI-TOFMS data indicates the major parts of the copolymer possess alternating structure, accompanied by a very small amount of consecutive sequences of EtMI. However, nearly alternating structure was successfully attained by regulating the monomer feed ratio. It has also demonstrated the synthesis of a glycopolymer from lactose-substituted VE and maltose-substituted MI in a similar way. Thus, RAFT copolymerization of a pair of saccharide-substituted VE and MI is promising synthetic strategy for obtaining alternating glycopolymers mimicking GAGs. In this study, specific interactions between the obtained glycopolymers and lectins were also investigated.

293. Development of a bioassay for the evaluation of synthetic neoglycopeptide based inhibitors of galectin carbohydrate recognition

S.I. Mirallai¹, C. Di Salvo¹, P.V. Murphy¹; ¹School of Chemistry, National University of Ireland Galway, Galway

In recent years, the significance of protein-carbohydrate interactions in several biological processes is becoming better understood. Cell-surface carbohydrates mediate the first contact between the host and the pathogen, for example, through binding to carbohydrate-recognizing proteins, thus being involved in several pathological events. Other interactions mediate cell adhesion or signal transduction and therefore carbohydrate binding proteins are targets for therapeutic intervention.

Galectins are a family of ®-galactoside-recognizing animal lectins, and they are involved in several biological activities such as inflammation, immunity, and cancer. The most ubiquitously expressed members of the galectin family are galectin-1 and galectin-3, and their expression in cancer cell lines is known to contribute to angiogenesis, tumour metastasis and neoplastic transformation. The development and identification of potent inhibitors with high affinity to galectins has been investigated in the Murphy group and glycocluster synthesis has been an area of interest.

This study involved the synthesis of neoglycopeptides which have the potential to self-assemble into structurally defined glycoclusters. In addition, the optimization and development of an effective bioassay, based on surface-immobilised glycoprotein, suitable for the evaluation of the new glycopeptides as ligands for galectin-1 and/or galectin-3 has formed part of the research work and will be presented.

294. LC-MS analysis of glycans in gastric cancer cells forcibly expressing CD44V9 by saccharide primer method

N. Miyoshi¹, H. Tsugawa², H. Suzuki³, T. Sato¹; ¹Department of Sci. and Tech., Keio Univ., Kanagawa; ²School of Med., Keio Univ., Tokyo; ³School of Med., Tokai Univ., Kanagawa

Cancer stem cell (CSC) is one of the causes of cancer recurrence or metastasis. Development of CSC targeting is desired because CSC is resistant to therapeutic agents and has high metastatic potential. CD44 is one of attractive targets for CSC treatment. CD44 is a glycoprotein and one of its isoforms is highly expressed in CSC. Furthermore, changes in glycosylation of CD44 play an important role in cancer recurrence and metastasis. In this study, CSC specific glycans were identified using cancer cells that forcibly expresses gastric CSC-specific CD44-variant9 (CD44v9) . MKN28 gastric cancer cells (wild-type) were employed as control and CD44v9-expressing cells were used to examine the changes in glycosylation. Comparative glycan analysis between wild-type and CD44v9-expressing cells was carried out using saccharide primer method. Saccharide primer is amphiphilic alkylglycoside and elongated glycans are obtained according to the glycan biosynthesis pathway of cells. The comparative glycan analysis using LC-MS revealed that changes in expression of several glycosylated products between CD44v9-expressing cells and wild-type. It was indicated that the expression level of sulfated glycosylated products is significantly increased in CD44v9-expressing cells. In addition, the expression level of glycosyltransferase was estimated by Real Time RT-PCR. The gene analysis revealed that the expression level of galactose-3-O-sulfotransferase that transfers a sulfate to position 3 of non-reducing beta-galactosyl residues was meaningfully increased in CD44v9-expressing cells. From these results, it is indicated that there is a correlation between expression of sulfated glycosylated products and gene expression of sulfotransferase. The sulfated glycosylated products and its glycan related factors such as glycan synthetase maybe candidates as target substances in CSC targeted treatment. In the future, the development of clinical application related to the sulfated glycans for CSC targeted treatment is expected.

295. Comparative study of structure-function relationship of a new family lectins from mytilus genus

T. Mizgina², A. Filshtein², I. Chikalovets², V. Molchanova², K. Hua¹, O. Chernikov²; ¹Department of Biotechnology and Animal Science, National Ilan University, Ilan, Taiwan, ²G.B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS, Vladivostok, Russia

There is considerable interest in glycobiology relating with marine organisms, because they have a rich resource of lectins.

A new lectin family has been proposed as a consequence of the isolation by us of the first member from the mussel Crenomytilus grayanus (CGL), which amino acid sequence does not match with any lectin family that have been reported. The second member of novel lectin family was isolated by Japanese scientists from the bivalve Mytilus galloprovincialis (MytiLec). Recently we have purified the third member from the mussel Mytilus trossulus (MTL). All three lectins consist of about 150 residues with three tandem-repeat domains, having 83-84% homology with each other.

Glycan array analysis was performed to understand more about detailed carbohydrate recognition capability. It was shown that CGL binding with <Gal-terminated glycans is stronger. CGL possessed slight affinity to ®Gal in highly branched glycans only. In the crystal structure, Gal is bound to CGL as an <-anomer, except for Site 3, which is recognized as a ®-anomer. Crystal structure of MTL is still not solved, but it was shown by glycan array analysis and hemagglutination assay preferable binding of ®Gal. The majority of lectins that have been studied for potential medical benefits are specific for ®-linked saccharides, so CGL and MTL are of interest for its unusual ligand specificity.

In view of the finding that Gal terminated glycans are expressed on the membrane of some tumor cells, we studied anti-tumor effect of the lectins. CGL recognized <Gal on the surface of Burkitt`s lymphoma Raji cells (high <Gal expression), leading to dose-dependent cytotoxic effect (IC50 6.8 ⎧g/mL), G2/M phase cell cycle arrest and apoptosis. CGL showed 35% of MCF7 cells viability inhibition at maximal (100 ⎧g/mL) lectin concentration tested (low <Gal, high ®Gal expression). CGL also reduced migration and colony formation of MCF7 cells.

It was found that MTL exhibited cytotoxic activity at a concentration of 100 ⎧g/ml against to MCF-7, MDA-231 and Daudi cells, the percentage of living cells after was 35%, 65% and 31% respectively.

Overall, our findings suggest the use of CGL and MTL in cancer diagnosis and treatment.

The investigation was partially supported by RFBR, project number 19-04-00157.

296. RAMP1 is c-mannosylated at TRP56 in cultured human cells

H. Mizuta¹, A. Takakusaki¹, T. Suzuki², K. Otake¹, K. Miura¹, N. Dohmae², S. Simizu¹; ¹Keio university, ²RIKEN

C-mannosylation is a unique type of glycosylation that is a binding between an <-mannose and the indole C2 carbon of the first tryptophan residue of the consensus sequence (Trp-Xaa-Xaa-Trp/Cys) via a C-C linkage. This modification was first reported in 1994, and about 30 proteins have been identified as C-mannosylated proteins in human so far. Whereas C-mannosylation affects protein secretory level, stability and intracellular localization, the association with C-mannosylation and disease including cancer has not been fully understood. Additionally, previous researches demonstrated dpy19 as a C-mannosyltransferase for C.elegans. Furthermore, both DPY19L1 and DPY19L3, mammalian homologs are also identified as C-mannosyltransferases. However, these transferases are common identified in the case of C-mannosylated in thrombospondin type-1 repeat domain. Thus, it is unclear whether DPY19 family proteins (DPY19L1-L4) have a C-mannosyltransferase activity against other type of proteins.

In this study we focused on receptor activity modifying protein 1 (RAMP1) as a candidate for C-mannosylated protein that has a putative C-mannosylation site at Trp56 (Trp56-Cys-Asp-Trp) in the extracellular domain (ECD). RAMP1 is known as a receptor for calcitonin gene-related peptide (CGRP) by forming a complex with calcitonin receptor-like receptor (CRLR). This RAMP1/CRLR complex regulates CGRP signaling. To elucidate whether RAMP1 is C-mannosylated or not, we first established HT1080 cell line overexpressing RAMP1-ECD. The reason is that it is easy to purify RAMP1-ECD from cell conditioned media compared with full length RAMP1 from cell lysates. Then we purified RAMP1-ECD and this samples were analyzed by LC-MS/MS. As a result, RAMP1 turned out to be C-mannosylated at Trp56 as we expected. Henceforth, to clear the role of C-mannosylation of RAMP1, we plan to establish the C-mannosylation-defect RAMP1-expresing cells.

297. N-glycan analysis of the microalga chlorella by MALDI-TOF and LC-ESI-MS

R.T. Mócsai¹, R. Figl¹, P. Kosma¹, F. Altmann¹; ¹Department of Chemistry, University of Natural Resources and Life Sciences, Vienna

Microalgae constantly prove to be a surprising branch of the plant kingdom, representing hitherto not seen N-glycan structures separating them not only from other eukaryotes but also from land plants. O-methylated carbohydrate structures have been found in various organisms like bacteria, fungi, non-vascular plants, nematodes and gastropods. However, apart from the impressive amount and diversity of O-methylation, Chlorellaceae show other specific features like arabinose (as both pyranose and furanose), galactose linked to mannose and the lack of plant-specific core alpha(1,3)-linked fucose.

In this work we aimed at the analysis of N-glycan structures of different Chlorella strains by MALDI-TOF-MS/MS, LC-ESI-MS/MS, and GC-MS.

Recently, we published that the N-glycome of Chlorella vulgaris consists exclusively of oligomannosidic structures that are methylated in the form of terminal 3-O-methyl-mannose and 3,6-di-O-methyl-mannose. Chlorella sorokiniana, however, contained a novel N-glycan with a monoisotopic mass of 1336.455 Da (without methylation) and a possible composition of Hex4HexNAc2Pent2. The most abundant glycan form (1378.502 Da) contained three O-methyl groups.

Interestingly, these closely related species show very different glycan profiles that could imply a wider genetic diversity than previously thought.

These novel structures foresee a large variety of unknown glycosyltransferases and new, potentially immunogenic glycan structures. N-glycan patterns suggest that these findings could constitute a valuable tool for taxonomic classification as well as for quality control and determination of origin of microalgal food supplements.

298. Heteromultivalent rotaxanes as pseudomonas aeruginosa antibiofilm agents

T. Mohy El Dine², R. Jimmidi², E. Gillon¹, A. Imberty¹, S.P. Vincent²; ¹CERMAV (UPR5301), CNRS and Université Grenoble Alpes, Grenoble (France), ²Laboratoire de Chimie Bio-Organique (CBO), Département de Chimie, Université de Namur, Namur (Belgium)

Our project aims at designing and synthesizing novel antiadhesives/antibiofilm agents based on heterovalent multimeric scaffolds (heteroglycorotaxanes) that would act as ligands of LecA and LecB of Pseudomonas aeruginosa (PA).

A variety of biological processes exploit the enhancement of binding affinities of a ligand for its receptors owing to the multivalent nature of this ligand. Protein-carbohydrate interactions are highly selective and frequently mediate the first step of the infection process for many pathogens including viruses, fungi, bacteria, and bacterial toxins.¹ However, individual interactions between sugars and proteins are very weak.² This poor binding affinity can be fully compensated by multiple simultaneous interactions between carbohydrate ligands and their receptors: cluster/multivalent effect.³

Taking advantage of this phenomenon, our laboratory has synthesized a variety of multivalent glycoclusters including pillar-arenes⁴ and rotaxanes⁵ which target the two bacterial lectins from the opportunistic PA.

Thanks to the inherent supramolecular nature of pillar-arene-based rotaxanes, we were able to efficiently synthesize a series of [2]-rotaxanes bearing 10 galactosides on the pillar central scaffold and a central axle displaying a tetrameric fucoside, on one side, and a polycationic peptide on the other side. The binding properties of these scaffolds towards the two lectins have been evaluated by Isothermal Titration Calorimetry (ITC). As a result, our ligands were capable of binding up to 6 LecA and 5 LecB monomers. Furthermore, the best molecules displayed antibiofilm properties against the PA PAO1 strain with a maximal of 90% inhibition.

References:

[1] P. I. Kitov, J. M. Sadowska, G. Mulvey, G. D. Armstrong, H. Ling, N. S. Pannu, R. J. Read and D. R. Bundle, Nature 2000, 403, 669-672.

[2] Y. C. Lee and R. T. Lee, Acc. Chem. Res., 1995, 28, 321-326.

[3] M. Lahmann, Top Curr. Chem., 2009, 288, 17-65.

[4] K. Buffet, I. Nierengarten, N. Galanos, E. Gillon, M. Holler, A. Imberty, S. E. Matthews, S. Vidal, S. P. Vincent and J.-F. Nierengarten, Chem. Eur. J., 2016, 22, 2955-2963.

[5] S. P. Vincent, K. Buffet, I. Nierengarten, A. Imberty, and J.-F. Nierengarten, Chem. Eur. J., 2016, 22, 88-92.

299. Analysis of recombinant follicle-stimulating hormone isoforms by capillary electrophresis

V. Monakhova¹, M. Sushkin¹, A.S. Simbirtsev¹; ¹State Research Institute of Highly Pure Biopreparations, Saint-Petersburg, Russia

Follicle-stimulating hormone (FSH) is a member of the glycoprotein hormone family and plays a key role in the development and functioning of reproductive systems. This hormone contains about 35% of saccharides that have effect on protein biological activity and pharmacokinetics. Different conditions, such as a source of hormone, purification procedures, and storage conditions may change the FSH isoforms.

Recombinant human FSH (rhFSH) is widely used in clinical practice, so it is important to control its glycosylation profile. Different methods have been already used for glycan analysis of rhFSH. Isoelectric focusing reveals the information about pI isoforms, high-performance chromatography provides data about composition of glycan brunches. Detailed glycan analysis can be implemented by mass spectrometry. This study is devoted to analysis of recombinant follicle-stimulating hormone isoforms by capillary electrophoresis.

The method of capillary zone electrophoresis was developed. Background electrolyte (BGE) for analysis of erythropoietin sialylated isoforms was initially tested. Resolution of six peaks was confirmed but the third peak of FSH isoforms interfered with a system peak. To solve this problem, different components of BGE were consistently removed and the optimal pH was chosen. Capillary isoelectric focusing was also used for pI isoform determination. rhFSH was found to be a highly heterological protein with the pI range 3.8-5.3.

Optimized methods of capillary electrophoresis allow ones to analyze rhFSH isoforms without time-consuming procedures of derivatization and could potentially be used for quality control tests or during process optimization.

300. Total synthesis of native cholesteryl phosphatidyl and lysophosphatidyl <-glucosides of helicobacter pylori

C.C. Chang¹, S. Mondal¹, C.H. Lin²,K.K. Mong¹; ¹Department of Applied Chemistry, National Chiao Tung University, Hsinchu, ²Institute of Biological Chemistry, Academia Sinica, Taipei

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the stomach of over 50% of the world population (1). Although majority of the carriers are asymptomatic, chronic infection with the bacteria is associated with peptic and duodenal ulcer, and gastric carcinoma. 25% of the total lipids in H. pylori are cholesteryl <-glucoside (<CG) derivatives (2). These <CG derivatives have been proven to play important roles in the bacterial infection and the subsequent pathogenic process (3). Thus, they have become attractive targets in organic synthesis (4).

Herein, we report the total synthesis of native cholesteryl 6’-O-phosphatidyl-<-glucosides (CPG) and cholesteryl 6’-O-lysophosphatidyl-<-glucosides (lyso CPG), which are the predominant <CG derivatives produced by H. pylori. A unique feature of the CPG and lyso CPG is the presence of a chiral dihydrosterculic acid (cyclo 19:0).

The key reactions in preparation of the chiral dihydrosterculic acid include Rh(II)-catalysed cyclopropenylation, chiral assisted resolution of enantiomers, and Julia-Kocienski olefination. 1-decyne and cycloheptanone are the starting substrates, which are converted to desired enantiomers of dihydrosterculic acid in overall eleven linear steps.

With the desired fatty acids in hands, 1-O-acyl sn glycerol or mixed 1,2-O-diacyl sn glycerol with the designated fatty acid components can be prepared, which are coupled with the cholesteryl <-glucoside via a phosphodiester bond (5). In this regard, the phosphonate chemistry is found effective. Ultimately, we are able to complete the first total synthesis of native CPG and lyso CPG compounds with the major fatty acid components. Currently, we are working with the collaborators to explore their biophysical properties.

References:

(1) K. E. L. McColl, N. Eng. J. Med. 2010, 362, 1597-1604.

(2) Y. Hirai, M. Haque, T. Yoshida, K. Yokota, T. Yasuda, K. Oguma. J. Bacteriol. 1995, 177, 5327-5333.

(3) H.-M. Jan, Y.-C. Chen, Y.-Y. Shih, Y.-C. Huang, Z. Tu, A. B. Ingle, S.-W. Liu, M.-S. Wu, J. Gervay-Hague, K.-K. T. Mong, C.-H. Lin, Chem. Sci. 2016, 6208-6216.

(4) H.-Q. Nguyen, R. A. Davis, J. Gervay-Hague, Angew. Chem. Int. Ed. 2014, 53, 13400-13403.

(5) J.-C. Hu, A.-F. Feng, B.-Y. Chang, C.-H. Lin, K.-K T. Mong, Org. Biomol. Chem. 2017, 15, 5345-5356.

301. A small library of compounds, containing the common disaccharide of streptococcus pneumoniae 19f and 19a capsular polysaccharides, as novel non-natural antibacterial saccharide antigens.

L. Morelli², F. Chiodo ¹, L. Lay ³, F. Compostella ²

¹Amsterdam Infection and Immunity Institute, Amsterdam UMC, The Netherlands, ²Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Via Saldini 50, 20133, Milano, ³Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133, Milano

Pneumonia is one of the most serious respiratory infection worldwide, and it is mainly caused by Streptococcus pneumoniae (SP) bacterium. The polysaccharide capsule (CPS) of SP is a major virulence factor. Commercially available vaccines are made of purified CPS fragments of the most virulent serotypes. Pneumococcal conjugate vaccines (PCVs) work well and, even if they are not able to prevent all infections, they have substantially reduced the incidence of invasive pneumococcal disease caused by the most virulent serotypes. However, ninety-eight different pneumococcal serotypes have been identified, classified into serogroups on the base of structural, biochemical and genetic differences in the CPS structure. Serotype replacement with emergent serogroups, not included in vaccine formulations, remains a concern. Since we are still distant from achieving a universal not serotype-dependent vaccine, we have considered to simplify PCV formulations designing novel non-natural saccharide antigens common to more than one serotype. In this context, we envisaged a potential antigen composed of common structures shared by different serotypes. In particular, SP 19F and 19A CPS repeating units share a common structure, the disaccharide ManNAc-®-(1→4)-Glc, that can be considered the parent motif for the selection of a “common epitope”. Therefore, we have synthesized a small library of compounds, containing different combinations of the common 19F/19A disaccharide and bearing an aminopropyl spacer at the reducing end to allow conjugation. Each compound will be biologically evaluated through glycan-arrays screening to reveal the presence of common antibodies protective both for 19F and 19A serotypes.

302. Analysis of polysia-ncam derived from embryonic and adult brains by sds-page/native-page map

A. Mori¹, Y. Yang¹, M. Hane¹, K. Kitajima¹, C. Sato¹; ¹Biosci. Biotech. Center, Nagoya Univ.; Grd. Sch. Bioagr. Sci., Nagoya Univ., Nagoya

Polysialic acid (polySia), which mainly occurs on the neural cell adhesion molecule (NCAM), is observed in whole embryonic brain as well as in restricted areas of adult brain, including hippocampus and olfactory systems. PolySia shows not only an anti-adhesive effect on NCAM-involved cell-cell interactions due to its bulky and hydrated properties, but also a reservoir function for neurologically active molecules. Recently, it has been reported that polySia-NCAM is involved in various mental disorders. The quantity and quality of polySia-NCAM are highly regulated and essential for normal brain functions. Therefore, to understand brain development properly, it is important to determine the structures of polySia-NCAM, which differ depending on development stages. Our objective is, thus, to elucidate the structures of polySia-NCAM from embryonic and adult brains, focusing on regulatory mechanisms of their biosynthesis.

First, we analyzed polySia-NCAM derived from mouse adult and embryonic brains using anion-exchange chromatography and gel filtration chromatography. Adult brain polySia-NCAM was found to include a larger negative charge as a monomer than embryonic brain polySia-NCAM. We also analyzed using the SDS-PAGE/Native-PAGE Map method, which was established to elucidate subtle differences of the polySia-NCAM structure, and revealed that polySia-NCAMs derived from adult and embryonic brains showed different properties. These results led us to assume that sulfation occurs on adult brain polySia-NCAM. Then, we subjected mouse whole brain homogenates of different developmental stages to Keratanase digestion and analyzed for polySia-NCAM structure by Western blotting using the polySia-specific antibody 12E3. As a result, the polySia staining was altered by the Keratanase digestion, suggesting that keratan sulfate (KS) chain exits on polySia-NCAM. All these data indicated that the structures of polySia-NCAM are different between adult and embryonic brains and suggest a new regulatory mechanism of polySia-NCAM mediated by KS chains.

303. Perinatal stress programs hippocampal glutamatergic synaptopathy and the n-glycome profile associated with reduction in polysialylated molecules

C. Clarisse⁴, S. Morley Fletcher², G. Van Camp², H. Bouwalerh², A. Harduin-Lepers⁵, S. Groux-Degroote⁶, F. Nicoletti¹, Y. Guerardel⁴, S. Maccari³; ¹Department of Physiology and Pharmacology “V. Erspamer”, University Sapienza of Rome, IT; IRCCS Neuromed, Pozzilli, IT, ²Univ. Lille, CNRS, UMR8576 UGSF Team Glycobiology of Stress-related disorders, FR, ³Univ. Lille, CNRS, UMR8576 UGSF Team Glycobiology of Stress-related disorders, FR; Department of Science and Medical – Surgical Biotechnology, University Sapienza of Rome, IT, ⁴Univ. Lille, CNRS, UMR8576 UGSF Team Structural Glycobiology of host-pathogen interactions, FR, ⁵Univ. Lille, CNRS, UMR8576 UGSF, Team Regulation of terminal glycosylation, FR, ⁶Univ. Lille, CNRS, UMR8576 UGSF, Team Regulation of terminal glycosylation, FR

In the Perinatal stress (PRS) rat model we have shown persistent hippocampal glutamate synaptopathy causally related to reduced risk-taking behavior. Interestingly, PRS rats present reduced hippocampal neurogenesis associated with a lower number of PSA-NCAM/BrdU+ cells. Polysialylated NCAM (PSA-NCAM) appears to be a hallmark of brain glycome that is modulated by chronic stress. The molecular nature of the PRS phenomenon has exclusively addressed the epigenomics related to maternal behavior with no investigation on epiproteomics such as glycomics. Indeed, there are no studies on brain glycosylation in the context of perinatal stress programming. Glycosylation of proteins and complex glycolipids plays a vital role in neuronal function and it may represent a linking bridge among all intracellular events. Therefore, we first performed a glycomic analysis using mass spectrometry of the glycolipids and glycans isolated from glycoproteins in the hippocampus of PRS male adult rats. This study revealed 24 different sialylated sphingolipids, all belonging to the ganglioside family, with no qualitative or quantitative difference between non-stressed and PRS rats. For proteins, mass spectrometry showed the presence of 62 different N-glycans which sequences were established by MS/MS. Relative quantification of individual signals showed that PRS increased the proportion of sialylated N-glycans but showed no effect on fucosylation. HPLC quantification indicated that PRS specifically increased sialylation of N-glycosylproteins while it reduced the levels of protein-associated polysialic acids, consistently with our data on PSA-NCAM. Second, we separately measured PSA levels in NCAM protein in order to link reduced polysialylation in PRS rats and hippocampal plasticity. Third, we investigated the expression of key sialyltransferase genes involved in the <2,3-sialylation (ST3GAL3, ST3GAL4, and ST3GAL6), in the polysialylation of NCAM N-glycans (ST8SIA2 and ST8SIA4), as well of sialidases (Neu1, Neu2, Neu3 and Neu4), to investigate the epigenetic machinery. The study of the sialylation status of proteins paves the way to the understanding of glycomic programming by early life stress of glutamate-dependent synaptic plasticity processes.

304. Glycans as biomarkers and therapeutic targets in prostate cancer

E. Scott², K. Livermore², E. Dennis², R. Garnham², S. Pitteri³, N. Wang¹, D. Elliott²,J. Munkley²; ¹Department of Oncology & Metabolism, University of Sheffield, UK, ²Institute of Genetic Medicine, University of Newcastle, UK, ³Stanford University School of Medicine, United States

Prostate cancer is the most common cancer in men, claiming over 350,000 lives worldwide annually. Current diagnosis relies on prostate-specific antigen (PSA) testing (which has low specificity and no prognostic value at diagnosis), and tissue biopsies (which are expensive, can be painful and only provide a snapshot of the tumour). A major problem is that it is difficult to distinguish indolent and aggressive prostate cancer. This leads to over-diagnosis, resulting in unnecessary biopsies and treatments for non-harmful cancers, but also means some aggressive cancers are missed. Upon diagnosis the growth of the majority of prostate cancers is driven by androgen steroid hormones, and the first line of treatment for advanced disease is usually androgen-deprivation therapy (ADT). Although this is usually initially effective the majority of men eventually relapse into a castrate resistant form of the disease that is ultimately lethal. Hence, there is an urgent unmet clinical need to identify new diagnostic and prognostic biomarkers, and to develop new treatments for advanced disease.

The prostate is an abundant secretor of glycoproteins of all types, and alterations in glycans are attractive as potential diagnostic and therapeutic targets. We recently identified glycosylation as a global target for androgen control in prostate cancer and further defined a set of glycosylation enzymes that are under direct control of the androgen receptor (AR). Importantly, these enzymes are linked to the synthesis of several cancer-associated glycans, which we predict can be exploited to improve the diagnosis and treatment of prostate cancer.

We are using in vitro and in vivo models to study how key glycosylation enzymes control important aspects of prostate cancer cell behaviour. Alongside this we are integrating multi-omic studies (of the transcriptome, proteome and glycome) to identify the mechanisms involved, testing the activity of existing glycan therapies on prostate cancer growth and spread, and monitoring the expression of glycosylation enzymes and downstream glycans in patient tissue, serum and urine. Here I will present examples of glycosylation enzymes we are investigating as diagnostic and therapeutic targets and discuss our exciting unpublished findings.

305. A novel structure of gpi anchored proteins; the ethanolamine phosphate on the second mannose bridges proteins

Y. Murakami¹, M. Ishida¹, T. Kinoshita¹; ¹Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

Glycosylphosphatidylinositol (GPI) is a glycolipid, which anchors 150 or more proteins to the mammalian cell surface. There are at least 27 genes involved in the biosynthesis and transport of GPI-anchored proteins (GPI-APs). Among them, PIGO is involved in the attachment of the ethanolamine phosphate (EthN-P) to the third mannose, which is transferred by PIGB. This bridging ethanolamine plays an important role by being covalently linked to the carboxyl terminal of the precursor proteins. Recently, we found that the HEK293 cells with complete knockout of PIGO or PIGB gene unexpectedly showed partial expression of GPI-APs, which is completely removed by further knockout of PIGG. PIGG is involved in the attachment of the EthN-P to the second mannose, which is removed soon after GPI is attached to the precursor protein by PGAP5. In PIGG knockout cells, GPI-APs are expressed with the normal structure at normal level, so physiological significance of this transient modification has been unclear. However, the PIGG deficient individuals showed intellectual disability, hypotonia, and early-onset seizure, suggesting the important role of PIGG function. Our results indicate that the EthN-P on the second mannose acts as the bridging ethanolamine. We speculate that this structure is also important in the normal individuals, and it is missing in PIGG deficient individuals, leading to cause the various symptoms. It has been believed that the precursor proteins are always linked to the EthN-P on the third mannose in mammalian cells and all other organisms except Entamoeba histolytica proteophosphoglycan that has only two mannoses. Our current data challenges this view. We are now analyzing the GPI structures expressed on the PIGO or PIGB knockout cells by mass spectrometry to demonstrate protein-linkage to the second mannose. Furthermore, using PIGG/PIGO double knockout cells, we have established a simple method for the functional analysis of PIGG. We can measure the PIGG activity with the flow cytometric analysis of GPI-AP-expression after transfecting the mutant PIGG expressing plasmid to PIGG/PIGO double knockout cells. This is a simpler and more sensitive assay than the previous analysis that utilizes radiolabeled mannose to detect the GPI intermediates.

306. Immunochemical method for identification of advanced glycation end products (ages)

M. Nagai¹, R. Ohno¹, J. Shirakawa¹, H. Sugawa¹, N. Katsuta¹, S. Sakake¹, S. Tanaka¹, Y. Tominaga¹, R. Nagai¹; ¹Laboratory of Food and Regulation Biology, Graduate School of Bioscience, Tokai University

Advanced glycation end-products (AGEs) were originally studied in a field of food chemistry as AGEs gives brown in color and savory flavor. AGE analysis has been broaden in the field of medicine since Monnier et al. (Proc. Natl. Acad. Sci. USA 1984) demonstrated that the AGE-like fluorescent intensity in the dura mater of the human brain increased by the pathogenesis of diabetes. This result indicates that accumulation of AGEs is related to pathogenesis of several age-related diseases.

To clarify the biological significance of AGEs in vivo, we obtained several epitope-identified monoclonal antibodies against AGEs. The antibodies have made it possible and easier to identify localization of AGEs, elucidate the formation pathways and screen multiple samples at once for the inhibitors of AGEs formation. One of the immunochemical detection in our previous study is to clarify the localization of GA-pyridine and CML in atherosclerotic lesion. CML was noted not only in the cytoplasm of formy macrophages, but also in the extracellular matrices. On the other hand, GA-pyridine was localized exclusively in the cytoplasm of formy macrophages.

Although there are other strong detection methods such as high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS), some AGEs are estimated lower than it should be due to its positive charge and stability against acid hydrolysis, whereas immunochemical measurement of AGEs in the physiological samples can be influenced by potential artifacts due to pretreatment steps, such as heating and alkaline treatment.

As each method has its own advantages and disadvantages, it is necessary to select a method depending on a purpose.

307. Identification of new ages as markers for metabolic abnormalities and their relation with diabetes

R. Nagai⁴, R. Ohno⁴, J. Shirakawa⁴, H. Sugawa⁴, R. Suzuki³, N. Katsuta⁴, S. Sakake⁴, S. Tanaka⁴, Y. Tominaga⁴, M. Nagai⁴, Y. Yamaguchi⁵, E. Ito¹, N. Taniguchi²; ¹Department of Diabetic Complications Diabetes Research Center, Research Institute National Center for Global Health and Medicine; ²Department of Glyco-Oncology and Medical Biochemistry, Osaka International Cancer Institute; ³Department of Orthopaedic Surgery, Jikei University; ⁴Laboratory of Food and Regulation Biology, Graduate School of Bioscience, Tokai University; ⁵Structural Glycobiology Team, Systems Glycobiology Research Group, Global Research Cluster, RIKEN Advanced Science Institute

Enzymatic glycosylation gives a variety of structural and functional roles to proteins, whereas non-enzymatic glycation of proteins can cause denaturation of structural proteins, decrease in enzyme activity, and dysregulation of metabolic pathways. In a non-enzymatic glycation process called Maillard reaction, carbonyls react with free amino residues of proteins and form Schiff bases and Amadori rearrangement products, which can undergo further changes to the form irreversible advanced glycation end-products (AGEs). Accumulation of AGEs is found in several tissues, including the kidneys of patients with diabetic nephropathy and chronic renal failure, and arterial walls of patients with atherosclerotic lesions. The level of AGEs was initially estimated based on the brown color, fluorescent intensity, and immunochemical methods using anti-AGEs antibodies. However, previous studies using fluorescent and immunochemical methods have not entirely identified the AGE structures. Subsequent investigations by instrumental analyses have revealed many AGE structures such as N^ε-(carboxymethyl)lysine (CML), one of the major antigenic and oxidation-derived AGE structures, and methylglyoxal-hydroimidazolone, generated from the glycolysis pathway through methylglyoxal. Both these compounds are used as markers for oxidation and defective carbohydrate metabolism. We previously demonstrated that GA-pyridine generated by the myeloperoxidase system through glycolaldehyde, accumulates in human atherosclerosis lesions. Furthermore, we also reported that S-(2-succinyl)cysteine (2SC), the product of chemical modification of thiol residues by the Krebs cycle intermediate, fumarate, generated during maturation of adipocytes, inhibits the secretion of adiponectin. 2SC can also be used as a marker for mitochondrial malfunction in vivo. Recently, we identified a fructose-derived AGE structure. Analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrated higher accumulation of fructose-derived AGE in lens proteins compared to CML in diabetic rats. Multiple measurements of several characteristic AGEs might, therefore, be useful biomarkers to identify metabolic abnormalities.

308. O-beta-n-acetylglucosaminylation modulates the binding specificity of odorant-binding protein to social odors and pheromones

A. Joly², M. Houdou¹, C. Spriet¹, F. Foulquier¹, P. Nagnan-Le Meillour², ¹Unité de Glycobiologie Structurale et Fonctionnelle, UMR8576 CNRS, Univ. Lille, ²Unité de Glycobiologie Structurale et Fonctionnelle, UMR8576 CNRS, Univ. Lille, USC INRA 1409

While being an essential sense, the molecular mechanisms by which olfaction occurs are not known. The discovery of olfactory receptors embedded in the membrane of olfactory receptor neurons twenty years ago gave hope and opportunities (Axel & Buck, Nobel Price 2002). However, and despite a growing interest, they are still orphan. Their ligand could either be the odorant molecule itself, or the odorant molecule bound to extracellular soluble proteins named Odorant-Binding Protein (OBP). OBP are indeed secreted by Bowman’s glands of the olfactory epithelium into the nasal cavity, where they constitute the major part of the olfactory secretome. To add an extra layer of complexity, the olfactory secretome composition varies according to the physiological status of animals (e. g. sex or puberty), suggesting that expression of specific proteins is under hormonal control (testosterone, estrogens). Thus, olfactory secretome can be considered as a phenotype of animal’s receptivity to odors and pheromones involved in social behaviors, such as reproduction, maintain of hierarchy, or care to the litter.

In pig species, the olfactory secretome is composed of c. a. 30 OBP isoforms coming from three different genes. Very interestingly we observed that OBP could undergo unusual post-translational modifications for secreted proteins, such as phosphorylation and O-GlcNAcylation (Nagnan-Le Meillour et al., 2014). We recently demonstrated that this diversity could govern/modulate the binding specificity of OBP isoforms to odorant and/or pheromone molecules (Bouclon et al., 2017; Nagnan-Le Meillour et al., 2019).

A ER-resident glycosyltransferase, EOGT, responsible for the O-GlcNAcylation of secreted proteins has been first characterized in Drosophila (Sakaidani et al., 2011), then in mouse (Sakaidani et al., 2012) and in humans (Ogawa et al., 2015). We have recently cloned the porcine gene homolog of Human EOGT (87% identity) in order to decipher the involvement of EOGT in OBP O-GlcNAcylation. To assess this point, porcine EOGT and its KHDEL mutant have been expressed in CHO and HeLa cells and were tested for their ability to transfer GlcNAc residues on recombinant, not O-GlcNAcylated OBP produced in yeast.

309. Identification and characterization of udp-mannose in human cell lines and mouse organs: differential distribution across brain regions and organs

K. Nakajima⁵, Y. Kizuka¹, Y. Yamaguchi⁷, Y. Hirabayashi⁶, K. Takahashi², Y. Yuzawa⁴, N. Taniguchi³; ¹Center for highly advanced integration of nano and life sciences (G-chain), Japan, ²Department of Anatomy II, Fujita Health University, ³Department of Glyco-Oncology and Medical Biochemistry, Osaka International Cancer Institute, Japan, ⁴Department of Nephrology, Fujita Health University, ⁵Research Promotion and Support Headquarters, Fujita Health University, Toyoake, Aichi, Japan, ⁶RIKEN Brain Science Institute, ⁷Tohoku Medical and Pharmaceutical University

Nucleotide sugars are donor substrates for glycosyltransferase and are thus vital regulators of glycosylation. Guanosine diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for mannosylation and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the synthesis of glycosylphosphatidylinositol-anchor. Yet, very few nucleotide sugars have been identified in mammals compared to other organisms. We hypothesized that mammals possess previously unidentified nucleotide sugars that regulate mammalian glycosylation.

Here, we identified low-abundant nucleotide sugars by using an improved LC-ESI-MS/MS method that we described previously (Nakajima et al. 2010, 2013). We for the first time detected low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate, exists in mammals. The cellular UDP-Man level was elevated in a mannose dose-dependent manner. Liquid chromatography-mass spectrometry analyses showed that mouse brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to other organs. These findings indicate that in mammals the minor nucleotide sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions. Because only a limited number of nucleotide sugars are known to be involved in glycosylation, the discovery of UDP-Man in mammals will provide new insights into our understanding of novel regulatory mechanisms of glycosylation.

Nakajima et al. Biochem. Biophys. Res. Commun. (2018) 495(1):401-407.

310. Design and synthesis of chemical probe for o-glcnacylated proteomic study.

G. Nam¹, E.J. Kim³, S.H. Seo², J.A. Honover⁴; ¹Center for Neuro-medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, South Korea, ²Convergence Research Center for Diagnosos, Treatment and Care System of Dementia, Korea Institute of Science and Technology (KIST), Seoul, South Korea, ³Daegu University, Department of Science Education, Gyeongsan-si, Gyeongsanbuk-do, South Korea, ⁴National Institute of Health (NIH), ICBB, NIDDK, Bethesda, MD, USA

O-GlcNAc modification of protein which is known as the addition/remove of N-acetylglucosamine one molecule to serine and threonine residues of proteins is one of the important post-translational modification of nucleocytoplasmic proteins. Dysregulation of O-GlcNAcylation has been implicated in diseases including type II diabetes, cancer, and neurodegenerative disease.

Identification of the modification sites of low-abundance O-GlcNAc modified proteins is a very challenging task and often necessitates the enrichment of target proteins/peptides from other nonglycosylated species. Among the several applied methods, most widely used bifunctional molecular probes containing a chemical-reporter-reactive moiety at one terminal and an affinity tag (such as biotin) at the other has some negative effects as such when the bound proteins/peptides are released because of harsh conditions required to interrupt such a strong interaction and when avidin-like molecules present in the samples. To overcome these problems, we designed and synthesized a chemical probe for a potential molecular tool for enriching O-GlcNAc proteins/peptides. Three-functional molecular probe consists of a chemical-reporter-capture group, a cleavable linker, and an affinity tag. We applied the azo linkers have recently attracted much attention since they are able to undergo cleavage following treatment with sodium dithionite (Na2S2O4), a mild and potentially bio-orthogonal reducing agent. In addition, we chose to use halo-tag ligand-protein chemistry which allows the covalent tethering of organic probes directly to halo-tag protein beads as an affinity tool. Here we describe the design and synthesis of this novel trifunctional molecular probe which is expected to be useful for O-GlcNAc proteomic study.

311. Glycome analysis of zebrafish embryos

S. Natsuka¹, K. Hanzawa¹, T. Sawa¹; ¹Niigata University

We had comprehensibly analyzed N-glycan structures of zebrafish embryos in several developmental stages (1). In this study, we performed analyses of N-glycome and morphogenetic abnormality of the embryos that were treated with glycosidase inhibitors. The treatment with kifunensin or 1-deoxymannojirimycin that inhibits ER-mannosidase-I presented quite similar morphogenetic abnormality in pharyngeal stage, and the embryos were lethal. Similar observation was also obtained from mgat1-morpholino treated embryos. In these glycosidase-treated embryos, complex-type N-glycans were drastically reduced. On the other hand, embryos treated with swainsonine, a Golgi-mannosidase-II inhibitor developed normally. Although complex-type N-glycans drastically decreased also in the swainsonine-treated embryos, hybrid-type increased, as if to compensate for complex-type. This results suggest that common antenna structure(s) on the complex and hybrid-type glycans is/are essential for normal embryonic development of zebrafish. The pharyngeal stage, in which the abnormality occurs, is the period when the shape characteristic of vertebrates is formed. Type 2-lactosamine structure (Galß1-4GlcNAc) common to complex and hybrid types is also specific in vertebrates. As a next step of the study in order to elucidate the biological function of complex-type glycans, we are trying to analyze comprehensively complex-type bearing glycoproteins in zebrafish embryo.

(1) Hanzawa, K., Suzuki, N., Natsuka, S. Structures and developmental alterations of N-glycans of zebrafish embryos. Glycobiology, 27 (3) 228-245 (2017).

312. Analysis of electrostatic interaction between insulin receptor and ganglioside gm3 using transmembrane peptide

Y. Nimura², K. Kabayama¹, Y. Asahina³, S. Hanashima², H. Hojo³, M. Murata², K. Fukase¹; ¹1)Grad. Sch. of Sci., Osaka Univ., Toyonaka, 2) PRC, Grad. Sch. of Sci., Osaka Univ., Toyonaka, 3)Inst. for Radiation Sciences, Osaka Univ., Suita, ²Grad. Sch. of Sci., Osaka Univ., Toyonaka, ³Inst. for Protein Research, Osaka Univ., Suita

The insulin receptor is a single transmembrane protein that regulates blood glucose level. The reduction of sensitivity to insulin leads to type 2 diabetes. It has been suggested that the overexpression and the accumulation of ganglioside GM3 on cell membrane reduces insulin signal in type 2 diabetes. The interaction of GM3 with the insulin receptor is considered to be due to an electrostatic interaction between a negative charge of GM3 and a positive charge of the insulin receptor. However, the detailed analysis has not been elucidated because of the complexity of the cell membrane.

In order to observe this interaction, we constructed a model system by incorporating the transmembrane peptide of the insulin receptor into liposomes.

First, in order to clarify interaction of these molecules in liposomes, we studied the optimal conditions of phase-separating liposomes containing GM3. We found that liposomes consisting of DOPC, GM3 and cholesterol did not undergo phase separation, whereas sphingomyelin containing liposomes showed phase separation. This result suggests that the charge repulsion between GM3 may affect the liposome phase separation.

Next, we synthesized the fluorescently labeled transmembrane peptide of insulin receptor. At the isolation of this peptide by HPLC system, the recovery rate was remarkably low because of its hydrophobicity. We improved this problem by using the isopeptide method that reduces hydrophobicity. The synthesized peptides were incorporated into liposomes and three-dimensionally observed by fluorescent microscopy. As a result, it became possible to observe the phase state of the whole liposome which was difficult to discriminate by usual 2D imaging. We successfully constructed an evaluation system for detailed analysis.

We also measured the self-quenching of the fluorescently labeled peptides in liposomes. Since the peptides in the liposomes associate in a certain proportion and causes self-quenching, the association state can be quantified from the percentage of self-quenching. As a result of the measurement, the amount of self-quenching peptides in GM3-containing liposomes was less than that in liposomes without GM3, which suggested that GM3 prevented the association of the peptides by the electrostatic interaction.

313. Smart nanomedicine targeting of lysosomal glycohydrolases through activated endocytosis of cancer cells

S. Nishimura¹, R. Koide¹; ¹Graduate School of Life Science, Hokkaido University, Sapporo 001-0021, Japan

Hexosamine biosynthetic pathway (HBSP) is a central de novo biosynthetic process of UDP-GlcNAc from D-glucose supplied through its transporters. Alternatively, GlcNAc is salvaged into HBSP from a variety of glycoconjugate turn over by the degradation processes in lysosomes. GlcNAc can be phosphorylated by GlcNAc-6-kinase and subjected directly to the synthesis of UDP-GlcNAc. UDP-GlcNAc is also utilized subsequently for the synthesis of CMP-Neu5Ac and UDP-GalNAc. Given accumulated evidence that cancer cells need large amount of UDP-GlcNAc for rapid synthesis of glycoproteins having hyperbranched N-glycans, mucins with dense O-glycans, and many glycosaminoglycans/hyaluronic acid during growth and proliferation, method to inactivate key glycosidases reside in lysosomes could critically contribute to the development of potent anticancer therapy. Nanoparticle design is one of potential approaches for targeted molecular delivery. However, delivery of inhibitors by nanoparticles remains a key challenge because of nonspecific “protein corona”. Surface protein coronas of nanoparticles affect strongly in vivo biodistribution, endogenous cellular uptake, and drug delivery efficiency. Here we present that highly engineered“nanosomes” made of core metals covered by antiadhesive mixed self-assembled monolayer allow for avoiding nonspecific protein adsorption and targeted molecular delivery through activated endocytosis, concurrently. Nanosomes carrying inhibitors for ß-hexosaminidases showed cancer specific and highly efficient inactivation of intracellular targets both in vitro and in vivo without any influence of protein corona. The present results provide evidence that lysosomal glycosidases responsible for GlcNAc salvage pathway are promising targets of anticancer nanomedicine inducing apoptotic cell death through lysosomal membrane permeabilization. Importantly, nanosomes enable to highlight functions of “weak interactions” between carbohydrate and proteins in physiological environments by avoiding nonspecific protein adsorption at their surface. Merit of nanosomes in potent nanomedicine design are evident, because chemistry and protocols needed for drug/ligand loading is very simple and generalizable for large-scale production and quality control of the products.

314. Fruit-body induction of pleurotus ostreatus by glucosylceramide and glyceroglycolipid analogous compounds

T. Nishimura¹, Y. Magae¹, S. Ohara¹, M. Sunagawa¹; ¹Forestry and Forest Products Research Institute, Tsukuba

The fruit body development of wood-rotting fungi is still unexplained. The hormones or external signal molecules, even though they are omnipresent in nature, have been not identified. In our trials to find chemical or natural substances that effectively stimulate the fruiting of Pleurotus ostreatus (oyster mushroom) on agar medium, several effective compounds were found, including the sucrose ester of fatty acids (SE), triterpenoid glycoside (saponin), the synthetic series of 3-O-alkyl-D-glucose^1),2). From the common structure of these substances, we hypothesized that a sugar linked to a hydrophobic compounds is essential structure to the fruit body development of P. ostreatus. If the true fruiting signals exist in nature, they might be glycoconjugate generally known to be involved in cell signaling and differentiation. Then, synthetic glyceroglycolipid analogs, 1,2-di-O-acyl-3-O-(ß-D-glucopyranosyl)-glycerol (GlcDAG) and 1,2-di-O-acyl-3-O-(ß-D-cellobiosyl)-glycerol (Glc₂DAG) with n-acyl chains ranging from 4 to 16 carbon atoms, and mushroom or plant-origin glucosylceramides (GlcCer) were investigated. GlcDAG10, GlcDAG14, Glc₂DAG10 and GlcCer from mushroom were found to stimulate the forming primordia of P. ostreatus. Furthermore, 1,2-di-O-acyl-3-O-(ß-D-mannosyl)-glycerol (ManDAG) with n-acyl chains ranging from 8 to 16 carbon atoms have been successfully synthesized from D-mannose (8 steps via allyl ß-D-mannopyranoside). ManDAG14 was found to be active. With above findings as a background, we are now trying to isolate an endogenous active substance from the mycelia of P. ostreatus (from the mycelia in vegetative growth stage, the primordia and the fruit-body).

Acknowledgment : This work is supported by JSPS KAKENHI Grant Numbers 18K19241

References

1) Magae Y., Nishimura T., Ohara S. (2005) Mycol. Res.109, 374-376,

2) Magae Y., Nishimura T., Ohara S. (2009) Current Chemical Biology 3, 231-237

315. Effects of a monogalactosyl diacylglyceride obtained from coccomyxa sp. kj, a green microalga, against mouse norovirus infection

Y. Nomura¹, K. Hayashi³, T. Hayashi¹, T. Kawahara¹, J. Lee⁴, S. Komatsu², M. Kanazashi², H. Kuno², K. Atsumi²; ¹College of Life and Health Sciences, Chubu University, Japan, ²DENSO CORPORATION, Japan, ³Graduate School of Engineering, Chubu University, Japan, ⁴Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Japan

Human norovirus is the leading cause of foodborne illness across the world, yet there are currently no vaccines and no antiviral agents available. Since the virus is shed in the feces and vomited matters of infected individual and stable in the environment over a prolonged period of time, effective inactivation strategies are necessary to control the spread of virus.

Coccomyxa sp. KJ (IPOD FERM BP-22254) is a green microalga that has received interest because it accumulates lipids at more than 30 % of dry weight under suitable culture conditions. From the alga, we recently isolated a monogalactosyl diacylglyceride (MGDG) as an antiviral compound via bioactivity-guided fractionation. In the present study, we investigated the efficacy of the MGDG using cell culture systems and also an animal infection model with mouse norovirus (MNV), a cultivable surrogate of human norovirus.

MNV was propagated in RAW264.7 cells and plaque-titrated on the cell monolayers. When the virucidal activity of the MGDG was evaluated by titrating the remaining infectivity of MNV following the treatment with the compound, it showed concentration- and time-dependent inactivation of MNV.

Using the MNV infection model, we studied the ability of the MGDG to suppress the viral replication in the intestinal tract when used prophylactically. BALB/c mice were orally administered twice a day with MGDG (5 mg/day) from 7 days prior to oral infection of MNV until 21 days post-infection. During the experiments, no mice showed MNV-induced diarrhea or the loss of body weight. MNV shedding in stool of mice was titrated from 8 hours until 21 days post-infection via a plaque assay. Reducing viral shedding was observed in MGDG-treated mice as compared with water-administered control ones. Higher neutralizing antibody titers in the sera were detected at 21 days post-infection in MGDG-treated mice than those in the control ones. In immunocompromised mice injected with 5-fluorouracil, longer shedding of MNV and lower neutralizing antibody titers were observed as compared with immunocompetent ones, where MGDG reduced the viral shedding and stimulated antibody production. We here demonstrate the benefit of prophylactic treatment of MNV infection with the MGDG.

316. Haemoglobin: an exceptional glycation target in senescence: results from the seniorlabor study

U. Nydegger⁵, B. Sakem⁴, Z. Stanga⁵, L. Risch⁵, P. Di Cesare¹, T. Lung³, M. Risch²; ¹InnoBoost SA, Lausanne, ²Kantonsspital Chur, Switzerland, ³Labormedizinisches Zentrum Dr. Risch, ⁴Labormedizinisches Zentrum Dr. Risch, Bern, Switzerland, ⁵University of Bern, Switzerland

Protein glycation extent reflects momentum of senescence and is increasingly listed as biological age estimator on the clock-face of aging allowing for distinction between chronological and biological age. In the SENIORLABOR study, 1362 healthy subjects were included (613 men and 749 women; age range 60–99 years). The crude prevalence of individuals unaware of having prediabetic fasting plasma glucose (FPG) or glycated haemoglobin A1c (HbA1c) levels, was 64.5% (n = 878). In analogy, unknown type 2 diabetes mellitus was found in 8.4% (n = 114). Considering HbA1c serum levels (Prediabetic Reference Interval: HbA1c 5.7 – 6.4%,42 – 47 mmol/mol see: www.diabetes.co.uk) alone, more subjects could be identified using glycated haemoglobin assessment than with FPG. The prevalence of prediabetes as well as of T2DM increased with age. Of 806 participants with a prediabetic HbA1c (59.2%, 95% CI 56.5– 61.8), as many as 549 subjects (68.1%, 95% CI 64.8–71.2) had a normal fasting plasma glucose level. The mean HOMA indices (homeostasis model assessment) for the different age groups, between 2.12 and 2.59, are consistent with clinically hidden disease in the SENIORLABOR study and are in agreement with the largely orderly Body Mass Indices found in the normal range. Participants with high fructosamine levels (> 283 ⎧mol/l) spread their HbA1c levels as wide as between 5.8%-11% whereas those with fructosamine < 205 ⎧mol/l had their HbA1c confined between 4.5 – 7.0%. Recent reports have found half-and-half elevated and low HbA1c serum levels at baseline of progressing age, both ends of the U-shaped distribution curve being associated with functional decline in the elderly, especially when the subjects exhibited circulating soluble interleukin-6 receptor. This currently allows one to only cautiously assign HbA1c as an age biomarker label and calls for further studies into proteins on their way to advanced glycation end products.

317. Synthesis of n-glycopeptides standards for mass-spectrometry based diagnostic of colorectal cancer

H. Oh¹, O. Zaitseva², D. Varon Silva¹; ¹Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am Muehlenberg 1, 14476 Potsdam, Germany, ²Glycoscience Research Laboratory, Genos, Borongajska cesta 83H 10000 Zagreb, Croatia

Colorectal cancer (CRC) is the second most common cancer in Europe and the survival rate of patient is dependent on detection at early stage. There is a high interest in developing novel methods for CRC diagnosis with better accuracy in early disease stages. The glycosylation pattern of proteins in sera is correlated with the different stages of CRC and therefore represents an alternative for CRC diagnosis. Immunoglobulin G (IgG) is the most abundant antibody isotype in human sera and is categorized into four subclasses, IgG1 - IgG4. IgG has two glycosylation sites on the Fc region and two in the VL of Fab region. A tryptic digestion of IgGs releases a Fc glycopeptide (EEQXNSTXR, X is either Y or F) useful for high throughput site-specific glycosylation analysis that shows potential application to CRC diagnosis. To understand the changes of glycosylation profile of IgGs and to obtain accurate quantitative information of the modification using mass-spectrometry (MS)-based methods, it is crucial to have highly defined standards as reference biomarkers.

Here, we report the synthesis of a set of glycopeptide standards for MS analysis of IgG glycosylation. A sialoglycopeptide was isolated from egg-yolk powder and modified to obtain a glycosyl asparagine building block for solid phase glycopeptide synthesis. We synthesized N-glycopeptides from the four IgG subclasses, and enzymatically modified the glycan structure of these glycopeptides to deliver a series of glycopeptide standards having differentiated glycan and peptide sequence. This library of 22 glycopeptides is a useful tool for optimization of MS methods and the quantification of IgG glycosylation in CRC patients and in animal models. Furthermore, we investigated the effect of glycan structure and amino acid sequence in the MS ionization properties of glycopeptides.

318. The role of o-linked-n-acetylglucosamine in the development of hepatocellular carcinoma

J.Y. Oh¹, E.R. Kim⁵, S.H. Kim¹, I.J. Oh¹, H.J. Chun¹, D.H. Han², J.W. Cho³, Y. Lee⁴; ¹Department of Internal Medicine, Yonsei University College of Medicine, Seoul, ²Department of Surgery, Yonsei University College of Medicine, Seoul, ³Department of Systems Biology, Glycosylation Network Research Center, Yonsei University, Seoul, ⁴Department of Systems Biology, Glycosylation Network Research Center, Yonsei University, Seoul; Department of Internal Medicine, Yonsei University College of Medicine, Seoul, ⁵Severance Biomedical Science Institute, Yonsei Biomedical Research Institute, Yonsei University College of Medicine, Seoul

O-linked-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification that regulates biological processes, such as signal transduction, transcription, epigenetic regulation and metabolism. Recent study has reported that O-GlcNAc transferase (OGT), a unique glycosyltransferase enzyme, plays a key role in promoting tumorigenesis of non-alcoholic fatty liver disease-associated hepatocellular carcinoma (HCC). In addition, O-GlcNAc has been reported to correlate with YAP in high glucose-associated liver cancer. HCC is the most common liver cancer and is well known to be caused mainly by chronic liver disease such as viral hepatitis and cirrhosis. However, the relationship between O-GlcNAc and HCC has not yet been clearly elucidated. Thus, the present study investigated the functional role of O-GlcNAc in hepatocellular carcinoma cells (HepG2 cells) and human HCC tissues.

ThiaMet-G (OGA inhibitor) treatment increased O-GlcNAc expression, and led to the proliferation in HepG2 cells. However, in normal hepatocyte cells (MIHA cells), although O-GlcNAc expression was increased by ThiaMet-G treatment, the proliferation did not significantly change compared to control cells. Conversely, the treatment with ST045849 (OGT inhibitor) decreased O-GlcNAc expression and the cell viability in MIHA cells. Sodium glucose cotransporter 2 inhibitor (SGLT2i), a recently approved medication for type 2 diabetes, has a mechanism to lower intracellular glucose levels by blocking glucose uptake from cells. SGLT2 is mainly expressed in kidney proximal tubular cells, but recently it has been reported that it is expressed in various tumor tissues. We found that SGLT2 expression was increased in human tissues of HCC. SGLT2i decreased O-GlcNAc expression and induced autophagy in HepG2 cells. Furthermore, O-GlcNAc and OGT expression levels were markedly enhanced in patients with advanced stage of HCC. These findings suggest that O-GlcNAc and OGT may have a key role in tumorigenesis of liver cancer.

319. In vivo gene therapy by utilizing modified ß-hexosaminidase b for gm2 gangliosidoses

Y. Ohnishi¹, D. Tsuji¹, N. Kamori³, S. Muramatsu², K. Itoh¹; ¹Department of Medicinal Biotechnology, Graduate School of Biomedical Sciences, Tokushima University, ²Department of Medicine, Jichi Medical University, ³Faculty of Pharmaceutical Sciences, Tokushima University

Introduction: GM2 gangliosidoses including Tay-Sachs disease (TSD) and Sandhoff disease (SD) are lysosomal storage disorders caused by the gene mutations of HEXA and HEXB encoding ß-Hexosaminidase (Hex) <- and ß-subunits, respectively. These diseases associate with the excessive GM2 ganglioside (GM2) storage in the central nervous systems (CNS) due to HexA (<ß heterodimer) deficiency, and show the serious neurological symptoms. There is currently no cure for GM2 gangliosidoses. In recent years, the adeno-associated virus (AAV) vector-mediated gene therapy (GT) for GM2 gangliosidoses has been studied. This in vivo GT is designed to introduce the HEXA and HEXB into the patients’ cells using the mono- or bicistronic AAV vector, and reduce the GM2 accumulation by HexA expression. On the other hand, by replacing the part of amino acid sequences of Hex ß-subunit with those of <-subunit, we succeeded in producing the modified HexB (modHexB, modß homodimer) with the GM2 degrading activity like HexA as well as higher biostability than HexA. The number of terminal mannose 6-phosphate (M6P)-carrying N-glycans per modHexB molecule (4) is greater than that of HexA (3). So, the GT using the single AAV-modHEXB is expected to be more effective than the previous trial. In this study, in order to develop a new treatment for GM2 gangliosidoses (HexA deficiency), we evaluated the therapeutic effects and brain distribution of our novel AAV9/3-modHEXB using the SD model mice (SD mice) and normal cynomolgus monkeys.

Methods: We administered the AAV9/3-modHEXB to SD mice by an intracerebroventricular (ICV) injection, and performed the behavioral test and biochemical analyses. Next, we administered AAV9/3-modHEXB intrathecally (IT) to the cynomolgus monkeys, and performed the hematological and biochemical analyses.

Results: In the AAV9/3-modHEXB-injected SD mice (SD-AAV) brain, the HexA-like activity was restored, and the GM2 accumulation was reduced. And the SD-AAV showed normal motor function, and the prolonged life span. IT administration to cynomorgus monkeys caused the mature modHexB expression and distribution in the wide range of CNS regions.

Conclusion: The in vivo GT based on an ICV or IT injection of AAV9/3-modHEXB will be effective as a novel treatment for GM2 gangliosidoses.

320. Role of glycosphingolipid balance regulated by sphingomyelin synthase 2 in inflammation and progression of cancer in murine models

T. Okazaki¹, C. Hashizume², M. Taniguchi²; ¹Ishikawa prefectural University, Research Institute for Bioresources and Biotechnology (1-308 Suematsu, Nonoichi, Ishikawa, Japan 921-8836) - Kanazawa Medical university, Department of Medicine (1-1 Daigaku, Uchinada, Ishikawa, Japan 920-0923), ²Kanazawa Medical university, Department of Medicine (1-1 Daigaku, Uchinada, Ishikawa, Japan 920-0923)

Sphingomyelin (SM) synthase is a key enzyme to generate SM and diacylglycerol by converting phosphocholine of phosphatidylcholine to ceramide. SM synthase (SMS) genes consist of three homologs, SMS1, SMS2 and SMSr. SMS1 acts as a homeostatic enzyme to maintain the level of SM in the membrane, whereas SMS2 seems to work when the SM level in the membrane was changed by a diverse stresses such as inductions of apoptosis and autophagy-related cell death. We established SMS2-deficient mouse embryonic fibroblasts (MEF) and conventional SMS2-KO mouse. SMS1-deficient MEF did not grow well, but SMS2-deficient MEF can grow similar to wild type MEF. SMS1 increases SM content, and SMS2 also increases SM but the level is lower than SMS1 does. In addition interestingly SMS2 can increase glucosylceramide content in MEFs. As compared to SMS1-KO mouse SMS2-KO mouse looks normal in terms of ordinary phenotypes.

Dextran sodium sulfate (DSS)-induced mouse acute colitis model has been used to investigate the mechanism of inflammation in colon and by addition of azoxymethane (AOM) adenomatous colon tumor is produced in mice. In SMS2-KO mice DSS-induced colitis was inhibited by increasing of ceramide/SM balance, which suppressed the expression of inflammatory cytokines such as IL-6 and TNFα and the recruitment of T lymphocytes to the inflammatory lesions. In addition DSS/AOM-induced polypoid tumors was also inhibited in SMS2-KO mice. Next we examined the effect of SMS2-deficiency in progression of T lymphoma (EL4)-xenograft model. EL4 cell growth was inhibited and overall survival time was prolonged in SMS2-KO mice. Then we investigated the mechanism of tumor inhibition by SMS2-deficient condition and found that the infiltration of host T cells into tumor-microenvironment was suppressed because of increase of ceramide in TIL (tumor-infiltrating lymphocytes) and decrease of SM.

Taken together it was shown that SMS2-deficiency might be an effective therapeutic condition to inhibit the inflammation and tumor growth through regulation of ceramide/SM/GC balance.

321. Relatioship between glycated haemoglobin, glycated albumin and fasting plasma glucose in diabetics and controls at university of ilorin teaching hospital, nigeria

A.B. Okesina¹, G.G. Aliyu¹, W.O. Oladosu¹, M.I. Abdulazeez¹; ¹UNIVERSITY OF ILORIN TEACHING HOSPITAL, ILORIN, KWARA STATE, NIGERIA

Background: In recent time there is increasing interest in using other markers of glycaemic control during the management of diabetic patients. Glycated serum albumin (GSA) is one of the markers which has not gained worldwide acceptability for use in this purpose, unlike fasting plasma glucose (FPG) and glycated haemoglobin (HbA1c). We measure GSA among diabetes patients and controls and compared the values with FPG and HbA1c. This is to determine the possibility of introducing GSA measurement for use in monitoring our patients; because it is more convenient than FPG which requires that patients must fast. Also, it may be better than HbA1c which is affected by red blood cells lifespan and heamoglobinopathy.

Method: This was a hospital based cross-sectional analytical study carried out over three months. A total of 300 participants, comprising of 150 type 2 diabetic participants with 150 age and sex matched non-diabetic apparently healthy adults as controls were recruited for this study. The fasting plasma glucose (FPG) was analysed using glucose oxidase method, GSA analysed using two steps that include ELISA method for determining glycated serum albumin (GSA) and colorimetry method for estimation of serum albumin. The HbA1c was assayed using nephelometry method.

Results: The age of the study population was between 40 and 68 years with a mean age of 54.92 ± 7.90 years. The mean values of HbA1c among the diabetic group (7.51 ± 2.50%) when compared to that of non-diabetic group (5.40 ± 0.51%) was significantly higher (p = <0.001). The mean FPG in the diabetic patients was significantly higher (8.53 ± 3.11mmol/L) compared to (5.35 ± 0.39mmol/L) in the control groups, p= <0.001. The mean GSA (23.48 ± 6.58%) was significantly higher in the diabetic group compared to controls (12.49 ± 1.98%) (p< 0.001). The correlation coefficients between the FGP and HbA1c is 0.764, between FPG and GSA is 0.671, and between HbA1c and GSA is 0.742

Conclusion: This study showed that markers of glycaemic control (FPG, HbA1c and GA) were positively correlated with each other indicating that GSA can conveniently be introduced to compliment the monitoring of our patients.

322. Induction of anti-oligosaccharide igg antibodies that recognize mammalian glycoproteins by immunization with novel artificial glycosphingolipids

T. Okuda¹, S. Hasaba², K. Shimizu², M. Date²; ¹Bio-Design Research Group, Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), ²Diagnostics Research Laboratories, Diagnostics Technical Service & Research Operations, Diagnostics Division, FUJIFILM Wako Pure Chemical Corporation

We previously found that artificial glycosphingolipids (artGSLs) containing very long-chain fatty acids behave as strong immunogens in mice, and promote the production of antibodies that recognize the oligosaccharide portion of artGSLs as the epitope. Here, we report that the oligosaccharide structure of artGSLs influences these immunogenic properties. We evaluated the antibody-inducing activity of artGSLs with different oligosaccharide structures in mice, and found strong IgG-inducing activity only with an artGSL ccontaining a core-fucosylated tetraoligosaccharide (Man®1,4GlcNAc®1,4[Fuc<1,6]GlcNAc). To characterize the immunogenic properties of this artGSL, we analyzed various derivatives, and found that the non-reducing terminal mannose structure was critical for the antibody-inducing activity. These artGSLs also exhibited IgG-inducing activity dependent on co-administration of lipid A adjuvant, but no cytokine-inducing activity similar to <-galactosylceramide was detected. Furthermore, repetitive immunization with the artGSL promoted the production of antibodies against a core-fucosylated <-fetoprotein isoform (AFP-L3) known as a hepatocellular carcinoma-specific antigen. These results indicate that the newly designed artGSLs specifically induce adaptive immune responses and promote antibody production by B cells, which can be utilized to develop anti-glycoconjugate antibodies and cancer vaccines targeting tumor-associated carbohydrate antigens.

323. Modified secretable alkaline phosphatase - new reporter protein for n-glysosylation analysis

M. Olczak¹, B. Bazan¹, T. Olczak¹; ¹Department of Biotechnology, University of Wroclaw, Poland

Glycosylation belongs to the most frequent posttranslational modifications of macromolecules. Among different types of protein posttranslational modification, N-glycosylation belongs to most common. In some cells, especially those with high activity of secretory machinery (ER-Golgi processing), most proteins are N-glycosylated. However, a detailed analysis of N-glycans covalently attached to glycoproteins in mammalian cell lines is not an easy task. Classical methods of analysis include detergent extraction of total protein pool followed by time-consuming purification steps, usually with relatively low recovery rates. Additionally, the most common problem in such methods is high amount of high-mannose type, not-mature types of glycans, abundant in ER or early Golgi compartments. The glycoprotein pool may also contain non-mature intermediates, which may produce unsatisfactory “background” in N-glycan profiles. Recently a few new methods, based mostly on removing of high-mannose structures, were introduced. However, most of them are still time-consuming and relatively complicated. To solve these problems many researchers use reporter, secreted proteins. We designed the new reporter, secretable protein, based psiTEST plasmid from Invivogen, which codes for the sequence of secretable alkaline phosphatase (SEAP). The main goal of this project was to design the secretable glycoprotein which: 1) may be synthesized and secreted to the media by transiently transfected mammalian cells of different types at high rates, 2) the secretion level may be monitored using simple colorimetric assay, 3) the purification procedure is simple, fast, and efficient, and is also not dependent on type of serum and culture media , 4) the reporter protein possesses at least 3 glycosylation sites. To achieve these requirements we constructed the modified psiTest plasmid coding SEAP. We attached the HA or 6xHis tag at the C-terminus of SEAP to facilitate purification. We also introduced new glycosylation sites. From 27 analyzed constructs 2 were chosen as the best in a view of secretion level and N-glycosylation pattern. The final constructs were tested in HEK 293, HepG2 and CHO cells. Our results are similar or better compared with other known reporter glycoproteins.

324. Biological significance of <2,6-sialyltransferases in early embryos of medaka fish

T. Omoto², D. Wu¹, H. Hashimoto¹, M. Hibi¹, C. Sato², K. Kitajima²; ¹Biosci. Biotech. Center, Nagoya Univ., ²Grad. Sch. Bioagr. Sci., Nagoya Univ.; Biosci. Biotech. Center, Nagoya Univ.

Sialic acids (Sias) are present at the non-reducing end of glycan chains of glycoproteins and glycolipids on cell surface, and play important roles in various biological phenomena such as immune system, neural system, fertilization and development. Sia is to date known to be important in vertebrate development; however, understanding of the importance at molecular level still remains to be pursued. In this study, we carried out the following experiments to understand how <2,6-Sia residues work in early embryos of medaka (Oryzias latipes), which is an excellent model vertebrate for developmental biology. First, we prepared the gene knockout medaka for two <2,6-sialyltransferases, ST6Gal I and ST6Gal II (ST6Gal I-KO and ST6Gal II-KO, respectively), using the CRISPR-Cas9 system. We found that ST6Gal II-KO medaka could survive until fully matured adult once hatched; on the other hand, ST6Gal I-KO medaka was lethal within two weeks after hatching. Second, we analyzed developmental stage-specific expression of ST6Gal I and ST6Gal II using real-time PCR. ST6Gal II was predominantly expressed within 2 days post fertilization (dpf), but ST6Gal I was predominantly expressed at 3 dpf and later. Finally, we sought to identify <2,6-Sia carrier proteins at 8 dpf using SNA lectin blotting. At 8 dpf, the expression of <2,6-Sia residues were detected on 85 kDa- and >150 kDa-proteins in ST6Gal I-KO, and >150 kDa-protein in ST6Gal II-KO. These results suggest that the 85 kDa-protein is a substrate of ST6Gal II, and the >150 kDa protein is a substrate of both ST6Gal I and ST6Gal II. Involvement of these glycoproteins in survival of medaka embryos is suggested.

325. Microfibril-associated glycoprotein 4 is c-mannosylated in human cells

Y. Osada¹, T. Suzuki², K. Miura¹, N. Dohmae², S. Simizu¹; ¹Keio University, ²RIKEN

C-mannosylation is a unique protein glycosylation. An <-D-mannose is attached to the indole C2 carbon atom in the N-terminal side tryptophan residue of consensus sequence (WXXW/C (X: represents any amino acid)) via a C-C linkage. This glycosylation was found by Hofsteenge’s group in 1994. They observed C-mannosylation of human ribonuclease (RNase2) in urine. Functions of C-mannosylated proteins have been reported such as the regulation of protein secretion, cell differentiation, signaling pathway, enzymatic activity, and subcellular localization of protein. Many C-mannosylated proteins contain thrombospondin type 1 repeat (TSR) domain or belong to type 1 cytokine receptor family. TSR domain is characterized by conserved Trp, Cys, and Arg residues and NH2-terminal part of TSR domain frequently contains WXXWXXW/C. Type 1 cytokine receptors are characterized amino acid motif (WSXWS). dpy19 is identified as a C-mannosyltransferase in C. elegans and mammalian have DPY19L1-L4 as four homologues of C. elegans, dpy19. Since DPY19L3 has C-mannosyltransferase activity in human cells, DPY19L1, 2, 4 are likely C-mannosyltransferase.

Human microfibril-associated glycoprotein 4 (MFAP4), consists of signal peptide and Fibrinogen C-terminal domain (32-255 amino acid residues), belongs to the fibrinogen-related protein superfamily. It expresses in vascular extracellular matrix fibers and binds to elastin, fibrillin, and collagen. Moreover, MFAP4 is associated with aortic aneurysm, pulmonary hypertension, and cirrhotic liver disease. MFAP4 contains the consensus sequence for C-mannosylation. Then, we transfected with an MFAP4-expressing vector to HT1080 cells and established an MFAP4-HT1080 cells, and then recombinant MFAP4 protein from conditioned medium was purified and revealed that MFAP4 is C-mannosylated at Trp235 by LC-MS/MS. In the future, to reveal the functions of C-mannosylation of MFAP4, we will establish an MFAP4 W235F-HT1080 cells.

326. Molecular docking and dynamics of vatairea guianensis lectin in complex with n-acetyl-d-galactosamine and tn antigen

V.J.S. Osterne³, V.R. Pinto-Junior¹, C.F. Lossio¹, L.A.G. Sousa⁴, M.V. Oliveira¹, M.T.L. Silva¹, J.L.A. Correia¹, E.J.M. Van Damme², K.S. Nascimento¹, B.S. Cavada¹; ¹Department of Biochemistry and Molecular Biology, Federal University of Ceara, Brazil, ²Department of Biotechnology, Ghent University, Belgium, ³Department of Biotechnology, Ghent University, Belgium / Department of Biochemistry and Molecular Biology, Federal University of Ceara, Brazil, ⁴National Institute of Amazonian Research, Manaus, Brazil

The transformation of normal cells into neoplastic cells is associated with changes in the glycan composition of cell membrane proteins, generating unusual carbohydrate structures, which determine characteristics related to cell differentiation, maturation and metastasis, suggesting, in turn, that these modifications are important throughout tumor progression. Lectins, proteins capable of binding to carbohydrates in a specific and reversible manner, have been used as tools to recognize these changes in glycan patterns, allowing to identify and differentiate benign and malignant tumors and evaluate the degree of glycosylation associated with metastasis. The Tn and sTn antigens are epitopes containing N-acetyl-D-galactosamine (GalNAc), they are present in the extracellular matrix of some carcinoma cells in humans and are often used as biomarkers. In this context, the present work aimed to characterize the interaction of Vatairea guianensis seed lectin (VGL) with N-acetyl-D-galactosamine, Tn antigen and its derivative sialyl-Tn antigen using molecular docking and molecular dynamics. A 3D model of the VGL structure was obtained via homology modeling using the X-ray structure of the Vatairea macrocarpa lectin as a template. The obtained structure was submitted to molecular docking with the previously cited carbohydrate/glycans. The docked complexes were used as inputs to run the molecular dynamics simulations and the free binding energies were estimated by molecular mechanics Poisson–Boltzmann surface area approach. VGL interacted strongly with GalNAc and Tn antigen with binding free energy of about -10 kcal/mol, contrary to that observed for sTn epitope. Our study also revealed new residues responsible for the binding with the carbohydrate structures, not previously identified in the crystals of Vatairea lectins in complex with carbohydrates. The present work indicated the capacity of VGL to differentiate between the two epitopes under study. Consequently, VGL will be an interesting tool for cancer research, diagnosis and therapy. This work further demonstrates the added value of bioinformatics in lectinology.

327. A BI-TO-MONO Sugar-binding transition in galectin-9 (gal-9) potentiates mesenchymal invasion of breast cancer epithelia

D. Pally¹, A. Srinivas¹, R.V. Kumar², R. Bhat¹; ¹Indian Institute of Science, ²Kidwai Cancer institute

Aberration in expressions and functions of glycans and their binding proteins (lectins) in cancer cells is one of their earliest ‘hallmarks’. Galectins are a conserved family of lectins that can bind to ®-galactosides. A special class of galectins known as tandem-repeat (GAL-4, -8, -9, and -12 in humans) can bind two distinct ®-galactosides simultaneously and play intricate roles in physiological and pathological contexts. In this study, we asked if tandem repeat galectins regulate breast tumor progression based on earlier cursory reports of their differential expression. Upon mimicking a progressive spectrum of the advancement from homeostatic breast- to invasive cancerous architectures by culturing HMLE (immortalised breast epithelial cell line), MCF7 (non-invasive breast cancer cell line), MDA-MB-231 (metastatic cell line) in laminin-rich ECM- and Type 1 collagen- rich scaffold gels, we observed that gene expression levels of GAL-9 tracked invasiveness of probed cells. Breast cancer patient samples (especially with a ‘triple negative’ (ER- /PR- /HER2-) histotype) also showed higher levels of GAL-9 when compared with matched histologically normal tissues. Further, perturbing GAL-9 levels showed a positive correlation with adhesion and invasion of cancer epithelia to and within laminin-rich matrices. Within a complex scaffold that mimics the epithelial-basement membrane-stromal matrix organization, GAL-9 enhanced the solitary but not the collective invasion of cancer epithelia. To dissect which carbohydrate recognition domain (CRD) is involved in regulation of cancer invasion, we generated individual domain-deleted mutants along with a linker-deleted bi-CRD GAL-9. Upon expression of the three mutants, we have observed that only N-terminal CRD of GAL-9 is able to increase cancer invasion similar to full length GAL-9 overexpression. We also observed that GAL-9 in human and murine cancer cells was cleaved into individual CRDs. . Our results, in the light of strongly predicted protease-susceptibility of the GAL-9 linker region suggest that the N-CRD of GAL-9 gets unbound to the C-CRD and potentiates the mesenchymal invasion of cancer epithelia through stromal-like milieu.

328. The cerebrospinal fluid (csf) n-glycome as a novel biomarker of parkinson’s disease. a mass spectrometry-based csf n-glycosylation study of patients affected by parkinson’s disease

A. Palmigiano¹, A. Messina¹, F. Esposito¹, R. Barone², G. Mostile³, A. Nicoletti³, L. Sturiale¹, D.A. Romeo¹, D. Garozzo¹, M. Zappia³; ¹CNR-IPCB UOS Catania, Institute for Polymers, Composites and Biomaterials, Via Paolo Gaifami, 18 – 95126, Catania, Italy, ²Department of Pediatrics, Child Neurology, University of Catania, Via S. Sofia, 78 – 95123 Catania, Italy, ³Department “G.F. Ingrassia”, Section of Neurosciences, University of Catania, Via S. Sofia 78, Catania, 95123, Italy

Parkinson’s Disease (PD) is a clinically heterogeneous, multifactorial, age-related neurodegenerative disorder. PD is characterized by some pathological features such as cytoplasmic Lewy bodies accumulation in substantia nigra pars compacta, loss of dopaminergic neurons, inflammation, mitochondrial dysfunctions, that lead to neuronal degeneration and death. Glycosylation is a common post-translational protein modification with multiple biological functions. Glycosylation changes have been recently found in serum of patients with PD. However, N-glycosylation profiling in PD cerebrospinal fluid (CSF glycome) is still almost unexplored.

We aimed to study CSF glycome in PD in order to identify potential glycosylation changes associated with PD.

We performed Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI MS) CSF N-glycosylation analysis of released and permethylated N-glycans from a cohort including 19 PD patients and 19 control subjects (age- and gender-matched). Control CSF spectra were characterized by a base peak at 2792.4 Da, corresponding to a biantennary A2 complex N-glycan.

Spectra from PD-CSF denote a significant increase of high-mannose 5 (M5, m/z 1579.8), agalactosylated biantennary (G0, m/z 1661.8), agalactosylated bisected biantennary (G0B, m/z 1906.9) bisected, agalactosylated core fucosylated N-glycans (G0BF, m/z 2081.0).

CSF N-glycome could shed new light on the pathological mechanisms that lead to the disease development and progression in PD.

329. A novel approach for the data interpretation of intact o-glycopeptide datasets

A. Pap¹, E. Klement¹, E. Hunyadi-Gulyas¹, K. F. Medzihradszky¹, Z. Darula¹; ¹Laboratory of Proteomics Research, Hungarian Academy of Sciences - Biological Research Centre, Szeged

Protein glycosylation is among the most frequent post-translational modifications (PTM). Our group focuses on the mass spectrometric analysis of intact O-glycopeptides. Our interest has been fueled by reports about the role of mucin-type O-glycosylation in protein processing, controlling protein activity, and promoting metastasis in various types of cancer. The main goal of our research is the site-specific characterization of this PTM in different body fluids for the better understanding of its role(s) in different physiological processes, and disease and perhaps to discover clinically-relevant biomarkers.

In this study urine samples were collected from healthy donors or from patients diagnosed with either superficial or muscle-invasive bladder cancer. Intact glycopeptides were enriched from tryptic digests by lectin affinity chromatography using wheat germ agglutinin (WGA). The resulting samples were analyzed in nanoLC-MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer, and spectra were recorded applying HCD-fragment-dependent EThcD activation using 15% normalized collision energy (NCE).

Data interpretation was performed in an iterative fashion. Full database searches were performed permitting only the most common urinary O-glycans. Then using the confidently identified sequences the HCD data were filtered for the presence of the Y₀ (gas-phase deglycosylated peptide) and Y₁ (peptide+GalNAc) ions in order to identify additional glycoform candidates. Glycan compositions were suggested from the mass difference between the mass measured and the peptide unmodified. These tentative assignments were manually validated. As a result of this new approach, several novel/unexpected glycoforms were identified, perhaps some of them may have significance in clinical diagnostics.

330. Mechanistic and epigenetic aspects of hyaluronan synthesis

I. Caon¹, D. Vigetti¹, M. Viola¹, E. Karousou¹, B. Bartolini¹, A. Parnigoni¹, E. Caravà¹,A. Passi¹; ¹University of Insubria

Epigenetic has emerged as a critical point in the control of gene expression and the hyaluronan (HA) synthase 2 shows some epigenetic aspects. Hyaluronan represents a perfect environment in which cells can migrate and proliferate as we described for human aortic smooth muscle cells (SMC). Smooth muscle cells (SMC) in the presence of different stimuli, as inflammation, oxLDL, mechanical stress, produced an altered ECM where HA is abundant. The control of the HA synthesis is critical not only in ECM assembly but also in various pathologies. In contrast with other glycosaminoglycans, which are synthesized in the Golgi apparatus, HA is produced on the plasma membrane by HA synthases (HAS1-3), using UDPGlcUA acid and UDPGlcNAc as substrates. UDP-sugar availability as well as the cellular energy are critical for the synthesis of HA and for HAS2 activity. The AMP activated protein kinase, a sensor of the energy status of the cell, leads to HAS2 T110 phosphorylation, which specifically inhibits HA secretion. However, the most general sensor of cellular nutritional status is the UDPGlcNAc produced by hexosamine biosynthetic pathway. This metabolic pathway is influenced by protein, fatty acid, nucleotide and glucose metabolisms and when activated leads to intracellular protein glycosylation (O-GlcNAcylation). We described that O-GlcNAcylation of serine 221 residue of HAS2 induces a dramatic stabilization of the enzyme on the membranes and an increase of HA production. Eventually we found a long non-coding RNA (NAT) positively controlsin cis the HAS2expressioninvolving p65 and NFkB pathway. This HAS antisense is also involved in the miRNA regulation acting as a sponge modulating the availability of some miRNA. Beside the antisense effect, another epigenetic control has been tested for P300 and histone acetylation. In fact transfection of P300 increased the HAS2 expression and HA synthesis whereas transfection of HDAC1 has opposite effects, indicating that this epigenetic control plays a role in this context. The sirtuins are also another part of the complex mechanisms involved in HAS 2 expression. The data produced in our laboratory indicate that epigenetic area is a key point of the hyaluronan metabolism and therefore of ECM composition.

331. O-glcnac on pkc( inhibits differentiation of mouse embryonic stem cells

F. Pecori¹, H. Ota¹,T. Miura³, S. Nishihara²; ¹Laboratory of Cell Biology, Department of Bioinformatics, Graduate School of Engineering, Soka University, ²Laboratory of Cell Biology, Department of Bioinformatics, Graduate School of Engineering, Soka University; Glycan & Life System Integration Center (GaLSIC), Faculty of Science and Engineering, Soka University, ³National Institute for Quantum and Radiological Science and Technology (NIRS); Laboratory of Cell Biology, Department of Bioinformatics, Graduate School of Engineering, Soka University

Mitogen-activated protein kinase (MAPK) activation <i>via</i> phosphorylation in the cytoplasm triggers mouse embryonic stem cells (mESC) differentiation. <i>O</i>-linked ®-<i>N</i>-acetylglucosaminylation (O-GlcNAcylation) is the sole glycosylation occurring in the nucleus and cytoplasm. <i>O</i>-GlcNAc is transferred by <i>O</i>-GlcNAc transferase (OGT) to serine and threonine residues of target proteins and removed by <i>O</i>-GlcNAcase (OGA) . One of the main <i>O</i>-GlcNAc molecular function is competing with phosphorylation. Therefore, O-GlcNAc is believed to regulate signaling by inhibiting protein phosphorylation. Previously, we demonstrated that OGT is necessary for mESC survival and <i>O</i>-GlcNAc is required for reversion from mouse epiblast stem cells to mESCs. However, the molecular mechanisms regulating intracellular signaling via </i>O<i>-GlcNAc in mESC remain unexplored. In this study, we performed knockdown (KD) of <i>Ogt</i> in mESC. Phosphorylation of Protein Kinase C (PKC) ( was promoted in <i>Ogt</i> KD mESCs. Immunoprecipitation using anti-PKC( antibody in mESC lysate and western blot analysis showed that PKC( is <i>O</i>-GlcNAcylated. PKC( is phosphorylated on threonine 410, the key phosphorylation site for PKC( activation, releasing it from autoinhibition and triggering subsequent phosphorylation of threonine 560. To identify the PKC( <i>O</i>-GlcNAcylated site we performed site mutagenesis analysis. As a result, we observed that T410, but not T560, is <i>O</i>-GlcNAcylated. Indeed, compared to the control, <i>O</i>-GlcNAc significantly decreased in T410A-mutated PKC(-FLAG expressing mESC, demonstrating that the T410 site of PKC( is <i>O</i>-GlcNAcylated. These results showed that <i>O</i>-GlcNAc competes with phosphorylation on T410 of PKC(. In addition, PKC( was observed to be a downstream component of FGF4 signaling. Indeed, MAPK was activated downstream of PKC( and promoted differentiation in <i>Ogt</i> KD mESCs. We confirmed these results using an <i>Ogt</i> KD inducible system. Currently, we are investigating putative candidate kinases that phosphorylate PKC(. In conclusion, we demonstrated a novel molecular mechanism regulating the maintenance of the undifferentiated state in mESCs through inhibition of PKC( phosphorylation by <i>O</i>-GlcNAc on T410.

332. Effect of deglycosylation method on obtained transferrin n-glycosylation profile

T. Petrovic¹, I. Trbojevic-Akmacic¹, G. Lauc¹; ¹Genos Ltd., Glycoscience Research Laboratory

Glycosylation is one of the most common and complex co- and post-translational protein modifications. The glycosylation status is crucial for protein functions, and it changes significantly during inflammation, sepsis and cancers. Human serum transferrin (Tf) is a glycoprotein synthesized mainly by hepatocytes and is involved in iron transport between sites of absorption and delivery. Changes in Tf glycosylation are seen in hepatocellular carcinoma, alcoholism and are a common marker for congenital disorders of glycosylation. Despite that, earlier studies have mostly been focused on presence or absence of sialic acid and Tf glycosylation hasn’t been studied in too much depth.

In general, glycoproteins must be isolated from complex samples for their detailed analysis. Even though many technologies have been developed recently, SDS-PAGE remains one of the most efficient ways to conduct parallel comparison and separate complex samples. It is specifically useful for samples where glycoproteins are available at lower quantities, such as Tf. After Tf separation by SDS-PAGE its N-glycans can be released directly from the gel band by performing in gel deglycosylation. Alternatively, Tf can be isolated from blood plasma by immunoaffinity purification and N-glycans released by in solution deglycosylation. Released N-glycans are then fluorescently labelled and analysed using ultra-high-performance liquid chromatography. Here, we compare these two approaches and show that the method of N-glycans release can change the obtained glycan profile of Tf.

333. Comparative microbial glycan binding specificities of three intelectins

J. Viola¹, J. Lee¹, M. Pierce¹; ¹University of Georgia

The X-type lectins contain members that function in innate immunity as well as fertilization. The first X-type lectin was identified in X. laevis oocyte cortical granules (XCGL-1 or XL-35) where it crosslinks the jelly-coat protein surrounding the oocyte. Following our identification of the coding sequence of XCGL-1, we identified two human homologs (subsequently termed hIntL-1 and -2). We interrogated synthetic and microbial glycan microarrays with recombinant hIntL-, -2 and native XCGL-1 and found that both hIntL-1 and -2 interacted with the surface glycans from several pathogenic microbes including: P. vulgaris, S. pneumoniae, P. mirabilis, and K. pneumoniae. hIntL-1 bound to microbial glycans containing ®-galactofuranose (®-Galf) and glycerol phosphate (GroP) moieties, as previously reported. By contrast, hIntL-2 showed minimal binding to glycans with ®-Galf, and only in a few instances did it bind GroP. XCGL-1 bound to the majority of the S. pneumoniae glycans, but not at the higher levels observed for hIntL-1 and -2. Molecular dynamic (MD) analyses were employed to validate the interactions between Galf and hIntL-1 and -2. A simulation of ®-Galf and hIntL-1 showed ®-Galf remained bound for the entire simulation with a root-mean-square deviation value of 1.04Å, indicating a stable interaction. hIntL-2, however, was unable to bind ®-Galf for the duration of the simulation. Amino acid sequence alignment of the three lectins showed several ligand-binding residue substitutions, suggesting these residues are critical for glycan specificity and affinity. Using an MD simulation, an hIntL-2 in which two of these residues were exchanged for those in hIntL-1, hIntL-2 was able to sustain binding of Galf. Exchanging these residues in the hIntL-1 sequence to those found in hIntL-2 resulted in loss of Galf binding by hIntL-1, demonstrating that the glycan binding specificities of the two human intelectins are dependent on at least two particular amino acids. A recent report suggests that intelectins are present not only in deuterostomes, but also in Placozoa, where the Trichoplax adhaerens genome contains over 30 putative intelectin-related sequences.

334. Modified sialylation is associated with synovial fibroblasts-dependent inflammation during rheumatoid arthritis.

Y. Wang², A. Khan², L. Bouché¹, M. Harnett², A. Dell¹, S. Haslam¹, M. Pineda²; ¹Imperial College London, ²University of Glasgow

In healthy joints, synovial fibroblasts (SFs) provide the required stromal support, but are recognized to adopt a pathological role in rheumatoid arthritis (RA), delivering region-specific signals to infiltrating cells that perpetuate local inflammation, pain and joint destruction. Interventions targeting SFs would improve current systemic therapies, as by directly modifying disease progression they would offer safer therapeutics. Unfortunately, our collective understanding of stromal immunology has not been translated to the clinic and new strategies are needed to find novel therapeutic targets. The vast, and yet unexploited amount of information contained in the SF glycome could offer such molecular targets, as glycans and glycan-binding proteins are being increasingly recognized as fundamental regulators of cellular interactions between stromal and immune cells.

We expanded SFs from healthy mice and mice undergoing experimental arthritis to evaluate their glycosylation by MALDI-TOF profiling of N- and O-glycans, describing an accurate molecular map of the SF glycome. Relative quantification of distinct glycan structures was then conducted to identify clusters of glycans differentially regulated during disease. Identified pathways were further investigated by qPCR. Likewise, human fibroblasts were isolated from RA patients, both at chronic and remission stages of disease.

Our results show that transformation of SFs into pro-inflammatory cells in RA is associated with glycan remodelling in response to pro-inflammatory mediators, a process that involved regulation of terminal sialylation. Interestingly, specific sialylation was modulated by different pro-inflammatory mediators, like IL-1ß, IL-17 or TNF<, which are key drivers of pathology in different disease phenotypes. This should provide further insight into the mechanisms underlying SF-dependent inflammation and its resolution and also, given the susceptibility of cell specific glycome to environmental changes, can provide crucial information that will help in patient stratification and discovery of novel glycosylated disease biomarkers.

335. Hyaluronic acid-nimesulide conjugates with high conjugation ratio for cancer therapy

L. Ping-Shan¹, J. You-Sin¹, L. Hua-Yang², C. Ching-Wen¹, Y. Hsiu-Ping¹; ¹Department of Chemistry, National Chung Hsing University, No. 250, Kuo-Kuang Rd., Taichung 402, Taiwan, ²Holy Stone Healthcare Co., Ltd, 4F, No. 88, Sec. 1, Neihu Rd., Neihu Dist., Taipei 114, Taiwan

Drug conjugated biopolymers as drug carriers have attracted much attention in drug delivery systems. We previously reported that nonsteroidal anti-inflammatory drug nimesulide conjugated hyaluronic acid (HA) can be acted as a potential therapeutic agent against colorectal cancer; however, the conjugation ratio of nimesulide on HA backbone is only 1 % (Int J Nanomedicine 2017 (27) 2315-2333). In this study, new synthetic approach in co-solvent system was carried out for higher nimesulide conjugation.

Our results showed that the conjugation ratio of nimesulide on HA can be increased from 1 % to more than 35 % using new synthetic method. For in vitro and in vivo studies, HA-nimesulide with 11% conjugation ratio was utilized in biological tests in order to maintain the targeting ability of HA. HA-nimesulide-11% conjugates revealed significant cell toxicity compared with free HA in HT-29 cells. For in vivo antitumor studies, 71 % of tumor growth inhibition (TGI) were significantly observed in HA-nimesulide-11% treated groups. Interestingly, apparent anti-angiogenesis based on CD 31 staining and characterization of microvessel was found in tumor area. Thus, HA-nimesulide conjugates with high conjugation ratio performed significant tumor growth inhibition, reduced angiogenesis and has a great potential as CD44 -targetable polymer drugs for cancer treatments.

336. Human mitochondrial voltage-dependent anion channels carry oxidized and over-oxidized methionine and cysteine residues as detected by high resolution mass spectrometry

M.G.G. Pittalà¹, R. Saletti², S. Reina¹, V. Cunsolo², V. De Pinto¹, S. Foti²; ¹Department of Biomedical and Biotechnological Sciences, University of Catania, Catania (Italy), ²Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Catania (Italy)

Voltage-dependent anion selective channels (VDACs) are the most abundant integral proteins found in the outer mitochondrial membrane, separating the intermembrane space (IMS) from the cytosol. They are a small family of pore-forming proteins (30-35 kDa) that allows the flow of hydrophilic molecules through the membrane. The VDAC isoforms, named VDAC1, VDAC2, and VDAC3 show sequence similarity but have a slightly different tissue-dependent expression pattern, a variable abundance (VDAC1 is the predominant form expressed in mitochondria of every tissue), a difference in cysteine content and are characterized by specific interactome partners. Since the IMS has an oxidative potential, we have investigated the oxidation state of methionine and cysteine residues.

The present work is part of a research line concerning the structural characterization of the VDAC proteins. Recently, we have reported the exclusive cysteine over-oxidation of VDACs in rat liver mitochondria (rVDACs), which it is not present in other transmembrane mitochondrial proteins, by means of “in solution” tryptic and chymotryptic proteolysis and UHPLC/High Resolution nanoESI-MS/MS, procedure originally developed by us.

In the present work, adopting the same previous developed procedure, we extended the analysis to the human VDACs (hVDACs), obtained by mitochondria from HAP1 cells.

Our results demonstrate that all three mitochondrial hVDACs, in physiological state, contain the methionines partially oxidized to methionine sulfoxide. Furthermore, cysteine residues are present both in the carboxyamidomethilated form and in the partially oxidized form to sulfonic acid.

The assignment of a functional role to these modifications of VDACs will be a further step toward the full understanding of the roles of these proteins in the cell.

337. GM2 gangliosidosis in a family of wild boars

S. Prioni¹, L. Cabitta¹, S. Grassi¹, V. Bertani², A.M. Cantoni², A. Corradi², V. Jagannathan³, C. Drögemüller³, S. Sonnino¹; ¹Department of Medical Biotechnology and Translational Medicine, University of Milano, Italy, ²Department of Veterinary Science, University of Parma, Italy, ³Institute of Genetics, Vetsuisse Faculty, University of Bern, Switzerland

Gangliosidosis are inherited lysosomal storage disorders caused by defective activity of a lysosomal hydrolase or an activator protein required for ganglioside catabolism, which results in the intra-lysosomal accumulation of undegraded metabolites. The molecular mechanisms linking the lysosomal accumulation to the pathology are still obscure.

We report on the biochemical features of a novel form of GM2 gangliosidosis in wild boar (Sus scrofa). Three littermate (3/3) wild boards, from a free ranging farm, presented neurological signs (dysmetria, ataxia, quadriplegia and lateral decubitus) at 6 months of age. Viral, bacterial and toxicological analysis were to exclude possible exogenous causes of the symptoms. Animals were euthanized at approximately one year of age. Necropsy revealed in all affected animals reduced consistency of cerebral and cerebellar parenchyma. Histology revealed enlarged foamy neurons, with diffusely severely vacuolated cytoplasm in brain, cerebellum, spinal cord, peripheral ganglia and retina. EM revealed the presence in neurons of numerous lysosomes, filled by membranous material. Biochemical studies revealed the presence of an elevate amount of GM2 ganglioside, confirming the diagnosis of GM2 gangliosidosis. In addition, genetic analysis revealed the presence of a recessively inherited missense variant (p.Arg499Cys) in the hexosaminidase subunit alpha (HEXA) gene located within the GH20 hexosaminidase superfamily domain of the encoded protein. In man and other species, pathogenic HEXA variants are known to be associated with the disease.

In conclusion, this HEXA-associated form of GM2 gangliosidosis, described for the first time in wild boars, is thus very similar to human disease.

338. Glycomic changes in parkinson’s disease (pd): complete characterisation of the human nigro-striatal n-glycome and alterations with the onset of PD

A.L. Rebelo¹, F. Gubinelli³, E. Brouillet³, R. Saldova², A. Pandit¹; ¹Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, Ireland, ²National Institute for Bioprocessing Research and Training (NIBRT), University College Dublin, Ireland; School of Medicine, College of Health and Agricultural Science, University College Dublin, Ireland, ³Neurodegenerative Diseases Laboratory, Université Paris-Saclay, and MIRCen, Institut François Jacob, CEA, France

Parkinson’s Disease (PD) is a neurodegenerative condition related with the death of dopaminergic neurons in the substantia nigra and an increased state of neuroinflammation due to the aggregation of aberrant proteins. Given that glycans may behave as regulators of protein stability and that PD is associated with the accumulation of proteins, an understanding of how the glycosylation correlates with the disease makes it highly attractive to explore. Here we aim to spatially characterise the nigro-striatal N-glycome profile and its changes in both the human brain (healthy/PD) and in a rat model of neuroinflammation (LPS induced).

Human and rat brain tissues were homogenised using RIPA buffer and the proteins were encapsulated in an acrylamide gel. PNGaseF was used to release the N-glycans and 2-Aminobenzamide to label them. These were analysed on Ultra Performance Liquid Chromatography (UPLC) and Mass Spectrometry. Further validation was performed using lectin histochemistry on tissue sections.

Our data shows similarities between glycan structures in the striatum and substantia nigra (around 30% of highly mannosylated, 47% of core fucosylated and 25% of outer arm fucosylated glycans). Besides, the global N-glycosylation traits in the human striatum and in the rat striatum are similar: there is a significant high percentage of tri-antenerary glycans, and glycans with core and outer arm fucose. Human brains present a small percentage of N-glycolylneuraminic acid (around 3%), which is a variation of the N-acetylneuraminic acid that is almost undetectable in humans (apart from tumour tissues) but is present in other mammals (e.g. rat brain). This highlights the conservation of the glyco-patterns in the brain of both species. The percentage of overall mannosylated glycans in the striatum and nigra increased significantly with PD and in the rat LPS model. This neuroinflammation model also showed a significant decrease in sialylation and fucosylation.

This study describes for the first time a complete characterisation of the N-glycome in human striatum and substantia nigra and provides an insight into the glycomic profile of PD. High mannosylated structures were shown to have an impact on the progression of the disease and play a role in the neuroinflammatory process.

339. A glycan tail as the anchoring point in the structure of novel sphingosine kinase inhibitors

B. Richichi¹, A. Papakyriakou⁴, L. Morelli³, E. Levati³, E. Puliti², F. Cencetti², P. Bruni², F. Compostella³; ¹Department of Chemistry, University of Florence, Via della Lastruccia 13, 50019 Sesto Fiorentino (FI), ²Department of Experimental and Clinical Biomedical Sciences, University of Florence, Viale GB Morgagni 50, 50134 Firenze, ³Department of Medical Biotechnology and Translational Medicine, University of Milan, Via Saldini 50, 20133 Milano, ⁴Institute of Biosciences & Applications, NCSR “Demokritos” Athens, Greece

Sphingosine kinases, SK1 and SK2, are enzymes localized respectively in the cytosol and in intracellular compartments, which catalyze the production of sphingosine-1-phosphate (S1P) in an ATP-dependent manner. S1P axis is involved in different pathological conditions (e.g. cancer, inflammatory disease, fibrosis), and aberrant S1P levels and/or SKs expression have been associated with diseases progression [1]. In this regards, different compounds have been exploited as chemical tools to alter the S1P axis either through inhibition of SK activity and by modulation of S1P receptors signaling. Specifically, a wide number of SK1 inhibitors have been developed to date. However, most of them are still limited by low potency and/or specificity among SK subtypes, indicating the need for new and more efficient SK1 inhibitors.

In this framework, we report here an original approach for the rational design of new SK1 inhibitors. It relies on the integration of the information coming from the SK1-co-crystal structure of known validated inhibitors into pharmacophoric models. This approach aims at increasing the activity of known inhibitors through the use of glycans as anchoring polar head group to hold inhibitors at the J-shaped hydrophobic binding pocket of SK [2]. In particular, in this communication we report on the rational structure-based design and stereoselective synthesis of glycan-containing PF543 analogues (Figure 1), one of the most potent SK1 inhibitors reported so far. Moreover, a preliminary evaluation of the role of the new synthesized SK inhibitors in TGF®-induced fibrosis of murine myoblasts to confirm the rationale of the proposed approach is described.

References

1. Pyne S, Adams DR, Pyne NJ. Sphingosine 1-phosphate and sphingosine kinases in health and disease: Recent advances. Prog Lipid Res. 2016, 62:93-106

2. Wang J, Knapp S, Pyne NJ, Pyne S, Elkins JM Crystal Structure of Sphingosine Kinase 1 with PF-543. ACS Med. Chem. Letters 2014, 5, 1329-1333

340. The recognition of paucimannosidic n-glycans by mannitou igm

S. Robakiewicz⁴, S. Serna², B. Echeverria², N. Reichardt², D. Charro³, N. Abrescia³, S. Delgado¹, A. Arda¹, J. Jiménez-Barbero¹, J. Bouckaert⁴; ¹Chemical Glycobiology Lab, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain, ²Glycotechnology Laboratory, CICbiomaGUNE, Paseo Miramón 182, 20014 San Sebastian, Spain, ³Structural Virology Lab, CIC bioGUNE, Bizkaia Technology Park, 48160 Derio, Spain, ⁴Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 du CNRS et Université de Lille, 50 Avenue Halley, 59650 Villeneuve d’Ascq, France

The importance of glycosylation in physiological and pathophysiological conditions is becoming increasingly recognised. Unfortunately, the quick detection of modifications in the expression of glycans associated with pathophysiological conditions is still difficult due to the lack of specific tools and markers. Mannitou IgM, a monoclonal murine antibody, was characterised as being able to specifically recognise the trimannosyl core structure of N-linked glycans called paucimannose. The unmodified paucimannose N-glycans are expressed abundantly in plants and invertebrates, whereas in healthy mammalian cells they are detectable in only very small amounts. However, they are highly present in numerous human cancer tissues, human adult pancreatic stem cells and inflamed mouse pancreata. We have managed to produce Mannitou by means of hybridoma technology as well as transient expression in HEK293T cells. The prediction of the Fab structure was done using homology modelling of the variable and constant domains. On the paucimannose glycan array, Mannitou exhibits the strongest binding to Man3GlcNAc2. Almost any substitution on this structure strongly reduces or even inhibits binding. The Mannitou - paucimannose interactions have been studied by means of SPR and STD-NMR which provide essential information on binding kinetics, specificity and the epitope. The up-regulation of paucimannosidic N-glycans under tumourigenic and inflammatory conditions makes the Mannitou antibody a promising diagnostic and therapeutic tool.

341. 3D bioprintable glycobiomaterials: design and synthesis of glycoconjugate polymers for 3d in vitro studies

L. Russo², S. Sampaolesi², S. Magli², L. Rabacchin², G. Risi¹, S. Bertini¹, C. Cosentino¹, F. Nicotra²;¹Ronzoni Institute for Chemical and Biochemical Research, ²University of Milano-Bicocca

Tissue engineering approaches are focussed on the development of scaffolds/biomaterials able to mimic Extracellular Matrix (ECM) composition and morphology in specific organs. Currently, it is well known that ECM is involved in the maintenance of cell functions in healthy conditions, and in the development and progression of pathological states. The glycosylation of cell microenvironment plays a fundamental role in cell-ECM and ECM-ECM interactions. The glycosignature of the ECM components actively participates in cell microenvironment regulation, in ECM remodeling and in organs functionality [1].

Specific organ morphologies and ECM glycosignatures influence the cell fate, inducing regeneration or pathological events.

We investigated the influence on the cell fate of different cell microenvironment glycosylation, in combination with different stiffness, generating 3D ECM bioprinted models. The 3D bioprinting technology represents today a transformative approach to generate customizable living scaffolds. The design of new synthetic strategies aimed to obtain new nano- and 3D bioprintable materials (bioinks) biologically inspired, has a high impact in different biomedical fields, from nanomedicine, to tissue engineering and cell biology studies [2,3]. Taking inspiration from ECM role in cell-cell and cell-ECM interactions, different glycoconjugates polymers have been synthesized in order to generate bioinks for 3D-bioprinting with different architectures.

References:

[1] G. Marsico, L. Russo, F. Quondamatteo, A. Pandit. Trends in Cancer. 2018, 4, 537-552

[2] S. Sampaolesi, F. Nicotra, L. Russo. Future Medicinal Chemistry, 2019, 11, 43-60.

[3] I. Donderwinkel, J.C.M. van Hest, N.R. Cameron. Polym. Chem., 2017, 8, 4451-4471.

Acknowledgments: Ministero della Salute, RICERCA FINALIZZATA 2016 RF-2016-02362946 ( 1/3/2018-28/2/2021) Theoryenhancing Projects. Title: Dissecting the link between pulmonary stromal changes and lung cancer progression for biomarkers discovery and therapeutic intervention

342. Deciphering the molecular code for fut9-dependent lewis x modification

T. Saito¹, H. Yagi¹, C. Kuo³, K. Khoo³, K. Kato²; ¹Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya; ²Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya and Exploratory Research Center on Life and Living Systems and Institute for Molecular Science (ExCELLS), National Institutes of Natural Sciences, Okazaki; ³Institute of Biological Chemistry, Academia Sinica, Taipei

Unlike DNA, RNA, and proteins, glycans of glycoproteins are supposed to be synthesized in template-independent manners. In other words, the glycan structures are not directly encoded in genome sequence and therefore generally unpredictable and heterogeneous. Consequently, one specific glycoprotein exhibits various glycoforms, while one specific glycotope is shared by diverse glycoproteins. In contrast, we have reported that Lewis X-containing glycans are specifically expressed on lysosomal associated membrane protein-1 (LAMP-1) in neural stem cells and fucosyltransferase IX (FUT9) is responsible for this Lewis X formation. Furthermore, the LAMP-1-specific Lewis X modification was observed in the CHO cells and HEK293T cells in which the FUT9 was overexpressed. These results raise a question as to how FUT9 selects LAMP-1 as an acceptor carrier. Herein, we address this selection mechanism.

LAMP-1 is composed of homologous N- and C-terminal domains, followed by transmembrane region. We conducted LC-MS/MS analysis for profiling the site-specific glycosylation of LAMP-1, indicating that the Lewis X-containing N-glycans were displayed almost exclusively in N-domain. We hypothesized that specific segment of LAMP-1 is responsible for the FUT9-dependent Lewis X modification. Using deletion and chimeric mutants of LAMP-1, we successfully identified a 22 amino acid sequence in N-domain, which is essential for the Lewis X formation. More surprisingly, FUT9-dependent Lewis X modification occurred in other recombinant glycoproteins, including erythropoietin and soluble Fc© receptor, when their C-termini were connected to the 22 amino acid segment. On the basis of these data, we conclude that the specific 22 amino acid sequence of LAMP-1 functions as a molecular code for the FUT9-dependent Lewis X modification.

343. High-efficiency detection of drug resistant-influenza virus with a fluorogenic sialidase probe

K. Sakakibara¹, Y. Kurebayashi¹, T. Takahashi¹, T. Otsubo², D. Kato¹, K. Ikeda², A. Minami¹, T. Suzuki¹; ¹Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, ²Department of Organic Chemistry, School of Pharmaceutical Sciences, Hiroshima International University

【Background】

Some influenza A viruses (IAVs) show resistant to sialidase inhibitors, which are clinically used for therapeutic against influenza. The monitoring of drug-resistant IAVs is important because drug-resistant IAVs have a risk that causes heavy damages to society and economy. Recently, we developed a fluorogenic sialidase probe “BTP3-Neu5Ac” that reacts to sialidase and deposits a fluorescence substance that visualized the sialidase activity. Here, we show a novel method for selective detection and isolation of drug resistant IAVs by imaging of sialidase activity.

【Result】

The drug resistant virus retains sialidase activity even in the presence of neuraminidase inhibitors (NAIs). Based on this principle, we developed a selective visualization method for drug-resistant influenza virus by using fluorogenic probe BTP3-Neu5Ac in combination with NAIs. Since BTP3-Neu5Ac enable the live imaging of the viral infected cells, drug resistant virus could be selectively isolated from the cells visvalized with BTP3-Neu5Ac.

Furthermore, we detected the virus concentrated on the membrane in a centrifugal filter unit using sialidase fluorescent probe. Fluorescence imaging in the presence or absence of NAI clearly distinguished drug resistant virus from drug sensitive virus. This assay could be performed within 15 minutes and the detection sensitivity was shown to be equal to or higher than the sensitivity of the commercial immunochromatography kits.

【Conclusion】

We developed a novel method for selective detection of drug resistant viruses using fluorogenic sialidase probe BTP3-Neu5Ac. This method has made it possible to detect drug resistant viruses more easily, quickly and sensitivity. Furthermore, this method is applicable to not only detection but also isolation of drug resistant viruses. Fluorescence imaging of drug-resistant viruses using BTP3-Neu5Ac will be a powerful tool for screening, monitoring drug-resistant influenza viruses, and the NAI-resistant mechanism. In addition, rapid detection of drug resistant influenza viruses will lead to a quick selection of effective antivirals at clinical fields.

344. Distinct glycosylation profiles of keratinocytes, fibroblasts and stem cells infected with different strains of herpes simplex type 1 virus

E. Samuelsson², K. Nyström², J. Nilsson¹, G. Larsson¹, R. Nordén²; ¹ Department of Clinical Chemistry and Transfusion medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, ² Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

During a viral infection, the Herpes Simplex Virus Type 1 (HSV-1) uses virus specific mechanisms to alter the host cell gene expression, resulting in an increased expression of genes in favor for the viral replication. We have previously found that among the genes with induced expression three fucosyltransferases organized in a cluster on chromosome 19 can be found, possibly resulting in increased expression of fucosylated N-acetylglycosamine residues. Altered expression of carbohydrates, either on viral proteins or on cellular counterparts may have implications for viral spread within the host.

In this work we have further investigated this phenomenon by defining the transcriptome in keratinocytes, fibroblasts and embryonal stem cells, all of which are natural targets of the HSV-1, infected with either a lab strain or an encephalitic strain of HSV-1. The resulting expression patterns of 89 different glycosyltransferases were used to reconstruct a hypothetical glycome. To confirm the theoretical glycome we characterized the resulting N- and O-linked glycans using LC-MS/MS.

The current study will possibly contribute with important insights about the viral mechanisms providing the cellular environment enabling lytic replication in keratinocytes and fibroblasts and a latent replication in neuronal cells, a phenomenon not fully understood. Further, this will derive information about the eventual variances in the induced glycome between viral strains known to induce distinct clinical symptoms.

345. A strategy for direct glycoconjugate preparations using a functional n-glycan

J.T. Sanes¹, H. Hinou¹, S. Nishimura¹; ¹Faculty of Advanced Life Science and Graduate School of Life Science, Hokkaido University, N21, W11, Kita-ku, Sapporo 001-0021, Japan

Glycosylation is a post-translational modification involved in protein folding, quality control, cell adhesion, and migration. The need to have correctly defined glycan components in biomolecules is important for glycan-activity studies to understand the mechanisms associated in various biological processes. With this, we turn to natural glycan sources as an alternative to chemically synthesized structures. Initial screening of glycans from natural sources is time-consuming. Natural glycan isolation from different samples demand numerous purification strategies typically requiring pure glycoproteins or glycopeptides, structure trimmings, and then cleaving the glycan component. The use of a well-established analytical and glycan enrichment procedure called glycoblotting hastens the initial screening process of searching for a particular glycan. With this, a highly relevant N-glycan that would serve as a starting structural template for different glycoconjugate preparations was isolated and conjugated to a glycosyl acceptor. The simple procedure developed eliminates the need for multiple isolation work. As a result of the strategy, an Fmoc-asparagine amino acid with a core N-glycan attached was chemo-enzymatically synthesized.

346. Synthesis of phosphonodisaccharide analogue from neisseria meningitidis a capsular polysaccharide

C.M. Santi¹, L. Auberger¹, L. Lay¹; ¹Università degli Studi di Milano

Neisseria meningitidis is one of the major causes of bacterial meningitis.

In particular, N. Meningitidis serogroup A (MenA) caused significant severe epidemics of meningococcal meningitis in the sub-Saharan Africa. Thanks to the introduction of the MenAfriVac vaccine, the serogroup A disease has almost vanished in 2017; however, it is important to persist with a strict control with a dedicated vaccination program.

MenAfriVac, compared to the previous vaccines, can be stored at temperatures up to 40°C for a maximum of four days. A further advancement could be the production of a more thermally stable vaccine, using MenA capsular polysaccharide synthetic analogues as antigens.

MenA capsular polysaccharide consists of (1->6)-linked-2-acetamido-2-deoxy-<-D-mannopyranosyl phosphate residues acetylated at C-3 to an extent of 70-90%. Since its stability issues are mostly due to the intrinsic lability of the anomeric phosphodiester linkages, our group synthesized isosteric, not acetylated phosphono analogues, where the anomeric oxygen of the phosphodiester is replaced by a methylene group. Their high stability to hydrolysis and ability to be recognised by anti-MenA serum were proved.

Considering that the 3-O-acetylation was revealed to be crucial for immunogenicity, we undertook the synthesis of 3-O-acetylated phosphono analogues of MenA polysaccharide. In particular, in this communication we describe the strategies we explored for an efficient synthesis of the phosphonodisaccharide.

347. Evaluation of methoxyacetate as protecting group for heparan sulfate synthesis

R. Schwoerer¹, K.J. Shaffer¹, O.V. Zubkova¹, P.C. Tyler¹; ¹Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand

Heparan Sulfates (HS) are a class of sulfated polysaccharides which function as dynamic biological regulators of the interactions of diverse proteins. We have previously developed a method for the synthesis of HS oligosaccharides (hexa-to dodecasaccharides) targeted at BACE1 (®-site APP cleaving enzyme 1) inhibition. In vitro activity assays identified several compounds as potent non-coagulant inhibitors of ®-secretase with potential for the development as leads for the treatment of Alzheimer’s Disease through lowering of A®-peptide levels. More recently we researched the transfer of this methodology to an automated synthesis platform.

In the pursuit to widen the applicability of the above methodology for the synthesis of structurally more diverse HS oligosaccharides, we had to expand our protecting group repertoire. Therefore we evaluated the use of methoxyacetate groups as an replacement for our preferred labile ester group chloroacetate, enabling the use of thiol reagents. We studied the assembly and deprotection of HS oligosaccharide precursors using methoxyacetate in the presence of acetoxymethyl benzoate, N-diacetates, acetate, benzoate and benzyl protecting groups.

348. Genetic association study and plasma protein n-glycan profiling in children newly diagnosed with type 1 diabetes and their healthy siblings

N. Selak², D. Kifer², V. Simunović², S. Kaur⁴, A. Cvetko², T. Keser², T. Pavić², L. Klarić³, F. Pociot⁵, G. Morahan¹, O. Gornik²; ¹Centre for Diabetes Research, University of Western Australia, Perth, WA, Australia, ²Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia, ³Genos Glycoscience Research Laboratory, Zagreb, Croatia; Institute of Genetics & Molecular Medicine, MRC Human Genetics Unit, University of Edinburgh, ⁴Steno Diabetes Center Copenhagen, Gentofte, Denmark, ⁵Steno Diabetes Center Copenhagen, Gentofte, Denmark; Faculty of Health and Medical Sciences, University of Copenhagen, Denmark; Copenhagen Diabetes Research Center (CPH-DIRECT), Department of Pediatrics E, Herlev Hospital, Denmark

Type 1 diabetes (T1D) is an autoimmune disease with an unknown cause. It is characterised by the destruction of pancreatic insulin producing beta cells. Glycosylation is a ubiquitous protein modification, but studies of glycosylation changes in T1D are scarce. We studied plasma samples of 1105 children and adolescents (0-18 years), collected within three months of T1D diagnosis through the Danish Registry of Childhood and Adolescent Diabetes. DNA samples were genotyped for 183,546 single nucleotide polymorphisms on the Immunochip. N-glycans of both total plasma proteins and IgG were enzymatically released from plasma proteins using PNGase F, fluorescently labelled, and profiled using hydrophilic interaction ultra-performance liquid chromatography with fluorescence detection. Genetic association analyses identified five genome-wide significant loci associated with total plasma proteins and/or IgG N-glycans. All identified loci, except for the complement C3 gene locus (C3), had been previously associated with N-glycosylation (MGAT3, MGAT5, ST6GAL1, and SYNGR1). In a further study, N-glycans from 187 children with T1D and their 244 unaffected siblings were compared. IgG N-glycans with both core fucose and bisecting N-acetylglucosamine (GlcNAc) were significantly increased in children with T1D relative to their healthy siblings. The most significant difference within total plasma proteins between children with T1D in comparison to their healthy siblings was observed for levels of triantennary disialylated N-glycans and those N-glycans with terminal mannose and GlcNAc residues. Further research is needed to elucidate the role of observed changes.

349. Immunogenicity of two polysaccharides from isatis indigotica as vaccine adjuvants

J. Shan¹, H. Li¹, K. Liu¹, P. Jia¹, B. Wang¹, C. Li¹, Y. Wang¹; ¹Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing, 100850, China

Objection To investigate the immunogenicity of Isatis indigotica polysaccharides (IIP-A-1 and IIP-2) as vaccine adjuvant. IIPA-1 was an <-glucan with the molecular weight of 3600 Da. Its backbone was <-(1→4)-D-glucan with (1→6) branch chain. IIP-2 has a molecular weight of 66,400 Da and is composed of arabinose and galactose at a ratio of 1.0:1.5 with ®-(1→3, 6)-galacan backbone. Methods (1) Keyhole limpet hemocyanin (KLH) was linked with IIP-A-1 and IIP-2 respectively to prepare immuno-antigen KLH-IIP-A-1 or KLH-IIP-2. Bovine serum albumin (BSA) was also linked with them respectively to prepare BSA-IIP-A-1 or BSA-IIP-2 as detecting-antigen. Rabbits were immunized with KLH-IIP-A-1 or KLH-IIP-2 (300 μg/rabbit) plus Freund adjuvant (0.65mL/rabbit) for 2 times. (2) Balb/C mice were immunized with IIP-A-1 or IIP-2 (500 μg/mouse) alone intramuscularly for 3 times. (3) Aftosa vaccine (2 μg/mouse), H1N1 influenza vaccine, or hepatitis B surface antigen (HBsAg, 2 μg/mouse) were respectively co-immunized with IIP-A-1 or IIP-2 for 2 times. Results (1) In the sera of rabbits immunized with KLH-IIP-A-1 or KLH-IIP-2 plus Freund’ adjuvant, the titers of KLH antibodies (1:50000), IIP-A-1 antibodies (1:20000) and IIP-2 antibodies (1:100000) were detected; (2) Both IgG1 (1:1000) antibody were detected in serum samples of mice after IIP-2 immunization as a antigen alone, but no IgG1 and IgM of anti-IIP-A-1 were measured; (3) The three vaccines were immunized respectively with IIP-A-1, no IgG1antibody of anti-IIP-A-1 was found. However, anti-IIP-2 IgG1 (1:400) was found with Aftosa vaccines, and a titer of 1:100 was found immunized with H1N1 vaccine or HBsAg separately with IIP-2. Conclusion IIP-A-1 doesn’t presented significant immunogenicity, but IIP-2 induced an anti-IIP-2 IgG1 production of in mice which was co-immunized with Aftosa vaccine, H1N1 vaccine or HBsAg.

350. Characterization of a novel galactosidase acting on arabinogalactan of mycobacterium tuberculosis

L. Shen⁵, A. Viljoen⁴, S. Villaume³, L. Chêne³, S.P. Vincent³, K. Takegawa², M. Joe¹, T.L. Lowary¹, L. Kremer⁴, Y. Guérardel⁵, C. Mariller⁵; ¹Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Canada, ²Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Japan, ³Laboratoire de Chimie Bio-organique (CBO), Université de Namur, Belgium, ⁴Mycobacterial Pathogenesis and Novel Therapeutic Targets (IRIM), CNRS UMR9004, Montpellier, France, ⁵Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), UMR8576 CNRS, Université de Lille, France.

Tuberculosis is a transmissible infectious disease that ranks in the second position among infectious disease after HIV/AIDS. Mycobacterium tuberculosis is the causative agent of tuberculosis. The synthesis of the mycolyl-Arabinogalactan-Peptidoglycan (mAGP) complex, which is the basis of cell wall architecture, is essential for bacterial viability and is often chosen as the target for anti-tuberculosis drugs. The remodeling of the mAGP complex is also essential for the physiology of mycobacteria during their division, their adaptation to the external environment or infection. This remodeling is accompanied by catabolism of arabinogalactan, which constitutes a new potential therapeutic target that deserves to be studied.

Here we show that protein Rv3096 is an exo-galactofuranohydrolase that releases alternate 5-D-Galf and 6-D-Galf residues from the arabinogalactan of M. tuberculosis. Enzymatic characteristics of Rv3096 such as kinetic constants, the need for cations and inhibition were studied by using a synthetic substrate, pNp-Galf. Furthermore, substrates mimicking the structure of arabinogalactan, as well as its endogenous substrate, were used to define its activity. Additionally, the 3D structure of the active site was generated by homology modeling. At last, the orthologue of Rv3096 in Mycobactederium smegmatis, MSMEG_5877 has been cloned and purified under the same conditions and its physiological functions in vivo were studied.

351. Development of a novel kit for o-linked glycan preparation

M. Sakaguch², M. Toyoda², Y. Miura²,H. Shimaoka², A. Kameyama¹; ¹Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST); ²Sumitomo Bakelite Co., Ltd.

Many proteins are regulated by glycosylation for their functions and dynamics in vivo, and it has been reported that alterations in glycan structures occurred on several glycoproteins during the onset and progression of many diseases. Therefore, a simple, rapid and accurate method to analyze their structures has been long awaited. Although a preparation method for analyzing N-linked glycans has been well established, there is currently no practical method for O-linked glycan that fulfills all the requirements. In general, O-linked glycan liberation from proteins including reductive beta-elimination and hydrazinolysis has been widely used, however there are shortcomings in each method to a greater or less extent, such as limitation on analytical equipment, generation of peeling products, yields, sensitivity, and complicated manipulations. To overcome the disadvantages, we have developed an innovative tool for preparing O-linked glycans that is far more practical than conventional methods in every aspect. Based on the novel O-linked glycan liberation method developed by Kameyama et al., we have designed and manufactured a time-saving O-glycan preparation kit, EZGlyco® O-Glycan Prep Kit. We have incorporated features such as simple operation, safety of reagents, rapid process of releasing O-linked glycans, a negligible amount of peeling, effective enrichment of released oligosaccharides, and easy fluorescent labeling of purified O-linked glycans. While conventional processes take 2-3 days to complete a series of treatments, the whole procedure of the kit could be carried out only in about 5 hours. The novel kit enables a high-throughput sample preparation due to its extremely simple processing. In this presentation, we will introduce the details of the novel kit. We believe that the tool has a great potential to be applicable to a wide range of O-glycan studies that requires a precise preparation of oligosaccharides from target glycoproteins. Thus, we expect that the kit would be widely employed for basic researches in glycobiology, O-glycan-related biomarker discovery, quality control of biopharmaceuticals, and development of new diagnostics in the near future.

352. Structural features of fngs and salt sensitivity of engase and engase/cpngase knocked-out arabidopsis thaliana

S. Shirai¹, R. Uemura¹, M. Maeda¹, R. Misaki², K. Fujiyama², Y. Kimura¹; ¹Department of Biofunctional Chemistry, Graduate school of Environmental and Life Science. Okayama Univ. Japan; ²International center for Biotechnology. Osaka Univ. Japan

In plant cells, the cytosolic peptide: N-glycanase (cPNGase) releases GN2 high mannose type free N-glycans (GN2-HMT-FNGs) from misfolded glycoproteins. Subsequently, endo-ß-N-acetylglucosaminidase (ENGase) hydrolyzes the ß1-4GlcNAc linkage in the GN2-HMT-FNGs to produce GN1-HMT-FNGs. These FNGs produced by cPNGase and ENGase were proposed to be plant specific signaling molecules associated with the plant differentiation and growth, although this idea has been unproven to date. As a part of study for unraveling the putative biological function of these FNGs in plants, in this study, we have constructed two kinds of transgenic A. thaliana, in which two ENGase genes (At3g11040, and At5g05460) are doubly knocked-out (DKO) and ENGase/cPNGase (At5g49570) genes are triply knocked-out (TKO), and analyzed the quantitative and qualitative changes in FNGs produced in each transgenic plant. Furthermore, we also analyzed the sensitivities to salt stress in these plants.

Structural features of FNGs: FNGs were extracted from leaves of wild type, ENGase DKO, and ENGase/cPNGase TKO with 0.1 M NH4OH aq. After dialysis of the crude extract against de-ionized water, FNGs were partially purified from the outer solution by a combination of cation-/anion-exchange, and a gel filtration. After pyridylamination of FNGs obtained, the structures were analyzed by SF-/RP-HPLC, ESI-MS analysis, and exoglycosidase digestions. As a result, it was found that GN1-HMT-FNGs occurred in wild type (3.9 nmol/g) only, and GN2-HMT-FNGs occurred in both ENGase DKO (10.6 nmol/g) and ENGase/cPNGase TKO (4.7 nmol/g). Interestingly, the GN2-HMT-FNGs were found even in the TKO line, suggesting that another mechanism involved in the production of this type FNGs works in plant ER.

Salt sensitivity on seed germination: Sterilized seeds were sowed on MS plate (pH 5.7) containing 3% sucrose and 0.3% gelrite with 0, 50, 100, and 130 mM of NaCl. After vernalization at 4°C for 3 days, the plates were incubated at 22°C under long-day conditions (16 h/8 h light/dark). The ENGase DKO was sensitive to the salt stress under 100 mM or 130 mM NaCl condition, although the wild type and the TKO line were not. These results suggested that the accumulation of GN2-HMT-FNGs in the DKO line may increase the salt sensitivity.

353. Involvement of c-mannosylation of isthmin-1 in the its secretion

S. Simizu¹, S. Yoshimoto¹, K. Katayama¹, T. Suzuki², K. Miura¹, N. Dohmae²; ¹Keio University, ²RIKEN

Isthmin-1 (ISM1) is a secreted 52-kDa protein that has an N-terminus signal peptide, a thrombospondin type-1 repeat (TSR1) domain, and a C-terminal adhesion-associated domain. It is an endogenous angiogenesis inhibitor and suppresses tumor growth. C-mannosylation, a rare type of glycosylation, is the attachment of a mannose to the N-terminal tryptophan residue in the consensus sequence of W-X-X-W/C via the C-C linkage. It occurs mainly in two groups, proteins containing a TSR1 domain and type I cytocike receptors containing a W-S-X-W-S motif. Although ISM1 has two predicted C-mannosylation sites (W²²³ and W²²⁶) in TSR1 domain, C-mannosylation of Ism1 has not been reported. In this studies, we performed to confirm C-mannosylation of ISM1 and to elucidate of roles or C-mannosylation on ISM functions.

To determine whether ISM1 is C-mannosylated or not, we established a C-terminal myc-his₆ (MH)-tagged ISM1-overexpressing HT1080 cell line (HT1080-ISM1-MH cells) and purified recombinant ISM1 (rISM1) proteins from the conditioned medium of HT1080-ISM1-MH cells. HT1080-ISM1-MH cells were cultured in serum-free medium for 24 h, and rISM1 was purified by cOmplete^TM His-Tag purification resin. Analyzing by LC-MS/MS, we revealed that ISM1 is C-mannosylated at both W²²³ and W²²⁶. Subsequently, we examined the effect of C-mannosylation on ISM1 with a mutant of ISM1. We constructed a mutant of ISM1 that W²²³ and W²²⁶ residues are replaced by phenylalanine residues (W223F/W226F:2WF) and established HT1080-ISM1/2WF-MH cells. Wild-type ISM1 was localized at Golgi apparatus, whereas 2WF ISM1 was localized in ER. These data indicate that C-mannosylation contributes to the intracellular trafficking of ISM1 from the ER to the Golgi apparatus. Furthermore, we masured the ISM1 secretion levels in conditioned mediat from HT1080-ISM1-MH cells and HT1080-ISM1/2WF-MH cells, respectively. Wild-type ISM1 was extracellularly secreted , however 2WF ISM1 was not detected. These data indicated that C-mannosylation on ISM1 affected not only intracellular trafficking but also efficient secretion.

354. Site-specific haptoglobin n-glycosylation changes in colorectal cancer

J. Simunovic⁴, V. Dotz¹, I. Trbojevic-Akmacic⁴, M. Novokmet⁴, K. Stavenhagen¹, L. De Neef¹, R.A. Tollenaar¹, W.E. Mesker¹, I. Kirac³, K. Vucic², M. Pezer⁴, M. Wuhrer¹, G. Lauc⁴; ¹Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands, ²Department for Safety and Efficacy Assessment of Medicinal Products, Agency for Medicinal Products and Medical Devices, Zagreb, Croatia, ³Department of Surgical Oncology, University Hospital for Tumours, Sestre milosrdnice University Hospital Centre, Zagreb, Croatia, ⁴Genos Glycoscience Research Laboratory, Zagreb, Croatia

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in Europe. Discovering less invasive diagnostic tools is essential for clinical practice, in particular for the early stage detection, but also for patient stratification. Previous studies showed significant changes of total plasma N-glycome in CRC. Assessing the site-specific glycosylation of an isolated carrier protein can provide valuable insights into CRC-associated glycosylation signatures. Previously, it was reported that elevated difucosylation of haptoglobin (Hp) tetraantennary glycans can be used to discriminate between early stage of hepatocellular carcinoma and cirrhosis. Hp is an acute-phase protein which is synthesized in the liver and is one of the major serum glycoproteins. We here present the first large-scale study of Hp glycosylation in CRC. Two different methods were applied on two independent clinical cohorts. A nano-LC-ESI-MS method for glycopeptide analysis of the four glycosylation sites was developed and applied on serum samples from a clinical cohort consisting of 186 CRC cases and 186 presumably healthy controls, in addition to 86 post-surgery CRC patients. To this end, Hp was captured from serum using affinity purification, and digested with trypsin, resulting in the detection of 75 different glycopeptide compositions in total. Data processing and quantification were performed in a semi-automated manner using the in-house built software LaCyTools. The second cohort consisted of 224 CRC cases and 268 healthy controls for which Hp was isolated from plasma followed by N-glycan release by PNGase F and measurement by UPLC with fluorescence detection. Using logistic regression, we tested for associations of glycopeptide features with case-control status. Our preliminary data indicate that Hp fucosylation and sialylation increase in CRC compared to controls. Site-specific analysis revealed specific alteration in synthesis of tetra-antennary structures, i.e. on asparagine 241. Moreover, the independent on-going replication setup will enable us to validate our findings and to compare two different analytical approaches for N-glycosylation analysis and further assess the potential of Hp glycosylation as a biomarker for patient stratification or disease outcomes in CRC.

355. Towards a new and convenient synthesis of group b streptococcus type ii (gbs ii) repeating unit

M. Singh¹, C. Colombo¹, L. Lay¹; ¹Department of Chemistry, University of Milan

Group B streptococcus (GBS) is a Gram-positive bacterium, which causes neonatal invasive diseases as well as severe infections in the elderly and immune compromised patients. ¹ On the basis of the capsular polysaccharide (CPS) structure, ten serotypes of GBS have been identified (Ia, Ib, II through IX). 2 Among these, type II GBS is one of the predominant GBS serotypes and responsible for 13% of early onset diseases. ³ Although the structure of GBS II was elucidated in 1983, ⁴ only one synthesis has been reported in literature. ⁵ The repeating unit of GBS type II is composed of <-Neu5Ac (2-3)-ß-D-Gal-(1-4)-ß-D-GlcNAc-(1-3)-[-ß-D-Gal-(1-6)]-ß-D-Gal-(1-4)-ß-D-Gal-(1-3)-ß-D-Glc.

In the present communication, we show our results of a novel approach to the synthesis of this heptasaccharide, based on the five subcomponents derived from retro synthetic analysis. Suitably protected lactosamine and lactose derivatives are pivotal building blocks in our synthesis. ⁶ The synthetic GBS II fragments will be used for glycan array and structural studies and immunochemical characterization with specific monoclonal antibodies.

References:

1. A. Nuccitelli, C. D. Rinaudo, D. Maione, Ther. Adv. Vaccines, 2015, 3, 76.

2. M. J. Cieslewicz, D. Chaffin, G. Glusman, D. Kasper, A. Madan, S. Rodrigues, J. Fahey, M. R. Wessels, and C.E. Rubens, Infection and Immunity, 2005, 73, 3096.

3. P. T. Heath, Vaccine, 2016, 34, 2876.

4. H. J. Jennings, K. G. Rosell, E. Katzenellenbogen, D. L. Kasper, J. Biol. Chem., 1983, 258, 1793.

5. S. Liming, Z. Han, L. Yaoyao, G. Guofeng, C. Feng, G. Zhongwu, G. Jian, J. Org. Chem.,2018, 83, 5920.

6. This project has received funding from the H2020-MSCA-ITN-2015 “Glycovax” under grant agreement No 675671.

356. Structural aspects of udp-n-acetyl-<-d-galactosamine:polypeptide n-acetylgalactosaminyltransferase from the snail biomphalaria glabrata

A. Turupcu¹, P. Poliak¹, C. Margreitter³, C. Oostenbrink¹, E. Staudacher²; ¹Institute for Molecular Modeling and Simulation, Department of Material Sciences and Process Engineering, University of Natural Resources and Life Sciences, Vienna, ²Institute of Biochemistry, Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, ³Randall Centre for Cell & Molecular Biophysics, King’s College London

Invertebrates show very complex glycosylation patterns. They combine structural characteristics known from other organisms and decorate their glycans with various additional features. Some snail species are intermediate hosts of parasites and control the intake, the development and the excretion of the parasite at least partly by recognition events based on glycosylation. Snails are therefore a valuable tool to understand the principle rules of glycosylation in general.

The mucin-type O-glycosylation initiating enzyme UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase (EC 2.4.1.41, ppGalNAcT, GenBAnk: KC18251) from the freshwater snail Biomphalaria glabrata has been cloned and characterised before [Taus et.al. Glycoconj. J. 30, 825-833 (2013), Taus et.al. Glycoconj. J. 31, 661-670 (2014)]. It transfers an N-acetylgalactosamine (GalNAc) residue from an activated donor (UDP-GalNAc) to a serine (Ser) or threonine (Thr) of an acceptor polypeptide chain and is the only characterised member of this enzyme family from mollusc origin.

Here, we compare the snail enzyme with the well investigated homologous human ppGalNAcT2 focussing on structural similarities and differences. An in silico homology model of the mollusc enzyme is created and various substrate peptides are modelled into the active site. This model of the mollusc enzyme is used to shed light on new and previously obtained data from in vitro experiments, such as substrate and cofactor specificity. We hypothesize about possible molecular interpretations of the available experimental data and offer potential explanations for observed substrate and cofactor specificity.

357. Missense mutations in the c-terminal portion of the b4galnt2-encoded glycosyltransferase are associated with sd(a-) histo-blood group phenotype

L. Stenfelt⁴, Å. Hellberg¹, M. Möller⁴, N. Thornton⁵, G. Larson², M.L. Olsson³; ¹Department of Clinical Immunology and Transfusion Medicine, Laboratory Medicine, Office of Medical Service, Skåne University Hospital, Lund, Sweden, ²Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Laboratory of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden, ³Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, Department of Clinical Immunology and Transfusion Medicine, Laboratory Medicine, Office of Medical Service, Skåne University Hospital, Lund, Sweden, ⁴Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, Lund, Sweden, ⁵International Blood Group Reference Laboratory, NHS Blood and Transplant, Bristol, United Kingdom

The histo-blood group antigen Sda, GalNAc®1-4(NeuAc<2-3)Gal®, is involved in pathogen invasion and suggested as a tumor marker. In the human population, ~90% carry Sda on their red blood cells, of whom a few display high expression, Sd(a++). Only 4% lack the antigen in all tissues and fluids, and may thereby produce anti-Sda, causing incompatible pre-transfusion tests. The genetic basis underlying human Sda deficiency has remained unexplained, although the ®1,4-N-acetylgalactos¬aminyl¬transferase encoded by the 11-exon gene B4GALNT2 was associated with Sda synthesis already in 2003 when the gene was cloned. We hypothesized that B4GALNT2 alterations result in the Sd(a-) phenotype.

B4GALNT2 was analyzed in nine Sd(a-) with anti-Sda in their plasma and the result compared to the B4GALNT2 reference sequence (transcript ENST00000300404) and eleven Sd(a+) individuals. Variants were evaluated using bioinformatic tools. Swedish (n=200) and Thai (n=183) cohorts were screened for two main variants.

Four SNPs of interest were identified in Sd(a-) individuals. Homozygosity for missense variant rs7224888T>C dominated the cohort (n=6) but we also found compound heterozygosity for rs7224888T>C and a splice-site mutation rs72835417G>A (n=1), compound heterozygosity for missense mutations rs148441237A>G (p.Gln436Arg) and rs61743617C>T (p.Arg523Trp) (n=1), and a sample with no deviations. The rs7224888T>C in exon 10 causes p.466Cys>Arg which is highly conserved evolutionarily. The rare rs148441237A>G (exon 10) and rs61743617C>T (exon 11) reside close by. In the European population rs7224888T>C occurs at 10.6-12%, which was confirmed in Swedish blood donors at 10%. The rs72835417G>A variant is predicted to affect splicing and had a frequency of 11%. The Sd(a++) phenotype has its highest rate known among the Thai, and accordingly both rs7224888 and rs72835417 had low frequencies (3.3% and 0.5%, respectively) in Thai donors. Taken together, the identified mutations correlate well with the frequency of Sd(a-). Defining the molecular background of Sda deficiency enables genotypic prediction of the Sd(a+) and Sd(a-) phenotypes and we now propose to establish a new blood group system, designated SID, in accordance with the International Society of Blood Transfusion guidelines.

358. Reduced breast tumor growth after immunization with a tumor-restricted muc1 glycopeptide conjugated to tetanus toxoid

N. Stergiou², N. Gaidzik³, A. Heimes¹, S. Dietzen², P. Besenius³, J. Jäkel⁴, W. Brenner¹, M. Schmidt¹, H. Kunz³, E. Schmitt²; ¹Department of Obstetrics and Women’s Health, University Medical Center Mainz, ²Institute of Immunology, University Medical Center Mainz, ³Institute of Organic Chemistry, Johannes Gutenberg University Mainz, ⁴Institute of Pathology, University Medical Center Mainz

Preventive vaccination against tumor-associated endogenous antigens is considered to be an attractive strategy for the induction of a curative immune response concomitant with a long-lasting immunological memory. The mucin MUC1 is a promising tumor antigen, as its tumor-associated form differs from the glycoprotein form expressed on healthy cells. Due to aberrant glycosylation in tumor cells, the specific peptide epitopes in its backbone are accessible and can be bound by antibodies induced by vaccination. Breast cancer patients develop per se only low levels of T cells and antibodies recognizing tumor-associated MUC1 and clinical trials with tumor-associated MUC1 yielded unsatisfactory therapeutic effects indicating an urgent need to improve humoral immunity against this tumor entity. Herein, we demonstrate that preventive vaccination against tumor-associated human MUC1 results in a specific humoral immune response, a slowdown of tumor progression and an increase in survival of breast tumor-bearing mice. For preventive vaccination, we used a synthetic vaccine containing a tumor-associated glycopeptide structure of human MUC1 coupled to Tetanus Toxoid. The glycopeptide consists of a 22mer huMUC1 peptide with two immune dominant regions (PDTR and GSTA), glycosylated with the sialylated carbohydrate STN on serine-17. PyMT (polyomavirus middle T-antigen) and human MUC1-double transgenic mice expressing human tumor-associated MUC1 on breast tumor tissue served as a preclinical breast cancer model.

359. Possible role of fungal glucosylceramide in the interaction with the host cell

T.G. Lacerda¹, M.S. Toledo¹, H.K. Takahashi¹,A.H. Straus¹; ¹Departamento de Bioquimica, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, SP, Brasil

Membrane microdomains present glycosphingolipids, sterols and specific glycoproteins and these molecules play a key role in several biological events such as cell adhesion and proliferation, morphogenesis, and infectivity. In order to investigate whether glucosylceramide present in pathogenic fungi could also modulate infectivity, it was performed experiments using Candida albicans which present glucosylceramide containing a specific ceramide moiety, common to most pathogenic fungi, not expressed in mammalian cells. Since fungi are surrounded by cell wall structures it was produced protoplasts using Zymolase T. Cell wall removal was confirmed by transmission electronic microscopy, and fungal viability analyzed by propidium iodide cell labeling.

By confocal microscopy the expression of glucosylceramide was analyzed using anti-glucosylceramide monoclonal antibody (mAb MEST-2). Differences of glucosylceramide distribution between protoplasts and control yeasts were observed. Thereafter, the expression of glucosylceramide was evaluated during interaction with macrophages. Protoplasts presented 40% of the rate of adhesion of control yeasts, suggesting that both the cell wall and components of the plasma membrane may be relevant to the infectivity process. For the control yeasts, no significant differences were detected regarding MEST-2 labeling of fungi isolated from cultures and those adhered to macrophages. Protoplasts incubated with macrophages were labeled with the mAb MEST-2, mainly in the contact region suggesting that microdomains of membranes and/or vesicles containing glucosylceramide are directed to the region of contact between fungi and mammalian cells, whereas in cell wall yeast glucosylceramide is uniformly expressed on the surface. Glucosylceramide polarization may be critical to infectivity processes. It is interesting to note that recent reports claimed that glucosylceramides, present in yeast forms of Cryptococcus neoformans, are involved in the formation and polarization of membrane microdomains and it is considered a virulence factor, helping yeasts in the invasion, as well as in the infectivity and survival processes in host cells.

Supported by FAPESP, CNPq and CAPES

360. Combined mass spectrometry methods for serum n-glycoprotein profiling enhance the awareness of the molecular patho-mechanism in ALG12-CDG

L. Sturiale³, S. Bianca⁵, A. Terracciano⁴, E. Agolini⁴, A. Messina³, A. Palmigiano³, F. Esposito³, C. Barone⁵, A. Novelli⁵, A. Fiumara⁶, D. Garozzo³, J. Jaeken¹, R. Barone²; ¹Center for Metabolic Diseases, UZ and KU Leuven, Leuven, Belgium, ²Child Neurology and Psychiatry, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy, ³CNR, Institute for Polymers, Composites and Biomaterials, IPCB, Catania, Italy, ⁴Laboratory of Medical Genetics, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy, ⁵Medical Genetics, Referral Centre for Rare Genetic Diseases, ARNAS Garibaldi - Catania, Italy, ⁶Referral Centre for Inherited Metabolic Diseases, Policlinico – University of Catania, Catania, Italy

Congenital disorders of glycosylation (CDG) are inherited metabolic diseases affecting the glycan biosynthesis of glycoconjugates. They represent an expanding group of multisystemic diseases with variable phenotypes and prevalent neurological involvement. More than 120 genetic disorders have been associated to defective glycosylation, mainly in the protein N-glycosylation pathway. Among these, CDG type I (CDG-I) occur in the cytosol or in the endoplasmic reticulum (ER) affecting dolichol-linked oligosaccharide synthesis, whereas CDG type II (CDG-II) involve the N-linked oligosaccharide processing in the Golgi.

ALG12-CDG (MIM: 607143) is caused by mutations of the human orthologue of the yeast asparagine-linked glycosylation (ALG)12 gene, encoding the mannosyltransferase which adds in the ER an <-mannosyl residue to the core Man(<1-6) of the dolichol-PP-oligosaccharide precursor, thus ensuring its correct shape and branching before the oligosyltransferase (OST) complex action.

Nine patients with ALG12-CDG have been reported so far. We investigated by MALDI MS and UHPLC-ESI MS the glyco-phenotype of a novel patient with unreported variants, sharing most of the clinical signs of patients with ALG12 deficiency, including severe recurrent infections with hypogammaglobulinemia and B cell dysfunction.

MALDI MS analysis of native transferrin showed underoccupancy of N-glycosylation sites, a typical feature of CDG-I. N-glycome analysis by MALDI MS, either on total serum N-glycan pool and on IgG released N-glycans, revealed the abnormal occurrence of high-mannose and hybrid N-glycan species. These accumulating glycoforms, further analyzed by UHPLC-ESI MS, corresponded to unbranched structures with <1,2-terminal mannose residues, previously identified in serum of patients with MAN1B1-CDG (CDG-II defect). Glycosylation analyses on the observed ALG12-CDG patient revealed a combination of CDG-I and CDG-II defects, these last associated with abnormal IgG N-glycan profile, consistent with the immunophenotype. Glycan characterization of target glycoproteins may endorse the molecular defect unraveling the complex clinical phenotype of CDG patients.

361. Altered surface n-glycosylation of melanoma-derived ectosomes influences their promigratory action exerted on recipient cells

M. Surman¹, A. Drożdż², E. Stępień², M. Przybyło¹; ¹Department of Glycoconjugate Biochemistry, Institute of Zoology and Biomedical Research, Jagiellonian University, Krakow, Poland, ²Department of Medical Physics, M. Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland

Ectosomes are heterogeneous population of extracellular vesicles that facilitate intercellular transfer of bioactive molecules including membrane and cytoplasmic proteins. In the case of tumour-derived ectosomes, their protein cargo may stimulate migratory properties of recipient cancer cells, contributing to disease progression. Little is known about mechanisms responsible for recruitment of particular proteins into ectosomes, however N-linked glycosylation is being investigated as possible sorting signal.

This study analyzed surface glycosylation and promigratory properties of ectosomes released by cutaneous melanoma (CM) cells treated with 1-deoxymannojirimycin hydrochloride (DMJ) – <-mannosidase I inhibitor. Primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cells were treated with 10 mM DMJ. Ectosomes released in vitro by DMJ-treated and control cells were isolated from conditioned media by sequential centrifugation, stained with a panel of lectins (PHA-E, PHA-L, SNA, MAA, GNA, AAA), and analyzed by flow cytometry. Migratory properties of less invasive CM cells (WM115, WM793) after were tested in wound healing assay after 18 h incubation with ectosomes released by DMJ-treated and control cells.

Flow cytometry studies revealed that DMJ treatment of CM cells resulted in increased number of high mannose structures (GNA staining) on the surface of corresponding ectosomes, and in depletion of complex N-glycans (PHA-L staining). Alternation in surface glycosylation of CM ectosomes was followed by impairment of their promigratory properties In wound healing assay incubation with ectosomes released by control cells (from all 4 CM cell lines) resulted in significant increase (from 2 up to 4.5-fold) of wound closure. After DMJ treatment only WM266-4 ectosomes caused any changes (1.5-fold increase) to migration rate of recipient WM115 cells. These results suggest the involvement of complex N-glycans in sorting of proteins involved in cancer cell migration into ectosomes, or in the interactions between ectosomes and recipient cells.

Acknowledgements: This work was supported by grants from the Jagiellonian University in Krakow (K/DSC/003978 and K/ZDS/008062).

362. Mice lacking core 1-derived o-glycan show acute multiple organ failure

R. Suzuki¹, Y. Nakamura¹, S. Fuseya¹, K. Hagiwara², T. Sato², H. Narimatsu², S. Takahashi¹, T. Kudo¹; ¹Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, ²Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology

Mucin-type O-glycosylation is a post-translational modification present on membrane-bound and secretory proteins. Its biosynthesis starts from transfer of a N-acetylgalactosamine (GalNAc) to the hydroxyl group of serine or threonine residues on proteins by polypeptide N-acetylgalactosaminyltransferases. This GalNAc residue is usually further modified by multiple glycosyltransferases. The major constituent of mucin-type O-glycan core structures is galactose-®1,3-GalNAc-<-serine/threonine, called core 1 structure, which is synthesized by core 1 ®3galactosyltransferase 1 (C1galt1). C1galt activity requires the expression of core 1 synthase specific molecular chaperone (Cosmc). C1galt1 knockout mice and Cosmc knockout mice both exhibited defective angiogenesis and fatal embryonic hemorrhage, indicating the indispensability of core 1-derived O-glycan for development and homeostasis. However, its physiological functions are still largely unknown.

In order to elucidate the physiological roles of core 1-derived O-glycan, we generated tamoxifen- inducible and ubiquitous Comsc knockout mice, CAG-CreER^TM::Cosmc^flox/Y mice (CAG-cKO). Tamoxifen was intraperitoneally injected into 6-week-old mice for 5 consecutive days to excise Cosmc gene. Ten days after induction, the body weight of CAG-cKO was lower than control mice (Cont). The organ weights of pancreas, white adipose tissue and thymus in CAG-cKO were 50% lower than Cont. The results of HE staining showed atrophy of pancreatic acinar cell and adipocyte in CAG-cKO. Protein casts with tubular lumina and glomerular sclerosis in CAG-cKO kidney were observed by PAS staining. CAG-cKO also showed severe thrombocytopenia and proteinuria. Biochemical analysis using serum showed higher levels of amylase activity and blood urine nitrogen, which implied pancreatitis and kidney dysfunction respectively. In order to confirm change of glycan structure, HPA lectin staining was performed. HPA recognizes terminal GalNAc which is expected to be increased in cKO mice. The intensity of HPA lectin in tissue above was higher than that of Cont. These data indicated multiple organ failure was caused by defective core 1 derived O-glycosylation.

363. Ngly1-deficiency: not just a defect in erad??

S. Tadashi¹; ¹RIKEN Cluster for Pioneering Research

The cytoplasmic peptide: N-glycanase (PNGase) is the enzyme widely conserved throughout eukaryotes[1]. This enzyme is involved in the degradation of misfolded/non-functional glycoproteins destined for the degradation process called ERAD (ER-associated degradation). In 2012, a patient harboring mutations of PNGase gene (NGLY1) was first reported. Symptom of these patients includes developmental delay, multifocus epilepsy, involuntary movement and liver dysfunction. From this report, it is clearly suggested that the cytoplasmic PNGase play a pivotal role in normal human development.

We analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 (B6) background[2]. Surprisingly, the additional deletion of Engase, encoding another cytosolic deglycosylating enzyme called ENGase (endo-beta-N-acetylglucosaminidase), resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of B6 mice could rescue the embryonic lethality of Ngly1-deficient mice[2]. Viable Ngly1-deficient mice in a B6 and ICR mixed background, however, showed a very severe phenotype. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the B6 and ICR mixed background.

We also showed that in Ngly1-KO cells, ERAD process was compromised[3]. Interestingly, not only delayed degradation but also the deglycosylation of a model substrate was observed in this cell. The unexpected deglycosylation was found to be mediated by ENGase. Surprisingly, the ERAD dysregulation in Ngly1-KO cells were restored by the additional KO of Engase gene[3]. These observations collectively suggest that the ENGase represents one of the potential therapeutic targets for this genetic disorder. In this symposium, we will overview our most recent progress on our NGLY1-research.

[1] T. Suzuki, et al., Gene. 2016, 577, 1-7.

[2] H. Fujihira, et al. PLoS Genet. 2017, 13, e1006696

[3] C. Huang, et al. Proc. Natl. Acad. Sci. USA 2015, 112, 1398-1403.

364. Novel bisgalactosylated type of n-glycans as decoy receptors for shiga toxin?

K. Szymczak-Kulus⁴, S. Weidler¹, A. Bereznicka⁴, K. Mikolajczyk⁴, R. Kaczmarek⁴, A. Urbaniak⁴, M. Olczak², E.Y. Park³, C. Unverzagt¹, M. Czerwinski⁴; ¹Bioorganische Chemie, Universität Bayreuth, Bayreuth, Germany, ²Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland, ³Laboratory of Biotechnology, Shizuoka University, Shizuoka, Japan, ⁴Laboratory of Glycobiology, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland

Shiga toxins (Stx) are the main virulence factors secreted by Shigella dysenteriae of serotype 1 and enterohaemorrhagic Escherichia coli (EHEC), which cause haemorrhagic colitis and hemolytic uremic syndrome. Stx produced by EHEC comprise Stx1, which is identical to the toxin secreted by S. dysenteriae of serotype 1, and more genetically distinct Stx2.

The main receptor recognized by Stx of both types is Gb3 (globotriaosylceramide, Gal<1-4Gal®1-4Glc-Cer), a neutral glycosphingolipid present in cell membranes of hematopoietic, digestive, nervous and urinary systems. It is also known as the P^k antigen belonging to the human P1PK histo-blood group system. Besides P^k, this system comprises two other glycolipid antigens: P1 and NOR. All these antigens are produced by human <1,4-galactosyltransferase (Gb3/CD77 synthase, P1/P^k synthase). This enzyme synthesizes terminal Gal<1-4Gal of Gb3 (P^k) and P1 antigen, while its mutein with p.Q211E substitution also generates terminal Gal<1-4GalNAc of the NOR antigen.

The terminal Gal<1-4Gal unit of Gb3 contributes to Stx binding. However, it has been demonstrated that Stx2e (causing pig edema disease), in addition to Gb3, binds also to Gb4 (globoside, globotetraosylceramide) and the Forssman antigen.

Recently we successfully galactosylated in vitro a synthetic N-glycoprotein, saposin D, using a recombinant catalytic domain of human <1,4-galactosyltransferase. Its activity towards N-glycans was further confirmed in vivo, by transfection of CHO-Lec2 cell line with a vector encoding the full-length enzyme and subsequent analysis of N-glycans. We show for the first time that human <1,4-galactosyltransferase, contrary to the popular belief, synthesizes Gal<1-4Gal not only on glycolipids but also on N-glycans. Moreover, we observed that the p.Q211E mutein shows substantially higher activity towards N-glycans in comparison to the consensus enzyme.

The N-glycans containing Gal<1-4Gal as the terminal disaccharide are recognized by Stx1, but not Stx2. In addition, experiments using Stx holotoxins indicate that Gal<1-4Gal on N-glycans do not function as true receptors, but instead capture the toxin and act as decoys preventing Stx internalization and cytotoxicity.

365. Structural characterization of neutral and acidic glycosphingolipids isolated from different aspergillus species

K.M. Rodrigues¹, W.A. Mazur¹, L.L. Guimaraes², M.S. Toledo¹, A.H. Straus¹,H.K. Takahashi¹; ¹Departamento de Bioquimica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brasil, ²Laboratorio de Ecotoxicologia, Faculdade Santa Cecilia, Santos, SP, Brasil

The incidence of serious fungal infections such as those caused by genus Aspergillus has increased in the last decade, mainly due to the increase in the number of immunocompromised patients, representing a serious cause of morbidity and mortality in this population. Glycosphingolipids (GSLs) of fungi may present important biological activities, acting as key signaling molecules in various cellular processes. Different Aspergillus species were cultured at 25°C. The lipids were extracted with isopropanol/hexane/water (55:20:25, v/v/v) and chloroform/methanol (2:1, v/v). The extracts were combined, treated with methylamine, and purified by silica gel 60 chromatography. Glycosylinositolphosphorylceramides (GIPCs) and monohexosylceramides (CMHs) from A. fumigatus, A. flavus, A. niger and A. nidulans were analyzed by mass spectrometry using electrospray ionization (ESI-MS/MS) in positive mode. Two peaks corresponding to CMHs, m/z 754 and 756, were identified in all species. The ion 754 m/z was detected in all Aspergillus species. These ions are consistent with CMHs containing ceramides d19:2/h18:1 and d19:2/h18:0. By high performance thin layer chromatography (HPTLC), A. fumigatus, A. flavus and A. niger express both GlcCer and GalCer. In the two species considered more pathogenic GalCer represents 70%. A. niger presents predominantly GlcCer (83%) and A. nidulans exclusively presents GlcCer. GIPCs containing 1-, 2- and 3-hexoses were identified by ESI-MS/MS, peaks of mass 1088, 1116, 1250, 1278, 1412 and 1440 m/z were detected in A. fumigatus, A. flavus and A. niger, these ions were derived from inositolphosphorylceramides (IPCs) 926 m/z and 954 m/z presenting ceramide t18:0/h24:0 and t20:0/h24:0, A. nidulans GIPCs derived only from IPC 926 m/z suggesting that t20:0 sphingoid base could represent a virulence factor. GIPCs of A. fumigatus, A. flavus, and A. niger present a reactive component with the monoclonal antibody MEST-1, indicating the presence of terminal residue of galactofuranose. These results indicate both CMHs and GIPCs have structures not expressed in mammals, these molecules as well as the enzymes involved in these sphingolipids biosynthesis can be considered as potential targets for antifungal therapy.

Supported by FAPESP, CNPq and CAPES.

366. Aberrant autophagy in lysosomal storage disorders is caused by decrease of snare proteins in lysosomes.

H. Tanaka¹, D. Tsuji¹, Y. Ohnishi¹, T. Matsugu¹, H. Teramoto², R. Nakae², K. Itoh¹; ¹Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate school of Pharmaceutical Sciences, Tokushima University, Tokushima, Japan, ²Faculty of Pharmaceutical Sciences, Tokushima University, Tokushima, Japan.

Lysosomal storage disorders (LSDs), which are caused by genetic deficiencies of lysosomal hydrolases or their related factors, are characterized by accumulation of substrates such as glycolipids, oligosaccharides, glycosaminoglycans and so on, and categorized by the neurological and/or peripheral symptoms. In the pathogeneis of LSDs, the abnormalities of autophagy have been suggested because autophagy is a major degradation pathway in cells and plays various important roles for homeostasis. In autophagy, intracellular components, including organelles, are separated by autophagosomes and delivered to lysosomes to be decomposed. In recent years, it has been reported that aberrant autophagy that is mainly impairment of lysosome-autophagosome fusion is caused by unknown molecular mechanisms in LSD cells, and should contribute to the progression of LSD’s symptoms. Therefore, we have tried to reveal the enigma leading to defective fusion of lysosomes-autophagosomes. In this study, we focused on SNARE proteins, which are necessary for membrane fusions in vesicle transport. Lysosome-autophagosome fusion also requires 5 SNARE proteins (lysosomal VAMP8 and STX7, autophagosomal STX17 and YKT6 and cytosolic SNAP29).

We examined the expression of these SNARE proteins and their distribution in lysosomes in control and LSD (GM2 gangliosidosis and galactosialidosis) cells by western blotting. As a result, there were not differences in total contents of VAMP8 and STX7. On the other hand, the amounts of VAMP8 and STX7 in lysosomal fraction separated by immunoprecipitation using anti-LAMP1 antibody were obviously decreased in both LSD cells. We demonstrated the same results by the analyses with the brains of GM2 gangliosidosis model mice in parallel to the course of neuronal cell death.

In conclusion, we revealed that VAMP8 and STX7 in lysosomes particularly in neurons are decreased due to the accumulated substrates including various glycoconjugates, and it may cause the impairment of lysosome-autophagosome fusion and following neurological symptoms in LSDs.

367. Synthesis of glycopolymers with pendant complex-type sialylglycopeptides and their binding affinity with a lectin and an influenza virus

T. Tanaka¹, K. Nakashima¹, K. Sakakibara², Y. Kurebayashi², T. Takahashi², T. Suzuki²; ¹Department of Biobased Materials Science, Graduate School of Science and Technology, Kyoto Institute of Technology, ²Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka

Glycopolymers consisting of synthetic polymers with pendant saccharides have attracted considerable attention as functional polymer materials that can be used in diverse fields, including biomaterials, biomedicines, and biosensors. The numerous approaches used for synthesizing glycopolymers reported to date can be classified into two categories: polymerization of glycomonomers and post-polymerization modification by saccharide derivatives. In post-polymerization modification approaches, polymer backbones bearing activated esters, such as N-hydroxysuccinimidyl (NHS) ester and pentafluorophenyl ester, are useful in synthesizing functional polymers for reaction with amine-containing compounds. In this study, glycopolymers were synthesized using synthetic polymers with pendant-NHS esters and a biantennary complex-type sialylglycopeptide (SGP), in which N-acetylneuraminic acids were presented at the nonreducing ends, by post-polymerization modification of an amino group of SGP with NHS esters on the polymer. The resulting glycopolymers bearing SGPs exhibited strong interaction with the corresponding lectin, Sambucus sieboldiana agglutinin, with higher association constant values of the order of 10^-7 M^-1. Moreover, the glycopolymers bearing SGPs interacted strongly with the human influenza A virus. Their binding affinities for the lectin and the influenza virus increased with increasing degree of polymerization and degree of SGP-substitution. Because SGP has been developed as a commercial resource recently and can be supplied for medicinal chemistry research among other purposes, the glycopolymers bearing oligosaccharides and the synthetic method proposed in this study will be useful for the development of biomedical and pharmaceutical technologies, including biomaterials and biosensors.

368. Property, metabolism and roles of sulfogalactosylglycerolipid in reproduction

N. Tanphaichitr¹, K. Kongmanas¹, A. Saewu¹, H. Xu¹, W. Kiattiburut¹, D. Burger¹, K. Faull⁴, F. Compostella², L. Panza³; ¹Ottawa Hospital Research Institute, University of Ottawa, ²Università degli Studi di Milano, ³Università del Piemonte Orientale, ⁴University of California, Los Angeles

Sulfogalactosylglycerolipid (SGG, aka seminolipid) is expressed selectively in mammalian male germ cells. SGG biosynthesis occurs in testicular germ cells (TGCs) in both pachytene spermatocytes and round spermatids. Alkylacylglycerol (mainly palmitylpalmitoylglycerol-PPG), SGG’s lipid backbone, is first galactosylated by ceramide galactosyltransferase to form galactosylglycerol (GG), which is then sulfated by cerebroside sulfotransferase to become SGG. Our biophysical studies indicate that SGG is an ordered lipid, having propensity to interact with cholesterol. Together with cholesterol, most of SGG on sperm exists in sperm lipid rafts, which have direct affinity for the egg zona pellucida (ZP). On the sperm surface, SGG co-exists with arylsulfatase A (ARSA), which also has direct ZP binding ability. Therefore SGG and ARSA likely act synergistically in sperm-egg interaction. Their co-existence on the sperm surface is due to the binding of SGG to the surface cleft of ARSA. SGG also interacts with high affinity to the active site pocket of lysosomal ARSA, resulting in SGG desulfation. In testicular seminiferous tubules (SFTs), ARSA exists in lysosomes of Sertoli cells, which are somatic cells that are important for development of TGCs into sperm. Sertoli cells are also engaged in phagocytosing residual bodies (excess cytoplasmic components of elongating spermatids) for mature sperm formation. As well, Sertoli cells phagocytose apoptotic germ cells, which amount to 50% of total TGCs. Both residual bodies and apoptotic germ cells contain SGG. Our studies using Arsa knockout mice indicate that phagocytosed SGG is targeted to Sertoli cell lysosomes for desulfation by ARSA. Presumably, galactosylceramidase would then degalactosylate GG to the backbone lipid, PPG, which can be shuttled to adjacent TGCs for a new round of SGG biosynthesis. In Arsa null mice, SGG desulfation does not occur resulting in its intracellular accumulation, a condition that is toxic to Sertoli cells. Arsa^-/- Sertoli cells produce higher levels of H₂O₂ than age-matched wild type mice. H₂O₂ would harm developing TGCs and Sertoli cells themselves. These accruing effects lead to decreased numbers of viable TGCs, increased numbers of abnormal sperm, decreased numbers of Sertoli cells and subsequently subfertility of aging Arsa^-/- male mice. Our results indicate the importance of sperm SGG in fertilization as well as SGG degradation in Sertoli cells in maintaining male fertility.

369. Pancreatic cancer therapy targeting a novel tumor marker using a lectin-drug conjugate

H. Tateno¹, O. Shimomura², T. Oda²; ¹National Institute of Advanced Industrial Science and Technology, ²University of Tsukuba

We performed comprehensive glycome analysis of six pancreatic cancer cell lines using high-density lectin microarrays (Shimomura et al. Mol Cancer Ther 2018). A human pluripotent stem cell (hPSC)-specific lectin rBC2LCN with specificity to Fuc<1-2Galß1-3GlcNAc/GalNAc showed higher binding to Capan-1 with a cancer stem cell-like property than other cell lines. rBC2LCN exhibited strong staining for all tumor tissues obtained from 69 patients and the staining was augmented by drug treatment. We then performed quantitative structural analysis of glycans expressed on tumors obtained from patient-derived pancreatic cancer xenograft mouse models using HPLC/MS, resulting in the detection of an O-glycan containing Fuc<1-2Galß1-3GalNAc (H type3) recognized by rBC2LCN. Since H type 3 was previously reported to be a hPSC marker (Tateno et al. J Biol Chem 2011), the glycan epitope was assumed to be closely related to cellular stemness. Interestingly, rBC2LCN was found to be internalized inside of Capan-1 upon binding. We then hypothesized that rBC2LCN might be utilized as a cargo to deliver drugs inside of pancreatic cancers. We therefore developed a lectin-drug conjugate (LDC, rBC2LCN-PE38) by fusing rBC2LCN with an anti-cancer agent, P. aeruginosa exotoxin A (PE38). rBC2LCN-PE38 exhibited a considerably high cytotoxicity against a pancreatic cancer cell line (in vitro) as well as various mouse xenograft models (in vivo). rBC2LCN-PE38 treatment prolonged the median survival time of pancreatic cancer xenograft mouse models. This presentation will introduce the current state of the art of pancreatic cancer therapy using LDC.

370. Intense physical exercise induces an anti-inflammatory change in igg n-glycosylation profile

M. Tijardović³, D. Marijančević¹, D. Bok², D. Kifer³, G. Lauc⁴, O. Gornik³, T. Keser³; ¹Endocrinology Laboratory, Department of Oncology and Nuclear Medicine, Sestre milosrdnice University Hospital Centre, Zagreb, Croatia, ²Faculty of Kinesiology, University of Zagreb, Zagreb, Croatia, ³Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia, ⁴Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia; Genos Glycoscience Research Laboratory, Zagreb, Croatia

Exercise is known to improve many aspects of human health, including modulation of the immune system and inflammatory status. Despite the general understanding that exercise reduces inflammation, the relation of the two is not yet fully understood. N-glycosylation of immunoglobulin G (IgG) and total plasma proteins was previously shown to reflect changes in inflammatory pathways, which could provide valuable information to further clarify the effects of exercise. In order to better understand the relationship between physical activity and inflammation, we examined the effect of intense exercise, in the form of repeated sprint training (RST), on IgG and total plasma proteins N-glycosylation.

Twenty-nine male physical education students were separated into treatment (RST, N = 15) and control (N = 14) groups. The RST group completed a 6-week exercise protocol while the control group was instructed to refrain from organized physical activity for the duration of the study. Three blood samples were taken at different time points: prior to the start of the training program, the final week of the exercise intervention, and at the end of the four-week recovery period.

Following the recovery period, IgG N-glycosylation profiles showed anti-inflammatory changes in RST group compared to the control group, which manifested as a decrease in agalactosylated and an increase in digalactosylated and monosialylated N-glycans. Observed changes show the potential of intense physical exercise to reduce levels of systemic basal inflammation, as well as the potential for IgG N-glycosylation to serve as a sensitive longitudinal systemic inflammation marker.

371. Lectin-based glycoprofiling of a prostate specific antigen for accurate prostate cancer diagnostics

J. Tkac¹, T. Bertok¹, L. Lorencova¹; ¹Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia

In this contribution we will describe advanced approaches based on application of magnetic particles for lectin-based glycoprofiling of a prostate specific antigen (PSA) for diagnosis of prostate cancer (PCa). The presentation will provide clinical usefulness of PSA glycoprofiling to distinguish healthy individuals from PCa patients. Ability of several lectins to differentiate between glycoforms of PSA from healthy individuals and those with PCa will be provided. Clinical performance of PSA glycoprofiling will be provided using only one lectin (single-parametric data evaluation) or using a combination of lectins (multiparametric data evaluation). In order to prove usefulness of PSA glycoprofiling for PCa diagnostics, in the clinical validation phase study samples from patients with a benign prostate disease (BPH) and patients with PCa will be included with PSA level in the grey zone (2-10 ng/mL). Such samples are the most challenging to analyse since the level of PSA cannot distinguish such samples. In this presentation we will provide evidence that PSA glycoprofiling can really discriminate BPH patients from PCa patients with high accuracy, while analysis of PSA level fail to do so. The presentation will provide clinical usefulness of PSA glycoprofiling for PCa diagnostics using one lectin or combination of several lectins. In order to enhance a diagnostic robustness of the assays, other parameters such as PSA level, fPSA% and age will be combined with the glycan analysis.

In the presentation a literature survey showing a great potential of PSA glycoprofiling not only as a diagnostic, but also as a prognostic (to distinguish aggressive PCa forms from non-aggressive ones) PCa biomarker will be provided. We will also provide usefulness of machine learning algorithms in analysis of large data sets.

The financial support from European Research Council (813120), the Slovak Research and Development Agency APVV 17-0300 and the Slovak Scientific Grant Agency VEGA 2/0137/18 is acknowledged.

372. Preparation and biological activities of chondroitin sulfate proteoglycan from salmon cartilage.

Y. Okamoto¹, T. Mano², T. Wada²,T. Toida¹; ¹Chiba University, ²Nihon Pharmaceutical Co. Ltd.

There have been several reports on physiological activities of salmon (Oncorhynchus keta) nasal proteoglycan which has shown immune-modulating activity in mice and curative properties for acute colitis induced by dextran sulfate sodium in rats. Also, Anti-aging effects of proteoglycans in heirless mice has been investigated. On the other hand, there are several reports on isolation and preparation of proteoglycan molecules from tissue materials, homogenization and extraction steps are important to obtain a large amount of an intact proteoglycan. In this presentation, a new extraction procedure in water containing sugar fatty acid ester as an edible detergent for preparation of the intact form of proteoglycan from salmon nasal cartilage will be introduced. This new isolation step suppressed the degradation of proteoglycan and simultaneously afforded to extract proteoglycan-type II collagen matrix. The prepared proteoglycan was compared with those prepared by different extraction procedures using gel filtration chromatography, and polyacrylamide gel electrophoresis. Additionally, the interaction between the proteoglycan and the human L-selectin was investigated by BIACORE and figured out the binding constant of the proteoglycan was 10 times higher than that of chondroitin sulfate chain. Furthermore, the productions of cytokines by mouse splenocytes under effects of proteoglycans on co-cultured condition were examined by using both RT-PCR and ELISA for anti- inflammatory cytokine levels.

373. Identification and characterization of glycosyltransferases from paramecium bursaria chlorella virus – 1

I. Speciale³, M.E. Laugieri⁵, A. Molinaro², J. Van Etten⁶,M. Tonetti⁴, C. De Castro¹; ¹Department of Agricultural Sciences, University of Napoli, Italy, ²Department of Chemical Sciences, University of Napoli, Italy, ³Department of Chemical Sciences. University of Napoli, Italy, ⁴Department of Experimental Medicine, University of Genova, Italy, ⁵Department of Experimental Medicine, University of Genova,Italy, ⁶Department of Plant Pathology, University of Nebraska

Paramecium bursaria Chlorella Virus – 1 (PBCV1) is a member of Phycodnaviridae, a family belonging to the Nucleo Cytoplasmic Large DNA Viruses (NCLDV) group. It infects the green alga Chlorella variabilis, an endosymbiont of Paramecium bursaria. PBCV-1 genome is 330 kb and it is predicted to encode 416 proteins, where 17 are enzymes that manipulate sugars at different levels, and at least 6 of them are putative glycosyltransferases (GTs). Several evidences have recently indicated that many giant viruses encode a glycosylation machinery that is independent from the infected host. Indeed, the glycans associated to Vp54, the major PBCV-1 capsid protein, display very unusual structures, not found in any other organism studied so far.

Sequence analysis of PBCV-1 putative glycosyltransferases suggests that some of them are multidomain proteins. In particular A064R comprises three domains; the crystal structure of the N-terminal domain has been already resolved, indicating a GT-A type structural fold. A typical SAM-dependent methyltrasferase fold is detected for the C-terminal domain, while no specific structural feature can be predicted for the second domain. Bioinformatic analyses of PBCV-1 mutants that display truncated Vp54 –associated glycans are consistent with the hypothesis that the first two domains of A064R represent a modular ®-L and <-L-rhamnosyltransferase, whilst the C-terminal domain is responsible for addition of the methyl groups capping the outermost rhamnose residue.

To confirm this hypothesis, A064R N-terminal and central domains were cloned and expressed together or separately. The enzymatic activity was tested using a synthetic acceptor and UDP-L-rhamnose as substrates, by UDP-Glo and HPLC assay. Structure of the final products was also confirmed by NMR analysis The results have led to the identification of the N-terminal domain as a ®-rhamnosyl transferase adding one residue to the core xylose unit of Vp54 glycan. Studies are ongoing to confirm the activity the central and C-terminal domain.

374. Study on in cellulo crystallization of human neuraminidase 1 and association with cathepsin A.

J. Tsukimoto¹, T. Iniwa¹, H. Yuto¹, S. Nishioka¹, K. Itoh¹; ¹Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate School of Pharmaceutical Science, Tokushima University.

Human neuraminidase 1 (NEU1) is a lysosomal glycosidase which hydrolyzes terminal sialic acids from the non-reducing termini of sialyloligosaccharides and is activated by interacting with cathepsin A (CTSA). CTSA itself is an acid carboxypeptidase but also acts as the molecular chaperone for NEU1 from endoplasmic reticulum (ER) to lysosome. Overexpression of single NEU1 in mammalian cells causes the intracellular crystallization of NEU1 protein. It is very rare case that human proteins crystalize in human cells. We performed some experiments to examine which parts of NEU1 molecule are important for interaction with CTSA and in cellulo crystallization.

First, we examined whether overexpression of CTSA prevents in cellulo NEU1 crystallization. We constructed an expression plasmid vector encoding CTSA and NEU1. The plasmid vector contains two CMV promoters for simultaneous expression of the two genes. The vector was transfected to CHO cells. Then, immunofluorescence analysis was performed to examine the subcellular distribution of NEU1, CTSA, and LAMP1. As a result, overexpression of CTSA could not completely prevent in cellulo crystallization, although a part of NEU1 was transported to lysosomes.

Next, we tried to find which regions of NEU1 molecules are important for in cellulo crystallization. We constructed plasmids which encode N or C terminal 6x histidine tagged (6His) NEU1. 6His-NEU1 (N terminal tagged) or NEU1-6His (C-terminal tagged) were transfected to CHO cells. Immunofluorescence analysis was performed for 6His-NEU1 or NEU1-6His to reveal whether they crystallize in CHO cells. As a result, we demonstrated that the NEU1-6His forms crystals, but the 6His-NEU1 did hardly in CHO cells.

In conclusion, N-terminal of NEU1 is important and C-terminal is not so important for in cellulo crystallization.

375. Glcnac-6-o-sulfotransferases regulate neuroinflammatory processes in neurodegenerative diseases and neuronal injury

K. Uchimura¹; ¹CNRS, Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, Villeneuve d’Ascq, France

C6-sulfation of GlcNAc residues in N-, O-linked glycans, and keratan sulfate (KS), is catalyzed by GlcNAc-6-O-sulfotransferase (GlcNAc6ST). Four members of the GlcNAc6ST family have been identified in mice. Microglia are key cellular mediators of neuroinflammatory processes. We have found that GlcNAc6ST1 is upregulated in adult central nervous system (CNS) tissues of neurodegenerative diseases, i.e., Alzheimer’s disease, amyotrophic lateral sclerosis, and that activated microglia become positive for sialylated KS-related oligosaccharides. Disruption in GlcNAc6ST1 resulted in a complete absence of the microglial sialyl 6-sulfo oligosaccharides and increased levels of phagocytosis and hyperresponsiveness to interleukin-4 in microglia. Injury to the adult CNS also causes neuroinflammation. We recently found that GlcNAc6ST1 and GlcNAc6ST4 are upregulated in activated microglia and macrophages in the injured adult spinal cord. The contribution of these GlcNAc6STs, and the enzymatic relationship between them, to neuroinflammation and functional recovery after CNS injury is unknown. Here we demonstrate that mice doubly deficient in GlcNAc6ST1 and GlcNAc6ST4, but singly deficient in either enzyme, show improved locomotor recovery after injury. The doubly deficient mice showed a lower abundance of activated microglia /macrophages in the lesion center and an increased level of serotonergic neural fibers in the cord. GlcNAc6ST1 and GlcNAc6ST4 are complementarily involved in the pathogenesis of spinal cord injury, likely by upregulating the detrimental differentiation of microglia/macrophages at the lesion site. The present study provides a new therapeutic basis for enhancing locomotor recovery after spinal cord injury, by targeting these two sulfotransferases.

376. A comparative analysis of affinities of antibodies to a high-mannose glycan by theoretical methods

K. Ueno-Noto¹, M. Kusumoto², K. Takano²; ¹College of Liberal Arts and Sciences, Kitasato Univ., ²Graduate School of Humanities and Sciences, Ochanomizu Univ.

Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein gp160 has been a target protein for the development of HIV-1 vaccines. It is a trimeric antigen which based upon glycoprotein gp120 linked to part of the transmembrane glycoprotein gp41. A series of broadly neutralizing antibodies called PGT have shown to be bound directly to HIV-1 via high mannose glycans on the glycoprotein gp120. Despite the similarity of the amino acids sequences of the PGT antibodies, their affinities to the glycan were experimentally shown to be different, but the causative factors remained elusive.

The objective of the present study is to establish a theoretical method to evaluate the experimentally shown affinities and elucidate the characteristics of the interaction between the high mannose glycan and the PGT antibodies. Quantum mechanical calculations considering electron correlation and solvation effects, were applied to the crystal structures of complexes of a series of PGT antibodies binding to a high-mannose glycan (PDB ID: 4TVP, 5T3S, 3TWC, and 3TV3), and detailed glycan-antibody interactions were analyzed. In addition, molecular dynamics simulations were performed with these glycan-antibody complexes, and the structural stabilities of them were systematically compared. The differences among structural stability of the glycan in the active site of the complexes and total interaction energies as well as binding free energies between the glycan and antibodies agreed well with the experimentally shown affinities of the glycan to the antibodies. The characteristics of the glycan-antibody interaction observed with the antibody having the highest affinities among them were as follows; the terminal saccharide of the glycan, Man D3, was structurally stable and responsible for the glycan-antibody binding through electrostatic and dispersion interactions. The structural stability of non-terminal saccharides such as Man 4 or Man C played substantial roles in the interaction via direct hydrogen bonds.

This study was supported in part by Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (18K11534 to K. U-N.).

377. The interaction of neisseria n-acetylhexosamine-1-phosphotransferases with their donor substrates

V. Veeramachineni¹, M. Battistel¹, J. Beaudet¹, H. Yu², X. Chen², W. Vann¹; ¹Center for Biologics Evaluation and Research, FDA, Silver Spring, Maryland, ²Department of Chemistry, University of California Davis / Davis, CA

Neisseria meningitidis is an important cause of bacterial meningitis worldwide. The serogroups of Neisseria meningitidis most commonly associated with disease are A, B, C, Y, W and X. Vaccines based on the capsular polysaccharides of serogroups A, C, Y, and W are available and effective at preventing disease, while a vaccine against group X is still in development. Serogroups A (NmA) and X (NmX) are 1-linked phosphodiesters of N-acetylhexosamines and are polymerized by N-acetylhexosamine-1-phosphotransferases from UDP-N-acetylhexosamines. Both enzymes share sequence homology, with conserved residues associated with genetic disease, in the catalytic domain of lysosomal N-acetylglucosamine-1-phosphotransferase in humans. We have investigated the interaction of the bacterial phosphotransferases with their sugar nucleotide donor substrates, by measuring the influence of substrate structure on direct binding and steady state kinetics of these enzymes. Our results show that modification of the acetamido group on their respective sugar nucleotide substrates has a dramatic effect on the kinetics of both NmA and NmX phosphotransferases. We have devised novel chemoenzymatic pathways to synthesize analogs of UDP-N-acetylglucosamine. Donor substrates with modifications at the 4-hydroxyl terminate NmX chain elongation. The 4-hydroxyl of N-acetylglucosamine may serve two purposes in the NmX enzyme active site, as an acceptor for the incoming sugar-1-phosphate and participating in binding of the sugar nucleotide to the enzyme. We have also synthesized analogs with modifications at the uridine to further investigate steric effects on acceptor and donor interactions with the NmX phosphotransferase.

378. Parasitic glycosylphosphatidylinositols as tools for biomedical research

A. Malik¹, R. Roller¹, M. Garg¹, M. Grube¹, M. Carillo¹, P.H. Seeberger¹,D. Varon Silva¹; ¹Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am Muehlenberg 1, 14476 Potsdam, Germany

Protozoan parasites cause multiple non-self-limiting infections and neglected diseases of high morbidity and mortality worldwide. Most of these parasites contain millions of copies of free glycosylphosphatidylinositol glycolipids (GPIs) or GPIs that anchor proteins to the cell membrane. GPIs contain a core structure with a glycan, a phosphoethanolamine, and a phospholipid and have cell-specific modifications. GPIs play an important role as immunomodulators of the host’s immune system during infections and induce the formation of anti-GPI antibodies. To investigate the potential of GPI glycolipids in diagnosis and the development of anti-parasitic vaccines, we established strategies to synthesize GPIs, GPI-derivatives and for the production of natural and non-natural GPI-protein glycoconjugates. Here, we present the synthesis and evaluation of GPIs glycolipids from Plasmodium falciparum, Trypanosoma brucei, Toxoplasma gondii and from humans. We evaluated the role of GPIs in the folding and activity of the naturally glypiated MSP1-19 protein of Plasmodium falciparum and of the human Thy-1 protein and produced GPI-glycoconjugates for in vitro and in vivo determination of immunogenic epitopes in P. falciparum GPIs. In addition, we present the development of a bead-based multiplex assay for detection of toxoplasmosis and the progress for a microarray-based detection of African human trypanosomiasis.

379. Recombinant fungal lectins as molecular tools for glycoprofiling

A. Cabanettes², S. Basheer², A. Breiman³, M. Henry⁴, A. Zitouni⁵, Y. Necib⁵, C. Unverzagt¹, J. Coll⁴, J. Le Pendu³, A. Varrot²; ¹Bioorganische Chemie, Bayreuth University, Bayreuth, Germany, ²CERMAV, CNRS UPR 5301, Univ. Grenoble Alpes, Grenoble, France, ³CRCINA, Inserm, Université d’Angers, Université de Nantes, Nantes, France, ⁴Institute For Advanced Biosciences, INSERM UGA U1209, CNRS 5309, La Tronche, France, ⁵Mentouri University, SNV, Microbiology and Biochemistry Department, Constantine, Algeria

The glycome exemplify the remarkable complexity and organismal diversity of glycans. It is the hallmark of cell identity and fate but also a signature of health and disease. Glycosylation is a complex non templated and highly variable process where defects or alterations are commonly linked with physiopathological states such as cancers or chronic inflammatory diseases. Glycosylation modulates the function of biomolecules (proteins, lipids) in many ways and controls properties such as immunogenicity, stability and bioactivity. Achieving the appropriate glycosylation of biotherapeutics is crucial since it is closely associated with drug’s quality, safety and efficacy, so it should be controlled and analyzed. Molecular tools are therefore necessary for rapid, easy and accurate glycoprofiling and for decoding the glycome information. Lectins can be such tools since those carbohydrate binding proteins are able to recognize glycan patterns in a highly specific manner. The ubiquity of their interactions with glycans opens great potential for their exploitation in medicine as well as in biomedical and biotechnological applications. We have biochemically and structurally characterized several fungal lectins for the exclusive detection and analysis of precise glycan epitopes We will present, here, PhosL from Pholiota squarrosa and TrfbL2 from the desert truffle Terfezia boudieri which recognizes <1,6 core fucosylation and TF antigen (Galß1-3GalNAc), respectively. Both glycan motifs are tumor associated carbohydrates antigen (TACA) and core fucosylation has also a great impact on antibodies bioactivities. We produce both lectins recombinantly to ensure steady availability and quality. We will present the identification, characterization and structure of TrfbL2 and our latest results on PhosL. Several constructs have been made and compared. Both lectins were fluorescently modified and could labelled whole cancers cells or tissue sections. The labelling is dependent of the glycan recognition so both lectins present a great potential as highly effective glycoprofiling tools.

380. Simple method for high throughput screening of fimh ligands

B. Roubinet¹, J. Bouckaert², C. Bridot², F. Vena¹, L. Landemarre¹; ¹GLYcoDiag, rue de Chartres, Bât. Physique-Chimie, 1er étage, 45067 Orléans (FRANCE), ²UGSF, Faculté des Sciences et Technologies Cité Scientifique - Bât. C9 59655 Villeneuve d’Ascq (FRANCE)

FimH lectin (or type 1 fimbrial lectin), found in Escherichia Coli (E.Coli), is a lectin which plays crucial role in bacterial adhesions and diseases through interactions with carbohydrate receptors. FimH lectin is known to have high and specific affinity with mannosylated structures. Since the 1970s, a large panel of synthetic inhibitors have been developped in order to modulate the bacterial colonisation. In this context, the development of smart and innovative tools to screen potential ligands/inhibitors of FimH or to highlight glycans specificity of a glycoconjugate recognized by this lectin have deep interests.

We have designed a robust and reliable lectin kit, called FimH LectPROFILE kit. The kit is composed of a 96-well plate immobilised with FimH, a specific tracer (neoglycoprotein) which has specific affinity for FimH and a revealing solution. Two type of tracers (either neoglycoprotein or neoglycocluster) of two different avidities have been developped to perform from sensitive to ultrasensitive screening of potential inhibitors of bacterial adhesion (or FimH ligands). FimH LectPROFILE kit have recently shown fast and efficient evaluations of crude or purified glycoconjugates interactions (i.e. synthetic molecules or glycoconjugates) and correct determination of IC50-values (concentration corresponding to 50% of lection inhibition). In this communication, we will shown the results obtained with the FimH LectPROFILE kit on a panel of common glycoconjugates and we will compare the results obtained (time, accuracy, ...) with the surface plasmon resonance (SPR) technology commonly used for the determination of biomolecular interactions.

381. Mapping the granule-specific n- and o-glycome of human neutrophils

V. Venkatakrishnan⁴, R. Dieckmann⁴, I. Loke¹, H. Tjondro¹, S. Chatterjee¹, J. Bylund³, M. Thaysen-Andersen¹, N.G. Karlsson², A. Karlsson⁴; ¹Biomolecular Discovery and Design Research Centre (BDDRC), Department of Molecular Sciences, Macquarie University, Sydney, Australia, ²Department of Medical Chemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, ³Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, ⁴Department of Rheumatology and Inflammation Research, Institute of Medicine Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

Protein glycosylation is implicated in diverse functions of the human innate immune system including in neutrophil biology. During neutrophil maturation in the bone marrow, distinct granule subsets containing a unique set of receptor and effector proteins are sequentially formed via a “targeting-by-timing” process. Apart from our recent reports documenting a set of immune-related paucimannosidic N-glycoproteins abundantly residing in the azurophil granules (AG), the existing knowledge of neutrophil glycans has unfortunately almost exclusively been derived from whole-cell preparations without granule-resolution. Thus, a detailed organelle-specific map of the neutrophil protein glycans remains to be established. To this end, the AG, specific (SG), gelatinase granules (GG), secretory vesicles and plasma membrane (SV+PM) were isolated from resting neutrophils obtained from three healthy donors and studied by glycomics. The N- and O-glycome were sequentially released from granule protein extracts and their fine structures were determined and quantitatively profiled using porous graphitized carbon-liquid chromatography-tandem mass spectrometry. Interestingly, the N-glycan profiles showed strong organelle-specific differences. For example, paucimannosidic N-glycans were abundant in the AG whereas the SG and GG comprised elongated N-acetyllactosamine-containing N-glycans with and without Lewis epitopes. Heterogeneity and the length of the N-glycans increased progressively from AG to SG and then to GG, suggesting time-dependent changes in the N-glycosylation process during the temporal granule formation associated with neutrophil maturation. In contrast, the SV+PM displayed more conventional oligomannosidic and complex N-glycans, possibly reflecting a successive addition of glycoproteins to the plasma membrane over time. O-glycans were surprisingly absent in AG while complex core 2 O-glycans were consistently identified in the other granule subsets. It appears there is less granule-specific O-glycosylation relative to N-glycosylation. This comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and provides a necessary first step towards understanding the role of neutrophil glycoproteins in the innate immune response.

382. Screening of polysialic acid positive cells during the development of fish larvae and during germ cell maturation in coregonus maraena.

M.T. Venuto², J. Martorell Ribera¹, A. Rebl¹, R. Bochert³, S.P. Galuska²; ¹Fish Genetics Unit, Institute of Genome Biology, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany, ²Junior research group Glycobiology, Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany, ³Research Station Aquaculture Born, Institute of Fisheries, Mecklenburg-Vorpommern Research Centre for Agriculture and Fisheries (LFA MV), Born/Darß, Germany

Whitefish Coregonus maraena is an anadromous freshwater fish in the Southern Baltic Sea and its connected rivers. C. maraena belongs to the family Salmonidae (subfamily Coregoninae) and the demand for this food fish has strongly increased during the last years, however, the natural stocks of C. maraena are low. Thus, various attempts have been made to develop strategies for extensive (ponds) and intensive (recirculating aquaculture systems (RAS)) farming. Nevertheless, due to its high susceptibility to stress and diseases, the aquaculture of C. maraena is a difficult endeavour and more knowledge of its physiology is necessary. For this study we used F2-generation from RAS to investigate the distribution of polysialic acid (polySia) during the development of fish larvae and during germ cell maturation.

Sialic acid polymers strongly modulate both neuronal as well as immunological events and, therefore, we are interested in the distribution of polySia in C. maraena. Interestingly, polySia appears during distinct stages of oogenesis and only selected cell populations were polySia-positive during embryonic development of the gills and other organs. The obtained data provide new insight in the role of polySia during the oogenesis and organogenesis in Salmonidae.

383. O-glcnac transferase silencing affects complex glycosylations in human colon cell lines: glycosphingolipid synthesis and sialylation of glycoconjugates are regulated by o-glcnacylation

A. Vercoutter-Edouart¹, M. Mortuaire¹, J. Biwi¹, C. Clarisse¹, Y. Guérardel¹, C. Biot¹, T. Lefebvre¹; ¹Université de Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, F-59000, Lille, France.

Heightened glucose metabolism is one of the molecular mechanisms stimulating tumorigenesis and promoting cancer cell growth. High glucose consumption upregulates the hexosamine biosynthetic pathway (HBP), resulting in an increase in UDP-GlcNAc concentration, the end-product of the HBP. UDP-GlcNAc is the donor-substrate of O-GlcNAc transferase (OGT) which is a unique glycosyltransferase catalyzing the O-GlcNAcylation of cytoplasmic and nuclear proteins. UDP-GlcNAc is also a substrate for the biosynthesis of other glycoconjugates. Moreover UDP-GlcNAc can be converted into UDP-GalNAc and CMP-NeuAc which are substrates for complex glycosylations of proteins and lipids. Aberrant cell surface glycosylation is a hallmark of various cancers, including colorectal cancer (CRC). These changes contribute to the progression of cancer by modulating cell-cell adhesion, extracellular matrix-cell interactions and signaling pathways. On the other hand, O-GlcNAcylation and OGT levels are significantly upregulated in CRC. We and other authors have previously shown that downregulation of OGT in cancer cells impedes their proliferation, migration and adhesion properties. Using three human colon cell lines (CCD841CoN fetal colon cells, HT29 adenocarcinoma cells, HCT116 carcinoma cells), we have further investigated by glycomics whether OGT is important for complex glycosylations, especially on N- and O-glycoproteins and glycosphingolipids (GSLs). We show that the GSLs composition is affected in OGT-silenced cells, albeit in a cell-dependent manner. Sialylation is also perturbed at the cell surface of these cells. In addition to lectin labeling, we used a biorthogonal metabolic strategy and click chemistry to detect sialylated glycans thanks to the metabolic uptake of ManNAl (alkyne analog of ManNAc), and incorporation into sialylated glycoconjugates. We have also compared the metabolic incorporation of ManNAl to the peracylated azido analog, Ac4-ManNAz. Thanks to this approach, identification of the labeled sialoglycoproteins that are affected by O-GlcNAcylation levels is possible. Altogether our results strongly suggest that OGT may regulate cellular interactions and signaling through fine regulation of cell surface complex glycosylation in colorectal cells.

384. Anti-muc1 monoclonal antibody recognizing a glycopeptide epitope

H. Wakui¹, Y. Tanaka², T. Ose¹, H. Hinou¹, M. Yao¹, S. Nishimura¹; ¹Graduate School of Life science, Hokkaido University, Sapporo, Japan, ²Graduate School of Life sciences, Tohoku University, Sendai, Japan

Mucin1 (MUC1) is a membrane-bound glycoprotein presenting on the epithelial cell surface. In many types of cancer cells, MUC1 bearing aberrant glycans are overexpressed and form neo-antigens called tumor-associated MUC1. Hence, numerous anti-MUC1 antibodies have been developed as candidates for diagnostic and therapeutic reagents. However, in our previous study, it was revealed that most of them do not have specificity for particular sugar chain structures. Glycan structures itself are biomarkers and strongly related to disease type and state. Therefore, glycan-specific anti-MUC1 monoclonal antibodies (mAbs) have potentials to improve the accuracy of diagnosis, and to be novel therapeutic antibodies.

Recently, our group obtained a novel anti-MUC1 monoclonal antibody (SN-101) targeting MUC1 that contains tumor-associated Tn antigen. To develop glycan-specific mAbs, a homogeneous synthetic MUC1 glycopeptide was immunized and the antibodies were screened by testing the ability to discriminate naked peptide and glycopeptide epitope.

In this study, we revealed, by using glycopeptide microarray analysis, that SN-101 needs a minimum epitope sequence VTSAPDT(Tn) and, interestingly, glycosylation at the VTSA region or replacing Tn antigen with T antigen in the PDT region blocked binding of SN-101. These binding features can be explained by a crystal structure of SN-101 complexed with a synthetic glycopeptide. Furthermore, it was demonstrated that SN-101 directly recognizes O4 and O6 position of GalNAc moiety, and that conformation of the glycopeptide within the SN-101 is quite similar to the solution NMR structure of MUC1 glycopeptide. This binding feature to the glycan is completely different from that of other conventional anti MUC1 antibodies such as SM3 and AR20.5.

Herein, we communicate the feasibility of a new strategy to establish glycopeptide antibodies.

385. The functional mechanism of c-type lectin receptor lox-1 in esophageal tumorigenesis

W. Lan¹, L. Can¹, X. Ling¹, G. Jianxin¹; ¹NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China

LOX-1 was initially found as a receptor of ox-LDL, so previous studies focused on endothelial cell surface LOX-1 and its biological function in atherosclerosis. Here, we investigated the expression pattern of LOX-1 in esophageal cancer, explored a new perspective how LOX-1 regulate cancer development and progression and provided a potential intervention.

In this study, we found LOX-1 expression had the maximum increasing discrepancy in esophageal cancer among 15 tumors from TCGA database, and genetic deletion of LOX-1 or inhibition of LOX-1 activity decreases tumor growth by modulating autophagy signature in clinical biopsies and mouse models. Mass spectrometry-based proteomic analysis combined with in situ Proximity Ligation Assay (PLA) identified a shuttling protein RACK1 recruited by LOX-1 as a key signal adaptor. This LOX-1-RACK1 interaction in esophageal cancer triggers MEK/ERK signaling to limit nuclear translocation of autophagy-related transcription factor. Furthermore, we offer a bioactive polysaccharide Fucoidan as an intervention strategy to inhibit esophageal tumorigenesis via reducing the stability of LOX-1 by ubiquitination degradation model. Our findings first establish the carcinogenesis role of LOX-1 in esophageal epithelial cells independent of apoptosis and highlight the intervention effect of LOX-1 exogenous ligand Fucoidan in esophageal cancer mouse model compared with the traditional chemotherapeutic drug 5-Fluorouracil.

386. N-glycosylation of human immunoglobulin g and rheumatoid arthritis risk in han chinese population: a mendelian randomization study

M. Sun², H. Hou³, D. Li³, W. Wang¹, Y. Wang²; ¹School of Medical and Health Sciences, Edith Cowan University, ²School of Public Health, Capital Medical University, ³School of Public Health, Taishan Medical University

Background We previously showed that plasma N-glycans is associated with rheumatoid arthritis. However, the causality of this association is uncertain. Hereby we investigated this by testing variants associated with levels of IgG N-glycosylation for association with RA susceptibility using a one-sample Mendelian randomization study.

Methods Patients were recruited in Tangshan, Hebei Province from January 2013 to January 2014. Sixteen SNPs showing association with IgG N-glycosylation were used as instrumental variables. Hydrophilic interaction high performance liquid chromatography-Ultra-performance liquid chromatography was used to test plasma N-glycans and Mass ARRAY system was used to get SNP genotyping. Two-stage least squares regression method was used to estimate the causal effect. Inverse variance weighted method was used to summarize the ratio estimates for the individual genetic variants.

Results Among the 16 SNPs, the genotypic frequencies of rs11710456 and rs2072209 (rs11710456, P=0.03, and rs2072209, P=0.04, respectively) as well as the allelic frequencies of rs11710456 and rs404256 (rs11710456, P=0.02, and rs404256, P=0.04, respectively) were significantly different between two groups. rs11710456 and rs404256 was associated with RA after adjusting for age and sex (rs11710456, adjusted odds ratio (AOR)=0.42, 95% confidence interval (CI): 0.22-0.80, P=0.01, and rs404256, AOR=1.58, 95% CI: 1.00-2.48, P=0.05, respectively). In these two SNPs, two-stage least squares regression method with rs404256 as IVs showed a causal association between N-glycans and risk of RA.

Conclusions Mendelian randomization analysis supports the causal relationship between IgG N-glycosylation and the susceptibility of rheumatoid arthritis, suggesting that IgG N-glycosylation might be the therapy target of RA.

387. Discovery of o-glcnac transferase inhibitors

Y. Wang¹, Y. Ma¹, X. Ye¹; ¹State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutics Sciences, Peking University, Beijing

O-GlcNAc transferase (OGT) is a key enzyme involved in dynamic O-GlcNAcylation of nuclear and cytoplasmic proteins similar to phosphorylation. Discovery of cell-permeable OGT inhibitors is significant to clarify the function and regulatory mechanism of O-GlcNAcylation. This will establish the foundation for the development of therapeutic drugs for relevant diseases. In our preliminary work, two cell-permeable non-competitive OGT inhibitors 1-(4-acetamidophenyl)-4-(naphthalen-2-yl)-1H-1,2,3-triazole (APNT) and 1-(4-acetamidophenyl)-4-[1,1’-biphenyl]-4-yl)-1H-1,2,3-triazole (APBT) were developed via tethering in situ click chemistry (TISCC). Both of them were able to inhibit O-GlcNAcylation in cells without significant effects on cell viability. APNT exhibited better inhibitory activity (IC₅₀ = 66.7 ± 0.8 ⎧M) and therefore it was selected as the lead compound to develop new OGT inhibitors. On the one hand, binding pocket of APNT derivative was expanded through traditional in situ click chemistry. Screening of 60 alkyne building blocks resulted in a novel OGT inhibitor with more potential activity. On the other hand, modifications of APNT were performed through medicinal chemistry. Several different linkers between the benzene ring and the naphthalene ring were attempted instead of the previous triazole ring to improve the solubility of the inhibitor. Next, the type and position of the substituents on the benzene/naphthalene ring were explored. This study provided a lead compound with the novel scaffold for subsequent exploration of OGT inhibitors and a potential tool for the regulation of O-GlcNAcylation. This work was financially supported by the National Natural Science Foundation of China (Grants 21302182, 21778007) and China Postdoctoral Science Foundation (Grants 2013M540133, 2014T70113, 2017M610721).

388. The promising effect of a chitosan–inulin conjugate on bacteria biofilm

Z. Wang¹, R. Li¹, G. Zhang¹, Y. Du¹; ¹State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, P.R. China

Chitosan, the only cationic polysaccharide in nature, has been demonstrated to have antimicrobial and anti-biofilm activities, which, however, require a relative high dosage of chitosan. Moreover, poor water solubility further restricts its applications on anti-infection therapy. Chitosan have been demonstrated to have antimicrobial and anti-biofilm activities, which, however, require a relative high dosage of chitosan. Inulins are a group of polysaccharides produced by many types of plants, and are widely used in processed foods. Compared to chitosan, inulin possesses a mild antibacterial activity against certain pathogenic bacteria. In order to develop an effective strategy to treat biofilm-related infections, we introduce a method by covalent conjugation of inulin to chitosan. The novel polysaccharide–polysaccharide conjugate significantly enhanced activities against broad-spectrum bacteria strains either in a biofilm or planktonic state. To be noted, the conjugate also showed low cellular toxicity to mammalian cells. These results suggested that chitosan conjugation of inulin was a viable strategy for treatment against biofilm-related infections. This finding may further spread the application of natural polysaccharides on treatments of infectious disease.

389. Glycans from plant glycoprotein lectins for designing targeted nanovaccine for cancer immunotherapy

V.U. Warrier¹, A.I. Makandar¹, B. Gupta¹, D. Sadaria¹, R.R. Shah¹, R.K. Gupta¹; ¹Protein Biochemistry Research Laboratory, Dr. D. Y. Patil Biotechnology & Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth (Deemed to be University), Tathawade, Pune - 411033, (Maharashtra), India

Cross-presentation is an important mechanism by which dendritic cells (DC) present exogenous antigens on MHC class I molecules that in turn activate CD8+ T cells to trigger cytotoxic T-lymphocyte(CTL) immune response. For effective anti-cancer immunotherapy, unleashing cross-presentation to trigger specific CTL response against the tumor cells is considered as an attractive option. Specific targeting of DCs in vivo can be achieved by targeting the DC-receptors such as C-type lectin receptors (CLRs). Most studied member of the CLRs is DC-SIGN, which is known to bind high mannose and fucose structures. Glycan modification of antigens has been reported to not only enhance the antigen presentation but also associated with strong CD8+ T cell response. In this study, we have done a systematic literature survey of plant glycoproteins lectins and build their N-and O-linked glycan structures using advanced GlycanBuilder2 software. Further we have purified glycosylated plant lectins from Glycine max and Erythrina indica using Lactosylamine-Bio-Gel P-100 column. Glycopeptides were prepared from purified lectins using pronase digestion and purification on Bio-Gel P-4 column. Fractions showing high carbohydrate content using phenol-sulphuric acid method selected for ESI-MS/MS analysis. Mass of glycopeptides obtained from SBA was found to be 2321.06 & 2259 Da, whereas, in E. indica lectin digest, mass was found to be 2078.73 & 2180.50 Da. Difference of 2 kDa per subunit between SBA and deglycosylated SBA confirmed the presence of Man9GlcNAc2 (molecular mass 1866 Da) in native SBA. Mass analysis also confirmed the presence of Man9GlcNAc2 in these glycopeptides. Hydrazinolysis of glycosylated lectins was performed for the complete release of unreduced N-linked oligosaccharides for 5 hr at 95°C, followed by hydrazine removal and re-acetylation and purification on DOWEX50W(H+) and Bio-Gel-P4 for purification of oligosaccharides. Thus, we have standardized and developed an innovative approach to purify glycopeptides and oligosaccharides. Our further studies are focused on studying the targeting of CLRs on DCs using these glycopeptides and oligosaccharides. (Financial assistance from DST-SERB (File No. ECR/2016/001187), DPU/106(18)/2015 and DPU/17/2016 is gratefully acknowledged)

390. Introduction of a novel labelling strategy to facilitate lc-ms analysis of released n-glycans

P. Widdowson³, Z. Szabo², S. Khan², J. Bones¹; ¹NIBRT, Dublin, IRE, ²Thermo Fisher Scientific, Oyster Point, USA, ³Thermo Fisher Scientific, Runcorn, UK

Glycosylation profiling and characterization is a key critical quality attribute (CQA) in the development of most protein therapeutics. Attached glycans have a marked effect on the immunogenicity, biological activity, pharmacokinetics and stability of a protein therapeutic and, thus, the comprehensive characterization of glycan content and structure is a regulatory requirement. Not all fluorescent glycan labels are compatible with mass spectrometric analysis, which is a highly desirable tool for comprehensive glycan identification.

A novel 3-aminobenzene sulfonic acid (3-ASA) fluorescent labelling approach has been developed and here we evaluate 3-ASA as a strategy for the labelling and subsequent LC-MS analysis of released N-glycans; facilitating enhanced identification and comprehensive glycan characterization.

Glycoprotein samples were prepared using a modified version of the Applied Biosystems™ GlycanAssure™ HyPerformance kit incorporating the 3-ASA label. Samples were heat denatured prior to deglycosylation using PNGase F and subsequently labelled with 3-ASA. Labelled glycans were then cleaned-up using GlycanAssure magnetic beads prior to LC-MS analysis. LC separation was performed using HILIC chromatography on a Thermo Scienfitic™ Vanquish™ Horizon UHPLC system. High resolution mass spectrometry was performed in negative ionization mode on an Orbitrap-based mass spectrometer controlled by Thermo Scientific™ Chromeleon™ Chromatography Data System (CDS) software.

Released and labelled N¬-glycans have been successfully analysed by LC-MS using the novel 3-ASA label. Glycan peaks were adequately resolved chromatographically and the relative quantities of the individual glycan species for all samples were highly comparable to those observed when using currently commercially available labels. Glycan labelling using 3-ASA has been shown to occur with high efficiency (> 90%) and no bias was observed for glycan labelling towards any specific glycan types.

12C and 13C-labelled isotopologues of 3-ASA have been prepared and utilized in the labelling strategy. The availability of both 12C and 13C-labelled isotopologues of 3-ASA has allowed the ability to quantify glycans based on isotopic ratios. This opens up the possibility for two-plexed glycan analysis to be carried out, whereby a 12C-labelled sample and a 13C-labelled sample can be mixed and analysed simultaneously with the ability to distinguish the samples using mass detection. This approach results in decreased overall run times by a factor of 2 which is particularly advantageous for high-throughput comparability studies such as clone screening and biosimilar comparability.

For characterization, mass spectrometry of 3-ASA labelled glycans was performed in negative ionization mode which proves advantageous when compared to positive mode ionization strategies. Analysis in negative mode was not accompanied by rearrangements such as fucose migration or in-source decay leading to loss of N¬-acetyl glucosamine residues leading to more confident identifications. In addition, MS2 fragmentation in negative mode generated more structurally informative spectra due to the increased propensity for cross-ring fragments to be formed. Released N-glycans from several glycoprotein sources have been labelled using 3-ASA and analysed in negative ionization mass spectrometry. More structurally relevant diagnostic ions were observed in glycans labelled with 3-ASA compared to other commercially available labels which led to enhanced identification and characterization glycan structures.

Enhanced structural characterization of released N-glycans using a novel fluorescent label in a manner accessible to the biopharma industry.

391. SLC35A2 has the potential to form complexes with functionally distinct glycosylation enzymes

W. Wiertelak¹, Y. Zadorozhna¹, B. Bazan¹, M. Olczak¹, D. Maszczak-Seneczko¹; ¹Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw,

Glycosylation is crucial for nearly all aspects of life, including the development of multicellular organisms. Substrates which are used in the glycosylation process are nucleotide sugars. They are synthesized in the cytosol. To be delivered into the ER and Golgi lumen, where glycosylation occurs, they need to be translocated across membranes of these organelles. Nucleotide sugar transporters (NSTs) play an essential role in this process. The mutations in the gene encoding SLC35A2 which has a UDP-galactose transporting activity, causes a variant of congenital disorder of glycosylation.

Here we employed a novel protein fragment complementation assay called NanoBiT to explore the potential of SLC35A2 to form complexes with selected glycosylation enzymes. We previously showed that SLC35A2 forms heteromers with N-acetylglucosaminyltransferases (MGATs 1-5). Here, for the first time, we demonstrate an interaction between SLC35A2 and B4GalT1, which is the second discovered interaction of this NST with a galactosylotransferase. Moreover, we show a previously unacknowledged interaction between SLC35A2 and a sialyltransferase (ST6Gal1). Furthermore, we show that Mgat2 interacts with B4GalT1. Given the fact that B4GalT1 interacts with ST6Gal1 and SLC35A2 interacts with Mgat2, B4GalT1 and ST6Gal1, we postulate that all listed proteins, which are crucial for process of N-glycosylation, could be in close proximity. We also demonstrate a close proximity between SLC35A2 and galactosyltransferases involved in O-glycosylation (C1GalT1) and keratan sulfate chain elongation (B4GalT4).

Our study suggests that the biological consequences of the SLC35A2 depletion in CDG patients might not be limited to a defect in UDP-galactose translocation across the Golgi membrane. Additionally, we suggest the existence of multiprotein glycosylation-related complexes, whose spatial organization might be orchestrated by NSTs.

392. Method development of o-glycan analyses for use in regenerative medicine

H. Wilkinson², A.L. Rebelo¹, A. Pandit¹, R. Fahey (saldova)²; ¹Centre for Research in Medical Devices, National University of Ireland Galway, Ireland, ²Centre for Research in Medical Devices, National University of Ireland Galway, Ireland | National Institute for Bioprocessing, Research and Training, Dublin, Ireland | UCD School of Medicine, College of Health and Agricultural Science and UCD Conway Insti

Glycosylation is the most common post-translational modification of proteins. Glycosylation patterns may differ between control and disease tissue and have potential to be used as biomarkers for these diseases. Regenerative implants and therapeutics developed from this knowledge may be utilised to re-establish a healthy glyco-environment. As glycans help determine protein stability and function, an understanding into the role of glycoproteins in Parkinson’s Disease (PD) is of great interest.

Two main classes of glycans present on these proteins are N-glycans and O-glycans. The enzyme PNGase F cleaves the common core of N-glycans from the glycoprotein for subsequent analysis, however, no single enzymatic approach exists to release O-glycans due to a lack of common O-glycan core, so chemical methods are used. The downside to this is a “peeling” reaction which degrades the glycan making analysis difficult and non-reproducible.

This work focuses on optimising a non-reductive ®-elimination for O-glycan release that utilises a base, 40% dimethylamine containing ammonium carbonate, to release and convert the glycan to a glycosylamine to prevent peeling. The addition of microwave radiation has been utilised to speed up a release reaction that typically requires 16-40 hours of incubation to 8-12 minutes. The optimisation of the method includes optimal release of O-glycans of fetuin from fetal bovine serum suspended in a bis-acrylamide gel by observing a range of sample preparation conditions such as reducing and alkylating the protein, limiting glycan heterogeneity by releasing N-glycans first, finding ideal reaction reagents, identifying ideal sample concentration and finding the most suitable microwave reaction conditions, as well as adjusting analytical techniques using hydrophilic interaction liquid chromatography-ultra performance liquid chromatography (HILIC-UPLC) and mass spectrometry (MS) for subsequent analysis.

After the method is developed, O-glycans will be released from nigro-striatal tissue regions of the human brain (healthy and PD) as well as from rat models with LPS induced neuroinflammation, forming the basis of a comparative glycan analysis for potential biomarker identification.

Acknowledgements

SFI and ERDF (Grant No. 13/RC/2073 and 17/RC-PhD/3480)

393. Glycan structures and their recognition roles in the human blood group abh/ii, lea, b, x, y and sialyl lea,x active cyst glycoproteins

A.M. Wu¹; ¹Glyco mics Res, Lab., College of Medicine, Chang-Gung University, Taoyuan, Taiwan.

Human ovarian cyst glycoproteins (HOC , cyst gps) isolated from pseudomucinous type of human ovarian cyst fluids is one of the most rich and pioneer sources for studying brosynthesis, structures and function roles of blood group ABH, Lea,b,x,y, sLea and sLex active glycoproteins. After 70+ years exploration, four top highlights are shared. (i) an updated concept of glycotopes and their internal structures in cyst gps was composited; (ii) the unknown codes of new genes in secreted cyst gps were unlocked as Lex and Ley ; (iii) recognition profiles of cyst glycans and a sialic acid rich (18%) glycan with lectins and antibodies were shown. (iv) co-expression of Blood Group A/ A-Leb/y and B/B-Leb/y active Glycotopes in glycans were isolated and illustrated. These are the most advanced achievements since 1980.

394. Both catalytic and non-catalytic domains of the cmp-sia synthetase are essential for embryogenesis

D. Wu¹, A. Fujita¹, H. Hashimoto¹, M. Hibi¹, Y. Taniguchi³, Y. Kamei⁴, C. Sato², K. Kitajima²; ¹Biosci. Biotech. Center, ²Biosci. Biotech. Center, Grad. Sch. Bioagr. Sci., ³Kyorin Univ. Sch. Med., ⁴Nat. Inst. Basic Biol.

Sialic acid (Sia) residues are expressed on glycan chains of vertebrate glycoproteins and glycolipids in a developmental stage-specific and organ-specific manners. It has been reported that disturbance of Sia biosynthesis leads to many human diseases, For example, Biallelic mutations in NANS gene encoding the sialic acid 9-phosphate synthase (SPS) lead to the intellectual developmental disorders (IDDs) with developmental delay and skeletal dysplasia. However, how the underlying mechanisms of these human diseases are related to the changes in Sia metabolisms is still mysterious. To identify the significance of the spatiotemporal expression of Sia at an animal level, we have not only generated medaka that contains point-mutations in the CMP-Sia synthetase (CSS) gene using the targeting induced local lesions in genomes (TILLING) method, but also established medaka fish with CSS-deficiency by transcription activator-like effector nucleases (TALENs) method. CSS catalyzes the synthesis of CMP-Sia, a donor substrate of all sialyltransferases that is prerequisite for the expression of Sia-glycoconjugates. Like other vertebrate CSSs, medaka CSS consists of the N-terminal catalytic domain and the C-terminal domains that may be important for the nuclear export of the enzyme.

After the TILLING library was screened for gametes with mutations in the exons of the CSS gene, we found that I230N and L303Q mutants that contain a single mutation in the N- and C-terminal domains of CSS, respectively. The in vitro enzyme activity of CSSs from both I230N and L303Q mutants was similarly low to N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), indicating the importance of the particular mutations in the enzyme activity. Interestingly, I230N mutant was lethal with significant abnormalities in pre-hatching embryos, while the L303Q mutant and CSS-null medaka could survive until young fry after hatching. The histological and immunohistochemical studies showed that apoptosis happened in the neural system of I230N medaka just before lethality. These results indicate the CSS domain-dependent significance for survival at the animal level. For L303Q and CSS-null medaka, reduction of sialylation in homozygous medaka was observed, indicating that sialylation is quite important for early development of medaka. To understand why sialylation was reduced only in L303Q, but not in I230N medaka, all the mutant CSSs were cloned and expressed in CHO cell and E. coli. As a result, although recombinant I230N and L303Q showed similar Vmax/Km values, L303Q was easily accumulated into insoluble fraction when it was expressed in both CHO and E. coli cells. It is thus indicated that C-terminal domain of CSS is quite important for keeping the enzyme stable.

395. The composition, chelation capacity and activity of lycium barbarum polysaccharides on sperm motility

T. Wu¹, C. Wang¹, F. Yang², N. Syifa¹, S. Hu¹, J. Liao², S. Wu²; ¹Division of Clinical Pharmacy, School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, TAIWAN, ²Institute of Biological Chemistry, Academia Sinica, Taipei, TAIWAN

High concentration of glucose (25 mM) increases the motility of mouse sperm. Extracts of Lycium barbarum fruits were reported to improve sperm generation at high temperatures and its polysaccharides (LBP) might be the main contributory agent. The purpose of this study was to investigate the composition, chelation capability and activity of LBP on sperm motility. To prepare the LBP, polysaccharides were extracted by deionized water, followed by size exclusion chromatography, and sugar composition analyses using GC/MS. Effects of LBP on sperm motility were determined in spermatozoa isolated from ICR mice epididymis. Motility analysis conducted by computer assisted sperm analysis was done at 0, 30, 60, 90 and 120 minutes. The result shows that the crude-LBP (c-LBP) with a concentration of 2.3 mg/mL could chelate 6 nmole of ferrous ion. c-LBP with a concentration of 10 mg/mL could protect sperm from insults of 40 microM of ferrous ion. The fractionated LBP₁, LBP₂ and LBP₃ were analyzed into seven kinds of monosaccharides and individual chelation capability of LBP₁, LBP₂ and LBP₃ were 18.2%, 62.1% and 95.8%, respectively. c-LBP (at equivalent to 25mM glucose) significantly maintained the sperm motility up to 90 mins compared to the normal controls. In conclusion, c-LBP (14.0 mg/mL) could prolong the sperm motility and protect sperm cells from iron insult mainly due to chelation activity of LBP3. Further studies are required to investigate the active components of LBP fractions and their mechanisms involving sperm motility maintenance.

396. Sulfatide promotes integrin <v expression through long non-coding rna ay in human hepatocellular carcinoma cells

B. Qi¹, C.L. Kang¹, Q.Q. Cai¹, C. Mei Hua¹, X.Z. Wu²; ¹Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Key Lab of Glycoconjugate Research, Ministry of Public Health, Shanghai, P.R. China, ²Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Key Lab of Glycoconjugate Research, Ministry of Public Health, Shanghai, P.R. China

xz_wu@shmu.edu.cn, Tel:8621 54237697

Abstract

It has been recently demonstrated that sulfatide regulates the expression of the integrin <V subunit in hepatoma cells, which enhances the metastatic ability of HCC cells. However, the regulation of expression and their downstream mechanisms are little known. In this study, we performed microarray analysis to identify lncRNAs whose transcription was stimulated with the treatment of exogenous sulfatide in human hepatocellular carcinoma cells. We noted that lncRNA AY, in SMMC-7721 HCC cells was significantly upregulated by exogenous sulfatide as compared to galactoceramide which carries no sulfo group in structure. The stimulating effect on AY transcription by sulfatide was further confirmed with quantitative polymerase chain reaction. In 55 HCC specimens, we also found that AY was elevated in HCC tumor tissue in comparison with adjacent non-tumor tissue and its levels were closely correlated with the expression of the integrin <V subunit. Cell biological assays revealed that AY konckdown with small interfering RNA-mediated inhibited HCC cells migration in vitro compared with control cells, and HCC cells with AY overexpressing significantly increased their migration rate. Interestingly, we further found that the dysregulation of AY had been shown to be associated with alterations of the expression of the integrin <V subunit. In order to demonstrate potential regulatory mechanisms, we observed lncRNA AY interacted with integrin <V subunit gene promoter and enhanced integrin <V transcription. Taken together, these results revealed the role of sulfatide in regulation of hepatocellular cancer cell migration and the integrin <V subunit gene expression through lncRNA-AY.

Keywords: Sulfatide; Metastasis; Liver cancer

397. Hydroxypropyltrimethyl ammonium chloride chitosan nanoparticles as adjuvants for inactivated newcastle disease vaccine

R. Xing¹, Y. Yang¹, P. Li¹; ¹Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences

Hydroxypropyltrimethyl ammonium chloride chitosan (HACC) is a water-soluble derivative of chitosan. To investigate adjuvants effects of inactivated Newcastle disease (ND) vaccine of HACC, NDV-loaded and blank HACC/CS nanoparticles were prepared. Then chickens were immunized with nanoparticles or commercial inactivated oil emulsion vaccine. After immunization, the humoral immunity levels of the chickens were evaluated by hemagglutination inhibition assay. The cellular immunity levels were determined by the quantification of cytokines, lymphocyte proliferation assay, the percentages of CD4+ and CD8+ T lymphocytes. Finally, the chickens were challenged with highly virulent virus. The results demonstrated that both the in vitro and in vivo experiments demonstrated that the nanoparticles are safe. Secondly, NDV-loaded HACC/CS nanoparticles and commercially inactivated oil emulsion vaccine could significantly activate the humoral immune response of chickens, the humoral immunity HACC/CS nanoparticles groups were lower than commercial vaccine. Thirdly, the significant increase of the cytokines (IL-2 and IFN-©), lymphocytes, and CD4+ and CD8+ cells in NDV-loaded HACC/CS NPs immunized chickens indicated the enhancement of cellular immunity levels. Moreover, the cellular immunity levels of NDV-loaded CS and HACC/CS nanoparticles were significantly higher than commercial oil emulsion vaccine, indicating the potential of nanoparticles as effective adjuvants promoting both humoral and cellular immunity. Finally, the protective rates of NDV-loaded HACC/CS nanoparticles against highly virulent NDV reached 100%, indicating the potential of HACC/CS nanoparticles as promising vaccine adjuvants.

398. Promotion of <2,3-sialylation of recombinant erythropoietin by tagging it with a 10-aminoacid sequence recognized by a cargo receptor complex

H. Yagi³, R. Honda¹, R. Yamada³, M. Yagi-Utsumi¹, K. Aoki², K. Kato⁴; ¹Exploratory Research Center on Life and Living Systems (ExCELLS) and Institute for Molecular Science, National Institutes of Natural Sciences and School of Physical Science, SOKENDAI, Okazaki, ²Exploratory Research Center on Life and Living Systems (ExCELLS) and National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, ³Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, ⁴Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya and Exploratory Research Center on Life and Living Systems (ExCELLS) and Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki

MCFD2 and ERGIC-53, which are the products of causative genes of combined factor V and factor VIII deficiency, form a cargo receptor complex responsible for intracellular transport of these coagulation factors in the early secretory pathway. Recently, we identified a 10-aminoacid segment in factor VIII that is recognized by the cargo receptor complex. Interestingly, we found that, in addition to secretion level, <2,3-sialylation level of recombinant erythropoietin was significantly elevated by tagging it with the 10-aminoacid segment. This raises the possibility that interactions of the secretory glycoproteins with the cargo receptor complex not only facilitate their vesicular transport from the ER to the ERGIC but also have impacts on the subsequent glycosylation pathways in the Golgi apparatus. We also found that several trans-Golgi resident <2,3-/<2,6-sialyltransferases were located in distinct subcellular zones. In addition, the erythropoietin tagged with the 10-aminoacid segment visited the <2,3-sialyltransferase zones along its secretory route. Based on these results, we propose that distinct localizations of sialyltransferases in the trans-Golgi can be determining factors of specific sialylation of glycoproteins on their secretory pathways. Our findings also suggest that the secretory pathways of glycoproteins can be switched just by tagging them with the specific aminoacid segment, offering a novel strategy for controlling glycoforms of recombinant glycoproteins of biopharmaceutical interest.

399. Genome-wide crispr screens identify novel glycan regulators

T. Yamaji², T. Sekizuka⁴, H. Hanamatsu¹, M. Kuroda⁴, J. Furukawa¹, K. Yahiro³, K. Hanada²; ¹Department of Advanced Clinical Glycobiology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, ²Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, ³Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, ⁴Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo

Proper glycosylation requires not only transcriptional regulation of glycosyltransferases, but also post-translational regulation of these enzymes, which is incompletely understood. Shiga toxin (Stx) and subtilase cytotoxin (SubAB) are virulence factors of enterohemorrhagic Escherichia coli. Both toxins utilize glycans for receptors: Stx recognizes a glycolipid Gb3, whereas SubAB binds sialoglycans on glycoproteins. In genetic screens against the cell death induced by these toxins, the identified host factors may be categorized into two groups: retrograde trafficking of the toxins and glycan biosynthesis for their receptors. In this study, we performed genome-wide CRISPR knockout screens using Stx- and SubAB-induced cytotoxicity, and identified various host cell genes that conferred resistance to these toxins as follows.

(1) Stx-resistance screening

Most of the enriched genes were sphingolipid- and membrane trafficking-related. As novel genes, LAPTM4A and TM9SF2 were identified that conferred strong resistance to Stx when they were disrupted. Disruption of either LAPTM4A or TM9SF2 genes reduced Gb3 biosynthesis, resulting in accumulation of its precursor, lactosylceramide. Loss of LAPTM4A decreased endogenous Gb3 synthase activity in a post-transcriptional mechanism, whereas loss of TM9SF2 did not affect Gb3 synthase activity, but instead disturbed localization of Gb3 synthase. Furthermore, the Gb3-regulating activity of TM9SF2 was conserved in the TM9SF family (TM9SF1–4).

(2) SubAB-resistance screening

Most were sialoglycan- and membrane trafficking-related genes. Interestingly, not only N-glycan synthesized by MGAT1 but also O-glycans synthesized by C1GalT1 were used as receptors for SubAB. Although most of the identified membrane trafficking-related genes (e.g. COG complex and GARP complex subunits) were observed in both screens, four genes, SLC39A9, KDELR1, KDELR2, and JTB, were enriched in SubAB screen but not in Stx screen. Among these genes, disruption of the SLC39A9 gene markedly reduced both complex-type N-glycans and core 1 O-glycans, and the O-glycan reduction was attributed to reduction of core 1 synthase (C1GalT1).

These two screens provide novel insights into the post-transcriptional regulation of glycosyltransferases.

400. Analysis of apngase-overexpressed tomato line (t2 generation): phenotype, pngase activity and structural features of fngs

C. Yamamoto¹, M. Ogura², C. Matsumaru¹, S. Murata¹, M. Maeda¹, Y. Kimura¹; ¹Department of Biofunctional Chemistry, Graduate School of Environmental and Life Science, Okayama University, Japan, ²Faculty of agriculture, Okayama University, Japan

Acidic peptide: N-glycanase (aPNGase) is a plant specific deglycosylation enzyme and releases GN2 type free N-glycans (GN2-FNGs) carrying the chitobiosyl unit from glycopeptides or glycoproteins. It has been believed that the aPNGase is involved in the turnover of glycoproteins in vacuoles or extracellular space. Although GN2-FNGs produced by aPNGase have been ubiquitously found in developing or growing plants, their physiological function(s) remain to be elucidated. Recently, we have found that the GN2-FNGs occur in the xylem sap of tomato and several glyco-enzymes involved in the degradation of GN2-FNGs, suggesting a migration of FNGs in plant. As a part of study to unravel the physiological functions of plant FNGs, we have already constructed a transgenic tomato (Micro-Tom), in which an aPNGase (aPNGase-Le) gene is overexpressed, and we confirmed the increase in the aPNGase activity and the amount of plant complex type-FNGs (PCT-FNGs).

In this study, we analyzed the rate of fruit-maturation of the aPNGase overexpressed tomato (T2 generation), aPNGase activities, and structural features of FNGs in fruits and leaves.

Phenotypic analysis: T-DNA insertion lines were checked by PCR using genomic DNA of aPNGase overexpressed tomato leaves (T2), then T-DNA non-inherited lines were used as control plants. The overexpression of aPNGase-Le gene was confirmed by RT-qPCR. The maturation rate of aPNGase-Le overexpressed tomato was significantly accelerated about 2 days as compared with the control plant in triplicate experiments.

aPNGase activity: Sialyl N-linked glycopeptides purified from egg yolk were used as a substrate. FNGs (NeuNAc2Gal2GlcNAc2Man3GlcNAc2) produced by aPNGase were pyridylaminated and analyzed by a combination of DEAE-/RP-HPLC. We found that the aPNGase activity significantly increased in both leaves and fruits of aPNGase-Le overexpressed tomato as compared with the control plant.

FNGs of the transgenic plant: Structural features and the amount of PCT-FNGs produced by the aPNGase-Le in the mature-green fruits and leaves of the transgenic and control plants were analyzed.

401. Analysis of glycans expressed in castration resistant prostate cancer cells by saccharide primer method

D. Yamamoto¹, K. Sasaki¹, T. Kosaka², M. Oya², T. Sato¹; ¹Faculty of Sci.and Tech.Keio Univ, ²School of Med.Keio Univ

Prostate cancer is the most commonly diagnosed malignant tumors in men.

The initial treatment for patient is androgen-deprivation therapy (ADT). Although ADT will eventually cease to work, most patients get castration resistant prostate cancer (CRPC). In the diagnosis prostate specific antigen (PSA) is used, but high false positive is a problem. It is well known that changes in the structure and expression of glycans are caused by canceration and malignant transformation of cells. In particular, increased expression of mucin type O-glycans has been reported in cancer cells. Therefore, in this study, glycans expressed in prostate cancer cells using LC-MS was analyzed, and aimed to elucidate expression level of glycans related to castration resistance. Saccharide primer method have been developed to evaluate glycans expressed in tumor cells. Saccharide primer is mimicry of intermediates in biosynthetic pathway of glycans and act as an acceptor for glycosyltransferase in cells. Saccharide primer method is useful for elucidating the kinds of glycans expressed in living cells. First, migration ability, which is one of the index of malignancy, and changes in malignancy due to castration resistance acquisition was evaluated by transwell migration assay. Next, glycans expression in prostate cancer cells was analyzed by saccharide primer method. Finally, gene of glycosyltransferase which expression level was particularly increased was analyzed by realtime RT-PCR.

As a result, the migration ability of prostate cancer cells increased according to the length of the androgen removal period. In addition, the expression of sLex type saccharide and core 4 type glycan were specifically increased. Furthermore, expression of glycosyltransferase involved in synthesis of these glycans was also increased. Thus, it is suggested that these glycans and glycosyltransferase are novel diagnostic markers and drug target candidates for CRPC.

402. Molecular mechanism and significance of activation of egfr signaling by sialidase neu3 in lung cancer

K. Yamamoto⁵, K. Takahashi¹, K. Shiozaki⁴, K. Yamaguchi³, H. Shima², T. Miyagi²; ¹Cell Recog. Study, Tohoku Med and Pharm. Univ., ²Divs. Cancer Chemother, Miyagi Cancer Ctr. Res Inst., ³Divs. Cancer Mol. Cell. Oncol, Miyagi Cancer Ctr. Res Inst., ⁴Faculty Fisheries, Kagoshima Univ., ⁵Faculty of Health, Saitama Med. Univ. / Divs. Cancer Chemother, Miyagi Cancer Ctr. Res. Inst.

The plasma membrane-sialidase NEU3, a key enzyme for ganglioside degradation, is involved in many cell surface phenomena. We previously demonstrated that a marked up-regulation of NEU3 in various human cancers with progress of malignant phenotypes, leading to increased cell invasion, motility and survival of cancer cells through activation of EGFR signaling. Its up-regulation is also important for promotion stage of tumorigenesis in vivo and in vitro. Our recent studies indicated that NEU3 potentiates EGFR-mediated oncogenic transformation through activation of EGFR and Src. Here, we have focused on NEU3 and EGFR, using human lung cancer tissues and cells, in order to elucidate the molecular mechanism of the activation of EGFR signaling induced by the sialidase. NEU3 was up-regulated in human lung cancer tissues, compared with non-cancerous tissues, especially in adenocarcinomas, and enhanced the phosphorylation of EGFR at Tyr-845 in non-small cell lung cancer (NSCLC) cells. When the catalytic activity of the sialidase was measured, NSCLC cells with EGFR mutations, H1650 and H1975 cells, showed higher endogenous sialidase activity towards gangliosides GM3 as compared to wild type A549 cells. The increased NEU3 activity was abrogated in the presence of the inhibitors for EGFR or/ and Src. Interestingly, the activation was hardly detectable in the NEU3 mutant of the tyrosine (Y346A). A fragment analysis of NEU3 and EGFR proteins showed that NEU3 seemed to directly interact with the tyrosine kinase domain of EGFR. These results suggest that NEU3 activates EGFR signaling thorough enhancement of Src-mediated phosphorylation in lung cancer, leading to potentiation of tumorigenesis and then acceleration of malignant phenotype of the lung cancer cells in a positive feedback manner.

403. Glycation reaction and physiological roles of the receptor for glycated proteins

Y. Yamamoto¹; ¹Kanazawa University Graduate School of Medical Sciences

The accumulation of advanced glycation end-products (AGEs) has been implicated in the pathogenesis of age-related chronic diseases such as type 2 diabetes, diabetic vascular complications, neurodegenerative diseases, and cancer. Among the AGE binding receptors, the receptor for advanced glycation end-products (RAGE) is well-characterized and is a member of pattern-recognition receptors (PRRs). It actively participates in the innate and adaptive immunity and inflammation.

We reported that RAGE could directly interact with lipopolysaccharide (LPS), which resulted in the induction of intracellular NF-│B activation and TNF-< secretion, and leaded to the septic shock to an excessive degree. The elevation of serum TNF-< and the tissue damages induced by LPS were attenuated in RAGE-null Ager^-/- mice. We also showed that RAGE could bind to phosphatidylserine and modulate apoptotic cell phagocytosis. Impaired phagocytosis of apoptotic thymocytes and defective clearance of apoptotic neutrophils were found in alveolar macrophages from Ager^-/- mice.

Recently, we showed RAGE-mediated transport of oxytocin from the intestinal tract into the blood circulation across the intestinal barrier and from the blood into the brain through blood brain barrier in mice. Oxytocin is a neuropeptide secreted from the pituitary gland into the blood and is known to mediate both physiological and psychosocial events surrounding mammalian birth, including uterine contractions, initiation of lactation, and maternal bonding. Our findings indicate that RAGE is critical to the behavioral actions of oxytocin associated with parenting and social bonding.

Furthermore, to understand structural features and physical properties of glycation reaction in proteins, we currently apply new techniques in nanotechnology and material science for visualizing the glycation reaction. Our recent advances may provide a new avenue in a research field of glycation biology.

404. Novel glyco-epitopes of free-living and parasitic nematodes

S. Yan², J. Vanbeselaere¹, F. Martini¹, B. Eckmair¹, C. Jin³, A. Joachim², I.B.H. Wilson¹, K. Paschinger¹; ¹Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria, ²Institut für Parasitologie, Veterinärmedizinische Universität, 1210 Wien, Austria, ³Institutionen för Biomedicin, Göteborgs Universitet, 405 30 Göteborg, Sweden

Nematodes constitute a large number of different species on earth and many of them parasitise animals and humans. Glycoproteins synthesised by nematodes possess glycan structures, which are rather different from those made by mammals. It is known that some epitopes of nematode glycans, such as the core <1,3-linked fucose on N-glycans, are immunogenic as they cross-react with antisera of infected mammals. Therefore, we set focus on analysing the N-glycomes of nematode species. In the past few years, we have studied the N-glycomes of free-living Caenorhabditis elegans and Pristionchus pacificus, of the sheep parasite Haemonchus contortus, the porcine parasites Oesophagostomum dentatum and the whipworm Trichuris suis, and more recently the canine heartworm Dirofilaria immitis. Using an off-line HPLC-MALDI-TOF-MS/MS approach coupled with glycosidase and chemical treatments, worm N-glycans were separated on HPLC columns and analysed by mass spectrometry in great detail. A repertoire of novel glyco-epitopes were revealed and characterised, for instance, different types of Gal-Fuc modifications of the chitobiose core found in the clade V nematodes, phosphorylcholine (PC) substitution of the LacDiNAc residues on the antennae (Trichuris), and long fucosylated HexNAc-based repeats as well as glucuronylated glycans (Dirofilaria). In addition, by using a novel PNGase A from rice or hydrazine, we were able to release N-glycans with heavily modified core structures from the wild type C. elegans, which carry up to five fucose or galactose residues. The findings of novel glyco-epitopes in nematodes not only provide clues about the molecular basis of host-parasite interactions, but also shed light on new diagnostic markers as well as potential therapeutic targets.

405. Identification of site-specific o-galnac glycosylation by ways of an o-protease-facilitated mass spectrometry analysis

C.H. Yeh¹, Y.C. Chien¹, K.H. Khoo¹; ¹Institute of Biological Chemistry, Academia Sinica, and Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.

Compared to N-linked glycosylation, identification of site-specific protein O-GalNAc glycosylation at Ser/Thr by mass spectrometry analysis is significantly more challenging. In particular, it has no distinctive sequon for a priori localization onto one or more of the many possible Ser/Thr sites typically carried on tryptic peptides derived from mucin domain. This is compounded by the lack of a pan-specific O-GalNAc glycan-releasing enzyme and facile loss of the entire O-glycans via common modes of CID fragmentation without incurring a scar, which defies MS/MS sequencing and site determination. A newfound endo-O-protease from Akkermansia muciniphila, commercially branded as OpeRATOR, is a potentially very powerful tool that would enable site-specific O-glycosylation mapping. OpeRATOR was recently reported to recognize O-GalNAc glycan and cleave its peptide carrier at the N-terminus of a glycosylated Ser/Thr. A complete digestion would in principle produce glycopeptides with O-glycosylated Ser/Thr located only at the N-termini and not elsewhere, thus making subsequent MS/MS analysis relatively easy. A caveat though is that any preference for or hindrance by specific O-glycan structures would bias the digest and resulting in non- or under-representative subsets of O-GalNAc glycopeptides. Incomplete or resistance to digestion would also produce O-glycopeptides carrying additional O-glycans at internal Ser/Thr other than at the N-termini. Based on applications to several glycoproteins with isolated single sites of O-GalNAc glycosylation, as well as those containing Ser/Thr rich mucin domains, we demonstrate in this work a biased OpeRATOR specificity/activity against O-glycans of larger size and/or sialylation. Detailed sequencing of recovered OpeRATOR-digested glycopeptide products by LC-MS/MS using both HCD and EThcD modes provided the requisite data, which were benchmarked against the gold standard bovine fetuin. Despite this shortcoming, OpeRATOR digestion can be judiciously incorporated into existing analytical workflow, to accompany that by trypsin and other proteases. It is useful to produce overlapping sets of O-glycopeptides spanning the heavily O-glycosylated mucin domain, to discover unexpected single sites, or to target identify suspected sites.

406. Oligosaccharides induce resistance to pseudomonas syringae pv. tomato dc3000 in arabidopsis thaliana via distinct n-glycosylation regulation pattern

X. Jia¹, H. Qin¹, M. Ye¹, H. Yin¹; ¹Dalian Institute of Chemical Physics, Chinese Academy of Sciences

As an essential co- and post-translational modification of proteins, glycosylation is involved in a number of biological processes. And evidence is accumulating that protein glycosylation play important roles in plant defense response. However, the function mechanism of N-glycosylation in plant defense response and chitosan oligosaccharide (COS) induced resistance still remains unclear.

In the present study, glycomics analysis of Arabidopsis proteins during Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) infection and COS induced resistance were carried out by LC-PGC-ESI-MS/MS. Sixty N-glycans were successfully quantified from three biological replicates of four treatment groups (mock, COS, mock+Pst and COS+Pst treated WT samples). Clustering analysis revealing that N-glycans were changed significantly during Pst DC3000 infection and COS pretreatment, moreover, COS pretreatment could reverse the effect of Pst DC3000 on some N-glycans, suggesting N-glycosylation is differently regulated during plant innate immunity and COS induced resistance.

In order to further confirm the involvement of N-glycosylation in plant innate immunity and COS induced resistance, and reveal the distinct N-glycosylation regulation pattern during this two processes, N-glycosylation impaired mutants ManI and stt3a were used for experiments. stt3a and ManI were shown more severe symptoms than WT when Pst DC3000 infection, and stt3a shown more impaired plant immunity than ManI, suggested that both high-mannose N-glycans and complex N-glycans are essential for Arabidopsis defense to Pst DC3000.

Unexpectedly, COS worked effectively on inducing resistance to Pst DC3000 in stt3a and ManI mutants, and the efficacy are almost similar with WT. Moreover, COS pre-treatment highly up-regulated both SA- and JA-mediated signalling pathways in N-glycosylation impaired mutants, similar with WT. Protein N-glycosylation deficiency in Arabidopsis has slight influence on COS induced resistance to Pst DC3000, implying that COS pre-treatment could avoid or offset the influence of N-glycosylation impairment on plant immunity.

In conclusion, COS regulating plant immunity in Arabidopsis-Pst DC3000 interaction by a distinct N-glycosylation modulation pattern compared with plant innate immunity, which makes COS inducing resistance in N-glycosylation deficient mutants effectively. This phenomenon provide strong evidence that COS functions as an effective and broad-spectrum plant immunity elicitor in multiple plants.

407. Conformational and functional impacts of dynamic o-glycosylation states on human notch1 egf domains

Y. Yokoi¹, S. Nishimura¹;¹Graduate School of Life Science, Hokkaido University, Sapporo, Japan.

Notch signaling pathway plays key roles on cell developing process. Human NOTCH protein contains 36 EGF-like repeats on extra cellular domain. Each EGF domain has conserved three disulfide bonds, C1-C3, C2-C4 and C5-C6. In many EGF domains, <-O-fucose [mature form is Neu5Ac<(2→3)Galß(1→4)GlcNAcß(1→3)Fuc<1→], ß-O-glucose [Xyl<(1→3)Xyl<(1→3)Glcß1→] and ß-O-GlcNAc [Neu5Ac<(2→3)Galß(1→4)GlcNAcß1→] are attached to Thr/Ser residues and these glycosylations regulate binding affinity with their ligands. However, the effects of O-glycosylation on protein structure and function, especially on its folding process are still unclear.

Considering that some variants carrying different and immature glycoforms can be produced during the biosynthetic processes and O-glycosylation site reside between two cysteine residues which participate in disulfide bonds, oxidative formation of the three disulfide cross-linkages may be affected significantly by dynamic O-glycosylation alteration. We hypothesized that the efficacy of the accurate folding of the NOTCH1 EGF domain depends on the interaction of individual glycoforms with the proximal peptide regions.

To examine this hypothesis, focusing on EGF12, EGF11 and EGF10, which are pivotal domains involving ligand binding site, we synthesized model glycopeptides and in vitro folding reactions from their unstructured status were assessed by monitoring HPLC profiles of the products. In addition, to check the interaction of O-glycans with peptide backbone, we also performed NMR structural analysis of each EGF domain. Our results revealed that O-glycosylations tend to facilitate proper folding of the EGF12 domain, while the nonglycosylated peptide affords large amounts of misfolded products. However, sugar extension of the Glcß1→ moiety with xyloses is a negative-regulation mechanism in the folding of the EGF12 domain. In EGF11, in contrast, oxidative folding processes afford only proper folding products independent of O-glycosylations. These results indicate characteristics of folding processes of each EGF domain, which are related to glycosylation states, are quite different. Structural analysis of synthetic EGF domains unveiled underlying novel regulation mechanism in the protein folding.

408. Possible involvement of kras in phospthatidylglucoside-mediated hl-60 cell differentiation. -kras promotes myeloid differentiation through wnt/®-catenin signaling

N. Yokoyama³, Y. Kim⁵, Y. Hirabayashi⁴, Y. Tabe², K. Iwabuchi¹; ¹ Laboratory of Biochemistry, Juntendo University Faculty of Health Care and Nursing; Infection Control Nursing, Juntendo University Graduate School of Health Care and Nursing; Institute for Environmental and Gender Specific Medicine Urayasu, Japan, ²Department of Laboratory Medicine, Juntendo University School of Medicine Hospital, Hongo Tokyo,Japan, ³Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, Urayasu Chiba,Japan, ⁴Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine,Urayasu Ciba Japan; Cellular Informatics Laboratory, RIKEN, Wako, Saitama, Japan, ⁵Laboratory for Neuronal Growth Mechanisms, Riken Brain Science Institutes, Wako, Saitama, Japan

Human promyelocytic leukemia HL-60 cells are neutrophilic promyelocytes that differentiate to granulocytic cells if exposed to compounds such as DMSO (dimethyl sulfoxide) and all trans retinoic acid (ATRA). Phosphatidylglucoside (PtdGlc) is a novel neutrophil differentiation marker, and anti-PtdGlc antibody DIM21 induces neutrophilic differentiation of HL-60 cells. The Ras genes encode small GTP-binding proteins involved in many cellular processes, including proliferation, differentiation and apoptosis. Wild-type Kras inactivates Ras growth-promoting signaling. Evidence suggests wild-type Kras is involved in other than oncogenic activity. However, the role of Kras in differentiation of myeloid cells remains unclear. In this study, we focused on novel functions of Kras in DMSO/anti-PtdGlc antibody-induced differentiation of HL-60 cells.

Kras was activated by either DMSO or DIM21 treatment in undifferentiated HL-60 cells. Inhibition of Kras attenuated CD11b expression during DMSO-induced differentiation of HL-60 cells. Phosphorylation of GSK3® was markedly enhanced during differentiation. Kras siRNA attenuated DMSO-induced phosphorylation of GSK3®. Moreover, inhibition of GSK3® enhanced CD11b expression and triggered accumulation of ®-catenin and Tcf in the nucleus in response to DMSO. Inhibitors of ®-catenin-mediated pathways blocked CD11b expression. Furthermore elevated expression of C/EBP< and C/EBP∑ accompanied by the expression of granulocyte colony-stimulating factor (G-CSF) receptor, was observed during differentiation. Induction of CD11b expression by treatment with DIM21 was weak. However, DIM21 in combination with low concentration of DMSO, which did not induce differentiation itself, greatly enhanced the expression of CD11b in HL-60 cells. Kras engaged in cross-talk with the Wnt/®-catenin pathway upon DMSO/anti-PtdGlc antibody treatment of HL-60 cells, thereby regulating the granulocytic differentiation of HL-60 cells. These results suggest that PtdGlc is involved in enhancing differentiation via the DMSO-induced pathway

409. Ligand recognition mechanism of siglec-7 as revealed by synthetic sialylpolymers

A. Yoshimura¹, Y. Asahina¹, H. Tanaka², K. Kitajima¹, C. Sato¹, ¹Biosci. Biotech. Center, Nagoya Univ., Nagoya, ²Chem. Sci. Eng., Tokyo Inst. of Tech., Tokyo

Siglec is a family of lectin-type receptors that recognizes sialic acid residues, a group of acidic nine-carbon sugars having a carboxyl group. Siglec regulates the immune response by binding to the sialic acid residues present on the own or adjacent cell surface. Of 15 species of human Siglecs, Siglec-7 is expressed in immune cells such as NK cells and macrophages, and specifically recognizes <2,8-linked disialic acid (diSia) structure. However, Siglec-7 also recognizes glycan structures different from diSia, such as 6-sulfo-sialyl Lewis x. Thus, the binding mechanism is too complicated to be explained by the evidence that has been reported to date. Therefore, in this study, in order to investigate the ligand recognition mechanism by Siglec-7 in detail, various binding analyses such as ELISA, Flow cytometry (FCM), and Bio-layer interferometry (BLI) methods were performed using the synthesized sialic acid-containing compounds. As a result, it was revealed that Siglec-7 showed the strong binding activity to polymer compounds in which diSia and monosialic acids (monoSia) were multivalently conjugated. On the other hand, Siglec-9 and Siglec-E, which have been reported to recognize mainly monoSia, showed little or no binding activity to these polymers. These differences in glycan recognitions suggest that, while Siglec-7 has high binding ability to the molecules containing multivalent sialic acid residues, the glycan structures carrying sialic acid residues are more important for Siglec-9 and Siglec-E. Moreover, it was shown that the polymer molecules could be used as efficient inhibitors for the Siglec-7-ligand binding and as probes of the regulatory mechanism of immune response by Siglec-7 in immune cells.

410. New paradigm for alveolar structural changes in lung disorders mediated by core fucosylated protein(s) in lung stem cells

J. Yu¹, T. Wu², E.S. Chen¹, S. Wang¹, Y. Tseng¹; ¹ Chang Gung Memorial Hospital, ²Chang Gung Memorial Hospital

We have developed an organotypic model for lung alveologenesis using isolated lung stem cells (LSCs) to elucidate if diminished core fucosylation can recapitulate the structural changes frequently observed in alveolar sacs of COPD and other lung disorders such as damages by PM2.5. In our studies, using quantitative site-specific MS proteomics, we have observed specific alterations at 355 core fucosylation sites with 186 corresponding protein candidates in LSCs after Fut8 knockdown. With significant decline of MS TMT ratio but similar mRNA array ratio in siFut8/siCon, we were interested particularly in a matricelluar protein CF01, which modulates cell-matrix interactions. Immunoprecipitation confirmed that core fucosylation of CF01 was a posttranslational modification and the association between CF01 and collagen I was significantly decreased upon Fut8 silencing. MS analysis indicated that CF01-293T WT CF01 was core fucosylated at N116, which was completely abolished in CF01-293T Fut8KO cell. In contrast, Biacore with CF01-293T Fut8KO exhibited a pronounced lower binding affinity to collagen 1. Molecular dynamics simulation showed that changes of core fucosylations caused pronounced alterations of the glycan conformation in CF01, leading to changes in its interactions with collagen I; but core fucosylation did not alter CF01 protein structure itself. Therefore, alveolar structural changes in lung disorders can be mediated by core fucosylated protein(s) in lung and collagen degradation may play a significant role in lung disorders.

411. Altered sialylation of igg from hashimoto’s thyroiditis patients influences antibody-dependent cellular cytotoxicity (adcc): the study on in vitro model

M. Ząbczyńska¹, K. Polak¹, E. Pocheć¹; ¹Department of Glycoconjute Biochemistry, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Kraków, Poland

Antibody-dependent cellular cytotoxicity (ADCC) is one of the mechanisms involved in a destruction of thyroid tissue in Hashimoto’s thyroiditis (HT). Three key components are involved in ADCC: the target cells expressing an antigen, IgG which recognizes antigen, and the effector cells with a surface receptor for IgG crystallizable fragment Fc (FcR). N-glycosylation of Fc affects the effector functions of IgG by enhancing or suppressing ADCC cytotoxicity effect. Our previous results demonstrated that IgG glycosylation is changed in HT in comparison to healthy individuals. The aim of the present study was to assess an impact of HT-specific IgG sialylation in ADCC using an in vitro model corresponding to immune process taking place in HT thyroid.

The normal thyroid Nthy-ori 3-1 cell line and the thyroid carcinoma FTC-133 cells were used as two variants of the target cells in ADCC model. Periphery blood mononuclear cells (PBMC) from healthy donors and HL-60 human promyelotic leukemia cell line served as the effector cells. IgG was isolated from sera of HT and healthy donors. The expression of thyroid peroxidase (TPO), the main autoantigen in HT autoimmunity, was confirmed in the target cells on gene and protein levels. FTC-133 cells showed the significantly higher TPO expression than normal cells. IgG isolated from HT and healthy donors were treated with <2-3,6,8-neuraminidase (NEU) to remove sialic acids from N-glycans. IgG (30 ug) were incubated with the target cells (105) for 2 h. Then the effector cells were added in an appropriate relation to the target cells: 1:500 or 1:12 for PBMC and HL-60 respectively, and incubated for 4 h. The cytotoxicity of thyroid cells was measured using CellTox™ Green Cytotoxicity Assay (Promega). The percentage cytotoxicity ratio of NEU digested IgG to untreated IgG corresponds to effect of IgG desialylation on target cell cytotoxicity. We observed a significant decrease of Nthy-ori cell cytotoxicity exposed to the desialylated IgG from control group, whereas the cytotoxic effect for HT desialylated IgG remained unchanged. The analysis demonstrated that the altered IgG glycosylation profile in HT results in a different functional effect.

This study was supported by the Jagiellonian University grant (No. DS/MND/WBiNoZ/IZ/40/2017).

412. Modulation of the innate immune responses with small synthetic glycans

A. Zamyatina¹; ¹Department of Chemistry, University of Natural resources and Life Sciences, Vienna

Immunomodulation has grown to promising strategy for treatment of acute and chronic diseases ranging from autoimmune disorders and cancer to antibiotic-resistant infections and sepsis. Coordinating the immune response to infection by targeting innate immune receptors is a complicated multistep process which requires precise balancing of the dysregulated intracellular inflammatory signaling pathways. We address the challenges of therapeutic immunomodulation by design, synthesis and functional studies of lipopolysaccharide (LPS) related glycans. LPS is a complex micro-heterogeneous Gram-negative glycan which mediates bacterial interaction with the mammalian innate and adaptive immune systems. “Pathogen-Associated Molecular Pattern” glycolipid lipid A – a membrane-anchored fragment of LPS - is responsible for the activation of the mammalian innate immune system in response to bacterial infection via Toll-like Receptor 4 (TLR4) and cytosolic protease caspase-4 (caspase-11 in mice). (1) We report on the discovery of first caspase-4/11 glycan-based ligands able to either activate or inhibit inflammatory caspases and on the invention of the unique immuno-modulating glycans which can dissect TLR4 and caspase-4/11 activation which is crucial for creation of novel immunotherapeutics targeted at the resolution of inflammation in infection and sepsis. (2) We developed glycan-based immunostimulants with picomolar affinity for TLR4 which induce highly potent and adjustable TLR4-mediated NF-│B signaling and, in contrast to bacterial LPS, do not provoke caspase-11 protease activity. We report on the development of first caspase-4/11 antagonists and show that the LPS-induced caspase-4/11 activation can be supressed by small synthetic glycans without inhibiting TLR4 signaling. Modification of the chemical structure of synthetic glycans allowed for development of powerful TLR4 antagonists which efficiently suppress TLR4-mediated LPS endotoxicity in vitro without interfering with the caspase-4/11 activation.

Besides, we demonstrate how the chemical synthesis of the key metabolic intermediates in the LPS biosynthesis, D-glycero-®-D-manno-heptose 1,7-bisphosphate (HBP) and ADP-heptose, enabled the discovery of Apha-kinase 1 as cytosolic immune receptor for bacterial ADP-Hep and ALPK1/TIFA-dependent axis of NF-│B activation.(3,4)

Financial support by Austrian Science Fund (Grant FWF P-28915) is gratefully acknowledged.

References

(1) Nature 2014, 514, 187

(2) Chem. Sci. 2018, 16, 3957

(3) EMBO Rep. 2018, 19, e46943

(4) Nature 2018, 561, 122

413. Neuroprotective effect and preliminary mechanism of oligoporphyran

Y. Liu¹, L. Geng¹, J. Wang¹, Q. Zhang¹; ¹Institute of Oceanology, Chinese Academy of Sciences

Oligoporphyran (OP) was prepared from porphyran through acidolysis reaction with a linear backbone of alternating 3-linked ®-D-galactose and 4-linked <-L-galactose-6-sulfate. In a LPS-stimulated BV-2 cell model, OP significantly reduced the proportion of amoebic-like microglia induced by LPS, inhibited the production of NO and major proimmunotary cytokines and the activation of BV2 cells. In a 6-OHDA-damaged PC12 cells, OP significantly ameliorated cell damage and attenuated the LDH release lesioned by 6-OHDA, restrained the overproduction of reactive oxygen species (ROS) and improved the antioxidant system, alleviated the mitochondrial membrane potential (MMP) loss and showed effective role on the increase of anti-apoptotic Bcl-2 proteins and the suppression of pro-apoptotic Bax proteins. OP also repressed the activity of caspase-3 and caspase-9. Tunel staining presented that OP inhibited apoptosis of PC12 cells. Additionally, OP inhibited the production of proimmunotary cytokines including Il-6, Il-1® and TNF-<. In a MPTP induced PD mice model, the behavioral deficits in spontaneous motor activity, latency to descend in a pole test, and suspension in a traction test were ameliorated, and excessive dopamine (DA) metabolism was suppressed after OP treatment. OP also protected dopaminergic neurons by preventing MPTP-induced decreases in dopaminergic transporter and tyrosine hydroxylase protein levels. OP increased the phosphorylation of PI3K/Akt/GSK-3 and inhibited the activation of caspase-3 and poly (ADP-ribose) polymerase,with changes in the Bax/Bcl-2 ratio. These results showed that OPmight promote DA neuron survival by regulating the PI3K/Akt/Bcl-2 pathway, thereby ameliorating the neurobehavioral deficits in a PD mouse model and suggesting OP as a neuroprotective treatment for PD.

414. Polypeptide n-acetylgalactosaminyltransferase 18 regulates the er homeostasis and o-glycosylation

A. Shan¹, J. Lu¹, Z. Xu¹, X. Li¹, W. Jia¹, H. Narimatsu²,Y. Zhang¹; ¹Ministry of Education Key Laboratory of Systems Biomedicine,Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai,China, ²Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Japan.

Mucin-type O-glycosylation, also known as O-GalNAc glycosylation, is among the most common and fundamental post-translational protein modifications and plays critical roles in various physiological and pathological processes. In human, mucin-type O-glycosylation is initiated by a family of 20 conserved UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), which catalyzes the transfer of the N-acetylgalactosamine (GalNAc) to the hydroxyl group of serine or threonine. Previously, we reported that ppGalNAc-T18 is only existed in vertebrates and predominantly distributed on the endoplasmic reticulum (ER), which is obviously distinct from others ppGalNAc-Ts localized to the Golgi apparatus and exhibits no classical catalytic activity in vitro. In this study, we found that silencing of ppGalNAc-T18 in cells decreased O-glycosylation levels and activated ER stress leading to cell apoptosis. After treatment with chemical chaperone 4-phenylbutyric acid (PBA) or forced expression of ppGalNAc-T18 in the ppGalNAc-T18 knockdown cells, these defects could be significantly alleviated, suggesting that ppGalNAc-T18 is important for ER homeostasis and protein O-glycosylation. Furthermore, we found both of the wild type and the Mn2+ binding site mutant ppGalNAc-T18 could rescue the O-glycosylation and alleviated the ER stress in knockdown cells. These results reveal a novel molecular role of ppGalNAc-Ts that the ER-localized ppGalNAc-T18 could regulate the O-glycosylation and ER homeostasis in a non-catalytic manner, and provide a new insight into the molecular mechanisms underlying ppGalNAc-T-mediated O-glycosylation and ER homeostasis.

415. O-glcnacylation regulates methionine cycle to promote pluripotency of stem cells

Q. Zhu¹, W. Yi¹; ¹College of Life Sciences, Zhejiang University

Methionine metabolism is critical for the maintenance of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) pluripotency. However, how methionine cycle is regulated to sustain ESC pluripotent state still remains poorly understood. Here we show that adenosylhomocysteinase (AHCY), an important enzyme in the methionine cycle, is critical in the regulation of maintenance and differentiation of mouse embryonic stem cells (mESCs). We show that mESCs and iPSCs exhibit high levels of methionine metabolism, whereas decreasing methionine metabolism via depletion of AHCY promotes mESCs to differentiate into all three germ layers. In addition, AHCY is post-translationally modified with an O-linked ®-N-acetylglucosamine sugar (O-GlcNAc), which is rapidly removed upon differentiation. O-GlcNAc modification of Thr136 in AHCY activates its activity and is important for the maintenance of H3K4me3 to sustain mESC pluripotency. Blocking glycosylation of AHCY decreases intracellular concentrations of S-adenosylmethionine verses S-adenosylhomocysteine (SAM/SAH), reduces the level of trimethylation of histone H3 lysine 4 (H3K4me3), and poises mESC for differentiation. Our findings reveal a critical role of AHCY and a mechanistic understanding of O-glycosylation in regulating ESC pluripotency and differentiation.

416. Galectin-8 promotes cytokine expression and metastatic tumor growth in mice

H. Shatz-Azoulay¹, Y. Vinik¹, R. Isaac¹, Y. Zick¹; ¹Weizmann Institute of Science, Rehovot, 76100, Israel

Secreted animal lectins of the galectin family are key players in cancer growth and metastasis. Here we show that galectin-8 (gal-8) induces in a number of cell-types the expression and secretion of cytokines and chemokines such as SDF-1 and MCP-1. This involves gal-8 binding to a uPAR/LRP1/integrin complex that activates JNK and the NFkB pathway. Cytokine and chemokine secretion, induced by gal-8, promotes migration of cancer cells toward cells expressing this lectin. Indeed, immune competent gal-8 knockout (KO) mice express systemic lower levels of cytokines and chemokines while the opposite is true for gal-8 transgenic animals. Accordingly, gal-8 KO mice experience reduced tumor size and smaller and fewer metastatic lesions when injected with cancer cells. These results suggest the existence of a ‘vicious cycle’ whereby gal-8 secreted by the tumor microenvironment, promotes secretion of chemoattractants at the metastatic niche that induce further recruitment of tumor cells to the metastatic site. These studies implicate gal-8 as a potential regulator of cancer progression and metastasis also in humans, because gal-8 mRNA levels positively correlate with those of SDF-1 in human bones.

417. Glycosylation masks terminal neo-epitope of a linear peptide to favor immune response toward the core peptide epitope producing parent protein bound antibodies

W. Zou¹, A. Marcil¹, W. Chen¹, R. Pon¹; ¹Human Health Therapeutics Research Center, National Research Council of Canada, Ottawa, Ontario, Canada

Glycosylation of peptides, particularly the hydrophobic peptides, at one terminus and conjugation to a carrier protein through the other can effectively make the peptides more soluble and their conjugates more immunogenic. More importantly, this approach may render the immune response shifting from the terminal neo-epitope towards the core region of the peptide, producing antibodies that recognize the parent protein. The proof of concept was established by the fact that the glycopeptide conjugates were able to generate specific antibodies targeting the core region of peptides from influenza hemagglutinin and neuraminidase, as well as antibodies reactive to the peptide in the dimerization loop of the human epidermal growth factor receptor 2.