Abstracts

August 27-September 1, 2017

Jeju, Korea

Dear Friends, Dear Sweet Friends,

Welcome to GLYCO XXIV, the 24th International Symposium on Glycoconjugates, an official biannual international symposium of International Glycoconjugate Organization (IGO).

Despite exaggerated News about precarious states, I respect you all who visited the Republic of Korea, which is in fact one of the safest countries in the world. Public peace in Korea is always well maintained that it is safe to walk around even at night. In fact Korea is much safer than Europe. Especially Jeju Island is known as “Island of no three kinds”. This reputation came from the fact that beggars, thieves and locked gates were hard to be found in Jeju Island. In other words, you are in the safest place in Korea. Juju Island therefore is the most suitable place for us, the sweet glycoscientists.

This symposium consists of 2 award lectures, 10 plenary lectures, 26 sessions with 31 key note speakers, and 2 poster sessions. It is expected to bring an opportunity for revival to the field of glycoscience in the Republic of Korea and Asia region.

In 1995, Professor William Lennarz, Dr. Yoshitaka Nagai, Dr. Naoyuki Taniguchi, Dr. Akemi Suzuki and other glycobiologist from Japan introduced the field of glycobiology for the first time in Korean Society of Molecular Biology Annual Meeting. After symposium Dr. Nagai mentioned that glycobiology in Korea was not even born yet.

22 years from then, however, glycobiology in Korea has grown well and about 150 Korean glycoscientists participate in this symposium. This could not be possible without help of countries like Japan and the United States of America that are advanced in the field of glycobiology. Korean glycoscientists always appreciate dedication of Dr. Naoyuki Taniguchi, Dr. Akemi Suzuki, and Professor Jürgen Roth who helped to develop Korean glycoscience.

Sugar is sweet and sticky. Likewise, I hope that we as carbohydrate researchers can make sweet friendship through active academic exchange in such a beautiful place, JeJu.

Jin Won Cho

Organizer of GLYCOXXIV

President Elect of IGO

SYMPOSIUM COMMITTEES

Organizer

Jin Won Cho (Yonsei University)

Local Organising Committee

Jong Shin You (KBSI)

Boyoun Park (Yonsei University)

Hyun Joo An (Chungnam National University)

Seonghun Kim(KRIBB)

Jin-Hong Kim (Seoul National University)

Doo-Byoung Oh (KRIBB)

Jeong Gu Kang (KRIBB)

Jeong-Heun Ko (KRIBB)

Yong-Sam Kim (KRIBB)

Yong-Ho Lee (Yonsei University College of Medicine)

Kyun Oh Lee (Gyeongsang National University)

Hyun Ah Kang (Chung-Ang University)

Eok-Soo Oh (Ewha Womans University)

In Kown Chung (Yonsei University)

Yong Il Park (The Catholic University of Korea)

Moonjae Cho (Jeju National University)

Jaehan Kim (Chungnam National University)

Hyun-soo Cho (Yonsei University)

Eugene C. Yi (Seoul National University)

Daesik Lim (KAIST)

Inn-Oc Han (Inha University)

Jae Kyung Sohng (SunMoon University)

Kisung Ko (Chung-Ang University)

Injae Shin (Yonsei University)

Glyco24 Scientific Advisory Board

Jin Won Cho (Korea), Chairman

Gordan Lauc (Croatia)

Nicolai Bovin (Russia)

Naoyuki Taniguchi (Japan)

Akira Kobata (Japan)

Anne Dell (United Kingdom)

Jianxin Gu (China)

Ken Kitajima (Japan)

Hakon Leffler (Sweden)

Sandro Sonnino (Italy)

Celso Reis (Portugal)

Hans Vliegenthart (The Netherlands)

Iain Wilson (Austria)

Els van Damme (Belgium)

Jaroslav Katrlik (Slovakia)

Jukka Finne (Finland)

Ken Kitajima (Japan)

Yehiel Zick (Israel)

Michaela Wimmerová (Czech Republic)

Kay-Hooi Khoo (Taiwan)

Hyun Joo An (Korea)

Guosong Chen (China)

Karen Colley (USA)

Christine Szymanski (USA)

IGO President

Gordan Lauc (Croatia)

IGO Immediate Past President

Jianxin Gu (China)

IGO President Elect

Jin Won Cho (Korea)

IGO Past Presidents

Eugene A Davidson (USA)

Paul A Gleeson (Australia)

Gerald Hart (USA)

Akira Kobata (Japan)

Jerzy Koscielak (Poland)

Jürgen Roth (Switzerland)

Harry Schachter (Canada)

Roland Schauer (Germany)

Avadhesha Surolia (India)

Guido Tettamanti (Italy)

Iain Wilson (Austria)

Hans Vliegenthart (The Netherlands)

Tamio Yamakawa (Japan)

M.C. Glick (USA)

R.W. Jeanloz (USA)

B. Lindberg (Sweden)

J. Montreuil (France)

N. Sharon (Israel)

IGO NATIONAL REPRESENTATIVES

Pedro Bonay (Spain)

Nicolai Bovin (Russia)

Inka Brockhausen (Canada)

Jin Won Cho (Korea, President elect IGO for 2017-2019)

Henrik Clausen (Denmark)

José Luis Daniotti (Argentina)

Anne Dell (UK)

Jukka Finne (Finland)

Paul Gleeson (Australia)

Gerald Hart (USA)

Thierry Hennet (Switzerland, Treasurer of IGO)

Jaroslav Katrlík (Slovakia)

Ken Kitajima (Japan)

Ute Krengel (Norway)

Gordan Lauc (Croatia, President of IGO for 2015 - 2017)

Hakon Leffler (Sweden)

Haojie Lu (China)

Jean-Claude Michalski (France)

Paulo A. S.Mourão (Brasil)

Andrei Petrescu (Romania)

Celso Albuquerque Reis (Portugal)

Werner Reutter (Germany)

Sandro Sonnino (Italy, Secretary of IGO)

Avadhesha Surolia (India)

Maciej Ugorski (Poland)

Els Van Damme (Belgium)

Johannes F.G. Vliegenthart (The Netherlands)

Iain B. H. Wilson (Austria)

Michaela Wimmerová (Czech Republic)

Albert M. Wu (Taiwan)

Yehiel Zick (Israel)

Seretary

Jihey Won

International Glycoconjugate Organisation Award 2017

figure b

Professor Pauline Mary Rudd

National Institute for Bioprocessing, Research and Training (NIBRT)

Fosters Avenue Mount Merrion Blackrock, Dublin, Republic of Ireland.

Reception: +353 1 215 8100 Direct Line: +353 1 215 8154 Mobile:+353 872682852

Email: Pauline.rudd@nibrt.ie

Webpage for Prof. Rudd at NIBRT

PERSONAL DATA:

Date of birth: September 17th 1942

Place of birth: Lymington, New Forest district of Hampshire, England

CURRENT POSITIONS:

• NIBRT Professor of Glycoproteomics and Glycobiology at University College, Dublin

• Visiting Investigator Biotechnology Institute, AStar Singapore

EDUCATION:

1954-1961 Bournemouth Grammar School for Girls

1961-1964 B.Sc. Special Chemistry Westfield College, University of London

1967 L.R.I.C. (Licentiate of the Royal Institute of Chemistry)

1995 Ph.D. The Open University, Milton Keynes

1998 MA Oxon Wolfson College

AWARDS/HONOURS:

2015 Honorary Doctorate: Sahlgrenska Academy, Gothenburg University, Sweden

2015 Short list American Chamber of Commerce award for innovation

2013 Adjunct Professor Trinity College, Dublin;

2012 Waters Global Innovation Award for contributions to Glycobiology

2012 Adjunct Professor National University of Ireland Galway;

2010 Agilent Thought Leadership award

2010 Adjunct Professor North Eastern University Boston;

2010 James Gregory Award (University of St Andrews)

2008 Fellow Royal Society of Medicine 2007 Honorary Professor, St Georges Hospital London

2006 Adjunct Professor University College, Dublin

2004 Erskine Visiting Fellow, Canterbury University, Christchurch, New Zealand

2001 Templeton/ASA Prize for Public Understanding of Science

1999 Visiting Professor of Biochemistry, Shanghai Medical University, PRC

1996 Wlodzimierz Mozolowski Memorial Lecture, Gdansk, Poland

CAREER:

1964-1969: Founding Scientist: Wessex Biochemicals/later Sigma London (Poole, Dorset) - manufacture of rare sugars and sugar phosphates from natural products

Restart of Career after bringing up 4 children:

1983-2006 Glycobiology Institute, University of Oxford; 2000-2006 University Reader

1998-1999 Visiting Research Associate, The Scripps Research Institute, La Jolla, CA

2001 Visiting scientist Ben Gurion University of the Negev, Israel

PATENTS AND PUBLICATION:

4 granted; 3 filed and 2 filed by UCD (2015)

330 academic publications; 56 trade publications in glycan analysis in e.g. BioProcessing and Analytical Journals

>400 international lectures (All Keynotes, Plenaries or invited)

NATURE PUBLICATION FEATURING PM Rudd’s analytical platform: A Spoonful of Sugar: Technology Feature Glycobiology. Nathan Blow. Nature, January 2009 457: 617-620

COMMERCIALISATION of analytical platform and software (UNIFI) by Waters Corporation.

The IGO Young Glycoscientist Award 2017

figure c

Dr. Yoann Rombouts

Researcher at the National Center for Scientific Research (CNRS)

Team "Mycobacterial Interactions with Host Cells"

Institute of Pharmacology and Structural Biology

UMR 5089 CNRS/University of Toulouse Paul Sabatier

BP64182

205 route de Narbonne

31077 Toulouse Cedex 04

Phone : +33 (0)5 61 17 59 10 - fax : +33 (0)5 61 17 59 94

HP: http://www.ipbs.fr/mycobacterial-interactions-host-cells

Personal data

Date of birth: October 12, 1984

Place of birth: Wattrelos, France

Education

2002-2005 Bachelor of Biochemistry, University of Lille, France

2005-2007 Master of Biology and Health Science, University of Lille, France

2007-2010 PhD in Analytical Biochemistry and Glycobiology, Structural and Functional Glycobiology Unit (UGSF), UMR 8576 CNRS, University of Lille, France

Professional Experience

October, 2007-December, 2010 PhD Fellow, UGSF, UMR 8576 CNRS, University of Lille, France (Director: Dr. Yann Guérardel; Co-director: Prof. Elisabeth Elass-Rochard)

October, 2010-August, 2011Temporary teacher and postdoctoral researcher, UGSF, UMR 8576 CNRS, University of Lille, France

September, 2011-December 2014Postdoctoral Researcher, Department of Rheumatology & Center for Proteomics and metabolomics, Leiden University Medical Center, The Netherlands (Supervisors: Prof. René Toes and Prof. Manfred Wuhrer)

January, 2015-PresentCNRS Researcher, Institute of Pharmacology and Structural biology, UMR 5089 CNRS- University of Toulouse, France (Laboratory of Dr. Olivier Neyrolles)

Professional societies

Groupe Français des Glycosciences (2008-2013; 2017)

Société Française de Biochimie et Biologie Moléculaire (2011-2012; 2017)

Dutch Society for Immunology (2012-2015)

Honors and Awards

2007-2010 National Science doctoral fellowship at University of Lille

2008Best Poster Award at the “22èmes Journées du Groupe Français des Glucides”

2012Best Oral Presentation Award at the “24èmes Journées du Groupe Français des Glucides”

2015Best Poster Award at the 11th jenner glycobiology and medicine symposium

Selected Publications

•Hafkenscheid L, Bondt A, Scherer HU, Huizinga TW, Wuhrer M, Toes RE, Rombouts Y. Structural Analysis of Variable Domain Glycosylation of Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Reveals the Presence of Highly Sialylated Glycans. Mol Cell Proteomics. 2017 Feb;16(2):278-287.

Rombouts Y, Willemze A, van Beers JJ, Shi J, Kerkman PF, van Toorn L, Janssen GM, Zaldumbide A, Hoeben RC, Pruijn GJ, Deelder AM, Wolbink G, Rispens T, van Veelen PA, Huizinga TW, Wuhrer M, Trouw LA, Scherer HU, Toes RE. Extensive glycosylation of ACPA-IgG variable domains modulates binding to citrullinated antigens in rheumatoid arthritis. Ann Rheum Dis. 2016 Mar;75(3):578-85.

Rombouts Y, Ewing E, van de Stadt LA, Selman MH, Trouw LA, Deelder AM, Huizinga TW, Wuhrer M, van Schaardenburg D, Toes RE, Scherer HU. Anti-citrullinated protein antibodies acquire a pro-inflammatory Fc glycosylation phenotype prior to the onset of rheumatoid arthritis. Ann Rheum Dis. 2015 Jan;74(1):234-41.

Rombouts Y, Brust B, Ojha AK, Maes E, Coddeville B, Elass-Rochard E, Kremer L, Guerardel Y. Exposure of mycobacteria to cell wall-inhibitory drugs decreases production of arabinoglycerolipid related to Mycolyl-arabinogalactan-peptidoglycan metabolism. J Biol Chem. 2012 Mar 30;287(14):11060-9.

Rombouts Y, Elass E, Biot C, Maes E, Coddeville B, Burguière A, Tokarski C, Buisine E, Trivelli X, Kremer L, Guérardel Y. Structural analysis of an unusual bioactive N-acylated lipo-oligosaccharide LOS-IV in Mycobacterium marinum. J Am Chem Soc. 2010 Nov 17;132(45):16073-84.

It is with great enthusiasm that I introduce to you the recipient of the IGO 2017 Young Glycoscientist Award: Dr. Yoann Rombouts is a Research associate for the French National Center for Scientific Research (CNRS) and is presently working at the Institute of Pharmacology and Structural Biology, University of Toulouse, France. Yoann Rombouts studied Cellular and Structural Biology followed by a PhD with Dr. Yann Guérardel on the structural characterization of mycobacterial glycolipids using NMR and MS. His PhD work has led to the publication of 10 articles in recognized journals in biochemistry and microbiology.

Yoann then moved to the Leiden University Medical Center (LUMC) to work at the Department of Rheumatology and the Center for Proteomics and Metabolomics on the glycosylation of rheumatoid arthritis (RA) specific autoantibodies. Dr. Rombouts analyzed these autoantibodies from healthy persons who have later developed RA and found that their Fc-glycosylation displayed significant changes towards a more pro-inflammatory phenotype only a few months before the RA diagnosis, thereby strongly suggesting that glycosylation could be involved in driving the disease process. Moreover, Dr. Rombouts discovered the frequent presence of additional N-glycans on unique sites (variable domain) of the autoantibodies. Dr. Rombouts’ postdoctoral research has been published in several high impact journals.

Dr. Rombouts has been involved as co-investigator in several studies related to infectious diseases, autoimmunity, cancer biology, immunology, plant physiology and glycobiology as documented in his impressive publication record. He has distinguished himself as a very successful and innovative scientist. I congratulate him with this award and hope that this will encourage him to continue advancing the frontiers of glycobiology and bridge to neighboring disciplines.

Manfred Wuhrer

Professor Proteomics and Glycomics

Head Center for Proteomics and Metabolomics

Leiden University Medical Center, Leiden, The Netherlands

ABSTRACTS

Plenary Lectures

1. Glycan microarray story from fabrication to applications

Injae Shin 1; 1Yonsei University, Department of Chemistry, 03722, Seoul, Korea

injae@yonsei.ac.kr

Glycan-associated recognition events in cells and organisms are not only critical for diverse physiological processes, but they are also involved in various pathological processes. Therefore, elucidation of glycan-mediated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to employ high-density glycan microarrays in efforts aimed at the high-throughput assessments of glycan-associated recognition events. As they include many glycans attached in a dense and orderly manner to a solid surface, glycan microarrays allow for multiple parallel analyses of glycan-protein binding events by utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this presentation, I will discuss our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces will be discussed. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan–protein interactions quantitatively, detect pathogens and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses.

2. Lectins from bacteria and fungi: therapeutical targets and research tools

Anne Imberty 1; 1CERMAV, CNRS and Université Grenoble Alpes, 601 rue de la Chimie, 38041 Grenoble, France

anne.imberty@cermav.cnrs.fr

A large number of pathogenic microorganisms display receptors for specific recognition and adhesion to the glycoconjugates present on human tissues. In addition to membrane-bound adhesins, soluble lectins are involved in infections caused by the bacteria Pseudomonas aeruginosa and Burkholderia cepacia and by the fungus Aspergillus fumigatus that are responsible for hospital-acquired diseases. Accumulated knowledge about the structures of the lectins and the interactions with host glycoconjugates has led to the design of powerful glyco-derived inhibitors that can serve as antimicrobial therapeutic agents, as a complement to or an alternative to antibiotic therapy. Design of glycosylated chips, liposomes, fullerenes and other nanoglycoparticles have provided information on multivalent interaction between receptors and cell surfaces. This also result in development of nanomaterials that can be used in diagnostic applications.

Furthermore, the multivalency of lectin is proposed to play a role in their strong avidity for glycosylated cell surfaces and also in their ability to affect membrane dynamics by clustering glycosphingolipids. Bacterial lectins are able to bind to glycoconjugates on human tissues and are therefore thought to be involved in the first step of infection. The role of lectins in membrane invagination indicates that they could also play a role in internalization of intracellular pathogens. Lectins as multivalent and specific compounds are also powerful tools for labeling tumor cells that present altered glycosylation. Engineering of lectins allows the creation of novel supramolecular tools, superlectins, neolectins and Janus lectins with modified architecture, valency and specificity.

3. Biological roles of glycans: from Drosophila to embryonic stem cells

Shoko Nishihara 1; 1Department of Bioinformatics, Graduate School of Engineering, Soka University, 192-8577, Hachioji, Japan

shoko@soka.ac.jp

The Golgi apparatus is the site of glycosylation by various glycosyltransferases, the expressions of which are developmentally regulated. Molecular evolutionary studies showed that a prototype set of glycosyltransferases existed before the protostome-deuterostome split, suggesting conservation of glycan function from Drosophila to mammals. Whole body-gene silencing of 72 Drosophila glycosylation-related genes resulted in lethality before eclosion for 78% of the genes. Tissue-specific silencing of these genes also showed abnormalities in the corresponding tissue, indicating that these genes were essential for development. Next, we performed detailed analysis of the biological function of each gene, such as the genes related to proteoglycan synthesis and encoding protein O-mannosyltransferases and core 1 β1,3galactosyltransferase 1 (dC1GalT1), and found that some of their biological roles were conserved between Drosophila and mammals. Recently, we showed that the dC1GalT1 mutant larvae exhibited excessive differentiation of hematopoietic stem cells, mislocalization of neuromuscular junction boutons and abnormality of muscle cell architecture. In addition the major O-glycan T antigen synthesized by dC1GalT1 localized within the proximal compartments of primary cultured neurons, whereas the N-glycan HRP antigen did not. These results suggested novel biological roles of mucin type O-glycans in Drosophila and possibly in mammals.

Since embryonic stem (ES) cells possess normal physiology and the ability to differentiate, we selected ES cells for analyzing the roles of glycans in mammals. Mouse and human ES cells are at different developmental stages, namely, the pre- and post-implantation blastocyst stages, which are also called naïve and primed states, respectively. Almost 100 glycosyltransferases were screened using gene silencing, followed by the alkaline phosphatase activity assay, which is an indicator of the naïve state, and followed by detailed analysis of the biological function of each selected gene. Our results revealed the biological roles of heparan sulfate, LacdiNAc (GalNAcβ1,4GlcNAc), and O-linked N-acetylglucosamine in naïve and primed ES cells. These structures were also detected in Drosophila, suggesting the species-wide functional conservation of these glycans.

4. Genomic events altering hominin sialic acid and Siglec biology predate the common ancestor of humans and Neanderthals

Ajit Varki 1; 1Departments of Medicine and Cellular and Molecular Medicine, UC San Diego, USA

varkiadmin@ucsd.edu

Human-specific pseudogenization of CMAH eliminated the common mammalian sialic acid (Sia) Neu5Gc, giving an excess of the precursor Neu5Ac, a major change in cell surface “self-associated molecular patterns”(SAMPs), which modulate innate immune cells by engagement of CD33-related Siglecs (CD33rSiglecs). The pseudogene appears to have been fixed ~2-3 million years ago (mya), possibly while contributing to the origins of the genus Homo. Genomic events altering several other genes of Sia biology have been reported, which also appeared to be human-specific, i.e., not found in available chimpanzee genomic data––suggesting a "hot spot" in hominin evolution. Availability of many more hominid genomes including ancient DNA of Neanderthals and Denisovans allows fresh analysis, and better evolutionary timing. All known human genomic changes in CD33-related SIGLECs (affecting 8 of 13 members of this class of genes) are found in current human African populations, indicating that they predated the common ancestor of modern humans. Comparisons with 147 “great ape” genomes indicate that these changes appear unique to hominins. There is no evidence for strong selection after the Human-Neanderthal/Denisovan common ancestor, ~500 kya. Consistent with these findings, Neanderthal and Denisovan genomes appear to include all the major changes found in modern humans. But genome level analyses can miss domain-specific rapid evolution within functional regions of proteins. Indeed, Sia-binding V-set domains of CD33rSiglecs prominent on innate immune cells harbor higher rates of evolution relative to those of the other hominids and of adjacent structural C2-set domains. CMAH pseudogenization induced SAMP changes likely triggered multiple selection processes involving CD33rSiglecs, emergence of human-specific pathogens preferentially targeting Neu5Ac, or Neu5Ac-containing pathogen molecular mimics of human glycans, to subvert immune responses. A “hot spot” in hominin biology likely occurred in Homo, prior to the Humans-Neanderthals common ancestor. A related consequence may be unusual human expression patterns of CD33rSiglecs in locations such as placental trophoblast (SIGLEC6), pancreatic islets (SIGLEC7), ovarian fibroblasts (SIGLEC11/16), amniotic epithelium (SIGLEC5/14) and microglia (SIGLEC11/16).

5. Glycocconjugate vaccines against bacterial infections based on synthetic glycans

Peter H. Seeberger 1; 1Max-Planck Institut for Colloids and Interfaces Am Mühlenberg 1, 14476 Potsdam (Germany)

peter.seeberger@mpikg.mpg.de

Most pathogens, including bacteria, fungi, viruses and protozoa, carry unique sugars on their surface. Currently, several glycoconjugate vaccines against bacteria are successfully marketed. Since many pathogens cannot be cultured and the isolation of pure oligosaccharides is difficult, synthetic oligosaccharide antigens are an attractive alternative. In this plenary lecture I will describe a medicinal chemistry approach to the development of semi- and fully synthetic glycoconjugate vaccines against severe bacterial infections, including resistant hospital microorganisms. This approach is fueled by oligosaccharides prepared by automated glycan assembly1,2 that has been commercialized.3 Quality control of synthetic oligosaccharides is ensured by ion mobility mass spectrometry (IM-MS).4

Vaccine programs aimed at protection from a series of Streptococcus pneumoniae serotypes, 5 Clostridium difficile6 and Klebsiella pneumoniae7 have progressed to the late preclinical stages and are now advanced to the clinic by Vaxxilon AG.

Synthetic oligosaccharides serve as basis for tools such as glycan microarrays and for the production of monoclonal antibodies.

1. Plante, O.J.; Palmacci, E.R.; Seeberger, P.H.; Science 2001, 291, 1523.

2. Seeberger, P.H.; Acc. Chem. Res. 2015, 48, 1450.

3. Hahm, H.S.; Schlegel, M.K.; Hurevich, M.; ….. Seeberger, P.H.; ProcNat Acad Sci USA, 2017, 114, E3385; www.glycouniverse.de

4. Hofmann, J.; Hahm, H.S.; Seeberger, P.H.; Pagel, K.; Nature 2015, 526, 241.

5. Parameswarappa, S.G.; Reppe, K.; Geissner, A.; .... Seeberger, P.H.; Cell Chem. Bio. 2016, 23, 1407; Schumann, B.; Hahm, H.S.; …. Seeberger, P.H.; Science Transl. Med. 2017, 9, eaaf5347; Seeberger, P.H.; Pereira, C.L.; Govindan, S.; Beilstein J. Org. Chem. 2017, 13, 164. Lisboa, M.P.; Khan, N.; Martin, C.; Xua, F.-F.; Reppe, K.; Geissner, A.; Witzenrath, M.; Govindan, S.; Pereira, C.L.; Seeberger, P.H.; submitted.

6. Martin, C.E.; Broecker, F.; Oberli, M.A.; …..Seeberger, P.H.; J. Am. Chem. Soc. 2013, 135, 9713; Broecker, F.; Hanske, J.;…. Seeberger, P.H.; Nature Comm. 2016, 7, 11224; Broecker, F.; Martin, C.E.; Anish, C.; Seeberger, P.H.; Cell Chem. Bio. 2016, 23, 1014.

7. Khan, N.; Xiao, G.; Diago-Navarro, E.; Fries, B.C.; Pereira, C.L.; Seeberger, P.H.; submitted for publication.

6. Glycan functions in development and glycan recognition by lectins and antibodies

Richard D. Cummings 1; 1Professor, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School Center for Glycoscience, Harvard Medical School, Boston, MA

rcummin1@bidmc.harvard.edu

Glycans in human and animal glycoconjugates are bound by a wide variety of glycan-binding proteins (GBPs), including animal cell receptors, microbial adhesins lectins and toxins, as well as functioning indirectly in glycoproteins to facilitate intra- and intermolecular interactions that maintain homeostasis and biological integrity. But glycans are also recognized by a wide variety of antibodies in human serum and other fluids, including saliva and cerebrospinal fluid. We have taken a multifaceted approach to explore glycan functions using human and mouse biology and genetics, and to explore protein-glycan interactions using a variety of technologies including shotgun glycomics and glycan microarray and glycans derivatized to microbeads. Our studies show that extended O-glycans are required for recognition by many types of GBPs and that loss of extended O-glycans due to engineered and acquired mutations in the molecular chaperone Cosmc lead to loss of function in almost all cell types, but we have identified many specific changes in lymphocyte function associated with normal O-glycans. In addition, we have identified many antibodies in animals and people that bind to simple or complex glycans, including microbial glycans, and the antisugar antibody repertoire (ASAR) is extensive but unique for each individual and indicates their history of exposure to different types of pathogens and commensal organisms. Overall, these studies reveal the complex recognition of glycans by endogenous GBPs and antibodies and the complex ways in which cellular glycans regulate many aspects of development and immunity

7. Chemical biology tools for O-GlcNAc: enzyme function to roles in cell physiology and disease

David. J. Vocadlo 1,2; 1Department of Chemistry, Simon Fraser University, Burnaby, Canada, 2Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, Canada

dvocadlo@sfu.ca

O-linked N-acetylglucosamine (O-GlcNAc) was first discovered 30 years ago and is now recognized as an abundant form of protein O-glycosylation found in the nucleus and cytoplasm of all multicellular eukaryotes. This modification is found on hundreds of proteins yet is regulated by only two enzymes; O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAc is in some cases reciprocal to serine/threonine phosphorylation and its levels fluctuate in response to nutrient availability and cellular stress. Consistent with the metabolic responsiveness of O-GlcNAc, this modification has been implicated in various essential biological processes including, for example, regulation of gene expression, satiety sensing, and cellular stress response. Accordingly, inappropriate regulation of O-GlcNAc has been proposed to play roles in diverse diseases including various cancers and neurodegenerative diseases including Alzheimer disease. In this presentation I will discuss our research focused on the development of chemical biology tools of these enzymes that are active in cells, tissues, and in vivo. I will also describe how we have recently used these tools, which including substrates, inhibitors, and metabolic probe molecules, to interrogate the functional roles of O-GlcNAc in various cellular processes. Topics of current interest include developing antibody-free methods for mapping O-GlcNAc to chromatin, evaluating the effects of altered O-GlcNAc on proteostasis, and determining its protective roles in animal models of diseases including Alzheimer’s.

8. Glycosylation with ribitol-phosphate

Tamao Endo 1; 1Tokyo Metropolitan Institute of Gerontology

endo@tmig.or.jp

Glycosylation is an essential post-translational modification in intermolecular and intercellular recognition. A defect in O-mannosyl glycan is the cause of aα-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant aα-dystroglycan (αa-DG) glycosylation. Recently, the entire structure of O-mannosyl glycan, [3GlcAaβ1,3Xylaα1,]n3GlcAbβ1,4Xylβ1,4Rbo5P,1Rbo5P,3GalNAcbβ1,3GlcNAcbβ1,4(phospho-6)Man, which is required for the binding of α-DG to extracellular matrix proteins, has been proposed. The tandem Rbo5P, a phosphoric ester of pentitol, is unique. We determined three enzymes to be involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing protein is CDP-Rbo synthetase, and fukutin and fukutin-related protein are Rbo5P transferases that use CDP-Rbo.

POMGnT1, a glycosyltransferase that participates in formation of GlcNAcβ1,2Man glycan, is causative for muscle-eye-brain disease (MEB), one of α-dystroglycanopathies. Although POMGnT1 does not catalyze any reactions involved in the above-proposed glycan, POMGnT1 KO mice and MEB patients lack the GlcA-Xyl repeat. This suggests that POMGnT1 plays important in processing of GalNAcβ1,3GlcNAcβ1,4(phosphate-6)Man. Fukutin forms a complex with POMGnT1, and POMGnT1 crystal structure reveals that the stem domain of POMGnT1 recognizes GalNAcbβ1,3GlcNAc. Since fukutin is required for the first Rbo5P modification of the GalNAcβ1,3GlcNAcβ1,4(phosphate-6)Man glycan, the POMGnT1–fukutin complex is important in forming a platform that requires further glycosylation. These findings expand our knowledge on glycosylation machinery, and reveal the pathogenesis of α-dystroglycanopathies.

9. Understanding catalytic and allosteric mechanisms in UDP-glucose pyrophosphorylases: towards utilizing conserved enzymes as novel drug targets

Rita Gerardy-Schahn 1 , Jana Indra Führing 1 , Johannes Thomas Cramer 1,2 , Ohm Prakash 1 , Francoise H. Routier 1 , Roman Fedorov 2; 1Institute for Cellular Chemistry, 2Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany,

gerardy-schahn.rita@mh-hannover.de

Allostery is the basis of a multitude of cellular signalling and regulatory processes. Because evolutionarily conserved enzymes are optimized to respond to cell- and/or system-specific needs, they differ in terms of allosteric networks. In our research we explore the links between catalysis and allostery and aim to exploit this knowledge for rational inhibitor design. Our target is the UDP-glucose pyrophosphorylase (UGP). The enzyme occupies a central position in the carbohydrate metabolism of cells from all kingdoms of life, because its product, UDP-Glc, is essential in a number of anabolic and catabolic pathways. Moreover, the significance of UGP as a virulence factor in protists and bacteria is well established and has given momentum to the search for pathogen specific inhibitors. While UGPs show high evolutionary conservation in all eukaryotes, the eukaryotic enzymes are unrelated to prokaryotic UGPs. Prominent aspects creating diversity between eukaryotic UGPs are quaternary organization and conformational flexibility. Both features are associated with catalytic functions. With the goal to understand the interplay of catalysis, allostery, quaternary organization and protein dynamics, we are using a combination of kinetic X-ray crystallography, biochemical, biophysical and theoretical chemistry. With the UGPs from Homo sapiens and Leishmania major as models, we reconstructed the enzymatic cycles. Obtained results enabled the description of structural changes during catalysis and explained the essential nature of the respective protein organisations. In our current work we extend structural and functional studies towards bacterial UGPs and the question how differences in the structure-function relationships can be exploited for specific inhibitor design. Eventually, our research shall establish a new basis on which UGPs can be exploited as drug targets.

10. Lipid rafts and glycosphingolipid-protein interactions

Sandro Sonnino 1; 1Department of Medical Biotechnology and Translational Medicine, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Italy

sandro.sonnino@unimi.it

Glycosphingolipids are ubiquity components of the plasma membranes, where they are inserted into the outer layer through the lipid moiety called ceramide, protruding the oligosaccharide chain into the extracellular medium. Glycolipids are not homogeneously distributed on the cell surface, but are highly enriched, together with sphingomyelin, cholesterol and dipalmitoylphosphatidylcholine in membrane domains called lipid rafts. Lipid rafts contain a few proteins, many of which have specific roles in the signaling processes.

Glycolipids through direct interactions with proteins or by modulation of the protein conformation by reorganization of the physical-chemical properties of the proteins environment are believed to play a specific role in modulating the signaling processes. Unlikely the studies on the glycolipid-protein interactions are complex and requires the use of chemical approaches and of experimental models not easily available in all laboratories.

In the late 80’s, taking advantage from the experience developed in the field of glycerophospholipids we developed a new chemistry to produce photoactivable glycosphingolipids from natural compounds. In the following years, we collected more and more detailed information on the glycolipid-protein interactions and how these are instrumental for the final yield of the signaling process.

In my lecture, I will present the history of the photolabeling experiments developed to study the glycosphingolipid-protein interactions and how these determine some cellular processes.

IGO Award Lectures

11. Glycosylation, complexity and the emergence of functional glycoproteins

Radka Fahey 1 , Roisin O’Flaherty 1 , Mark Hilliard 1 , Gordon Greville 1 , Sinead Hallinan 1 , Pauline M. Rudd 1,2; 1National Institute for Bioprocessing Research and Training, Dublin Ireland, 2 Bioprocessing Technology Institute, AStar, Singapore

pauline.rudd@nibrt.ie

An appreciation of the multiple systems that underpin biology is a necessary step towards deciphering the complexity of organisms and identifying disease related pathways for multiple drug targeting. This talk will present our attempts to gain some kind of top down and bottom up overview of the factors that alter the glycosylation of glycoproteins and potentially their function. The tipping points that are reached at each node from the constraints of the genome and epigenetics, the transcriptome, lipidome and metabolome, protein synthesis and folding, through to the proteome and glycan processing pathways may direct the neoglycoprotein along particular routes. Disease pathogenesis usually involves extensive disruption to many pathways and processes so it is unsurprising that alterations in glycans have been noted in most diseases, ranging from congenital disorders of glycosylation to autoimmune disease, neurological disorders, heart disease, sepsis and cancer. In many cases these changes are involved in disease pathogenesis or host response. Glycosylation processing itself reflects the detailed processing pathways available in individual cells, the environment of the cells and the phases of cell growth and death. Much of this is explored in bioreactors producing glycosylated therapeutics. Working from the bottom up, modelling of the 3D structures of glycoproteins enables us to visualise the detailed structural features of oligosaccharides as well as their dynamic range, the conformational space that they occupy and the constraints that the protein imposes on their location, orientation and potential functional interactions.

• Haakensen VD et al., Serum N-glycan analysis in breast cancer patients--Relation to tumour biology and clinical outcome. Mol Oncol. 2016 Jan;10(1):59-72

• Greville G et al., Epigenetic regulation of glycosylation and the impact on chemo-resistance in breast and ovarian cancer. Epigenetics. 2016 (12):845-857. Review

• Hayes JM et al., Fc gamma receptors: glycobiology and therapeutic prospects. J Inflamm Res. 2016 Nov 16;9:209-219. Review.

• Walsh I et al., Quantitative profiling of glycans and glycopeptides: an informatics' perspective. Curr Opin Struct Biol.2016 Oct;40:70-80. Review

12. Glycosylation of anti-citrullinated protein antibodies in rheumatoid arthritis: a bitter sweet symphony

Yoann Rombouts 1,2,3 , Hans U. Scherer 2 , Lise Hafkenscheid 2 , Tom W. Huizinga 2 , Manfred Wuhrer 3 , René E.M. Toes 2; 1Institut de Pharmacologie et Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, Toulouse, France, 2Department of Rheumatology, Leiden University Medical Center, The Netherlands, 3Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

yoann.rombouts@ipbs.fr

Anti-citrullinated proteins antibodies (ACPA) are the most relevant prognostic and diagnostic biomarker for rheumatoid arthritis (RA), an autoimmune disorder affecting around 1% of people worldwide. ACPA-positive RA patients are characterized by a progressive disease, a high rate of joint destruction, and a low chance to achieve remission. Despite strong and well-defined associations with clinical phenotype, the molecular mechanisms underpinning the ACPA-mediated immune response are incompletely understood. Glycosylation of antibodies, especially the N-linked glycosylation of the constant fragment (Fc) of IgG, has been shown to modulate the interactions with different classical and non-classical Fc receptors, thereby regulating IgG effector functions. This, together with the well-established fact that RA patients produce IgG with a skewed glycosylation profile, lead us to focus our research on the structure, function and clinical relevance of ACPA-IgG glycosylation. This lecture will describe our efforts to characterize the dynamic changes in Fc- and Fab-linked glycosylation of ACPA-IgG and determine how these glycosylation features could drive the disease process.

Session 1. Glycans in Development/Signaling

Keynote Lectures

13. Deletion of B3GLCT disrupts craniofacial, skeletal, and cardiac development in mice

Robert Haltiwanger 1 , Bernadette Holdener 2 , Diana Rubel 2 , Richard Grady 2 , Sardar Uddin 3 , David Komatsu 3 , Simon Bamforth 4 , Jurgen Schneider 5 , Takashi Sato 6 , Hisashi Narimatsu 6; 1University of Georgia, Complex Carbohydrate Research Center, Athens, GA USA, 2Stony Brook University, Department of Biochemistry and Cell Biology, Stony Brook, NY USA, 3Stony Brook University, Department of Orthopedics, Stony Brook, NY USA, 4Newcastle University, Institute of Genetic Medicine, Newcastle, UK, 5Wellcome Trust Centre for Human Genetics, BMRU, Oxford, UK, 6National Institute of Advanced Industrial Science and Technology in Japan

rhalti@uga.edu

Peters plus syndrome (PPS) is a rare genetic disorder characterized by the presence of anterior eye segment abnormalities, short stature, brachydactyly, and developmental delay. In addition, cleft palate, congenital heart defects, and/or urogenital defects are present in 50% of patients. The disease is caused by recessive loss-of-function mutations in β3-glucosyltransferase (B3GLCT). B3GLCT transfers a glucose to O-fucosylated thrombospondin type I repeats (TSRs). TSRs with O-Fucosylation consensus sequences are tandemly repeated within 49 predominantly extracellular matrix (ECM) associated proteins. The ADAMTS class of proteins (A Disintegrin and Metalloproteinase with ThromboSpondin motifs) makes up nearly 50% of these proteins, and is implicated in controlling the structural properties of the ECM, influencing cell migration, organogenesis, tissue organization and cell signaling. We used a mouse B3glct knockout to gain insight into the developmental origin of PPS and identify B3GLCT targets responsible for PPS anomalies. B3glct mutants showed reduced neonatal viability. MicroCT and MRI imaging identified potential ventricular septal and myocardial wall defects in some homozygotes. The survivors were runted and had broadened and domed heads. Skeletal preparations, 3D microCT renderings, and histological analyses identified defects in cranium structure, endochondral ossification, and hydrocephalus. Finally, reducing the copies of Adamts9 in B3glct homozygotes resulted in 100% neonatal lethality. These results provide evidence that defects in PPS patients result, at least in part, from abnormalities in ADAMTS9 function, and demonstrate that the B3glct mutant mouse will provide an invaluable resource for understanding how changes in the ECM structure or composition can lead to the collection of common congenital abnormalities seen in PPS patients. Funding sources: NIH GM061126 to RSH, and BHF FS/11/50/29038 and RE/08/004 to JES, and NIH RO1HD070888 to DEK.

14. Regulation of cell signaling by ganglioside GD3/GD2 in gliomas

Koichi Furukawa 1,2 , Yuki Ohkawa 1 , Kei Kaneko 2 , Noboru Hashimoto 2 , Zhang Pu 1,2 , Robiul H. Bhuiyan 1 , Yuhsuke Ohmi 1 , Keiko Furukawa 1; 1Chubu University College of Life and Health Sciences, Kasugai, 487-8501, Japan, 2Nagoya University Graduate School of Medicine, Nagoya, 466-0065, Japan

koichi@isc.chubu.ac.jp

Gangliosides have been reported to play roles in the regulation of cell signaling on cell surface, and consequently of cell phenotypes. In particular, they are involved in the regulation of crucial signaling in cancers. A number of studies on phenotypic changes in the transfectant cells of glycosyltransferase cDNAs have been reported. However, mechanisms by which gangliosides exert regulatory functions on cell membrane remain to be clarified.

In order to clarify mechanisms for gangliosides to enhance malignant properties of cancer cells, enzyme-mediated activation of radical sources (EMARS)/MS have been performed, leading to identification of ganglioside-associated molecules. The most representative molecule is Neogenin defined as a GD3-associated molecule in melanomas. Neogenin was significantly found in lipid rafts of only GD3+ cells, and enhanced cell growth, invasion and motility. To our surprise, gamma-secretase was also recruited to lipid rafts, and efficiently cleaved intracytoplasmic domain of Neogenin (NeICD), promoting various gene expression after entering into nucleus. ChIP-sequence revealed that various target genes might be promoted to play as actual effectors of melanoma-related GD3.

In glioma cells, GD3/GD2 were specifically found in PDGFB-induced mouse gliomas with RCAS-tv-a system. Expression of GD3/GD2 enhanced malignant properties of gliomas, such as invasion activity. PDGFB induced expression of GD3 and PDGF receptor (PDGFR) α, and formation of ternary complex consisting of PDGFR α, GD3 and Yes. This complex seemed to be essential for the enhanced invasion activity of gliomas. In DNA array, increased expression of several series of genes was detected in wild-type mouse-derived gliomas compared with GD3 synthase (S)-KO-derived gliomas. Among them, MMP families showed low expression in GD3S-KO-derived gliomas. Thus, molecules activated under cancer-associated gangliosides in vivo might be good targets for therapeutics of glioma patients.

Lectures

15. Tunicamycin: a dual action glycotherapy to treat breast cancer

Dipak Banerjee 1,2 , Krishna Baksi 3 , Juan Martinez 1 , Aditi Banerjee 1 , Zhenbo Zhang 1 , Jesús Serrano-Negrón 1,4 , Eva Romero 1 , Neysharie Sanchez 1; 1Department of Biochemistry, School of Medicine and 2Institute of Functional Nanomaterials, University of Puerto Rico, San Juan, USA, 3Department of Anatomy & Cell Biology, School of Medicine, Universidad Central del Caribe, Bayamón, USA, 4Department of Natural Sciences & Mathematics, Interamerican University of Puerto Rico, Bayamón, USA

dipak.banerjee@upr.edu

Breast cancer kills ~400,000 – 500,000 women per year and is expected to rise to 747,802 in 2030, if a new therapeutic is not forthcoming. ER-/PR-/HER2- breast cancer (i.e., triple negative cancer; TNBC) accounts for 15% of all breast cancers and has a disproportionate share of mortality. The etiology of breast cancer is complex and its progression is multi-factorial. Since, asparagine-linked (N-linked) glycoproteins have been found to play a critical role in angiogenesis and tumor progression we have targeted the process with a homolog of protein N-glycosylation inhibitor tunicamycin. The result is a quantitative inhibition of angiogenesis (in vitro & in vivo), non-metastatic & metastatic human cancer cells, and double- & triple negative breast tumors in athymic nude mice. The animals exhibited no behavioral and/or skeletal toxicity. Mechanistic details indicate development of ER stress, cell cycle arrest in G1, and induction of unfolded protein response (upr)-mediated apoptosis. There was no expression of ER chaperone GRP78 on the cell surface. Tunicamcin gold nanoparticles are three-times more potent, cause ER stress, inhibit cell cycle progression but do not induce apoptosis. Tunicamycin does no harm to non-proliferating cells. Once inside, tunicamycin self amplifies the ‘death signal’ including down regulation of dolichol phosphate mannose synthase (DPMS) activity. Thus, the target cells have ‘point of no return’ whether they express “complex” or “high mannose” N-glycans. Partly supported by grant HIH/NIMHD 2G12MD007583 (KB).

Session 2. Glycans in Metabolism/Glycogene expression and regulation

Keynote Lectures

16. ENGase as a potential drug target for NGLY1-deficiency

Tadashi Suzuki 1; 1RIKEN, Japan

tsuzuki_gm@riken.jp

The cytoplasmic peptide:N-glycanase (PNGase) is the enzyme widely conserved throughout eukaryotes. 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. Surprisingly, the additional deletion of Engase, encoding another cytosolic deglycosylating enzyme called ENGase (endo-β-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, produced by crossing the mice with an outbred mice strain (ICR) could rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a B6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. 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. 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. These observations collectively suggest that the ENGase represents one of the potential therapeutic targets for this genetic disorder.

References

1. Fujihira, H., et al. (2017) PLOS Genetics 13: e1006696.

2. Huang C, et al. (2015) Proc Natl Acad Sci U S A 112:1398-1403

17. Expression of N-Glycan branching enzymes and their products in disease implication.

Naoyuki Taniguchi 1; 1Systems Glycobiology Research Group, RIKEN Global Research Cluster, Wako, Japan,

dglycotani@riken.jp

N-glycan branching enzymes like α1,6 Fucosyltransferase, N-Acetylglucosaminyltransferase III (GnT-III) and β-Galactoside α2,6-sialyltransferase-1 (St6gal1) and their products are implicated in various disease onset, diagnosis and therapeutics. Core fucose is a target for various cancer1, but antibody therapy is also implicated in COPD (chronic obstructive pulmonary disease). We found that a keratan sulfate disaccharide designated L4 attenuated alveolar destruction, reduced neutrophil influx and inflammatory cytokines, inactivated matrix metalloproteinase and myeloperoxidase in bronchoalveolar lavage fluid of two murine COPD models2. Bisecting GlcNAc, a GnT-III product was high in Alzheimer’s disease patients. Analysis of GnT-III KO mouse revealed that decreased cleavage of APP (AB-precursor protein) by BACE1 (β-site amyloid precursor protein cleaving enzyme-1) as well as decreased AB plaque. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less co-localized with APP, leading to accelerated lysosomal degradation3. St6gal1 was most downregulated in VATs (visceral adipose tissues) from obese mice and differentiated adipocyte model cells. Integrin-β1 was identified as one of the target proteins of St6gal1 in adipose tissues, and phosphorylation of focal adhesion kinase (FAK) was decreased after HFD (high fat diet) feeding. St6gal1 overexpression in differentiating 3T3-L1 cells inhibited adipogenesis while KO mice showed enhanced adipogenesis. The downregulation of St6gal1 during adipogenesis was found to be involved epigenetic DNA methylation in St6gal1 silencing4. In conclusion, findings of various glycosyltransferase functions will open a new avenue toward promising druggable candidates.

References:

1) Taniguchi N, Kizuka Y. Ad Cancer Res. 2015. 126:11-51

2) Gao C, et al. Am J Physiol Lung Cell Mol Physiol. 2017. 312(2):L268-L276

3) Kizuka Y, et al. EMBO Mol Med. 2015. 7(2):175-89

4) Kaburagi T, et al. J Biol Chem. 2017. 292(6):2278-2286

Lectures

18. Hematopoietic-derived galectin-3 causes cellular and systemic insulin resistance

Shuainan Liu 4 , Pingping Li 4 , Min Lu 3 , Gautum Bandyopadhyay 5 , Takeshi Imamura 1 , Zhufang Shen 4 , Steven M. Watkins 2 , Martin Brenner 3 , Bing Cui 4 , Jerrold Olefsky 5; 1State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China, 2Diabetes Early Discovery, Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115, USA, 3Division of Endocrinology and Metabolism, UC, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA, 4Pharmacology, Department of Medicine, Shiga University of Medical Science, 1 Tsukinowa, Seta, Otsu-city, Shiga 520-2192, Japan, 5Lipomics Technologies, Inc., West Sacramento, CA 95691, USA

lipp@imm.ac.cn

In obesity, macrophages and other immune cells accumulate in insulin target tissues, promoting a chronic inflammatory state and insulin resistance. Galectin-3 (Gal3), a lectin mainly secreted by macrophages, is elevated in both obese subjects and mice. Administration of Gal3 to mice causes insulin resistance and glucose intolerance, whereas inhibition of Gal3, through either genetic or pharmacologic loss of function, improved insulin sensitivity in obese mice. In vitro treatment with Gal3 directly enhanced macrophage chemotaxis, reduced insulin stimulated glucose uptake in myocytes and 3T3-L1- adipocytes and impaired insulin-mediated suppression of glucose output in primary mouse hepatocytes. Importantly, we found that Gal3 can bind directly to the insulin receptor (IR) and inhibit downstream IR signaling. These observations elucidate a novel role for Gal3 in hepatocytes, adipocytes and myocyte insulin resistance, suggesting that Gal3 can link inflammation to decreased insulin sensitivity. Inhibition of Gal3 could be a new approach for the treatment of insulin resistance and diabetes.

19. MGAT3 gene promoter methylation correlates with IgG glycosylation in inflammatory bowel disease

Dora Markulin 1 , Vlatka Zoldos 1 , Marija Klasic 1 , Aleksandar Vojta 1 , Irena Trbojevic Akmacic 2 , Gordan Lauc 2 , Nicholas Ventham 3 , Vito Annese 4 , Salome Pinho 5 , Snjezana Dzijan 6; 1University of Zagreb Faculty of Science, Zagreb, Croatia, 2Genos Glycoscience Research Laboratory, Zagreb, Croatia, 3University of Edinburgh, Scotland, UK, 4University of Carregi, Florence, Italy, 5IPATIMUM, Lisbon, Portugal, 6University of Osijek School of Medicine, Osijek, Croatia

vzoldos@biol.pmf.hr

Inflammatory bowel diseases (IBD) are intestinal disorders that occur due to inappropriate immune response to intestinal microbiome in susceptible individuals. Genome-wide association studies identified several genes to be important for both IgG glycosylation and IBD, including the MGAT3, a glyco-gene encoding for the enzyme (GnTIII) which adds bisecting GlcNAc to a glycan structure. MGAT3 promoter methylation was analysed from whole blood of IBD patients from two large independent cohorts using bisulfite pyrosequencing. Methylation level was generally high, with significant difference between healthy controls (HC) and Crohn’s disease (CD) or ulcerative colitis (UC). In addition, the MGAT3 promoter showed differential methylation between HC and CD at some CpG sites in B lymphocytes, as well as in CD3+ T-cells from inflamed bowel tissue of UC patients with active disease state from the third independent cohort. The MGAT3 promoter methylation was correlated to glycan structures found on IgG molecules from the same blood samples of the two large individual cohorts. Significant correlations were found between MGAT3 promoter methylation and the FA2B/FA2 ratio, suggesting that activity of the GnT-III enzyme (the MGAT3 gene product) might be altered in IBD. Structure FA2 was positively correlated with MGAT3 promoter methylation, suggesting that the substrate abundance decreases when the enzyme is more actively transcribed due to decreased methylation. Significant correlation was found between the MGAT3 promoter methylation and galactosylated and sialylated structures as well, suggesting that these processes could be co-regulated with MGAT3 expression.

20. Modulation of glyco-gene expression using CRISPR/Cas9-based molecular tools for epigenetic editing

Aleksandar Vojta 1 , Paula Dobrinić 1 , Vanja Tadić 1 , Goran Josipović 1 , Vladimir Zanki 1 , Marija Klasić 1 , Vlatka Zoldoš 1; 1University of Zagreb, Faculty of Science, Department of Biology, 10000 Zagreb, Croatia

vojta@biol.pmf.hr

Gene expression can be regulated by epigenetic mechanisms, such as DNA methylation at CpG dinucleotides in gene promotes. This is particularly important for glyco-genes, since protein N-glycosylation seems to be epigenetically regulated. We have constructed molecular tools for targeted DNA methylation and demethylation by repurposing the CRISPR-Cas9 system. The Cas9 protein can be targeted to almost any 20 bp sequence by co-expression of a guide RNA. To facilitate targeted CpG methylation, catalytic domain of the DNA methyltransferase DNMT3A was fused to catalytically inactive Cas9 (dCas9). This fusion enabled silencing of gene expression by methylation of CpG sites in regulatory regions of the targeted genes – the BACH2 and IL6ST, involved in IgG glycosylation. Targeted gene promoter demethylation was accomplished by an analogous construct comprising dCas9-TET1 catalytic domain fusion, validated in the MGAT3 and LAMB1 gene promoters that are also involved in IgG glycosylation. Rigorous characterisation of our constructs in the HEK293 cell line showed peak methylation and demethylation activity about 30 nucleotides downstream from the targeted site within a ~35 bp wide window. Changes in methylation and demethylation at the target were up to 60%, with values around 30% being typical. Activity was highly specific for targeted regions and heritable across cell divisions. Multiple guide RNAs could target the dCas9-DNMT3A construct to multiple sites, which enabled hypermethylation of a wider genome region. We demonstrated that the gene IL6ST decreased expression level following promoter hypermethylation, which served as a proof of the concept of artificial epigenetic silencing by targeted CpG methylation in vivo. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodelling the aberrant epigenetic landscape. Epigenetic editing at specific sites could alter the gene expression pattern selectively, unlike the widely used epigenetic inhibitors. Thus, our system represents promising molecular tools for unravelling details of molecular processes in a living cell, especially protein glycosylation. Development of a comprehensive regulatory toolbox is underway, with potential uses in gene therapy and glyco-engineering.

Session 3. Asian Community of Glycoscience and Glycotechnology Guest (ACGG) Session I

Keynote Lectures

21. A Stepwise approach using MS platform for glycosylation CQAs

Hyun Joo An 1; 1Chungnam National University, Graduate School of Analytical Science and Technology, 34134, Daejeon, Korea

hjan@cnu.ac.kr

Glycosylation of therapeutic proteins can significantly affect drug’s quality, safety, and efficacy. The glycosylation pattern cannot be prophesied from the genetic pathway, and the nature glycosylation depends on host cell line and culture condition. It can lead to display diverse glycans with macro- and micro-heterogeneity. Thus, glycomic analyses are necessary to assess biotherapeutic quality and establish the similarity of biosimilars now coming to market. Many regulatory agencies recommend a stepwise approach throughout the development step, starting with a comparability assessment of quality attributes for establishing biosimilarity. Current glycome analyses involve the use of complementary methods for assessing specific glycosylation attributes including glycan composition and structure, abundance, and glycosylation site with microheterogeneity. Here, we illustrated a stepwise approach using MS platform for glycome quality assessment, namely glycan analysis by glycomic approach, site-specific analysis using glycoproteomic approach, and intact protein analysis.

22. Dissecting the site-specific N- and O-glycosylation of the ectodomains of receptor protein tyrosine phosphatase functionally implicated in extracellular matrix binding and intracellular signaling

Kay-Hooi Khoo 1 , Chu-Wei Kuo 1 , Yu-Chun Chien 1 , Deepa Sridharan 1 , Takashi Angata 1 , Tzu-Ching Meng 1; 1Academia Sinica, Institute of Biological Chemistry, Taipei, Taiwan

kkhoo@gate.sinica.edu.tw

It is well appreciated that a heavily O-glycosylated mucin domain is intrinsically disordered but the density and spatial distribution of different O-glycan stubs along a somewhat extended peptide backbone will dictate its overall conformation and how the presented glycotopes may mediate functional biomolecular recognition and interactions. Most family members of the transmembrane receptor-like protein tyrosine phosphatases (RPTP) carry one or more such stretches of potentially heavily O-glycosylated region within their extracellular domains in addition to the N-glycans. The ectodomains of the R4 subtype RPTP, namely PTPRA and PTPRE, contain a mere 120 and 25 amino acid residues, respectively. Yet the former carries 7 potential N-glycosylation sites and the latter 2 sites within this stretch of amino acid sequence that is also rich in Ser/Thr/Pro. Virtually nothing is known of their detailed site-specific glycosylation pattern due to technical difficulties in sequencing such glycopeptides. We hypothesize and seek to provide evidence that the glycosylated ectodomain of PTPRA render its catalytic cytosolic domains responsive to external environment by engaging components of the extracellular matrix, and/or by dictating the permissible tightness of inter-molecular biophysical contacts via its glycosylation pattern. Indeed, we showed for the first time unequivocal mass spectrometry (MS)-based evidence for high density of O-glycosylation, the complete elaboration of which is essential for the expression of a mature transmembrane form on cell surface, enabling proper interactions with fibronectin that is dependent on its N-glycans. This is consistent with previous work demonstrating a positive role of PTPRA in promoting the aggressiveness of fibroblast-like synoviocytes in rheumatoid arthritis. We further show that the N-glycans are essential for PTPRA-mediated signaling. The advances and current pitfalls in MS/MS sequencing and identification of multiply O- and N-glyocsylated peptides along with the ways forward will be emphasized in this presentation.

Lectures

23. Uncovering the catalytic mode of heparan sulfate N-deacetylase/N-sulfotransferase-4

Yi-Jun Li 1 , Fen-Shan Wang 1 , Ju-Zheng Sheng 1; 1Shandong University,Institute of Biochemical and Biotechnological Drug & National Glycoengineering Research Center School of Pharmaceutical Sciences, 250012, Jinan, China

shengjuzheng@sdu.edu.cn

N-Deacetylase/N-sulfotransferase (NDST), responsible for heparan sulfate (HS) N-sulfation, is a key enzyme directing further modifications including O–sulfation and C5-epimerazation1. The arrangement of the sulfate groups in the HS sugar chains determines its ability to bind to the protein factor, which in turn affects the biological activity of the proteoglycans containing the sugar chains2. Therefore, the systematic study of the catalytic mode of NDST is not only important to reveal the mechanism of HS biosynthesis, but also can directly guide the enzymatic synthesis of HP oligosaccharides in vitro. There are four NDST isozymes in human cells, which have different substrate specificity to regulate the N-sulfation modification of heparan sulfate. The modification mode of NDST1 has been fully elucidated 3. Here, highly purified recombinant NDST-4 and a selective library of structural-defined oligosaccharides were utilized to simulate the N-sulfation of HS biosynthesis in vitro. The substrate specificity and catalytic mode of NDST-4 were obtained by analyzing the structure of the reaction product. We discovered that recombinant NDST-4 containing full amino acid sequence of wild type is lack of the N-deacetylase activity, while only displayed N-sulfotransferase activity; Unlike the dominating role of NDST-1 in forming domain structures in HS, which carries out the modification in a highly ordered fashion, the directional characteristic unretained for NDST-4 while the N-deacetylase domain was inactive. Furthermore, we employed biacore SPR systems to analysis the kinetics of individual oligosaccharides binding to NDST-4, suggesting that the affinity for this enzyme of substrate molecules carrying only the N-acetyl groups within glucosamines is much lower than ones containing GlcNH2 residues. It’s the possible reason for the lack of N-deacetylase activity for NDST-4.

(1) Zhang, X., Wang, F., and Sheng, J. (2016). Carbohydr. Res. 428, 1–7

(2) Gama, C. I., Tully, S. E., Sotogaku, N., Clark, P. M., Rawat, M., Vaidehi, N., Goddard, W. A., Nishi, A., and Hsieh-Wilson, L. C. (2006). Nat. Chem. Biol. 2, 467–473

(3) Sheng, J., Liu, R., Xu, Y., and Liu, J. (2011). J. Biol. Chem. 286, 19768–19776

24. Serum GalNAc-associated glycan as a biomarker for diagnosis and prognosis of cholangiocarcinoma

Atit Silsirivanit 1 , Waraporn Saentaweesuk 1 , Chaisiri 1 , Sopit Wongkham 1; 1Khon Kaen University, Department of Biochemistry, Research Group for Glycosciences and Glycotechnology, Faculty of Medicine, Cholangiocarcinoma Research Institute, 40002, Khon Kaen, Thailand

atitsil@kku.ac.th

Aberrant glycosylation has been demonstrated in cholangiocarcinoma (CCA), the CCA-associated glycans are potentially used as biomarkers for diagnosis and monitoring of the disease. Our study using lectin histochemistry revealed the specificity of Sophora japonica agglutinin (SJA), an N-acetyl galactosamine (GalNAc) binding lectin, to abnormal bile ducts and cholangiocarcinoma. In this study, SJA-based sandwich ELISA was successfully developed for the detection of serum GalNAc-containing glycan. SJA-specific Glycan (SJG) was highly detected in serum of CCA patients, comparing with patients of other hepatobiliary diseases and healthy persons (P < 0.05). Moreover, high level of serum SJG indicates the shorter survival of CCA patients. In conclusion, we have disclosed here the potential of GalNAc-associated glycan as a new diagnostic and prognostic serum marker for CCA, which can be detected by the newly developed sandwich ELISA system.

Acknowledgements: We would like to thank the supports from Senior Research Scholar Grant of TRF and Khon Kaen University (KKU) (RTA5780012); KKU research grant (592401); and Faculty of Medicine, KKU (RG60201). CW and WS was grateful to the support from the RGJ-PhD Program, TRF (PHD/0021/2556).

25. Galactofuco-oligosaccharides derived from undaria galactofucan induce apoptotic death of human prostate cancer cells in vitro and in vivo

Jisun Lee 1 , Seul Lee 1 , Chang Won Lee 1 , Ji Won Choi 1 , Sarang Cho 1 , Seong Cheol Kim 1 , Woo Jung Kim 2 , Yong Il Park 1; 1Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi-do 14662, Korea, 2Biocenter, Gyeonggido Business & Science Accelerator (GBSA), Suwon, Gyeonggi-do 16229, Korea

yongil382@catholic.ac.kr

In this study, the potential in vitro and in vivo anti-cancer effects of low-molecular-weight mannogalactofucans (LMMGFs, < 4,000 g/mol) and high-molecular-weight Undaria galactofucan (GF) in human prostate cancer were evaluated and compared. RT-PCR and Western blot analyses demonstrated that LMMGFs more significantly induced cell cycle arrest at the G0/G1 phase and cell death via suppression of the Akt/GSK-3β/β-catenin pathway than GF in human PC-3 prostate cancer cells. The LMMGFs upregulated the mRNA expression of death receptor-5 (DR-5), the ratio of Bax to Bcl-2, the cleavage of caspases and PARP proteins, the depolarization of mitochondrial membrane potential (MMP), and ROS generation. Moreover, LMMGFs (200-400 mg/kg) effectively reduced both tumour volume and size in a xenografted mouse model with PC-3 cells. These results have demonstrated that LMMGFs attenuate the growth of human prostate cancer both in vitro and in vivo, suggesting that LMMGFs can be used as a potent functional ingredient in health-beneficial foods or therapeutic agent to prevent or treat the androgen-independent human prostate cancer.

Session 4. Glycotechnology I

Keynote Lectures

26. Glycan function in simple cells and simplified tissues

Hans Wandall 1; 1Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, Denmark.

hhw@sund.ku.dk

Our molecular understanding of how glycans influence cellular functions is limited. This is in part due to the complexity and technical difficulties associated with the analysis of glycans. We have taken a genetic approach, combined with sophisticated mass spectrometry, to systematically dissect the functional role of glycans in biological functions, including tissue formation, cancer formation, and host-pathogen interactions. As the first proof-of-concept for our discovery strategy, we have used genetic engineering to deconstruct N- and O-glycosylation in CHO cells and developed a design matrix that facilitates the generation of cells with desired glycosylation. This engineering approach will aid the production of glycoproteins with defined and homogenous glycosylation and allow genetic dissection of glycan functions in many different cells. As an example, we will present the use of genetic engineered cells as a novel production platform for allergy vaccines. Also, we have pioneered a next generation approach using organotypic tissue models to decipher glycan functions. The tissue model has provided the first evidence that aberrant glycosylation in cancer directly induces oncogenic features. We now extend these studies to characterize the impact of site-specific glycosylation on epithelial differentiation and malignant transformation. Using organotypic model systems equipped with cells with and without GalNAc-T1, -T2, -T3, or T6 we identify distinct phenotypes and selective effects on specific cellular pathways identified via differential transcriptomic, phosphoproteomic, and proteomic analyses. Furthermore, we have performed a step-by-step genetic deconstruction of glycosylation capacities and provide comprehensive cell and tissue models for discovery and dissection of structure-function relationships of glycans.

27. Inter-individual differences in the IgG glycome; What did we learn from analyzing 40,000 IgG glycomes

Gordan Lauc 1; 1University of Zagreb, Croatia

glauc@pharma.hr

Immunoglobulin G is one of the mostly studied glycoproteins and an excellent example of importance of modulation of protein function by alternative glycosylation. Glycan structures attached to the IgG Fc fragment are essential for function of the immune system and among other things, they significantly contribute to the decision whether a pro-inflammatory or an anti-inflammatory immune response will be activated. Changes in IgG glycosylation were found to associate with a number of different diseases, as well as chronological and biological ages of an individual, but the genetic and environmental factors that underlie these changes are not well defined. An intriguing element of these associations is the fact that structural details of glycans attached to IgG are of great physiological significance. Changes in IgG glycosylation affect many aspects of the immune system and thus may actually contribute to the development and/or progression of diseases and/or age-associated degeneration. Individual variability in IgG glycome composition is very large, but glycosylation of an individual protein seems to be under strong genetic influence, with heritability up to 80%, indicating strong genetic component in the regulation of IgG glycosylation and its changes.

Session 5. Asian Community of Glycoscience and Glycotechnology Guest (ACGG) Session II

Keynote Lectures

28. Carbohydrate-based macromolecular self-assemblies and their immunological functions

Guosong Chen 1; 1Fudan University, China

guosong@fudan.edu.cn

Carbohydrates are the most abundant organic species in the world and also one of most important biological macromolecules with nucleic acids and proteins. The self-assembly of DNA and proteins make a significant contribution to our lives and they have been employed to make functional self-assembled materials. Compared to the development of DNA and proteins, our knowledge and manipulation to the self-assembly of carbohydrates as well as their functionality are quite limited. The major obstacle is the complicated chemical structure of oligosaccharides, i.e. perplexing glycoforms and microhetrogeneity on proteins, which make the research a problematic and long-term task. Under this circumstance, macromolecular self-assembly might provide an alternate insight to this problem. In this talk, I will present: 1) developement of precise protein array with regular shape at μm scale controlled by protein-carbohydrate interaction; 2) construction of polymeric vesicles mimicking glycocalyx, structure, self-assembly and immunological functions; 3) control of macromolecular self-assembly by chemical reactions related to sugars.

Lectures

29. Essential roles of O-GlcNAcylation in B cell activation

Jung-Lin Wu 1 , Pan-Hung Hsu 2 , Yu-Ju Chen 3 , Takashi Angata 4 , Kuo-I Lin 1; 1Academia Sinica, Genomics Research Center, 115, Taipei, Taiwan, 2National Taiwan Ocean University, Department of Life Science and Institute of Bioscience and Biotechnology,Keelung 202, Taiwan, 3Academia Sinica, Institute of Chemistry, 115, Taipei, Taiwan, 4Academia Sinica, Institute of Biological Chemistry, 115, Taipei, Taiwan

kuoilin@gate.sinica.edu.tw

O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) is a post-translational modification that adds a GlcNAc to serine or threonine residue of nuclear and cytosolic proteins. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT) and is removed by O-GlcNAcase (OGA). O-GlcNAcylation regulates many aspects of proteins, including protein stability, localization and activities, and plays important roles in many diseases such as diabetes, neurodegenerative diseases and cancer. The functional role and molecular mode of action of protein O-GlcNAcylation in B cells is not well understood. B cell activation is triggered by the engagement of B cell receptor (BCR) with antigen, thereafter inducing the differentiation of antibody-secreting plasma cells or memory B cells. We showed that protein O-GlcNAcylation accumulates after cross-linking of BCR by anti-IgM in mouse splenic B cells and that inhibition of OGA by a specific inhibitor, thiamet G, promotes anti-IgM-mediated activation of B cells. Comparative phosphoproteomic analyses reveled several O-GlcNAc-dependent phosphoproteins in B cell activation. We further created a mouse line in which Ogt is deleted in a B cell-specific manner to demonstrate that Ogt ablation impairs BCR cross-linking induced cell activation and survival. These mice showed severe defects in the production of antigen-specific antibody following immunization. These results demonstrate that B cells rely on O-GlcNAc to efficiently induce BCR signaling and evoke antibody response.

30. Glyco-chemical biology using fucose analogs

Yasuhiko Kizuka 5 , Naoyuki Taniguchi 5 , Sho Funayama 1 , Miyako Nakano 2 , Kazuki Nakajima 3 , Yoshiki Yamaguchi 4 , Shinobu Kitazume 5 , Shien-Tai Ren 6 , Tsui-Ling Hsu 6 , Chi-Huey Wong 6; 1RIKEN, Disease Glycomics Team, 351-0198, Wako, Japan, 2Osaka University, Department of Disease Glycomics (Seikagaku Corporation), 567-0047, Suita, Japan, 3Hiroshima University, Graduate School of Advanced Sciences of Matter, 739-8530, Higashihiroshima, Japan, 4Fujita Health University, Division of Clinical Research Promotion and Support, 470-1192, Toyoake, Japan, 5RIKEN, Structural Glycobiology Team, 351-0198, Wako, Japan, 6Academia Sinica, Genomics Research Center, 115, Taipei, Taiwan

y.kizuka@riken.jp

Fucosylated glycans are known to be involved in various diseases such as cancer and chronic obstructive pulmonary disease. However, therapeutic application of basic glycobiology knowledge has not progressed well probably due to the lack of easy tools for the detection, imaging and manipulation of glycans. To address this issue, sugar analogs are promising candidates to develop new compounds for glycobiology research.

We recently developed a novel alkynylated fucose called “7-alkynyl-fucose” as a high sensitive probe for fucosylated glycans (Kizuka et al, Cell Chem. Biol. 2016). Although several fucose analogs were developed as a probe, they showed high toxicity or low sensitivity. Our newly developed probe, in combination with click chemistry, detects cellular fucosylated glycans with higher sensitivity and lower toxicity than existing probes. Detailed enzymatic assays revealed that all fucosyltransferases tested utilize 7-alkynyl-fucose more efficiently than other fucose analogs. By MS analysis, we also found that 7-alkynyl-fucose is metabolically converted to GDP-form in cells and that 7-alkynyl-fucose is mainly incorporated into core position of N-glycans, indicating that 7-alkynyl-fucose is a powerful tool to detect and visualize core fucose.

We also discovered that “6-alkynyl-fucose”, which is now widely used as a detection probe, strongly inhibits cellular fucosylation. We showed that addition of 6-alkynyl-fucose almost completely depleted GDP-fucose pool. Moreover, our enzymatic assays revealed that GDP-6-alkynyl-fucose directly inhibits a key enzyme for GDP-fucose synthesis, FX, but not GMDS or fucosyltransferases. Furthermore, the inhibition potency was much higher than an exiting fucosylation inhibitor, 2-fluoro-fucose, and the treatment with 6-alkynyl-fucose was found to halt hepatoma invasion. These results indicate that 6-alkynyl-fucose is a promising tool for both basic glycobiology and therapeutic use.

31. Wisteria floribunda agglutinin, a unique lectin, as a probe for serum biomarker research

Takashi Sato 1 , Atsushi Kuno 1 , Akira Togayachi 1 , Yasunori Chiba 1 , Hiroyuki Kaji 1 , Hisashi Narimatsu 1; 1Glycoscience and Glycotechnology Research Group, Biotechnology Research institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology(AIST), 1-1-1 Umezono, Tsukuba, Ibaraki, JAPAN

takashi-sato@aist.go.jp

The glycan structure of glycoproteins is known to reflect the cell differentiation status. Therefore, glycan structures produced by diseased cells are distinguishable from those produced by nondiseased cells. Proteins can be markers of the tissues that secrete them. Thus, we previously proposed a strategy for identification of novel serum biomarkers using a combination of several glycoscience-based technologies. Using this concept, we first searched for lectins that could be used as probes for discovering altered glycan structures.

We describe the identification of Wisteria floribunda agglutinin (WFA) as a good probe recognizing glycan-altered glycoproteins secreted by diseased cells. We characterized the natural and recombinant WFA by the glycoconjugate microarray and frontal affinity chromatography. Interestingly, WFA could be applied as a probe to several diseases, i.e., liver fibrosis, liver cirrhosis, prostate cancer, ovarian cancer, and IgA nephropathy. WFA shows advantages for application as a serum biomarker because serum contains very few WFA-positive glycoproteins; therefore, WFA signals show low background in serum.

Based on findings to date, WFA-positive serum biomarkers can be measured without prepurification of target glycoproteins and may have applications as serum biomarkers in patients with various diseases.

32. Golgi, O-glycosylation and cancer development

Frederic Bard 1; 1IMCB / A-STAR

fbard@imcb.a-star.edu.sg

It has been known for many years that various cancer cells dramatically up-regulate production of the one sugar glycan called Tn and the related two sugars glycan TF. The Golgi apparatus is athe cell’s glycosylation factory. Yet, how this factory produces different glycans in a disease state such as cancer is still poorly understood. In recent years, we have identified a mechanism regulating the initiation of O-glycosylation and production of Tn through the relocation of Tn producing enzymes (GALNTs) from Golgi to ER. Our recent results indicate that this GALNTs activation strongly promotes the growth of liver tumors and their capacity to form tumors. I will discuss the mechanisms underlying this tumor promoting activity and illustrate how cell surface proteins become hyper-glycosylated.

Session 6. Glycotechnology II

Keynote Lectures

33. Plant based glycan engineering for the production of therapeutic proteins

Herta Steinkellner 1; 1University of Natural Resources and Life Sciences, Department of Applied Genetics and Cell Biology, Vienna, Austria

herta.steinkellner@boku.ac.at

Plants are increasingly being recognized for the production of recombinant therapeutic proteins and recently it was shown that plants are highly amendable to glycan engineering approaches. Paradoxically, the limited endogenous glycosylation repertoire has turned out to be an advantage for generating homogeneous glycans, in contrast to the large glycome and the resulting glycan heterogeneity in CHO cells that impedes the targeted manipulations. We have developed a plant based expression platform enabling extensive engineering of glycans towards complex targeted structures. The system is based on the use of a glyco-engineered Nicotiana benthamiana line lacking plant specific glycosylation, instead synthesizes human-type complex N-glycans, i.e. GnGn structures (doi: 10.1111/j.1467-7652.2008.00330.x.). This structure, common in all higher eukaryotes, serves as acceptor for further N-glycan diversifications. The plant based approach uses a modular switch of glyco-engineered N. benthamiana mutants in combination with transient expression modules harboring mammalian glycosylation enzymes. Using this approach we were able to produce proteins with predefined glycosylation profiles, up to a complexity level of poly-sialylated structures (>40 sialic acid residues) (doi: 10.1073/pnas.1604371113.). Such unusual polymer formation with its strong negative charge not only changes a protein's functionality but may also be used per se in therapeutic applications (e.g. in inflammatory settings). Harvesting of recombinant proteins one week post DNA construct delivery allows high speed and flexibility (doi: 10.1016/j.copbio.2014.06.008.). This presentation provides an overview of the current front in plant based glycan engineering processes, bottlenecks and challenges, which a special focus on generation of different antibody formats (IgG, IgA, IgE and IgM).

Lectures

34. An efficient and versatile whole-cell biocatalyst based on respiring Escherichia Coli culture: useful for the synthesis of fucosylated oligosaccharides

Rachel Chen 1 , Ningzi Guan, Hyun-Dong Shin; 1Georgia Institute of Technology, USA

rchen@chbe.gatech.edu

Microbial catalysis has recently emerged as one of the most promising approaches in oligosaccharide synthesis. However, despite significant progresses, microbial synthesis still requires much improvement in efficiency and in reduction of process complexity. Additionally, in light of the stunning natural diversity and many varied applications, broadening the range of glycans accessible via microbial synthesis is of paramount importance to many scientific fields and medical, food, diagnostic applications. Major challenges in microbial synthesis include catabolite repression and high cellular energy requirement. Here we demonstrated a new approach to overcome these challenges by directly tapping into the cellular “power house”, the TCA cycle, to sustain biocatalysis. This approach not only circumvents catabolite repression but also eliminates acidic glycolysis byproducts. As such, the whole-cell biocatalysis can be carried out without sophisticated fed-batch feeding, pH control. The system could achieve several grams per liter titer (3-4 g/L) within a 24 hours period in shaker flask cultivation for three targets, 2’-Fucosyllactose and two novel trisaccharides, fucosyl lactulose and fucosyl cellobiose, demonstrating both the efficiency and the versatility of the biocatalyst developed. To the best of our knowledge, this is the first use of respiration for oligosaccharide synthesis and the first description of successful synthesis of fucosyl lactulose and fucosyl cellobiose.

35. Novel LPMO enzymes identification, characterization, structural study and application on polysaccharides degradation

Huiyan Zhang 1 , Xin Zhang 1 , Jiu Ju 1 , Xiaochen Jia 1 , Yong Zhao 1 , Haichuan Zhou 1 , Zuochen Yu 1 , Heng Yin 1; 1Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 ZhongshanRoad, Dalian 116023, China.

yinheng@dicp.ac.cn

Polysaccharides such as chitin, cellulose, alginate are abundant in nature. These polysaccharides could be hydrolyzed to soluble oligosaccharides or monosaccharides for further application. In previous work of my lab, several high-activity polysaccharides degrading enzymes like chitinase, alginate lyase, inulinase were identified and characterized. Some of these enzymes were successful industrial applied for oligosaccharides production.

A new kinds of polysaccharides degradation enzyme LPMO has attracted vast attention due to its unique catalytic mechanisms and clear significance in biomass conversion. Two chitin-active LPMO and 4 cellulose-active LPMO was obtained from Bacillus thuringiensis, Magnaporthe grisea and Myceliophthora thermophile in this work.

Recently, we have determined the crystal structure of BtLPMO10A for the first time. Also, we obtain from the same bacterium another highly conserved LPMO, BtLPMO10B, and compare its biochemical feature with BtLPMO10A. The results indicated that BtLPMO10B is active on α-chitin, β-chitin and colloidal chitin consistent with BtLPMO10A. Deletion of CBM domains of BtLPMO10B did not lose its oxidative ability, however, lowered its binding to amorphous substrates significantly. BtLPMO10A and BtLPMO10B have clear difference in copper coordination environments. ITC analysis shows that BtLPMO10A has higher copper binding affinity than BtLPMO10B. Both BtLPMO10A and BtLPMO10B have synergistic effect with chitinase to degrade chitin. The LPMO and chitinase co-degradation effect could be 10 times higher than chitinase application only.

36. “Neo-Glycoenzymes” produced by utilizing transglycosylation activity of endoglycosidases and synthetic N-glycans and its application to development of glycobiologics for lysosomal storage diseases

Kohji Itoh 4 , So-ichiro Nishioka 4 , Isao Kobayashi 1 , Yuji Matsuzaki 2 , Akira Harazono 3 , Akiko Ishii 3 , Tomo Hidaka 4 , Daisuke Tsuji 4 , Hideki Sezutsu 1; 1Tokushima University, Graduate School of Pharmaceutical Sciences, Department of Medicinal Biotechnology, 770-8505, Tokushima, Japan, 2Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki 305-8602, Japan, 3Tokyo Chemical Industry Co., LTD., 114-0003,Tokyo, Japan, 4National Institute of Health Sciences, Division of Biological Chemistry and Biologicals, 158-8501, Tokyo, Japan

kitoh@tokushima-u.ac.jp

Lysosomal storage diseases (LSDs) caused by genetic defects of lysosomal enzymes are a group of congenital metabolic disorders, which present excessive accumulation of glycoconjugate or lipid substrates and various neurovisceral symptoms.

In recent years, enzyme replacement therapy (ERT) by periodically dosing a recombinant human lysosomal enzyme formulation, which is produced by a cultured mammalian cell line, to the patients are clinically applied. The ERT is based on a series of mechanism consisting of the binding of terminal mannose 6-phosphate (M6P)-containing high mannose-type N–glycans, which are selectively added to lysosomal enzymes, with cell surface M6P receptor (M6PR) as delivery target, its endocytosis and transport into lysosomes.

A novel production method of glycobiologics, which can overcome the problems of high cost or pathogen contamination, etc, is desired. In this study, a neo-glyco IDUA (active human α- iduronidase) was created by transglycosylation technology using a modified endo-glycosidase M (endoM N175Q) and chemically-synthesized terminal M6P-containing high mannose-type N-glycan derivatives as N-glycan donors. N-glycan structures attached to the recombinant human IDUA purified from the silk glands and cocoons of transgenic silkworm overexpressing IDUA in the silk glands (12 mg and 3.6 mg of enzymatically active IDUA proteins purified from 200 pairs of silk glands and 200 cocoons, respectively) as glycan acceptors contained high-mannose, pauci-mannose, and hybrid-type oligosaccharides, although the glycan compositions were different at six N-sequon sites, which could be partly replaced to terminal M6P-carrying synthetic N-glycans by transglycosylation technology.

The neo-glyco IDUA was internalized into cultured fibro-blasts derived from a patient with IDUA deficiency (mucopolysaccharidosis type 1, MPS1) via M6P receptor. The enzymes were further transported into lysosomes, and then the deficient enzyme activity was recovered successfully. Using this technology, the neo-glycoenzyme carrying synthetic terminal M6P-containing N-glycans to recombinant human lysosomal enzymes, which are produced safely by transgenic silkworms in a low cost manner. These neo-glycoenzymes are expected to be applied as the active ingredient of ERTs for LSDs widely.

Session 7. Glycobiology in Animal Model System/Glycoprotein Quality Control and Trafficking

Keynote Lectures

37. Recurrent infection progressively disables host protection against intestinal inflammation

Won Ho Yang 1,2,3 , Douglas M. Heithoff 1,3 , Peter V. Aziz 1,2 , Markus Sperandio 4 , Victor Nizet 5 , Michael J. Mahan 1,3, Jamey D. Marth 1,2,3; 1Center for Nanomedicine, 2SBP Medical Discovery Institute, 3Department of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, Santa Barbara, California 93106, 4Walter Brendel Center for Experimental Medicine, Ludwig-Maximilians-University, Munich, Germany, 5Department of Pediatrics and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093

jmarth@sbpdiscovery.org

Intestinal inflammation is the central pathological feature of colitis and the inflammatory bowel diseases. These syndromes arise primarily from unidentified environmental factors. We have found that recurrent low-titer infections with Salmonella enterica Typhimurium (ST), a major source of human food poisoning, caused a progressive inflammation of the murine intestinal tract, predominantly the colon, which persisted following pathogen clearance and escalated with repeated infections. Recurrent ST infection disabled a host mechanism of protection by accelerating the molecular aging and clearance of nascent intestinal alkaline phosphatase (IAP). Administration of lipopolysaccharide (LPS) endotoxin recapitulated host Neu3 neuraminidase induction and IAP deficiency. A genetic defect in IAP sialylation in ST3Gal6 deficiency was also pathogenic while absence of the Toll-like receptor-4 provided protection. Oral administration of IAP or the marketed neuraminidase inhibitor Zanamivir were similarly therapeutic with reductions in LPS-phosphate. Repeated exposure to an environmental and food-borne pathogen caused intestinal inflammatory disease by disabling a previously unknown constitutive anti-inflammatory mechanism of the host.

38. Analysis of ER glucosyltransferase using chemically synthesized substrates

Yukishige Ito 1; 1RIKEN, Japan

yukito@riken.jp

Structures of glycoproteins are characterized by their complexity and diversity. To clarify their functions, synthetic approaches are considered to be promising. Development of synthetic methodologies useful for efficient and facile preparation of oligosaccharides is a focal issue in carbohydrate chemistry. In light of their structural diversity, practical strategy to facilitate the synthesis of oligosaccharide is expected to be highly valuable. Glycoprotein glycans are known to play numerous biological roles, both intra- and intra-cellularly, through their interaction with various proteins such as lectins, glycosidases, and glycosyltransferases. However, their precise analysis has been hindered by structural heterogeneity of glycoproteins.

Given these circumstances, our effort has been directed to synthesis of glycoprotein glycans and related probes for analysis of their intracellular functions. This talk will summarize our recent efforts to analyze properties of ER glucosyltransferase called UGGT (UDP-Glc: glycoprotein glucosyltransferase) which plays a pivotal role in glycoprotein folding cycle (Calnexin/Calreticulin cycle) in the ER. Our study extensively employed structurally defined substrates including high-mannose-type glycan derivatives and glycoproteins, which are obtained by chemical or chemoenzymatic synthesis, which successfully clarified various that affect reactivity to UGGT (Ito et al., Sem. Cell Dev. Biol., 41, 90-98, 2015). By using a synthetic probe that was able to label UGGT, putative folding-sensing region of this enzyme was proposed (Ohara et al., Biochemistry, 50, 4909-4917, 2015). Furthermore, both isoforms of UGGT (UGGT1 and UGGT2) were revealed to be enzymatically active (Takeda et al., Glycobiology, 24. 344-350, 2014). Data that suggest roles of their domains will also be presented (Takeda et al., Glycobiology, 26, 999-1006, 2016).

Lectures

39. Structural basis of quality control and trafficking of glycoproteins

Koichi Kato 1,2 , Takumi Yamaguchi 2,3 , Saeko Yanaka 1,2 , Tatsuya Suzuki 1,2 , Hirokazu Yagi 2 , and Tadashi Satoh 2; 1Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki 444-8787, Japan, 2Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan, 3School of Materials Science, Japan Advanced Institute of Science and Technology, Nomi 923-1292, Japan

kkatonmr@ims.ac.jp

Glycoproteins in the early secretory pathway are subject to sophisticate quality control, in which their N-linked glycans play key roles as quality tags. In the endoplasmic reticulum (ER), the newly synthesized polypeptides are modified with a triantennary high-mannose-type oligosaccharide embedding ‘glycotopes’ as protein-fate determinants, which are sequentially exposed by a series of glycosidases and thereby recognized by the intracellular lectins operating as ER chaperones, cargo receptors, and degradation mediators.

We provide structural and dynamical views of the N-glycan dependent quality control and trafficking of glycoproteins using X-ray crystallography and NMR spectroscopy in conjunction with other biophysical techniques. We characterized 3D structures of the N-glycan processing enzymes exemplified by glucosidase II and UGGT, which create the monoglucosyl glycoforms capable of interacting with the ER chaperones.

We also develop a paramagnetism-assisted NMR technique combined with computational approach for exploring conformational spaces occupied by the high-mannose-type oligosaccharides carrying the protein-fate determinants. Based on the structural data, we discuss the molecular recognition mechanisms of the dynamic conformational ensembles of the high-mannose-type oligosaccharides by the ER chaperones and the cargo receptors.

40. Glycomic approach to explore the species-specific glyco-biosynthesis for the development of humanized mouse model

Dan Bi Park 1 , Sumin Kim 1 , Jeong Gu Kang 2 , Jeong-Heon Ko 2 , Jong Shin Yoo 3 , Hyun Joo An 1; 1Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea, 2Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea, 3Biomedical Omics Group, Korea Basic Science Institute, Ochang, Chungbuk, Korea

danbi.park87@gmail.com

Mouse models have been a mainstay in biomedical and biopharmaceutical research for decades. Currently, mouse models are mostly used in non-clinical trials for drug’s safety and efficacy. However, mouse model has a different environment in vivo from humans, which results in poor reliability and unexpected immune response in clinical trials applied to the human. Interestingly, essential genes have close to 100% preservation between human and mouse, but the homology of genes related to glycosylation reveals great difference between two species. Glycosylation represents common post-translational modification and plays primary role in many biological processes including cell-cell interaction and modulation of membrane protein function. Therefore, glycosylation monitoring is an important aspect because glycans can strongly affect drug’s stability, immunogenicity, and pharmacokinetics. Herein, we explored the difference of glycosylation between human and mouse on the most representative blood cells and plasma level. Approximately 100 glycans were obtained on blood cells and plasma of human and mouse, respectively and the results showed qualitative as well as quantitative differences between human and mouse. Using common and top 50 N-glycans based on the normalized absolute peak intensity in each sample of human and mouse, we built up the glycome map based on the biosynthetic pathway. We could determine the species-specific glycan structures and enzymes involving in glycosynthesis. These results would be useful as the reference data to design glycan-humanized mouse model by glycoengineering. Glycoproteomic approaches are undergoing to figure out which protein is attached to species-specific glycans. And then CRISPR/Cas9 system will be applied to produce the transgenic mouse. Glycan-humanized mouse model built on the multi-omics data will be a powerful tool in understanding of human biological pathways.

Session 8. Glycobiology in Plant

Keynote Lectures

41. Expression and glycomodification of immunotherapeutic proteins in plant

Kisung Ko 1; 1Therapeutic protein Engineering Laboratory, Department of Medicine, Medical Research Institute, College of Medicine, Chung-Ang University, 84 Heukseok-Ro, Dongjak-Gu, Seoul, Korea

ksko@cau.ac.kr

Therapeutic proteins can prevent or treat wide ranges of diseases such as cancer, and viral or bacterial infections. Plants have advantages including the lack of animal pathogenic contaminants, low cost of production, and ease of agricultural scale-up compared to other currently available systems. However, plants are ideal expression systems for the production of biopharmaceutical proteins, due to capability of the authentic N-glycosylation process. The majority of therapeutic proteins are glycoproteins harboring N-glycans, which are often essential for their stability, folding, and biological activity of glycoproteins. Thus, the effective plant system for recombinant therapeutics requires the appropriate expression machinery with optimal combination of transgene expression regulatory conditions such as control of transcriptional and post transcriptional events. We are interested in developing a plant system to express a large amount of recombinant therapeutic proteins in plant cells using so called cell-reprogramming and glyco-engineering strategies in plants, particularly including glycoprotein subcellular targeting, inhibition of plant specific glycosyltranferases and addition of human specific glycosyltransferases. Currently, we have successfully developed plant expression systems for production of recombinant vaccines and monoclonal antibodies for immunotherapy.

Lectures

42. N-acetylglucosaminyltransferaseII (GnTII) is required for cellular responses to environmental and developmental cues in plants

Kyun Oh Lee 1; 1Gyeongsang National University, Division of Life Science, 52828, Jinju, Korea

leeko@gnu.ac.kr

N-acetylglucosaminyltransferase II (GnTII) is involved in the formation of GlcNAc2Man3XylFucGlcNAc2 structure in plants. Although the biochemical function of GnTII has recently been reported, its physiological roles are poorly understood at present. To investigate physiological significance of GnTII function in plants, Columbia-0 (Col-0), gnt2, 35S:GnTII and hex1,2&3 (a triple mutant lacking the activity of all three N-acetyl-β-D-hexosaminidase genes) were exposed to several stresses and hormones, and their responses were analysed. Prolonged treatment of sodium chloride (NaCl) and tunicamycin (TM) leads to up-regulated expression of GnTII in Col-0. Loss-of-function mutation in GnTII results in impaired growth and increased callose deposition in roots under stress conditions. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis shows that altered N-glycan structures in gnt2 induces unfolded protein response (UPR) under stress conditions. Loss-of-function mutation in GnTII results in accelerated leaf senescence and altered expressions of senescence- and cytokinin-related genes under dark conditions. 6-Benzylaminopurine (BAP) treatment indicates that GnTII function is implicated in the cytokinin recognition. Taken together, our results indicate that addition of the 6-arm GlcNAc residue to plant N-glycans by GnTII is important for cellular responses to environmental and developmental cues.

43. Loss of N-acetylglucosamine phosphate mutase impair protein glycosylation, plant growth and defense response in arabidopsis

Xiaochen Jia 1 , Hongyan Zhang 1 , Xiaoyan Liu 1 , Hongqiang Qin 1 , Mingliang Ye 1 , Heng Yin 1; 1Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

yinheng@dicp.ac.cn

In our previous work, the first plants N-acetylglucosamine phosphate mutase (AGM), the third enzyme in hexosamine biosynthesis pathway has been characterized in Arabidopsis.

In this work, we found that the T-DNA insertional mutants Atagm1 has an obvious reduction in UDP-GlcNAc, results in the impairment of protein glycosylation, including N-glycosylation and O-GlcNAcylation according to western-blot and monosaccharide composition results.

Furthermore, glycomics, glycoproteomics, and proteomics experiments were conducted to validate these results. The overall profiles of N-glycans isolated from WT and Atagm1 leaves were similar, however, the complex-type glycans were reduced sharply in Atagm1. 334 glycoproteins were identified, some glycoproteins were significantly down-regulated in Atagm1, as well as the N-glycan occupancy of some glycoproteins were reduced. 1306 proteins were identified, the significantly up-regulated proteins in Atagm1 are mainly involved in the process which related to transcription, translation, protein biosynthesis and protein folding. All these results further confirmed the impairment of protein glycosylation in Atagm1.

Atagm1 showed temperature-dependent growth defects in seedlings, with lignin and cellulose deposition, these phenotypes further confirmed by proteomics data, the relative proteins which involved in plant growth, metabolism and defense process was significantly down-regulated.

Atagm1 was more susceptible to Pst DC3000 than wide type, suggested the impaired plant defense system in Atagm1. However, chitosan oligosaccharide (COS) was still effectively on Atagm1 disease control. COS pretreatment inhibited Pst DC3000 multiplication in Atagm1 obviously, via highly activated both SA and JA signaling pathway, which was similar to the effect and mechanism of COS on WT plants.

Taken together, AtAGM1 is essential for protein glycosylation, plant growth and defense response in Arabidopsis.

44. Novel features of sugar-binding properties of hevein family chitin-binding proteins

Yoko Itakura 1 , Sachiko Nakamura-Tsuruta 1 , Junko Kominami 1 , Hiroaki Tateno 1 , Jun Hirabayashi 1; 1AIST, Biotechnology Research Institute for Drug Discovery, 305-8568, Tsukuba, Japan

jun-hirabayashi@aist.go.jp

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc-binding domains. Although they have been used as probes for glycotopes expressed on animal cells, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins representing the family, i.e., Datura stramonium agglutinin (DSA), Lycopersicon esculentum (currently renamed Solanum lycopersicum) lectin (LEL; tomato lectin), Phytolacca americana mitogen (PWM; pokeweed mitogen), Solanum tuberosum lectin (STL; potato lectin), Urtica dioica agglutinin (UDA), and Triticum vulgaris agglutinin (WGA; wheat germ agglutinin), by quantitative frontal affinity chromatography (FAC). As a result, it was found that their sugar-binding properties significantly diverged, and some novel features became evident for them: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied significantly among them. These observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations using lectin microarray, an advanced method for glycan profiling.Y. Itakura, S. Nakamura-Tsuruta, J.Kominami, H. Tateno, and J. Hirabayashi (2017) Int J Mol Sci, in press

Session 9. Glycolipid

Keynote Lectures

45. New paradigm for chronic inflammation mediated by ganglioside molecular species

Jin-ichi Inokuchi 1 , Hirotaka Kanoh; 1Tohoku Medical and Pharmaceutical University, Japan

jin@tohoku-mpu.ac.jp

The presence of a large number of molecules of gangliosides and cell and tissue-specific regulation of their expression are remarkable characteristics but we do not understand the molecular mechanisms controlling their expression and the biological functions achieved by these characteristic ganglioside molecules. Normal human sera contain total gangliosides at the concentration of 15 μg/mL and 90% of the total gangliosides are GM3. We found that GM3 functions as a physiological regulatory factor of the balance between homeostatic and pathological states in adipocytes by modulating insulin signaling (1) and GM3 concentration in human sera increases in individuals with the metabolic syndrome (2). Further analysis using LC-MS/MS reveals that these individual sera increase in GM3 containing very long acyl chains with or without α-hydroxy modification, and decrease in GM3 carrying long acyl chains (2). Inflammatory cytokines, hypoxic conditions, and ER stress produced in the state of diabetes mellitus increase the biosynthesis of GM3 in the adipocytes, and GM3 synthase KO mice suppress inflammatory reactions when they are fed with high fat diets (1). Based on these findings, we focused molecular mechanisms involved in these responses. We have found a surprising evidence that GM3 containing very long acyl chains with or without α-hydroxy modification (C24:0 and αOH-C24:0) induces the secretion of inflammatory cytokines when monocyte or macrophages are stimulated by TLR4 ligand, LPS or HMGB1, while GM3 containing long acyl chains and an unsaturated acyl chain (C16:0, 18:0 and 24:1) suppresses the secretion of inflammatory cytokines. These effects are triggered by inducing the multimer formation of TLR4-LRR/MD2 complex and the up-regulating of signal transduction (submitted). Collectively, we propose a novel inflammation loop triggered by GM3 molecular species.

1. Nagafuku M et al., (2015) Control of homeostatic and pathogenic balance in adipose tissue by ganglioside GM3. Glycobiology 25:303-318.

2. Veillon L et al., (2015) Identification of Ganglioside GM3 Molecular Species in Human Serum Associated with Risk Factors of Metabolic Syndrome. PLoS ONE DOI:10.1371/journal.pone.0129645

46. Neutral glycolipids are key players in immunological functions of human phagocytes

Kazuhisa Iwabuchi 1,2; 1Juntendo University, Infection Control Nursing, Graduate School of Health Care and Nursing,279-0023, Urayasu, Japan, 2Juntendo University, Institute for Environmental and Gender-Specific Medicine, Graduate School of Medicine, 279-0021, Urayasu, Japan.

iwabuchi@juntendo.ac.jp

Professional phagocytes, such as neutrophils, are essential components of the innate immune system. Several studies have indicated this system depends on specific interactions of molecules, which are expressed on cell surfaces of host and invading microorganisms. Phagocytes express several specific receptors and adhesion molecules (pattern recognition receptors; PRRs) on their surface, including Toll-like receptors and αMβ2 integrin that recognize glycoconjugates (pathogen-associated molecular patterns; PAMPs) of microorganisms. The microorganisms are engulfed by phagocytes when sensed by PRRs under nonopsonized conditions. The engulfed microorganisms are trapped within intracellular vacuolar compartments called phagosomes which subsequently fuse with lysosomes to kill and digest the microorganisms.

Here I would like to introduce the organization and functions of neutral glycolipids of human neutrophils. Lactosylceramide (LacCer) forms lipid rafts coupled with Src family kinase Lyn on plasma membranes of neutrophils. LacCer mediates neutrophil chemotaxis, phagocytosis and superoxide generation. Pathogenic mycobacteria, such as Mycobacterium tuberculosis, have been shown to avoid fusion with lysosomes, resulting in their escape from killing by phagocytes. Neutrophils phagocytose mycobacteria via the carbohydrate-carbohydrate interactions between LacCer-enriched lipid rafts and lipoarabinomannan (LAM) expressing on mycobacteria. Importantly, pathogenic mycobacteria utilize LacCer-enriched microdomains to arrest phagolysosome formation. As in the case of LacCer, phosphatidylglucoside (PtdGlc) is expressed on neutrophils. PtdGlc located in the Fas-enriched domains, which are different from LacCer-enriched domains, initiate Fas-dependent neutrophil apoptosis. These observations suggest that LacCer and PtdGlc form different types of functional microdomains on plasma membranes of neutrophils, and mediate different functions in different manners.

Lectures

47. Immune recognition of self and non-self glycolipids via C-type lectin receptors

Sho Yamasaki1; 1Osaka University, Japan

kyamamoto@biken.osaka-u.ac.jp

C-type lectin receptors (CLRs) comprise a family of proteins that share a common structural motif and are involved in various immune responses, and identified as pattern recognition receptors (PRRs) following TLRs, NLRs and RLRs. We found that Mincle (Macrophage-inducible C-type lectin) is an FcRγ-coupled activating receptor that recognizes dead cells cells and induces damage-induced inflammation. In addition, Mincle recognizes mycobacterial glycolipid, trehalose dimycolate, to promote protective immunity. Related CLRs, such as MCL, Dectin-2 and DCAR, also recognize characteristic mycobacterial glycolipids and mediates their adjuvanticity. Furthermore, we recently found that an intracellular metabolite, β-glycosylceramide, released from damaged cells activates myeloid cells via Mincle. These results suggest that CLRs may act as sensors for damaged-self (DAMPs) and non-self pathogens (PAMPs) to maintain immune homeostasis. In this symposium, the physiological advantage and potential risk of the immune responses triggered by CLRs will be discussed.

48. LC-MS of glycosphingolipids

Akemi Suzuki 1 , Masakazu Nagafuku 1 , Takahiro Nitta 1 , Kei-ichiro Inamori 1 , Jin-ichi Inokuchi 1; 1Tohoku Medical and Pharmaceutical University, Institute of Molecular Biomembrane and Glycobiology, 981-8558, japan

akmszk@gaea.ocn.ne.jp

Glycosphingolipids (GSLs) are composed of a glycan chain and a ceramide. Ceramides are able to anchor GSLs into the outer leaflet of biomembranes by hydrophobic interaction and hydrogen bonding with other membrane lipids and proteins, and play a critical role for microdomain formation and functions. Recent advances of LC-MS provide an excellent analytical tool for structural characterization of GSLs including their ceramide structures, highly sensitive detection, and possibly quantitation. Structural characterization of GSLs in crude lipid mixtures by LC-MS becomes successful using the same samples as those for TLC and almost same sample amount for one lane of TLC analysis with a chemical detection. Therefore, a combination of TLC and LC-MS is an excellent protocol for routine characterization of GSLs. In this presentation, several typical examples of such ganglioside analysis we have done are presented. Mouse CD8+ T cells are characterized by containing more extended GM1b (Galb3GalNAcb4(NeuNGca3)Galb3GalNAcb4Galb4GlcbCer) as one of major gangliosides than CD4+ T cells. Mouse spleen shows a complex of ganglioside profile due to the presence of gangliosides containing NeuNAc as well. Brain of St3gal5 null mouse contains GM1b(d18:1-18:0 and d20:1-18:0), GD1α (d18:1-18:0 and d20:1-18:0), and GD1c(d18:1-18:0 and d20:1-18:0) as the major gangliosides. GM3 is the major ganglioside in human sera and is composed of a number of molecular species such as GM3(d18:1-16:0, 18:0, 20:0, 22:0, 23:0, 24:1, h24:1 and h24:0). As the next step of investigation, we need to look at the relation of these gangliosides with functions under physiological and pathological conditions. The drawback of this protocol is the difficulty of quantitation. To clear this problem, we need to collaborate with chemists and to chemically synthesize stable-isotope labeled GSLs used as internal standards.

49. Lipid-driven membrane organization and signaling in myelin repair

Sara Grassi 2 , Simona Prioni 2 , Yana Zorina 1 , Livia Cabitta 2 , Sandro Sonnino 2 , Alessandro Prinetti 2; 1University of Milano, 2Acorda Therapeutics, Inc.

alessandro.prinetti@unimi.it

In the central nervous system (CNS), myelin development and functional maintenance require tightly regulated metabolism of galactosphingolipids, galactosylceramide and sulfatide (3-O-sulfogalactosylceramide). In particular, sulfatide plays a role as a physiological gatekeeper, controlling the timely formation of mature, myelin-forming oligodendrocytes (OL) at the appropriate stage of CNS development. In addition, in mature myelin, sulfatide drives the lateral organization of myelin membrane, thus affecting the sorting, lateral assembly, membrane dynamics and also the function of specific myelin proteins in different substructures of the myelin sheath. Remarkably, sulfatide-driven lateral organization of specific subdomains seems to be altered in pathologies, including multiple sclerosis, the most common CNS demyelinating disease.

In this study, we demonstrated that the recombinant human antibody rHIgM22, which is able to bind to myelin and the OL cell surface and effectively promotes myelin repair in mouse models of multiple sclerosis, specifically binds to sulfatide in vitro, suggesting that sulfatide-driven OL membrane organization could be the target of rHIgM22 effect. On the other hand, we demonstrated that rHIgM22 is able to recognize a lipid (whose structure is under determination) expressed in glial cells other than OL, in particular in astrocytes and microglia. Treatment with rHIgM22 is able to inhibit the activity of acidic sphingomyelinase in mixed glial cultures, resulting in the stimulation of cell proliferation. Thus, the myelin-repairing effect of rHIgM22 might be mediated by multiple alterations in sphingolipid metabolism and sphingolipid-driven membrane organization in several different glial cell populations, whose cooperative action is essential for the functional recovery of myelin after lesion.

50. Lysoglycosphingolipids induce neuronal cell death via PI3K/Akt signaling

Ryosuke Watanabe 1 , Daisuke Tsuji 3 , Hiroki Tanaka 3 , Michael Shintaro Uno 3 , Nozomu Okino 2 , Makoto Ito 2 , Kohji Itoh 3 ;1Tokushima University, Department of Medicinal Biotechnology, Institute for Medicinal Research, Faculty of Pharmaceutical Sciences, 7708505, Japan, 2Tokushima University, Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate School of Pharmaceutical Sciences, 7708505, Japan, 3Kyushu University, The Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, 8128581, Japan

kitoh@tokushima-u.ac.jp

Sphingolipidoses are inherited metabolic disorders caused by the deficiencies of lysosomal hydrolases involving sphingolipids degradation. Although these diseases show severe dysfunction in the central nervous system due to neuronal cell death, the pathophysiological mechanism is little known in detail, and the fundamental therapy has not been established. We studied the pathophysiological mechanism of GM2 gangliosidosis, one of the sphingolipidoses, using neurons induced from the iPS cell line derived from a patient with Tay-Sachs disease, a β-hexosaminidase A (HexA) deficiency. As a result, it was found that PI3K/Akt signaling was down-regulated in the neuronal cells with the GM2 gangliosidosis. It is also known that not only glycosphingolipids (GSLs) but also their lyso-forms, lysoglycosphingolipids (lysoGSLs) lacking the fatty acid moiety of GSLs accumulate in tissues of the patients with sphingolipidoses. To study the contribution of lysoGSLs to pathogenesis of sphingolipidoses, we also compared the effects of lysoGSLs with those of GSLs on survival and signaling in cultured human neuroblastoma cell line (SH-SY5Y) as neuronal cells.

When SH-SY5Y cells were treated with GM1 and GM2 as GSLs as well as lysoGM1 and lysoGM2 as lysoGSLs, the GSLs did not influence cell viability, whereas lysoGSLs decreased cell viability. In addition, lysoGSLs reduced the intracellular level of phosphorylated Akt. Cytotoxicity of lysoGSLs was found to be attenuated by simultaneous treatment with FTY720, a PI3K/Akt signal activator.

These results suggest that neuronal cell death in sphingolipidoses should be caused by lysoGSLs down-regulating PI3K/Akt signaling. Furthermore, we believe that FTY720 can be a therapeutic agent for sphingolipidoses.

The neuropathogenic mechanism of sphingolipidoses due to lysoGSLs via signaling abnormality will be further elucidated

Session 10. Proteoglycan and Sialic Acid

Keynote Lectures

51. Proteoglycan neofunctions: regulation of inflammation & beyond

Liliana Schaefer 1; 1Pharmazentrum Frankfurt/ZAFES, Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Goethe-Universität Frankfurt am Main, Frankfurt am Main, Germany

schaefer@med.uni-frankfurt.de

Inflammatory processes in the body can be triggered not only in response to external signals, but also in response to the release of endogenous molecules called damage-associated molecular patterns. One such molecule, the small leucine-rich proteoglycan biglycan, is ubiquitously distributed in the body and can be proteolytically released from the extracellular matrix following tissue injury. In its soluble form, biglycan acts as a ligand of Toll-like receptors (TLR)-2 and -4, leading to the activation of NF-κB and subsequently the transcription and secretion of several pro-inflammatory cytokines and chemokines in macrophages.

In continuation of this work, we generated a novel transgenic mouse model, in which biglycan was constitutively overexpressed and secreted by hepatocytes (BGNTg), thereby providing a constant source of biglycan released into the blood stream. Secondary polycythemia, a disease characterized by a selective increase in circulating mature erythrocytes, is caused by enhanced erythropoietin (Epo) concentrations triggered by hypoxia-inducible factor-2α (HIF-2α). While mechanisms of hypoxia-dependent stabilization of HIF-2α protein are well established, data regarding oxygen-independent regulation of HIF-2α are sparse. We discovered that although the BGNTg mice were apparently normal, they harbored an increase in mature circulating erythrocytes. In addition to erythrocytosis, the BGNTg mice showed elevated hemoglobin concentrations, hematocrit values and enhanced total iron binding capacity, revealing a clinical picture of polycythemia. In BGNTg mice markedly enhanced Epo mRNA expression was observed in the liver and kidney, while elevated Epo protein levels were found in liver, kidney and blood.

Mechanistically, we showed that the transgenic animals had an abundance of HIF-2α protein in the liver and kidney. Finally, by transiently overexpressing circulating biglycan in mice deficient in various Toll-like receptors (TLRs), we determined that this novel function of biglycan to promote Epo synthesis was specifically mediated by a selective interaction with TLR2. Thus, we discovered a novel biological pathway of soluble biglycan inducing HIF-2α protein stabilization and Epo production presumably in an oxygen-independent manner, ultimately giving rise to secondary polycythemia.

52. Role of sialic acids and sialylation changes in regulating cell interactions and signaling

Philippe Delannoy 1; 1Structural and Functional Glycobiology Unit, UMR CNRS 8576, University of Lille, Villeneuve d

philippe.delannoy@univ-lille1.fr

Sialylation is one of the most important modifications of proteins and lipids, and cell surface sialoglycoconjugates are thought to play important roles in a variety of biological functions including cell-cell and cell-substrate interactions, bacterial adhesion, cell immunogenicity and cell signaling. Alterations of sialylation are observed in number of diseases such as cancer and inflammation. In that context, pro-inflammatory cytokines have been shown to modulate cell surface sialylation by regulating the expression of sialyltransferases involved in the biosynthesis of carbohydrate chains. The sialylation changes induced by pro-inflammatory cytokines and their consequences on cell interactions and signaling were clearly demonstrated in several types of cancer and in cystic fibrosis, showing that these changes in cell surface sialylation could also regulate cell signaling and contribute to the disease progression.

53. New structural insight of proteoglycans related to melanoma cell migration and metastasis

Ji-Hye Yun 1 , Youngsil Choi 2 , Bohee Jang 2 , John R. Couchman 3 , Eok-Soo Oh 2 , Weontae Lee 1; 1Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Korea, 2Department of Life Sciences, Division of Life and Pharmaceutical Sciences and the Research Center for Cellular Homeostasis, Ewha Womans University, Korea, 3Department of Biomedical Sciences, University of Copenhagen, Biocenter, 2200 Copenhagen, Denmark

wtlee@yonsei.ac.kr

Syndecans are type I transmembrane heparan sulfate proteoglycans found on most eukaryotic cell surfaces, which comprising a large extracellular domain, a single transmembrane domain, and a short cytoplasmic domain. The extracellular domain is heavily glycosylated with heparan sulfate chains and cytoplasmic domain functionally interacts with numerous soluble ligands/proteins for its optimum function. The syndecan cytoplasmic domain forms a compact intertwined dimer with a symmetrical clamp shape and two antiparallel strands are twisted with a cavity. NMR data have determined the detailed inter-molecular interactions between transmembrane domains of syndecans in solution. Since syndecans lack any intrinsic signaling capacity, the interactions of their cytoplasmic domains with various adaptor proteins can play essential regulatory roles. These adaptor proteins include the postsynaptic density protein, disc large, and the zonula occludens (PDZ) domain-containing scaffold protein, syntenin-1, which can link the cytoplasmic domain of certain syndecans to the cytoskeleton. Syndecan/syntenin-1 complex shows an unusual symmetrical pair of dimers anchored by a syndecan-4 dimer, inhibiting the functions of syndecan-4. We have shown that multi-merization of both syndecan and syntenin-1 is crucial for the negative regulation of proteglycan signaling. I will also discuss structure and dynamics of homo- and hetero-oligomerization of syndecan mediated by transmembrane domains in solution, lighting molecular insight into the cancer signaling mechanisms by syndecan oligomerization. Our findings propose that three-dimensional structure of syndecan proteoglycan mainly governs its cellular function such as cytoskeletal organization and cell migration.

Lectures

54. Chemical synthesis of higher-carbon sugars

Junchang Wang 1 , Jiazhe Wang 1 , Wenjing Fan 1 , Xuemeng Mi 1 , Yan Chen 1 , You Yang 1; 1East China University of Science and Technology, Department of Pharmaceutical Sciences, 200237, Shanghai, China

yangyou@ecust.edu.cn

Higher-carbon sugars are a large family of cell surface carbohydrates that mediate a series of biological processes such as cell growth, cell differentiation, adhesion, cell signaling, pathogen recognition, viral infection, cancer growth and metastasis. Chemical synthesis of pure and well-defined higher-carbon sugars is required for understanding the structure and function of this unique type of sugars. Thus, a cascade aldol/hemiketalization reaction is described for the construction of D-manno-heptulose. Green synthesis of chloro(organophosphine) gold(I) complexes based on the employment of 4,4’-dihydroxydiphenyl sulfide (DHDPS) as reductant enables the preparation of antiarthritic drug auranofin. Under the promotion of the gold(I) catalyst prepared by the new method, glycosylation of Kdo ortho-hexynylbenzoate with alcohols leads to β-Kdo glycosides. Also, a rapid and efficient conversion of sialyl thioglycosides to sialyl esters is discussed.

Session 11. Society for Glycobiology (SFG) Guest Session I

Keynote Lectures

55. Glycoprotein substrate recognition in mammalian polysialylation: molecular mechanism and inhibition

Karen Colley 1 , Shalu Mendiratta 1 , Deidre Foley 1 , Matthew Thompson 1 , Joseph Zapater 1 , Gaurang Bhide 1; 1University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics, 60607, Chicago, Illinois USA

karenc@uic.edu

Polysialic acid (polySia) is a unique oncofetal glycopolymer that is added to the glycans of a select group of mammalian glycoproteins by two Golgi localized polysialyltransferases (polySTs), ST8Sia-II and ST8Sia-IV. Due to its negative charge and large size, polySia blocks cell-cell adhesion, modulates the signaling of its carriers, and serves as a reservoir for key biological ligands like growth factors and neurotransmitters. PolySia is crucial for the proper development of the nervous system, plays roles in tissue regeneration and immune cell function, and is highly expressed in several types of cancer where it promotes migration and metastasis. The process of polysialylation is unusual because it is protein specific and requires that the polySTs recognize their substrates through an initial protein-protein interaction that allows the enzymes to dock and modify substrate glycans. Our laboratory has elucidated the mechanism of this recognition event for both the neural cell adhesion molecule (NCAM) and neuropilin-2 (NRP-2). Evaluating the recognition and polysialylation of enzyme and substrate mutants and their direct interactions using ITC and NMR, we have identified specific basic residues in a conserved polyST polybasic region (PBR) that directly interact with acidic residues on the surface of a substrate recognition domain. For NCAM, Asp520, Glu521 and Glu523 on the surface of the first fibronectin type III repeat interact with Arg82 and Arg93 in the PBR of ST8Sia-IV, or the analogous residues in ST8Sia-II, to dock and polysialylate two N-glycans in the adjacent Ig5 domain. For NRP-2, Glu652 and Glu653 on the surface of the meprin-A5 antigen-u tyrosine phosphatase (MAM) domain and Arg82 and Lys99 of ST8Sia-IV PBR are required for NRP-2 recognition and the polysialylation of O-glycans in an adjacent linker region. Understanding the molecular details of this recognition process and the differences in the recognition requirements for each polyST substrate, will allow us to devise strategies to inhibit substrate recognition and polysialylation. This work was supported by NIH RO1 GM101949 (to KJC).

56. The ST6Gal-I sialyltransferase imparts a cancer stem cell phenotype, driving tumor initiation and progression

Susan L Bellis 1; 1University of Alabama at Birmingham, Dept of Cell, Developmental and Integrative Biology, 35294 Birmingham, AL, USA

bellis@uab.edu

Cancer cells upregulate distinct glycosyltransferases and their cognate glycan structures, however the functional role of glycans in regulating tumor cell behavior remains poorly-understood. One prevalent tumor-associated glycosyltransferase is the ST6Gal-I sialyltransferase, which adds α2-6 sialic acids to critical N-glycosylated receptors including select integrins and death receptors. Through this activity, ST6Gal-I acts as a master regulatory molecule conferring an invasive, apoptosis-resistant cell phenotype. Immunohistochemical studies by our group have revealed that ST6Gal-I is strongly expressed in colon, ovarian and pancreatic cancers, whereas levels are negligible in the normal differentiated epithelium of these organs. Moreover, high ST6Gal-I levels are associated with metastasis and reduced survival of ovarian cancer patients. Animal studies employing ST6Gal-I transgenic mice, syngeneic transplant models, and limiting dilution assays have shown that ST6Gal-I promotes tumor initiation and progression. As examples, ST6Gal-I overexpression dramatically increases tumorigenesis and metastasis in a genetic model of pancreatic cancer (K-ras driven) and a chemically-induced colon cancer model (AOM/DSS). One mechanistic clue regarding ST6Gal-I’s pro-tumorigenic function was yielded from analyses of normal human tissues. Unexpectedly, ST6Gal-I expression was found to be highly enriched in normal stem cell compartments, and furthermore, ST6Gal-I was strongly upregulated upon cellular reprogramming into induced pluripotent stem cells. These data prompted our hypothesis that ST6Gal-I drives tumorigenesis by endowing cells with cancer stem cell (CSC)-like characteristics. Our collective studies using colon, pancreatic and ovarian cancer cells with forced ST6Gal-I overexpression or knockdown, or primary ovarian cancer patient cells sorted for high or low ST6Gal-I activity, have confirmed that ST6Gal-I imparts all of the landmark CSC features evaluated to date including CSC marker expression (ALDH1, CD133), tumorspheroid growth, chemoresistance, and resistance to hypoxia and serum-deprivation. Taken together, our results highlight a critical function for ST6Gal-I in conferring a CSC phenotype that fosters tumor progression in multiple cancer types.

57. Significance of α2,6-Sialylation in early development of medaka fish as revealed by reverse genetic approaches

Ken Kitajima 3 , Yan Ge 2 , Di Wu 1 , Katsue Tajima 1 , Emi Maruyama 1 , Hisashi Hashimoto 2 , Masahiko Hibi 2 , Chihiro Sato 3; 1Nagoya University, Bioscience and Biotechnology Center and Graduate School of Bioagricultural Sciences, 4648601, Nagoya, Japan, 2Nagoya University, Bioscience and Biotechnology Center, 4848601, Nagoya, Japan, 3Nagoya University, Graduate School of Bioagricultural Sciences, 4648601, Nagoya, japan

kitajima@agr.nagoya-u.ac.jp

Sialic acid (Sia) commonly exists in vertebrate glycoconjugates, and is involved in various biological phenomena. In mouse, Sia has been shown to critically important for early development, because gene deficiency of the UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE), a key enzyme for de novo synthesis of Sia, causes embryonic lethality at animal level (Schwarzkopf et al., 2002). However, why Sia is required for early development still remains unknown. To answer this question, we carried out the following experiments using medaka fish, Oryzias latipes, as a model vertebrate. (i) Early embryos were subjected to lectin staining with SSA (Siaα2,6-Gal) and MAA (Siaα2,3-Gal), showing that expression of the SAA epitope was prominent in blastodisc, although both epitopes were detected. To gain an insight into the significance of Sia residues, effects of microinjection of SSA and MAA lectins into the perivitelline space of fertilized eggs were examined. SSA-injected embryos were lethal prior to embryo body formation, while MAM-injected embryos normally developed; (ii) ST6Gal I and II, which catalyze the synthesis of Siaα2,6Gal, were cloned from embryos and suppressed in their embryonic expression using the morpholinooligonucleotide method. In the single or double morphants for these genes, embryonic development stopped at the gastrulation stage. It is thus concluded that the expression of Siaα2,6Gal residues synthesized by ST6Gal I and II on the blastodisc is important for gastrulation; (iii) Medaka fish deficient in the ST6Gal I or II gene was generated using the CRISPR-Cas9 method. Both null-mutant fish were lethal in early stages of fry, although severity was slightly different between null-mutants. Lethality was more delayed in the null-mutants than in the morphants, probably due to effects of maternal RNAs. These results suggest that expression of both ST6Gal I and II was necessary for development and survival of medaka fry.

Session 12. Glycopathology: Glycans in Cancer I

Keynote Lectures

58. The role of glycosylation in the modulation of cancer cell aggressiveness

Ana Magalhães 2 , Celso Reis 1 , Stefan Mereiter 2 , Catarina Gomes 2 , Daniela Freitas 2 , Henrique Duarte 2 , Diana Campos 2 , Joana Macedo 2 , Meritxell Balmaña 2 , Joana Gomes 2; 1i3S – Instituto de Investigação e Inovação em Saúde, and IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto; Portugal, 2i3S – Instituto de Investigação e Inovação em Saúde, and IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto. Faculty of Medicine, UP. Institute of Biomedical Sciences of Abel Salazar ICBAS, University of Porto, Portugal

celsor@ipatimup.pt

Alterations of glycosylation are common on the cell surface during malignant cell transformation and are associated with cancer progression and poor prognosis of the patients. These glycosylation modifications have been shown to impact the biology of cancer cells, as well as the tumour microenvironment, the process of metastasis formation and cancer progression [1].

The characterisation of the glycosylation modifications occurring in cancer is of high interest and represents a source of biomarkers for cancer detection, patient stratification and therapeutic intervention. This presentation reports the application of glycomics and glycoproteomics for: (a) the characterisation of the effects of increased expression of terminal sialylated structures in cancer cells, and (b) the evaluation of the activation of tyrosine kinase receptors, MET (HGFR, Hepatocyte growth factor receptor) and RON (MSPR, Macrophage-stimulating protein receptor), in human gastric cancer cells [2,3,4], that lead to the activation of downstream intracellular signalling pathways and induction of cancer cell aggressive phenotypes [3,4]. We finally discuss the application of glycans as cancer biomarkers [5].

References:

1. Pinho S.S. and Reis C.A. Glycosylation in cancer: mechanisms and clinical implications. Nat Rev Cancer 2015, 15, 540-555.

2. Mereiter S. et al. Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer. Biochim Biophys Acta 2016, 1860, 1795-1808.

3. Gomes C., et al. Expression of ST3GAL4 Leads to SLe(x) Expression and Induces c-Met Activation and an Invasive Phenotype in Gastric Carcinoma Cells. PLoS One 2013 8(6), e66737.

4. Magalhães A., Mereiter S., Reis C. Reciprocal Modulation of Terminal Sialylation and Bisecting N-Glycans: A New Axis of Cancer-Cell Glycome Regulation? J Biol Chem 2016, 291, 8308.

5. Campos D, et al. Probing the O-glycoproteome of gastric cancer cell lines for biomarker discovery. Mol Cell Proteomics. 2015, 14(6):1616-29.

Lectures

59. Defect of galactosylation of IgG is a predictor of disorders of the immune system

Shifang Ren 1 , Zejian Zhang 1 , Wenjun Qin 1 , Ruihuan Qin, Jing Han 1 , Jianxin Gu 1; 1Fudan University, School of Basic Medical Sciences, 200032, Shanghai, China

renshifang@fudan.edu.cn

Immunoglobin G (IgG), a highly abundant glycoprotein in serum, is known to mediate a variety of blood immune responses. Serum IgG glycosylation, particularly galactosylaltion, has been found highly associated with some diseases including cancer and autoimmunity disease. To investigate whether the alteration of IgG galactosylatio is a common feature in diseases resulting from disorders of the immune system, the value of serum IgG galactosylation in normal physiology and disease needs systematically evaluated under the same analysis system. In our studies, the quantitative distribution of IgG glycosylation in 12 types of cancers, autoimmune diseases, inflammatory diseases, benign tumors and healthy conditions in large cohorts were assessed employing the same analytical method and evaluation model. The distribution of IgG galactosylation (referred to as Gal-ratio) was measured by calculating the relative intensities of agalactosylated (G0) vs mono- galactosyl (G1) and digalactosyl (G2) N-glycans according to the formula of G0/(G1 + G2 × 2) in each sample. The alteration of serum IgG Gal-ratio in these diseases will be described and compared here. The results indicated that IgG Gal-ratio could be a pan-cancer biomarker as well as be a complementary pre-clinical and clinical marker for immune-system related disease differentiation, diagnosis as well as for the evaluation of disease therapies. Take all the results together, we found that IgG Gal-ratio could be a potential biomarker for readout of immunological status, which would be of great significance for diagnosis, prevention and treatment of immune-related diseases as well as providing new insights in the complex mechanism of the underlying disorders of the immune system.

60. Exploring the glycome of serum haptoglobin using nano LC/MS and LC/MS/MS: aberrant glycosylation in gastric cancer

Sung Hyeon Lee 1 , Jae-Han Kim 2 , Se Hoon Park 3 , Jung Hoe Kim 4 , Hyun Joo An 5; 1GLYCAN Co., Ltd., 13605, Seongnam, Korea, 2Department of Food and Nutrition, Chungnam National University, Daejeon, Korea, 3Division of Hematology-Oncology, Samsung Medical Center, Seoul, Korea,, 4Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea, 5Asia-pacific Glycomics Reference Site, Chungnam National University, Daejeon, Korea

biolee81@glycan.kr

Aberrant glycosylation of protein is a major post-translational modification (PTM) in a wide variety of human diseases, including immune disorders and cancers. In particular, a variety of cancers display changes in glycan structures of serum proteins such as IgG, α-fetoprotein (AFP) and haptoglobin (Hp). Gastric cancer (GC) is one of the most commonly occurring cancers and has the highest overall mortality rate worldwide. Unfortunately, the non-invasive tests with carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19-9) are limited due to their lack of sensitivity and specificity in GC diagnosis. Therefore, many researchers try to find new types of biomarkers with high diagnostic value for gastric cancer. Hp, a major acute-phase glycoprotein, consists of two α- and two β-subunits whose glycosylation level changes in various types of cancer and inflammation. Here, we investigated the indirect (released N-glycan) and direct (intact glycoprotein) glycomic profiles of serum Hp as one of potential candidates for gastric cancer using chip-based nano LC-QTOF MS and MS/MS analysis following targeted antibody-assisted purification. Initially, N-glycans bound on serum Hp were released enzymatically, and then the expression profile of N-glycans was monitored by nano LC Chip Q-TOF system. As a result, we observed clear and clinically significant deviations between gastric cancer patients and controls suggesting the potential glycan biomarkers for GC diagnosis. Based on these N-glycan signatures of serum Hp, we have developed new analytical strategy using intact glycoprotein analysis, and rapidly explored the N-glycan alteration of serum Hp without releasing and collection of glycan. The diagnostic efficacy based on P-values and AUC was evaluated in 88 serum samples (44 healthy controls and 44 patients). As a result, we found several predictive m/z signals with the AUC range 0.81 to 0.93 showed high efficiency diagnostic values as screening markers for GC. Beyond a precision analytical platform, the MS technique that is able to directly measure the aberration of serum Hp with glycan heterogeneity will be introduced as a potential diagnostic platform for GC screening.

61. Changes in the glycosylation of kininogen can be used for the early detection of hepatocellular carcinoma

Anand Mehta 1 , Mengjun Wang 1 , Harmin Herrera 2; 1Medical University of South Carolina, Department of Cell and Molecular Pharmacology, 29425, Charleston, SC, USA, 2Drexel University, Department of Microbiology and Immunology, 19102, Philadelphia, PA, USA

mehtaa@musc.edu

Background: Hepatocellular carcinoma (HCC) has the greatest increase in mortality among all solids tumors in the United States and is associated with over 700,000 deaths worldwide, almost solely related to low rates of early tumor detection. Development of non-invasive biomarkers for early detection of HCC may reduce HCC-related mortality.

Methods: We have developed an algorithm that combines routinely observed clinical values into a single equation that in a study of > 3,000 patients from 5 independent sites, improved detection of HCC as compared to the currently used biomarker, α-feto-protein (AFP), by 4-20%. However, this algorithm had limited benefit in those with AFP <20 ng/mL. To that end, we have developed a secondary algorithm that incorporates a marker, fucosylated kininogen, to improve the detection of HCC, especially in those with AFP <20 ng/mL and early stage disease.

Results: The ability to detect early stage AFP negative (AFP< 20 ng/mL) HCC increased from 0% (AFP alone) to 89% (for the new algorithm). Glycan analysis revealed that kininogen has several glycan modifications that have been associated with HCC, but often not with specific proteins, including increased levels of core and outer-arm fucosylation and increased branching. LC MS/MS glycopeptide analysis identified one N-linked glycan site (N294) that was specifically altered in HCC with 100% accuracy.

Conclusions: An algorithm combining fucosylated kininogen, α fetoprotein, and clinical characteristics is highly accurate for early HCC detection. In addition, site specific analysis of glycan sites allow for greater biomarker accuracy as compared to analysis of whole protein.

62. Structure-activity relationship within fucosylated chondroitin sulfates from several sea cucumber species

Elizaveta Yu. Borodina 1 , Nadezhda E. Ustyuzhanina 1 , Maria I. Bilan 1 , Anatolii I. Usov 1 , Nikolay E. Nifantiev 1; 1N.D. Zelinsky Institute of Organic Chemistry, Leninsky prospect 47, 119991, Moscow, Russia

borodina.lisa@gmail.com

Thrombosis is a common pathology underlying ischemic heart disease, ischemic stroke, and venous thromboembolism, which are the leading causes of death worldwide. Many patients with cancer are in hypercoagulable state. These conditions are required the treatment with anticoagulants such as heparinoids, which cause severe side effects. Therefore the searching for new safe and effective anticoagulants represents a challenging task. Anionic polysaccharides fucosylated chondroitin sulfates (FCS) from body wall of sea cucumbers were shown to possess of anticoagulant and antithrombotic activities. The aim of our work is to assess the effect of FCS on hemostasis and to make their structure-activity relationship clearer.

FCS were isolated from the body walls of sea cucumbers Massinium magnum, Cucumaria japonica, C. frondosa, Eupentacta fraudatrix, Apostichopus japonicus, Actinopyga mauritana [1-5]. These biopolymers were shown to build up of alternating→4)-linked β-D-glucuronic acid and→3)-linked N-acetyl β-D-galactosamine residues in a backbone. Structural variations were determined in positions of fucosyl branches (at O-3 of GlcA or O-6 of GalNAc) and in pattern of sulfation of Fuc (Fuc2,4SS or Fuc3,4SS), GlcA (GlcA3S or GlcA2,3SS) and GalNAc (GalNAc4,6SS or GalNAc4S) residues. Besides, several oligosaccharides related to FCS were synthesized [6,7].

To assess the effect of FCS and related oligosaccharides on fibrin clot formation, the aPTT assays with normal plasma were performed. Influence of the tested compounds on interaction between thrombin or factor Xa and ATIII was measured in the assays with pure proteins. To assess the ability of FCS to inhibit platelets aggregation, experiments with PRP were performed. As a result, the value of the biological effect was shown to depend on structural features of FCS. The most active samples were FCS bearing sulfate groups both at O-4 and O-6 of GalNc and fucosyl branches at O-3 of GlcA. This work was supported by RSF grant 14-13-01325.

1. Carbohydr. Polym. (2017), 167, 20

2. Carbohydr. Polym. (2017), 165, 7

3. Carbohydr. Polym. (2017), 164, 8

4. Carbohydr. Polym. (2016), 153, 399

5. Glycobiology (2016), 26, 449

6. Marine Drugs (2015), 13, 770

7. Carbohydr. Res. (2017), 437, 9

Session 13. Society for Glycobiology (SFG) Guest Session II

Keynote Lectures

63. Manipulating bacterial glycoconjugate pathways for the reduction of infectious diseases

Harald Nothaft 1 , Cory Q. Wenzel 1 , Christine M. Szymanski 2; 1University of Alberta, Department of Biological Sciences, T6G 2E9, Edmonton, Canada, 2University of Georgia, Complex Carbohydrate Research Center, 30602, Athens, USA

cszymans@ualberta.ca

Regardless of whether in 1967 the United States Surgeon General actually uttered the words, “It is time to close the book on infectious diseases, and declare the war against pestilence won.” or whether the statement simply became an urban legend, there was a shift in the following years to reduce efforts in antimicrobial discovery and to increase focus on chronic diseases. We are now battling with emerging and re-emerging infectious diseases, particularly multidrug resistant pathogens, with very few effective weapons remaining in our arsenal. This is resulting in a resurgence of applying old and new approaches toward therapeutic discovery including screening large compound libraries, sampling untouched natural environments for new antibiotics, reviving bacteriophage therapy and developing new methods for glycoconjugate vaccine biosynthesis.

One great advantage scientists have exploited in the fight against infectious agents is the fact that microbes are capable of synthesizing diverse glycoconjugates that provide unique targets for antimicrobial therapy. For example, several highly successful vaccines are currently administered to young children that are comprised of bacterial capsular polysaccharides conjugated to diphtheria toxoid allowing immunoglobulin class-switching and long term memory responses.

Although glycoconjugate vaccines have been used for decades in humans, the associated production costs have excluded their use in livestock, and particularly poultry. Campylobacter jejuni is a common foodborne pathogen that is part of the normal gut microbiota of chickens. C. jejuni infection has also been associated with growth-stunting in infants and with the development of the autoimmune paralysis known as Guillain-Barré syndrome. The first bacterial N-linked protein glycosylation system was described in C. jejuni and subsequently it was demonstrated that the pathway can be functionally transferred into Escherichia coli. These discoveries provide novel opportunities for inexpensive glycoconjugate vaccine development against C. jejuni and other infectious agents creating a new era in bacterial glycoengineering

64. New paradigms in bacterial - host glycointeractions provide new opportunities to treat and prevent disease

Michael Jennings 1; 1Institute for Glycomics, Griffith University, Gold Coast, QLD 4222, Australia.

m.jennings@griffith.edu.au

Glycans are important structures in many host - pathogen interactions. Bacterial lectins such as adhesins and toxins exploit host glycans as targets. From the host perspective, human lectins can recognise bacterial glycans in innate immune processes. The molecular details of many bacterial - host interactions remain to be discovered. Understanding these processes is key for the development of novel strategies for prevention and therapeutics. We have applied glycan array to discover novel interactions between bacterial and human cells. Using the Institute for Glycomics glycan arrays we have discovered novel glycan targets for the archetypal cholesterol dependent cytolysin toxins, such as pneumolysin and streptolysin O. Previously it had was believed that cholesterol rich membrane of host cells were the only receptor of these toxins. We report high affinity binding to glycan structures. For example pneumolysin binds to sialyl Lewis X with a equilibrium dissociation constants (KD) of 18.8nM. Binding to this structure is required for efficient red blood cell lysis by pneumolysin. In further recent studies we have discovered a new role for bacterial surface glycans in binding to host cells via glycan-glycan interactions. Glycan microarrays reveal that lipooligosaccharide/lipopolysaccharide of four different bacterial species can bind directly to host glycan structures. We identified numerous high affinity host-glycan:bacterial-glycan pairs with KDs ranging between 100 nM and 50 μM. Assays with human cells show that glycan:glycan interactions can mediate bacterial adherence to cells, and that this adherence can be competitively inhibited by either host glycans or bacterial glycans. The discovery of large numbers of glycan:glycan interactions suggests that these interactions are important in bacterial infection.

65. Microbial control strategies

Viliam Pavliak 1; 1International Vaccine Institute, Department of Science, 08826, Seoul, Korea

viliam.pavliak@ivi.int

Bacterial, parasitic, and viral infections constitute one of the major health problems worldwide. Many of these infectious agents express saccharide molecules at their surface to enhance their ability to cause disease. Pathogenic bacteria have coats of capsular polysaccharide (CP), which help them evade the immune system by protecting the bacteria from phagocytosis, or uptake of the bacteria by immune cells.

For vaccine development, capsular polysaccharides, who are traditionally viewed as T-independent antigens, are poor immunogens especially in infants and immunocompromised persons. Coupling these to proteins converts them into T-dependent antigens capable of eliciting robust immune responses. The current commercially available conjugate vaccines are produced by chemical linking of a polysaccharide to a protein in vitro. Alternative conjugate vaccines approaches are being developed and tested in clinic.

The introduction of a conjugate vaccine approach to vaccine development has had a rapid and positive effect in preventing infections by a series of main global human pathogens including, Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis. They are a potent tool for the prevention of life-threatening bacterial infectious diseases like meningitis and pneumonia and are among the safest and most efficacious vaccines. Conjugate vaccines targeting other important pathogenic bacteria causing disease in the developed and developing world are currently in clinical development.

Session 14. Glycopathology: Glycans in Cancer II

Keynote Lectures

66. Oncogenesis alters glycan regulation of colon cancer stem cell populations via multiple mechanisms

Hua-bei Guo 1 , Michael Pierce 1; 1Complex Carbohydrate Research Center and University of Georgia Cancer Center, Athens, GA USA

hawkeye@uga.edu

We investigated the O-GlcNAc-mediated epigenetic regulation of human colon cancer stem cells (CCSC). Significant reduction of the CCSC population was observed in the tumor cells after OGT knockdown, whereas tumor cells treated with the O-GlcNAcase inhibitor showed an increased CCSC population, indicating that O-GlcNAc levels regulated the CCSC compartment. When grown in suspension, tumor cells with OGT knockdown showed a reduced ability to form tumorspheres, indicating a reduced self-renewal of CCSC due to reduced levels of O-GlcNAc. ChIP-sequencing experiments using an anti-O-GlcNAc antibody revealed significant chromatin enrichment of O-GlcNAc-modified proteins at the promoter of the transcription factor MYBL1, which was also characterized by the presence of H3K27me3. RNA-sequencing analysis showed an increased expression of MYBL1 in tumor cells with OGT knockdown. Forced overexpression of MYBL1 led to a reduced population of CCSC and tumor growth in vivo, similar to the effects of OGT silencing. Moreover, two CpG islands near the transcription start site of MYBL1 were identified, and O-GlcNAc levels regulated their methylation status. These results strongly argue that O-GlcNAc epigenetically regulates MYBL1, functioning similarly to H3K27me3. The aberrant CCSC compartment observed after modulating O-GlcNAc levels is therefore likely to result from the epigenetic regulation of MYBL1 expression by O-GlcNAc, thereby significantly affecting tumor progression. We have also studied the regulation of CCSC by altered GnT-V levels, which are commonly up-regulated in colon carcinoma. Decreased expression levels of GnT-V down-regulated the population of CCSC, affecting their ability for self-renewal and tumorigenicity in NOD/SCID mice. Nuclear translocation of β-catenin and expression of Wnt target genes were positively associated with expression levels of GnT-V, indicating the regulation of canonical Wnt/β-catenin signaling. FZD-7 receptors expressed N-linked β(1,6) branching, indicating that FZD-7 can be modified by GnT-V. The aberrant Wnt signaling observed after modulating GnT-V levels is likely to result from altered N-linked β(1,6) branching on FZD-7, thereby affecting Wnt signaling, the compartment of CCSC, and tumor progression.

Lectures

67. N-acetylglucosaminyltransferase V (GnT-V) confers the anoikis resistance during cancer metastasis

Kyoung Jin Song 2 , Jeong Gu Kang 2 , Nam-Soon Kim 2 , Yong-Sam Kim 2 , Jeong-Hwa Lee 1 , Jeong Heon Ko 2; 1KRIBB, Genome Editing Research Center, P.O.Box 115, Daejeon Korea, 2The Catholic University of Korea, Department of Biochemistry, College of Medicine, 06591, Seoul, Korea

jhko@kribb.re.kr

Causal roles of aberrant glycosylation have been widely investigated in the development and progression of various cancers. One of the glycosyltransferases responsible for aberrant glycosylation in cancer is N-acetylglucosaminyltransferase V (GnT-V), which catalyzes an addition of β1,6-N-acetylglucosamine (GlcNAc) to the core N-glycan, and many lines of evidence have demonstrated the role of N-acetylglucosaminyltransferase V (GnT-V) in cancer development. Tissue inhibitor of metalloproteinase-1 (TIMP-1) and protein tyrosine phosphatase kappa (PTPk) were suggested to be involved in cancer malignancy upon aberrantly glycosylation by GnT-V.

On the one hand, anoikis is a form of anchorage-dependent apoptosis, and cancer cells adopt anokis-resistance molecular machinery to conduct metastasis. Here, we report that N-acetylglucosaminyltransferase V gene expression confers anoikis resistance during cancer progression. Overexpression of N-acetylglucosaminyltransferase V protected detached cancer cells from apoptotic death, and suppression or knockout of the gene sensitized cancer cells to the apoptotic death. The gene expression also stimulated anchorage-dependent as well as anchorage-independent colony formation of cancer cells following anoikis stress treatments. Importantly, treatment with the lectin from Sambucus sieboldiana significantly sensitized anoikis-induced cancer cell deaths in vitro as well as in vivo. We propose that the lectin alone or an engineered form could offer a new therapeutic treatment option for cancer patients with advanced tumors.

68. Functional crosstalk between platelet attachment and activation via podoplanin and TGF-β3 enhances hematogenous metastasis in HCC

Weicheng Wu 1 , Yuanyuan Ruan 1 , Jianxin Gu 1; 1Key Laboratory of Glycoconjugate Research Ministry of Health, School of Basic Medical Sciences, Fudan University, Shanghai, China

cakarote@163.com

Platelets play critical roles in hematogenous metastasis in various cancers and podoplanin has been implicated in tumor-induced platelet activation and metastasis in several kinds of cancers. However, the role of podoplanin in hepatocellular carcinoma (HCC) hematogenous metastasis and the precise mechanism how platelets involves in podoplanin-associated metastasis are largely unknown. Herein, we analyzed podoplanin expression in human HCC samples and found that positive podoplanin expression was detected in 107/255 (42.0%) HCC cases and significantly correlated with malignancy-associated thrombosis. Low level of podoplanin in HCC cells was sufficient to induce the binding and activation of platelets, thus promoting tumorigenesis and metastasis in vitro and in vivo. Meanwhile, podoplanin-related platelet attachment and activation played different roles in HCC metastasis and growth. Platelet attachment enhanced tumor growth, while platelet activation preferred to elevate metastasis through TGF-β3 secretion from platelets and tumor cells sequentially. Podoplanin-mediated platelet attachment and activation also synergistically induced a functional cross-talk to further drive TGF-β3 expression and metastasis in HCC. The expression of podoplanin and TGF-β3 were also strongly correlated in HCC cases. Conclusions: Tumor-derived podoplanin mediates the functional cross-talk between platelet attachment and platelet activation to promote TGF-β3 expression and hematogenous metastasis in HCC, and suggest that podoplanin-mediated tumor-platelet interaction may build a TGF-β3 abundant niche to trigger the metastasis of more HCC cells.

69. α2,6-Sialylation mediates hepatocellular carcinoma growth in vitro and in vivo by targeting the Wnt/β-catenin pathway

Yujie Zhao, Anwen Wei, Jianing Zhang, Shujing Wang 1; 1Dalian Medical University, China

wangshujing@dmu.edu.cn

Abnormal sialylation due to overexpression of sialyltransferases has been associated with tumorigenesis and tumor progression. Although ST6Gal-I influences cancer persistence and progression by affecting various receptors, the underlying mechanisms and mediators remain largely obscure, especially in hepatocellular carcinoma (HCC). We found that ST6Gal-I expression was markedly upregulated in HCC tissues and cells, high levels being associated with aggressive phenotype and poor prognosis. Furthermore, we examined the roles and mechanisms of ST6Gal-I in HCC tumorigenesis and metastasis in vitro and in vivo. ST6Gal-I overexpression promoted proliferation, migration, and invasion of Huh-7 cells, whereas its knockdown restricted these abilities in MHCC97-H cells. Additionally, in a mouse xenograft model, ST6Gal-I-knockdown MHCC97-H cells formed significantly smaller tumors, implying that ST6Gal-I overexpression can induce HCC cell malignant transformation. Importantly, enhanced HCC tumorigenesis and metastasis by ST6Gal-I may be associated with Wnt/β-catenin signaling promotion, including β-catenin nuclear transition and upregulation of downstream molecules. Together, our results suggest a role for ST6Gal-I in promoting the growth and invasion of HCC cells through the modulation of Wnt/β-catenin signaling molecules, and that ST6Gal-I might be a promising marker for prognosis and therapy of HCC.

Session 15. Glycoimmunology (Immunity & Sugar Chain) I

Keynote Lectures

70. Mycobacterial tuberculosis ManLAM regulates host innate and adaptive immune responses

Xiao-Lian Zhang 1; 1Wuhan University School of Medicine, China

zhangxiaolian@whu.edu.cn

Pathogen-surface glycans have been implicated as immunosuppressive epitopes and might contribute to chronic or latent infection and immune invasion. Mannose-capped lipoarabinomanan (ManLAM) is a lipoglycan serving as a major cell wall component in Mycobacterium tuberculosis (M.tb). Here, we demonstrate a novel ManLAM mediated immune evasion mechanism by inhibitory effects on the dendritic cells (DCs), polarizations of M1 macrophages (innate immune) and Th1 cells (adaptive immune) via CD44 and Mannose Receptor (MR). IL-10-producing B (B10) cells were increased in peripheral blood mononuclear cells (PBMC) from patients with active pulmonary tuberculosis (TB). We also demonstrate a novel mechanism in which M.tb ManLAM induces IL-10-producing B cells, then inhibits CD4+Th1 immunity and negatively regulates anti-TB immunity in vivo. We also provide data supporting that an aptamer specifically against ManLAM with low and no toxicity, holds a potential application as a new potential antimycobacterial agent and tuberculosis vaccine immune adjuvant.

Lectures

71. Anti-α-Gal xeno-antibodies: old myths and new facts

Nicolai Bovin 1 , Daniel Bello-Gil 2 , Rafael Máñez 2; 1Institute of Bioorganic Chemistry RAS, Moscow, Russia, 2Infectious Pathology and Transplantation Division, IDIBELL, Hospitalet de Llobregat, Spain

professorbovin@yandex.ru

Using printed glycan array (400 glycans+200 bacterial polysaccharides) we investigated the natural anti-glycan antibodies in humans and αGal-transferase KO mice. We worked with both native sera and antibodies isolated on Galα1-3Galβ1-4GlcNAc-sepharose affinity adsorbent. As the result, we do not confirm a number of old myths, in particular:

Myth 1. ~2% of immunoglobulins in healthy donors are antibodies against Galα1-3Galβ1-4GlcNAc. In fact, the content of specific anti-Galα1-3Galβ1-4GlcNAc immunoglobulins is at least 10-fold smaller.

Myth 2. All humans have anti-Galα1-3Galβ1-4GlcNAc antibodies. In fact, ~10% of donors have a low or very low level of the antibodies, as, for example, one of authors (NB).

Myth 3. αGal-transferase KO mice have anti-α-Gal antibodies similar to humans. In fact, the specificity of mice antibodies is much lower than in humans. Thus, the KO mice are not an adequate model in the study of antibody-dependent rejection.

Finally, genetically identical mice of the same age, housed in the same cage, unexpectedly demonstrated non-identical profiles of the glycan-binding antibodies.

1. P. Obukhova, R. Rieben, N. Bovin. Normal human serum contains high levels of anti-Galα1-4GlcNAc antibodies. Xenotransplantation, 14, 627-635 (2007).

2. D. Bello-Gil, N. Khasbiullina, N. Shilova. Repertoire of mice natural antibodies: mice vs. humans difference, and otherness of individual animals. Front. Immunol. (submitted). Supported by grants #14-50-00131 of the Russian Science Foundation and #603049 (TRANSLINK) from European Union FP7.

72. N-Glycan of B cell maturation antigen prevents gamma secretase-mediated cleavage

Han-Wen Huang 1 , Chen-Chun Chen 1 , Kuo-I Lin 1 , Chi-Huey Wong 1; 1Genomics Research Center, Academia Sinica, Taipei, Taiwan

chwong@gate.sinica.edu.tw

B cell maturation antigen (BCMA), a member of tumor necrosis factor receptor (TNFR) family, is essential for plasma cell survival and acquired by malignancies and inflammatory accessory cells to gain additional survival signaling. Therefore, specific targeting BCMA by chimeric antigen receptor T cells (CAR-T) and disrupting ligand-receptor binding by antibody or recombinant proteins were considered as potential therapeutic treatments. Recently, glycosylation of BCMA was newly identified by aid of sugar alkynyl probes coupled with mass spectrometry and further proved by glycosidase hydrolysis and glycosite mutation. However, the function of glycan on BCMA is still not addressed well so far. Previous study reported that the terminal sialic acid of glycosylated BCMA regulates surface expression of BCMA and raises a question that is how N-glycan controls surface expression of BCMA. Here, we showed that removing glycan from BCMA by glycosite mutation or glycosidase hydrolysis exhibited decreased surface retention of BCMA and increased soluble BCMA in culture medium. Interestingly, the soluble BCMA collected from culture medium were almost non-glycosylated. Addition of gamma secretase inhibitor (GSI) which prevents BCMA shed from membrane by gamma secretase obviously elevated the surface level of BCMA on glycosidase-treated or glycosite-mutated cells and largely reduced the soluble BCMA. Comparing to the sialidase treatment, the effect of GSI on surface expression and soluble level of BCMA was overwhelming. Besides, we also found that BCMA increased by GSI treatment was almost non-glycosylated form, consequently protecting cell from dexamethasone and TRAIL-induced apoptosis. These results suggest that N-glycan on BCMA regulates surface expression of BCMA to optimize the appropriate cellular response by preventing gamma secretase-mediated cleavage.

73. Harnessing glycosylation-dependent pathways to control joint inflammation

Margaret Harnett 1 , Laura Bouché 2 , Stuart Haslam 2 , Anne Dell 2 , Andrew Filer 3 , Christopher Buckley 3 , Miguel Pineda 1; 1University of Glasgow, Institute of Infection, Immunity and Inflammation, G128TA, Glasgow, UK, 2Imperial College London, Department of Life Sciences, SW7 2AZ, London, UK., 3University of Birmingham, Institute of Inflammation and Ageing Birmingham, B15 2WB, Birmingham, UK.

miguel.pineda@glasgow.ac.uk

Background: Rheumatoid Arthritis (RA) is chronic inflammatory condition of the joint for which current treatment consists primarily of immunosuppressive drugs with substantial side-effects. In healthy joints, synovial fibroblasts (SFs) provide the required stromal support, but are recognized to adopt a pathological role in RA, delivering region-specific signals to infiltrating cells that perpetuate local inflammation and joint destruction. Interventions targeting SFs would improve current 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. Glycomic studies focusing on defining the structures and functional roles of glycans in stromal cells could offer such molecular targets, as glycans and glycan-binding proteins are being increasingly recognized as fundamental regulators of cellular interactions and immunity.

Methods: SFs from naïve mice and mice undergoing Collagen-Induced Arthritis (CIA) were expanded ex vivo to evaluate their glycosylation profile, as characterised by plant lectin reactivity in flow cytometry and immunohistochemistry studies, following their stimulation with pro-inflammatory cytokines. MALDI-TOF profiling of the N- and O-glycans from such SFs allowed us to describe an accurate molecular map of the SF glycome. Importantly, to map SF glycosylation signatures associated with disease progression in RA, we isolated matched SFs and dermal fibroblasts from long duration RA patients at joint replacement surgery, as exemplars of inflammatory and non-inflammatory derived lineages.

Results: Our results suggest that transformation of SFs into pro-inflammatory cells in RA is associated with glycan remodelling in response to pro-inflammatory mediators. Intriguingly, distinct glycosylation signatures are detected between different RA patient samples. 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 personalized medicine.

Session 16. Glyco(proteo)mics analysis I

Keynote Lectures

74. Glycomics and glycoproteomics underpinning glycobiology research

Stuart Haslam 1; 1Imperial College London, United Kingdom

s.haslam@imperial.ac.uk

Glycans, in the form of polysaccharides or glycoconjugates (bound to proteins and lipids), are found on the cell surface of every cell in nature. Glycans engage with receptors to mediate cell-cell recognition and multiple important biological processes. Mass spectrometry (MS), with its ultra-high sensitivity and ability to analyse complex mixtures of glycans, is the most powerful tool currently available for glycan structure analysis. Our laboratory is engaged in numerous world-wide collaborations in which we exploit high sensitivity mass spectrometric methodologies for the structural characterization of glycans found in a diverse range of biological material. This presentation will review MS strategies incorporating MALDI-TOF-TOF MS/MS and nanoLC-ES-MS/MS for defining the glycomes of cells, tissues and purified glycoconjugates as well as establishing glycoprotein site-specific glycosylation. The broad range of our research activities will be illustrated by data from ongoing collaborative projects embracing host pathogen interactions and human disease.

Lectures

75. The analysis of the mannose receptor of breast cancer

Jing Fang 1 , Tao Tao 1 , Haojie Lu 1,2; 1Institutes of Biomedical Sciences and Department of Chemistry, Fudan University, Shanghai, 200032, P. R. China, 2Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032, P. R. China

luhaojie@fudan.edu.cn

As one of the most common malignant tumors in female patients worldwide, breast cancer is recognized as a heterogeneous cancer. The membrane surface markers expression analysis can make breast cancers fall into at least 4 subtypes (LuminalA,LuminalB,HER-2,TNBC).In which Triple-negative breast cancer (TNBC) conventional hormonal or anti-HER2 targeted therapies have no favorable value for TNBC which lacks known common therapeutic targets. Therefore, aggressive clinical behavior and poor prognosis make it urgent to search for appropriate biomarkers for diagnosis. According to the preliminary experimental data, we discover that the mannose receptor was differential expressed in the serum of breast cancer. In this study, we use rigorous biological research methods and mass spectrometry to analysis this glycoprotein. We found that the sMR and membrane type MR not only in normal human serum and breast cancer patients have obvious differences, membrane type MR expressed in four types of breast cancer also has difference. We also discovery of five new glycosylation sites, and the ability to distinguish the four types of breast cancer by the detection of site information.

76. A LC-MS/MS method for the quantitative and qualitative analysis of Aβ glycopeptides – applied on AD and control cerebrospinal fluid samples

Jonas Nilsson 1 , Gunnar Brinkmalm 2 , Lisa Gilborne 1 , Henrik Zetterberg 2 , Kaj Blennow 2 , Anders Wallin 2 , Johan Svensson 2 , Xuefei Huang 3 , Göran Larson 1; 1University of Gothenburg, Department of Clinical Chemistry and Transfusion Medicine, Sweden, 2University of Gothenburg, Department of Psychiatry and Neurochemistry, Sweden, 3Department of Chemistry, Michigan State University, USA

jonas.nilsson@clinchem.gu.se

Amyloid precursor protein (APP) is a transmembrane glycoprotein that undergoes processing along the amyloidogenic and the non-amyloidogenic pathways. In the amyloidogenic pathway, β- and γ-secretase cleave APP into several Aβ peptide variants where the 42-amino acid long Aβ, Aβ1-42, is regarded to have a direct link to Alzheimer’s disease (AD) since it is a major constituent of AD brain plaques. In the non-amyloidogenic pathway, APP is cleaved by α-secretase in the middle portion of the Aβ sequence resulting in shorter peptides, and is thought to protect from amyloid deposition in the brain. In general, the proteolytic destiny and lifetime of proteins are not only governed by the availability and specificity of proteases but also by glycosylations, which may modulate the accessibility of proteolytic sites.

To this end, by the use of liquid chromatography – tandem mass spectrometry (LC-MS/MS) we have previously identified several glycosylation sites of APP from human cerebrospinal fluid (CSF) including one at Tyr-10 of shorter Aβ peptides such as Aβ1-15 (DAEFRHDSGYEVHHQ). This was the first known example of mammalian Tyr glycosylation of an extracellular protein. Recently we showed that LC-MS/MS together with high-energy collision induced dissociation (HCD) was beneficial to use for the discrimination between GalNAc and GlcNAc by analysis of the relative abundances of HexNAc generated oxonium ions. Here we have used this methodology to assign the core glycan structure of Aβ O-glycopeptides to Galβ3GalNAcα1-O-Tyr. Further support was accomplished by the use of a synthetic Galβ3GalNAcα1-O-Tyr substituted Aβ1-15 glycopeptide, which was isotopically labeled and added to the CSF prior to the Aβ immunopurification protocol. We quantitated the absolute concentrations of the Aβ1-15 glycopeptides and peptide in 100 μL-CSF samples from 20+20 AD patients and controls to determine the possible perturbation of Aβ processing due to the Tyr glycosylation.

77. A Glycoproteomic approach reveals that the S-layer glycoprotein of Lactobacillus kefiri is O- and N-glycosylated

Gustavo Cavallero 1 , Mariano Malamud 2 , Adriana Casabuono 1 , María Serradell 2 , Alicia Couto 1; 1Buenos Aires University, Faculty of Sciences, Organic Chemistry Department, 1428, CABA, Argentina,, 2La Plata University, Faculty of Sciences, Department of Biological Sciences , LaPlata, Argentina

acouto@qo.fcen.uba.ar

In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive β-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequence SSASSASSA, substituted on average with eight glucose residues and decorated with galacturonic acid and another O- glycosylated site on peptide 471-476, with a Glc5-8GalA2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI analysis in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures.

78. Isoform-specific mucin type O-glycosylation maintain epithelial homeostasis

Ieva Bagdonaite 1 , Emil M.H. Pallesen 1 , Kirstine Lavrsen 1 , Sergey Y. Vakhrushev 1 , Lars Hansen 1 , Hiren J. Joshi 1 , Eric P. Bennett 2 , Sally Dabelsteen 2 , Hans H. Wandall 1; 1University of Copenhagen, Department of Cellular and Molecular Medicine, 2200, Copenhagen N, Denmark, 2University of Copenhagen, Department of Odontology, 2200, Copenhagen N, Denmark

ieva@sund.ku.dk

Mucin type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Deficiencies of individual GalNAc-Ts cause subtle distinct phenotypes in insect and murine models, and altered expression patterns of GalNAc-Ts have been identified as prognostic cancer markers. Yet, we have little understanding of the cell and tissue specific functions of the individual isoforms. We have recently used precise genetic engineering to target the human C1GalT1 chaperone COSMC to generate stable cells and tissue models with homogenous truncated GalNAc O-glycans, demonstrating a malignant phenotype. We now extend these studies to characterize the impact of site-specific glycosylation on epithelial differentiation and malignant transformation. Using an organotypic model system equipped with cells with and without GalNAc-T1, -T2, or -T3, we identify distinct phenotypes, and selective effects on specific cellular pathways identified via differential transcriptomic, phosphoproteomic, and proteomic analyses. In addition, we find non-redundant O-glycosylation performed by single isoforms using quantitative differential O-glycoproteomics and identify isolated sites on proteins involved in cell adhesion, differentiation, and stress response. Importantly, knock-out of GalNAc-T1, -T2, or -T3 does not lead to malignant transformation, in contrast to homogeneous truncation of O-glycans. While GalNAc-T1-T3 are abundantly expressed in most healthy tissues, we found that GalNAc-T6 was highly upregulated in colon adenocarcinomas but absent in normal-appearing adjacent tissue samples, suggesting that it plays a role in colon carcinogenesis. GalNAc-T6 expression was associated with a cancer-like growth pattern, whereas GalNAc-T6 knockout cells showed more normal differentiation patterns, normalized cell-cell adhesion, and formed crypts in tissue cultures. In addition, several GalNAc-T6 specific targets were identified by O-glycoproteomic analysis. Taken together, these data strongly suggest that individual GalNAc-T isoforms glycosylate a subset of specific targets that play important roles in tissue differentiation, homeostasis and oncogenesis.

Session 17. Glycoimmunology (Immunity & Sugar Chain) II

Lectures

79. Novel approach for the discovery of N-glycan variation on Bechet’s disease: tracing the isomeric distribution and their biological aspects

Nari Seo 1 , Nguyen Thi My Tuyen 2 , Joong Kyong Ahn 3 , Hoon-Suk Cha 4 , Kyoung Heon Kim 5 , Hyunjoo An 1 , Jaehan Kim 2; 1Chungnam National University, GRAST, Deajeon, 03134, Korea, 2Chungnam National University, Department of Food and Nutrition, 04134, Daejeo, Korea, 3Kangbuk Samsung Hospital, Sunkyunkwan University School of Medicine, Department of Internal Medicine, Seoul, Korea, 4Samsung Medical Center School of Medicine, Sungkyunkwan University, Department of Medicine, Seoul, Korea, 5Department of Food Bioscience and Technology, Korea University, Seoul, Korea

jaykim@cnu.ac.kr

Bechet’s disease (BD) is an immune disease which showed a chronic and relapsing systemic vasculitis of unknown etiology. It is characterized by recurrent ulcers and inflammations on oral, genital, uvea area and further vascular, central nervous system (CNS), and gastrointestinal tracks. Bechet’s disease has a wide spectrum of symptoms ranging from benign episodes to more serious complications such as blindness. Although many diagnostic criteria have been developed and revised by experts in the field, diagnosing BD is still complicated and challenging. To discover the diagnostic biomarkers, serum N-glycans from Bechet patient (n=100) and healthy control (n=100) have been compared. Instead of conventional global profiling of serum N-glycome, quantitative ratio between structural isomers were analyzed by UPLC-triple quadruple mass spectrometry. Three acidic glycans with the composition of 5:4:0:1, 5:4:0:2, and 5:4:1:1 (Hex:HexNAc:Fuc:NeuAc) were monitored in MRM mode, then six, two and three isomers were found, respectively. Among eleven bi-antennary glycan isomers, four of them showed high diagnostic efficacy having AUC of ROC curve over 0.98. When the sensitivity was fixed to 90%, corresponding specificities were over 92% to 96%. Another four isomers showed the AUC from 0.84 to 0.94 showing 64%~84% specificity with the sensitivity of 90%. Marker glycans exhibits unique correlations with biological characteristics of cohorts. Two isomers of 5:4:0:1 and one of 5:4:0:2 isomer were able to completely distinguish male and female groups in healthy control cohort. Glycan expression level in female groups were 6 to 10 times higher than those observed in male groups resulting 1.0 AUC in ROC curve. Expression level in patient cohort were 5 to 10-folds higher than those in control group showing complete segregation. Correlation between the glycans were also clear. Groups of isomers with each composition exhibits Pearson’s product over ±0.90 within or between the groups. This study present the novel approach for the disease marker discovery that tracing the isomeric structure of specific N-glycan. As results, the markers with undoubtable quantitative deviation and the correlations reflecting their biological characteristics have been found.

80. Glycoconjugate vaccines based on carbohydrate antigen from actinobacillus pleuropneumoniae as potential tool in the fight against animal pleuropneumonia

Sylwia Przybyl 1 , Joanna Bajzert 2 , Wojciech Jachymek 1 , Tadeusz Stefaniak 2; 1Hirszfeld Institute of Immunology and Experimental Therapy PAS, Department of Immunochemistry, Weigla 12, 53-114 Wroclaw, Poland, 2Wroclaw University of Environmental and Life Sciences, Department of Immunology, Pathophysiology and Veterinary Preventive Medicine, Norwida 31, 50-375 Wroclaw, Poland

sylwia.walesa@iitd.pan.wroc.pl

A. pleuropneumoniae is microaerophilic, Gram-negative bacteria belonging to the Pasteurellaceae family. This pathogenic microorganism is a major cause of porcine pleuropneumonia and fibrinous pleurisy, highly contagious diseases of respiratory tract, affecting predominantly young pigs. Several virulence factors of A. pleuropneumoniae have been found, including carbohydrate antigens such as capsular polysaccharides and lipopolysaccharides. Carbohydrate antigens expressed by A. pleuropneumoniae are similar to antigens expressed by other mucosal pathogens, such as P. multocida. A. pleuropneumoniae infections can be difficult to diagnose, due to the fact of frequent asymptomatic disease. Pleuropneumonia and fibrinous pleurisy can be diagnosed due to cough combined with a high mortality, especially in growing pigs, but can occur in all ages of swine. Animals suffer from a fever, respiratory ailments, swelling lung and tonsils, lung with hemorrhagic areas, presence of blood-stained froth around the nose and mouth. A. pleuropneumoniae is a significant factor for economic losses in the swine. Prophylaxis (such a vaccines) is the best way to avoid the expensive implications of A. pleuropneumoniae infections. Intensive research in the A. pleuropneumoniae vaccination area resulted in the development of several immunogenic preparations from bacterins based on whole cells to more sophisticated ones such as subunit vaccines. Presented studies described the process of developing glycoconjugate vaccines based on carbohydrate core from A. pleuropneumoniae with three different protein subunits. High performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) was performed to optimize the conjugate production process and pre-analyze the molecule. Mass spectrometric data of neoglycoconjugate and proteins were acquired for precise characteristics. Sera (before and after immunization) was studied in enzyme-linked immunosorbent assay (ELISA) to determine the concentration of protective antibodies. Serum bactericidal test was performed on A. pleuropneumoniae, P. multocida and M. haemolytica, which are primary pathogens of respiratory tract of cattle and pigs. Protective efficacy of cell-free-antigen carbohydrate vaccines was studied.

81. Synthetic furanoside-containing glycoconjugates: useful tool for immunological studies

Vadim Krylov 4 , Dmitry Argunov 4 , Dmitry Vinnitskiy 4 , Andrey Matveev 1 , Nina Tikunova 1 , Johannes Huebner 2 , Hans-Christian Siebert 3 , Nikolay Nifantiev 4; 1N.D. Zelinsky Institute of Organic Chemistry Russian Academy of Sciences, Moscow, Russia, 2Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia, 3Hauner Children’s Hospital Ludwigs-Maximilian University, Munich, Germany, 4RI-B-NT Research Institute of Bioinformatics and Nanotechnology, Kiel, Germany

vadimkrilov@yandex.ru

Biomolecules containing hexofuranosyl constituents are essential for the viability and virulence of various pathogenic microorganisms (bacteria, protozoa, fungi) however are absent in mammals. The acute need for new therapeutic approaches stimulates numerous biochemical investigations such as cell-to-pathogen interactions, immune response, cell adhesion, etc, which require well-defined probes and neoglycoconjugates. Unfortunately, the chemical synthesis of furanoside-containing ligands is very laborious which limits their use for detailed glycobiological studies.

Pyranoside-into-furanoside (PIF) rearrangement recently discovered in our laboratory permits facile access to complex furanoside-containing oligosaccharides and neoglycoconjugates thereof which has been demonstrated on derivatives related to polysaccharides from the bacteria Klebsiella pneumoniae, Enterococcus faecalis, and fungus Aspergillus fumigatus. Herein we report the application of the synthetic oligosaccharides for (1) generation of monoclonal and polyclonal antibodies and characterization of their specificity; (2) antigenic characterization of pathogenic cell-wall; (3) characterization of the topology of protein-carbohydrate interactions, (4) structural analysis of native polysaccharides etc.

This work was supported by RSF grant 14-23-00199 (NEN).

[1] V.B. Krylov, D.A. Argunov, D.Z. Vinnitskiy, S.A. Verkhnyatskaya, A.G. Gerbst, N.E. Ustyuzhanina, A.S. Dmitrenok, J. Huebner, O. Holst, H.-C. Siebert, N.E. Nifantiev, Chem. Eur. J., 2014, 20, 16516.

[2] V. B. Krylov, A. G. Gerbst, D. A. Argunov, A. S. Dmitrenok, A. S. Shashkov, Z. Kaczynski, J. Huebner, O. Holst, and N. E. Nifantiev, Chem. Eur. J., 2015, 21, 1749.

[3] D.A. Argunov, V.B. Krylov, N.E. Nifantiev, Org. & Biomol. Chem., 2015, 11, 3255.

[4] D.A. Argunov, V.B. Krylov, N.E. Nifantiev, Org. Lett., 2016, 18, 5504.

[5] R. Zhang, L. Wu, T. Eckert, M. Burg-Roderfeld, M.A. Rojas-Macias, T. Lütteke, V.B. Krylov, D.A. Argunov, A. Datta, P. Markart, A. Günther, B. Norden, R. Schauer, A. Bhunia, M.A. Enani, M. Billeter, A.J. Scheidig, N.E. Nifantiev, H.-C. Siebert, Q. Rev. Biophys., 2017, 50, doi:10.1017/S0033583517000075.

Session 18. Glyco(proteo)mics analysis II

Lectures

82. Proteome-scale identification of O-GlcNAcylated proteins using a free radical generating N-terminal chemical tagging probe

MiRa Bang 1 , ByungKyu Cho 1 , MinJueng Kang 1 , HanSol Jang 1 , JinWoo Jung 1 , SangDuk Yang 1 , Eugene C. Yi 1; 1Seoul National University, Department of Molecular Medicine and Biopharmaceutical Sciences, Seoul, Korea

euyi@snu.ac.kr

The O-linked β-N-acetyl glucosamine (O-GlcNAc) modification, a dynamic post-translational modification (PTM) to serine or threonine residues of nuclear and cytoplasmic proteins, is involved in many different cellular processes including signal transduction and gene regulation. Protein O-GlcNAc modification is known to be associated with several human disease states, such as diabetes, cancer and neurodegenerative disorders. Despite the vital functional roles of protein O-GlcNAcylation in many cellular processes, the area of O-GlcNAc research field has been hampered, mainly due to the lack of techniques for the identification, quantification and site mapping of O-GlcNAc modification in proteins. In this study, we describe the development of an N-terminal chemical probe to induce a radical-driven peptide fragmentation while retained the O-GlcNAc moiety in the conventional CID mass spectrometry analysis. This free-radical initiating N-terminal chemical labeling allowed us to acquire normal tandem MS/MS patterns without a neutral loss of O-GlcNAc moiety, which is suitable for the precise assignment of O-GlcNAc modification sites. More importantly, we found that the chemical probe also allows a proteome-scale analysis of O-GlcNAcylated proteins as the radical-driven peptide fragmentation for O-GlcNAc-modified peptides is CID-fragmentation dependent. Collectively, combined with the radical generating N-terminal chemical tagging and with the conventional CID mass spectrometry analytical platform, we can confidently identify O-GlcNAc-modified proteins with an accurate determination of O-GlcNAc modification sites in a high-throughput manner. This approach can be used in a variety of applications for O-GlcNAc research, which will provide insights into important functions of protein O-GlcNAcylation in many biological processes.

83. Automated identification and quantitation of site-specific glycoproteins with mass spectrometry: glycoProteome analyzer (IQ-GPA)

Jong Shin Yoo 1; 1Biomedical Omics Group, Korea Basic Science Institute, Ochang, Korea

jongshin@kbsi.re.kr

Protein glycosylation, one of the most prevalent posttranslational modifications in proteins, plays important roles in biological systems via various processes, such as adhesion, signaling through cellular recognition, and response to abnormal biological states. However, due to the complexity and heterogeneity of glycoprotein, current glycoprotein analyses focus mainly on either the identification of glycosites or the released glycans. We have developed MS based high-throughput method for intact N- and O-glycopeptides analysis, named IQ-GPA. It can automatically identify and quantify the glycopeptides including glycan compositions and amino acid sequences. The efficiency of IQ-GPA was demonstrated by the analysis of standard α1-acid glycoprotein, hemopexin and benchmark glycoprotein mixture. Additionally, O-GPA allows direct analysis of site-specific O-glycopeptides from glycoprotein mixtures using the O-GPA-DB from Uniprot with estimated FDR ≤ 1%. O-GPA is capable of automatic identification and quantification of O-glycopeptides using HCD and ETD-MS/MS spectra. IQ-GPA was applied for the various analyses of N- and O-glycoproteins present in cell, tissue, and blood from human and mouse. The results show how useful IQ-GPA to automatically identify and quantify N- and O-glycoproteins at the proteome level is.

84. DIA SWATH LC-MS/MS quantification of glycopepides in liver disease

Radoslav Goldman 1 , Miloslav Sanda 1 , Nathan Edwards 1; 1Georgetown University, Department of Oncology, 20057, Washington DC, USA

rg26@georgetown.edu

Data independent (DIA) SWATH analysis of peptides is an established method but DIA quantification of glycopeptides is far less common because of altered CID fragmentation of this complex modification. We describe adjusted fragmentation conditions that enable efficient glycopeptide SWATH workflows as well as LC-MS/MS-MRM or HR-PRM targeted analyses. Glycopeptide fragmentation was optimized for maximum recovery of informative ions using isolated glycoprotein standards. Construction of fragment ion libraries enabled project-specific quantification of the glycopeptides using a glycopeptide SWATH workflow optimized for quantification in complex background. Using this workflow, we quantified simultaneously 125 glycoforms of 25 glycopeptides in human serum without glycopeptide enrichment. Quantification of the fragment ions increases sensitivity approximately 100-fold compared to precursor ion measurements under the optimized CID conditions. Resolution of linkage is facilitated by structure-specific fragments and further assisted by glycosidase digests. Comparison of the glycoforms in healthy controls and cirrhotic patients shows an average 1.5-fold increase in the fucosylation of bi-antennary glycoforms and 3-fold increase in the fucosylation of tri- and tetra- antennary glycoforms. Targeted analysis of specific classes of glycoforms identified additional changes in protein glycosylation associated with the progression of liver disease. In summary, we show that DIA SWATH quantification of glycopeptides is feasible under optimized CID conditions in complex samples. Analyte-specific adjustments maximize the sensitivity and specificity of quantification under clinically relevant conditions.

85. Disturbance of macrophage glycosylation induced by Mycobacterium bovis BCG infection

Clément Delannoy 1 , Elisabeth Elass 1 , Yann Guerardel 1; 1Univ.Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, F 59 000 Lille, France

cl-ment.delannoy@ed.univ-lille1.fr

Macrophages mediate innate immune system through the initiation and regulation of inflammation and contribute to adaptive immunity via antigen processing. Cell surface glycosylation has been widely described to be involved in different physiological or pathological processes, such as host defense, immunological and inflammatory responses. Following pathogens infection, glycosylation machinery can be also disturbed by the cellular environnement. In recent years, several studies have been shown that infection or stimulation of human macrophages with mycobacteria could modulate gene expression related to glycometabolism. These data establish that during mycobacterial infection, glycosylation of macrophages is finely regulated by the cellular environment or by the pathogen. The purpose of our study is to determining the structural modifcations of glycosylation after mycobacterial infection.To accomplish this, macrophages derived from THP-1 and from PBMC were infected with mycobacterium bovis BCG. Once the macrophages are infected, glycans were released by chemical extraction and enzymatic digestion. After purifcation, differents glycans were analyzed by MALDI-TOF spectrometry. To correlate our results, we have also performed FACS analysis using plant lectins. Following M. bovis BCG infection, macrophages present different pattern of N-glycosylation. Analysis of data obtained by mass spectrometry shows antennarisation modification of complex type N-glycans and variations of fucosylation level.

86. MS-Based quantification method for monitoring N-glycosylation process in the cells: SILAC coupled to NanoUPLC-PRM mass spectrometry

Chia-wei Lin 1 , Ilaria Affolter 1 , Markus Aebi 1; 1Institute of Microbiology, D-Biol, ETHZ, 8093, Zurich, Switzerland

linc@micro.biol.ethz.ch

N-glycosylation, involving consecutively enzymatic reactions occurring in the ER and Golgi, is one of the most important post-translational modifications on newly synthesized proteins in the cells. Most of the enzyme components of the biosynthetic machinery, hydrolases and glycosyltransferase, are known. However, detailed analysis of a few N-glycoproteins reveals a glycosylation site-specific microheterogeneity of glycan structures, suggesting a determining role of the covalently attached in the processing pathway.

In order to quantify this site-specific processing, we developed a novel analytical workflow that is composed of mass spectrometry (MS)-based approach and stable isotope labelling of amino acids in cell culture (SILAC) that made it possible to monitor site-specific N-glycan structures of a given N-glycoprotein. With the recent development of fast data acquisition high-resolution and accurate-mass (HR/AM) mass analyzers, targeted MS/MS can be used for reliable quantification of target proteins in complex matrices. Among the different targeted MS/MS approaches, parallel reaction monitoring (PRM) implemented on a Q Exactive quadruple Orbitrap mass spectrometer was demonstrated to have comparable performance as traditional SRM method in sensitivity, reproducibility, accuracy and dynamic range in proteomic applications. Since all fragment ions are generated by higher energy dissociation (HCD) following by Orbitrap measurement, the PRM method can provide rich and reliable structural information for glycopeptide quantification. In this study, we applied the PRM methodology on glycopeptide quantification and examined its sensitivity by using commercial glycoproteins as standards. To address the biosynthetic process of glycosylation, SILAC was introduced into our workflow and applied to monitor in vivo processing of IgG glycans in the time-resolved manner. We demonstrate that the newly developed platform provides the necessary analytical tools to visualize the pathway of N-glycosylation synthesis in a precise way.

87. Glycome of stem cells and application to regenerative medicine

Hiroaki Tateno 1 , Kayo Suzuki 1 , Jun Hirabayashi 1; 1National Institute of Advanced Industrial Science and Technology

h-tateno@aist.go.jp

Human pluripotent stem cells (hPSCs) and human mesenchymal stem cells (hMSCs) have immense potential as cell sources for regenerative medicine due to their differentiation and proliferation abilities. We developed glycan profiling technology called “high-density lectin microarray” containg approx. 100 lectins and performed comprehensive glycome analyses of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs) generated from different tissue types, and hMSCs of different passages isolated from various somatic tissues. To confirm the results obtained by lectin array, we also performd a quantitative glycome analysis targetting both N- and O-glycans. We found that three glycan epitopes, α2-6Sia, α1-2Fuc, and type1 LacNAc (Galβ1-3GlcNAc), are highly expressed on hPSCs. Among 96 lectins used for lectin array, a lectin called rBC2LCN with specificity to Fucα1-2Galβ1-3 motif containing the above two characteristics (α1-2Fuc and type1 LacNAc) was found to specifically bind to hPSCs, but not to non-hPSC cells. rBC2LCN is a practical reagent, since it can be used for live staining of hPSCs. Furthermore, we successfully developed practical technologies to detect and eliminate hPSCs resided in transplanting cells in order to overcome the issues of the tumorigenicity of hPSCs. The glycome of hMSCs related to their differentiation ability was also clarified. We demonstrated that α2-6sialylated N-glycans are marker of the differentiation potential of stem cells such as hMSCs. I will show the basics and applications of the glycome of stem cells.

References:

1. Tateno et al. J Biol Chem. 2011 Jun 10;286(23):20345-53

2. Onuma et al. Biochem Biophys Res Commun. 2013 Feb 15;431(3):524-9

3. Hasehira et al. Mol Cell Proteomics. 2012 Dec;11(12):1913-23

4. Tateno et al. Sci Rep. 2014 Feb 12;4:4069

5. Tateno et al. Stem Cell Reports. 2015 May 12;4(5):811-20

6. Tateno et al. Glycobiology. 2016 Dec;26(12):1328-1337

7. Tateno et al. Regenerative Therapy 2017; 6:1-8

Session 19. Structural Glycobiology

Keynote Lectures

88. Employing rapid throughput and quantitative glycomic and glycoproteomic toward understanding nutrition and cancer

Carlito B. Lebrilla 1; 1University of California, Davis, CA USA

cblebrilla@ucdavis.edu

The new tools for profiling glycans and glycopeptides make it possible to quantitate yielding profiles that can be used to monitor large samples sets. These methods based on advanced liquid chromatography and high mass accuracy mass spectrometry provide high repeatability to render even small changes observable. In this presentation, the development of these methods is described as well as the attempts to ensure that they are accurate while providing the largest number of analytes. For oligosaccharide analysis, as many as 800 oligosaccharides have been observed in a single run. For glycoproteins, thousands of glycopeptides and the corresponding proteins are observed while a subset of proteins and their site-specific glycosylation are quantitated. These tools open new possibilities in biology. They are currently employed toward understanding the biological functions of human milk components. Many of the oligosaccharides are found to prebiotic and serve as food for a very specific microbe that colonizes the infant gut. Peptides and glycopeptides function as antibacterial and immunomodulators, while glycoproteins function as pathogen block. The methods are also employed on cancer cells and tissues. Glycans are observed to change in cancer cells and in blood. Changes in glycosylation point to the enzymes that are affected by transition from normal to cancer and suggest possible therapeutic targets.

Lectures

89. The role of carbohydrate binding proteins in host pathogen interactions

Gita Jancarikova 2 , Eva Fujdiarova 1 , Petra Sykorova 1 , Atul Kumar 2 , Josef Houser 2 , Martina Pokorna 2 , Anne Imberty 3 , Michaela Wimmerova 2; 1Masaryk University, Central European Institute of Technology, 62500, Brno, Czech Republic, 2Masaryk University, Faculty of Science, Kotlarska 2, 61137, Brno, Czech Republic, 3CERMAV-CNRS, 38041 Grenoble cedex 9, France

michaw@chemi.muni.cz

Lectins are ubiquitous carbohydrate-binding proteins, which play a key role in various processes including cell-cell communication and host-pathogen interaction, but also serve as a valuable tool for medicine and life sciences research. Carbohydrate-mediated recognition plays an important role in the ability of pathogenic bacteria to adhere to the surface of the host cell in the first step of their invasion and infectivity. Lectin-carbohydrate interactions are usually characterised by a low affinity for monovalent ligands that is balanced by multivalency resulting in high avidity for complex glycans or cell surfaces.

Contribution is focused on structure-function studies of several examples of microbial lectins participating in the host-pathogen interaction as well as lectins from bacteria that may be involved in nematobacterial complexes highly pathogenic for a broad range of insects.

This work was supported by the Czech Science Foundation (GA13-25401S) and received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 692068.

90. Cooperative ligand binding of a C-type lectin like receptor Dectin-1

Hari Dulal 1 , Yoshiyuki Adachi 2 , Naohito Ohno 2 , Yoshiki Yamaguchi 1; 1RIKEN, Structural Glycobiology Team, 351-0198, Wako, Japan, 2Tokyo University of Pharmacy and Life Science, 192-0392, Hachioji, Japan

yyoshiki@riken.jp

Recent advances in structural analyses of mammalian lectin receptors reveal the atomic details to attain the fine specificities toward diverse endogenous and exogenous glycans. C-type lectin is one of the largest lectin families in mammals and we are extending the knowledge of ligand binding mechanisms (1). Dectin-1 is a mammalian C-type lectin like receptor and engaged in immunological defense against fungal pathogens. Upon binding with fungal β-glucans, the cytoplasmic tail of Dectin-1 receives phosphorylation and this triggers secretion of certain inflammatory cytokines such as tumor-necrosis factor (TNF) and IL-12. However the mechanism is unknown how the β-glucan binding to Dectin-1 induce such intracellular signaling. To address this question, we first investigated the ligand binding of Dectin-1 in detail. A series of NMR study revealed that β-glucan-Dectin-1 interaction is enhanced upon increasing β-glucan chain length (2). Furthermore, a β-glucan ligand laminarin (degree of polymerization ~ 25) strongly binds to Dectin-1 and induces oligomerization of Dectin-1 (3). Dose-response curve revealed that wild-type CTLD cooperatively binds to laminarin with the Hill coefficient of 3.1, while some Dectin-1 mutants abolish or decrease the cooperativity with the Hill coefficient from 0.9 to 2.1. We suggest that the ligand-induced cooperative oligomer formation of Dectin-1 is physiologically relevant in sensing exogenous β-glucan and triggering subsequent intracellular signaling.

References

(1) Nagae and Yamaguchi, Curr. Opin. Struct. Biol. 34, 108-15 (2015)

(2) Hanashima et al, Glycoconj. J. 31, 199-207 (2014)

(3) Dulal et al, Protein Expr. Purif. 123, 97-104 (2016)

91. Structural bioinformatics in glycosciences - from uncovering small motives in large datasets to validation and on-line visualization of extra large biological objects

Jaroslav Koca 1 , David Sehnal 1 , Radka Svobodova Varekova 2 , Lukas Pravda 1 , Zuzana Zufanova 2 , Martin Novacek 2; 1CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic, 2National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic

jkoca@ceitec.cz

Thanks to high throughput methods for molecular structure determination, a huge large amount of data about biomacromolecular structures is currently available. This is also the case of glyco related proteins, e.g., glycoproteins and lectins. For example, a number of protein structures in PDB database that involve a glycan is currently more than 25,000. A need for solving key questions (e.g., discovering binding sites of selected glycans, studying their variability in different organisms, examining changes caused via mutations, investigating protein/carbohydrate recognition and regions important for it, etc) calls for an application of structural bioinformatics approaches.

The structural bioinformatics offers several methodologies, which help to analyze the data and answer important biological questions. It makes it possible to detect regions in biomacromolecules, which are important for a biological function (binding sites, cavities, etc.). It enables validation of the obtained data, recognizing structural problems and allows to remove wrong structures from an analyzed dataset. Afterwards, one can compare the obtained data uncovering their similarities and differences. Last but not least, using tools of structural bioinformatics, one can perform on-line visualization of the results even for extra large biological objects with millions of atoms.

A set of structural bioinformatics tools developed in our lab and focused on the above mentioned tasks will be presented in this paper. We will show PatternQuery1 for biomacromolecule fragment detection, ValidatorDB2 for structure validation, SiteBinder3 for fragment comparison and LiteMol4 for the on-line visualization. Using these tools, an application of structural bioinformatics to several biologically important glycoscience case studies (e.g., analysis of lectin binding sites, involving CH-pi stacking interactions in sugar recognition, saccharide ligand validations etc) will be shown.

References:

1. Sehnal,D., Pravda,L., …, Koča,J. (2015) Nucleic Acids Res., 43, W383–W388

2. Sehnal,D., Svobodová Vařeková,R., … Koča,J. (2015) Nucleic Acids Res., 43, D369–D375

3. Sehnal,D., Svobodová Vařeková,R., … Koča,J. (2012) J. Chem. Inf. Model., 52, 343–359

4. www.litemol.org

92. Dual binding modes of thio-digalactosides with human galectins-1, -3 and -7 as the structural basis of potent and selective inhibitors

Hsien-Ya Lin 1 , Tsai-Chen Yang 1 , Shang-Te Danny Hsu 1 , Chun-Hung Lin 1; 1Academia Sinica, Institute of Biological Chemistry, 11529, Taipei, Taiwan

chunhung@gate.sinica.edu.tw

Human galectins serve as appealing targets in cancer immunotherapy and fibrotic diseases. They represent a family of small soluble lectins characterized by a conserved β-galactoside-binding site within the carbohydrate recognition domain (CRD) and their versatile abilities to form multivalent lattices with membrane-associated glycosylated ligands. The CRD has about 130 amino acid residues, and the resolved 3-D structures indicate that it consists of a highly conserved β-sandwich fold, slightly bent forming a groove on the concave side. Serving as the carbohydrate-binding site, this groove was defined to contain subsites A-E on the basis of the residues in contact with the bound glycan. We investigate the binding interactions of three thio-digalactoside (TDG) derivatives (including TDG, TD139 (3,3’-disubstituted TDG, the recently approved drug for idiopathic pulmonary fibrosis), and TAZTDG (3-substituted TDG)) with human galectins-1, -3 and -7 by X-ray crystallography, isothermal titration calorimetry and 19F NMR spectroscopy. The arene-arginine interactions were confirmed for the first time at subsite E. In addition to the interactions contributed by sugar residues at subsites C and D, the substituent of TAZTDG was found to preferentially bind to subsite B of galectin-3, whereas the same group was in favor of subsite E of galectins-1 and -7. The observed dual binding modes not only demonstrate how the potency can be improved from Kd values of μM to nM, but also offer useful insight to develop selective inhibitors for individual galectins.

93. Role of glycolization in orientation of ectodomain of bitopic membrane proteins

Tomasz Rog 1 , Edouard Mobarak 1 , Karol Kaszuba, 2 , Anirban Polley 2 , Ilpo Vattulainen 1; 1University of Helsinki, Department of Physics, FI-00560, Helsinki, Finland, 2Tampere University of Technology, Department of Physics, Tampere, Finland

tomasz.rog@helsinki.fi

Bitopic membrane proteins with a single transmembrane helix are the largest group of membrane proteins, as approximately half of the integral membrane proteins belong to this group. Typically extracellular domain of bitopic proteins is heavily glycosylated. In this study we performed molecular dynamics simulations of three proteins: epidermal growth factor receptor (EGFR), T-cell surface antigen CD2, and tool like receptor 4 (TLR4) in glycosylated and non-glycosylated form. For the case of EGFR [1] and CD2 [2] extracellular domain tends to collapse at the membrane surface while in the presence of glycosylation upright orientation is adopted. This is due to steric effects and due to shielding patches of positively charge residues at the protein surface which fewer interactions of proteins with lipids. For the case of TLR4 we observed large tilting of the extracellular domain for the case of glycosylated form of the protein in active state (with lipopolysaccharide bound) while upright position was observed when carbohydrates branches were removed. To summarize, glycosylation is one of important determinants of the orientation of the extracellular domains of bitopic membrane proteins.

[1] K. Kaszuba et al. 2015. Role of glycosylation on the structure and dynamics of EGF receptor. Proc. Natl. Acad. Sci. USA 112, 4334–4339.

[2] A. Polley, et al. 2017. Glycosylation and Lipids Working in Concert Direct CD2 Ectodomain Orientation and Presentation. J. Phys. Chem. Letter, 8, 1060−1066.

94. Accelerated structure determination of oligosaccharides with three-dimensional NMR spectroscopy

Xiaoya Qiao 3 , Xiujuan Chi 3 , Jingyu Yan 1 , Xinmiao Liang 1 , Ying Liu 2 , Xuanjun Ai 3; 1Dalian Institute of Chemical Physics, Chinese Academy of Sciences, National Laboratory for Clean Energy, 116023, Dalian, China, 2Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Key Laboratory of Separation Science for Analytical Chemistry, 116023, Dalian, China, 3National Center for AIDS/STD Control and Prevention, Division of Virology & Immunology, 102206, Beijing, China

xai@dicp.ac.cn

The structure information of oligosaccharides is usually a prerequisite for understanding their functions at various biological processes and for developing new drugs. NMR spectroscopy is one of the most used techniques in structure determination of oligosaccharides, either in free forms or in glycoconjugates or complexes. However, restricted to natural resources, controllable biosynthesis and detection sensitivity, two-dimensional (2D) NMR spectra have been commonly applied in the studies of oligosaccharides, resulting in serious signal overlapping and time-consuming data analysis.

A new NMR pulse sequence of the three-dimensional (3D) HSQC-TOCSY, which separates the overlapping 1H-1H through-bond correlation signals with a third 13C dimension, was developed in our lab by some modification of the original version. Comparing to no detectable signal from the original pulse program, a satisfied data could be obtained within 50 hours for a model sample of 2 mM lactose by performing the modified 3D HSQC-TOCSY in the same acquisition condition. The sensitivity-improved new method was then applied to a fucose-containing hexaose from human milk, to elucidate its structures in free form and in complex with Pseudomonas aeruginosa lectin LecB. Combined with the well-established 2D NMR spectroscopy such as 13C-1H HSQC and H2BC and 1H-1H ROESY, atom chemical shifts of the hexaose were undoubtedly assigned, significantly accelerating the process in elucidation of the primary structure with priori knowledge from mass spectroscopy. Furthermore, based on the almost full NMR assignment of the hexaose, its model complex structure with pathogenic LecB was generated by a combination of NMR experiments and docking calculation.

The new 3D NMR spectroscopy provides a promising solution on accelerating structure determination of oligosaccharides. Further methodology development of 13C-edited multidimensional ROESY spectroscopy is in progress.

Session 20. Microbial Glycobiology

Keynote Lectures

95. Rise and fall of mycobacterial cell wall

Yann Guerardel 1; 1Lille1 University, Institute for Structural and Functional Glycobiology, CNRS UMR 8576, Villeneuve, France

yann.guerardel@univ-lille1.fr

The core of the Mycobacterium tuberculosis cell wall consists of a mycolyl-arabinogalactan-peptidoglycan (mAGP) skeleton that is regarded as an attractive drug target. However, despite the impressive progress made over the past decades in understanding mAGP biogenesis, the mechanisms by which the tubercle bacillus adapts its cell wall structure/composition in response to various environmental conditions, especially during infection, remain poorly understood. Indeed, the catabolic pathways of the major cell wall-associated components remain obscure. It is expected that deciphering the molecular basis of cell wall degradation may shed new light on the physiology of M. tuberculosis and the ability of the bacilli to regulate and adapt its envelope architecture in the infected host. In addition, because of their uniqueness, enzymes involved in the catabolic process and mAGP disassembly may offer great promise for future therapeutic intervention against tuberculosis.

In this context, we are investigating whether catabolism of the mycobacterial cell wall as a whole represents a strategy employed by mycobacteria to regulate its cell wall composition during various developmental stages. Here, we will present our most recent work concerning the identification of several mycobacterial glycosidases potentially involved in the remodeling of cell wall. Using complementary biochemical and genetic approaches, we have identified distinct arabinan- and galactan-degrading enzymes that present exquisite specificity toward mycobacterial arabinogalactan. We also demonstrated that some of these enzymes are targeted by anti-tubercular drugs and are required by mycobacteria for optimal growth into the host cells. We believe that these results will provide new insights into the mycobacterial survival toolbox and may ultimately lead to the development of intervention strategies against M. tuberculosis.

Lectures

96. The effect of chitosan oligosaccharide and its antibiotic conjugate inhibits bacteria biofilm

Zhuo Wang 1 , Ruilian Li 1 , Xiafei Zhang 1 , Yuguang Du 1; 1State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China.

wangzhuo@ipe.ac.cn

With the widespread use of antibiotics, pathogens have been becoming increasingly serious because of drug resistance. Recent studies found that the resistance of pathogens to antimicrobial drugs is closely related to the formation of biofilms, and more than 80% of human bacterial infections are associated with biofilm. Biofilm is an existence formation of bacteria for self-protection, and the bacteria in biofilm was considerably more resistant to antibiotics than planktonic cells. Biofilm greatly hamper the effect of antibiotic treatment on bacterial infection, anti-biofilm drugs are required to treat biofilm-related infections. Chitosan, the N-deacetylated product of chitin, was reported with anti-biofilm activity which was related to its electric charge amount. Chitosan oligosaccharide (COS) is the low molecular weight degradation product of chitosan. It was reported that COS has antibacterial effect, and COS with higher degree of deacetylation and lower degree of polymerization had stronger antibacterial activity. However, the study on anti-biofilm activity of COS was rare. Herein we demonstrate the anti-biofilm effect of chitosan oligosaccharide (COS) on Pseudomonas aeruginosa and Staphylococcus aureus. The anti-biofilm activity of COS against bacteria biofilm is related to the structure of COS including the pattern of deacetylation and degree of polymerization. We also tested the anti-biofilm effect of the conjugate of COS and streptomycin, which was synthesized after the reduction of Schiff base formed by COS and streptomycin. Studies suggested that COS- streptomycin conjugate greatly improved the anti-biofilm activity of the antibiotics, through interfering the drug-efflux system as well as manipulating expression level of genes related to biofilm polysaccharide biosynthesis. Thus, this work presents an innovative strategy for treating recalcitrant bacterial infections.

97. Exploring the role of Campylobacter coli GT42 enzymes on lipooligosaccharides biosynthesis

Alejandra Culebro 2 , Warren Wakarchuk 1 , Mirko Rossi 2; 1University of Helsinki, Department of Food Hygiene and Environmental Health, PL66, Helsinki, Finland, 2Ryerson University, Department of Chemistry and Biology, M5B 2K3, Toronto, Canada

aleja.culebro@helsinki.fi

Sialic acid (N-acetylneuraminic acid, Neu5Ac) is the most naturally abundant nonulosonic acid. Sialylated glycoconjugates, which have been shown to influence pathogenesis through immune evasion, adhesion, and invasion, are synthesized by sialyltransferases. Functional and structural studies on C. jejuni LOS associated sialyltransferases (CstII and CstIII) have been carried out due to their association to the onset of the autoimmune Guillian-Barré syndrome. The CstII variants can either be monofunctional (α-2,3-sialyltransferase activity) or bifunctional sialyltransferases (α-2,3- and α-2,8- activity), while CstIII is monofunctional (α-2,3 activity). Although C. coli is the second most frequently isolated Campylobacter species, the activity of C. coli distant CstII homologues is unknown. Therefore, in the present study we explore the role of two C. coli putative sialyltransferases (CstIV and CstV) in LOS biosynthesis by combining genetic, mutational, recombinant protein, and LOS structural studies. Results show that despite several substitutions at amino acids deemed important for CstII activity, CstIV and CstV play an active role in LOS biosynthesis as deletion of cstIV and cstV result in LOS truncation. Nevertheless, no sialic acid has been detected in the intact LOS of C. coli strains expressing either CstIV or CstV by CE-MS and EA-OTLC-MS analysis. Furthermore, recombinant CstIV and CstV have not shown sialyltransferase activity on BODIPY-labelled acceptors so far. Nevertheless, LOS associated sialic acid biosynthesis gene (neuB1) appears to be involved in CstV substrate biosynthesis. Conversely, strains expressing CstIV lack neuB1 orthologue. Therefore, other genes potentially associated to CstIV substrate synthesis (neuB2 and neuB3) are being investigated. Preliminary studies suggest that neuB2 does not play a role in the biosynthesis of CstIV substrate. Finally, the LOS structures synthesized by CstIV and CstV are currently being studied in detail.

98. An investigation into an unusual glycan branching enzyme in mycobacterium tuberculosis

Seung Seo Lee 1 , Jessica Gusthart 1 , Kyeong Kyu Kim 2; 1University of Southampton, School of Chemistry, SO17 1BJ, Southampton, UK, 2Sungkyunkwan University School of Medicine, Department of Molecular Cell Biology, 16419, Suwon, Korea

s.s.lee@soton.ac.uk

Tuberculosis (TB) is described by the World Health Organisation as a ‘major global health problem’ which, alongside HIV, is one of the leading causes of death worldwide. The causative agent of TB is Mycobacterium tuberculosis and drug resistant strains are threatening the fight to bring the disease under control.

Mycobactera produce unique polymethylated carbohydrates such as methylglucose lipopolysaccharides (MGLPs).1-2 The genes involved in the biosynthetic pathway of MGLPs have been identified and many have been found to be essential to cell survival.2-3 One such essential gene is Rv3031 which codes for an enzyme that catalyses an early step in MGLP synthesis, presumably adding a glucose unit to glucosyl glycerate (GG) to form di-glucosyl glycerate (DGG)(Fig. 1).3

Sequence analysis suggests that the enzyme encoded for by Rv3031 is a glucan branching enzyme (GBE), though this enzyme is unusual in that appears to be working on small substrates rather than the typical large substrates that known GBEs act upon. Thus, this raises the possibility of using small molecule inhibitors upon this enzyme.

We characterised this unusual glucan branching enzyme to understand its molecular mechanism. We synthesized acceptor substrates, and potential inhibitors, and used them to demonstrate the activity of this Mycobacterium tuberculosis GBE (MtGBE). Also, the conventional activity of MtGBE on long chain glucans was tested to show a flexibility of its activity. Presently, MtGBE appears to only act on α-1,6-branched glucans such as amylopectin, not on linear glucans such as amylose. To elucidate the structural determinants of such unusual activity, a crystal structure was determined and compared with those of known GBE in the GH family 57. Amino acid sequence alignment of MtGBE with GH57 GBEs suggested a change of crucial tyrosine to histidine, which was reflected in the x-ray structure. Altogether, our results are shedding a light into the unusual activity of a GBE in Mycobacterium tuberculosis, expanding the nature’s repertoire of handling glycosidic bonds.

99. EntS-an iterative O- and S-glycosyltransferase of Enterococcus faecalis

Rupa Nagar 1 , Alka Rao 1; 1CSIR-Institute of Microbial Technology, Protein Science and Engineering, 160036, Chandigarh, India

raoalka@imtech.res.in

Glycosylation of proteins and peptides is common in bacteria. While N-linked and O-linked glycosylation is universal and widespread, S- linked glycosylation is rare and observed only in bacteria. Glyocin F and Sublancin 168 are the two naturally S-glycosylated antimicrobial peptides. SunS, a glycosyltransferase (GT) of GT-2 family (Bacillus subtilis 168) is identified to S-glycosylate sublancin 168, whereas its homologue ThuS (Bacillus thuringiensis serovar andalousiensis BGSC 4AW1) exhibits both O- and S- linked glycosylation activities, in vitro. Here, we report a second homologue of SunS in Enterococcus faecalis TX0104 (EntS) as a nucleotide diphosphate (UDP/GDP) dependent GT having additional glycosylation capabilities as that of SunS and ThuS. EntS, encoded by ORF 0417 in E. faecalis TX0104 was expressed and purified in Escherichia coli and its enzyme action was reconstituted in vitro using UDP-hexose as donor substrate and synthetic enterocin 96 peptide/variants as acceptor. Using mass spectrometry methods, EntS is characterized as an O- and S- glycosyltransferase that can catalyze the transfer of at least two hexoses sequentially on to serine or cysteine residue of an acceptor peptide thereby producing an O- linked or S- linked di-glycosylated product. EntS is a metal-dependent, iterative GT that transfers the glycan onto acceptor peptide in sequential dissociative manner. Further, EntS can catalyze at least three different linkages in the disaccharide glycan in one pot, where proximal linkage is defined as O-linked or S- linked to the monosaccharide and terminal linkage is identified as β, implying EntS is a multifunctional GT.

Session 21. O-GlcNAc Modification I

Keynote Lectures

100. Nutrient regulation of transcription & signaling by O-GlcNAcylation

Gerald Hart 1; 1Department of Biological Chemistry Johns Hopkins University School of Medicine, Baltimore, MD USA 21205

gwhart@jhmi.edu

O-GlcNAcylation cycles on and off thousands of nucleocyto-plasmic proteins and has extensive crosstalk with protein phosphorylation. O-GlcNAc cycles on nearly all proteins involved in transcription, where it regulates gene expression in response to nutrients. O-GlcNAc also regulates the cycling of the TATA-binding (TBP) protein on and off DNA during the transcription cycle and is required for TBP to bend DNA.

Targeted, inducible, deletion of the O-GlcNAc Transferase in αCAMKII positive (excitatory) neurons of adult mice results in a morbidly obese mouse with a satiety defect. Thus, O-GlcNAcylation not only serves as a nutrient sensor in all cells, but also is directly involved in appetite regulation. O-GlcNAcylation also plays an important role in the trafficking of the AMPA receptors in neurons and in the development of functional synaptic spines. Recent studies have shown that more than one-half of all human protein kinases are modified by O-GlcNAc and all kinases that have been tested are indeed regulated in some way by the sugar. Abnormal O-GlcNAcylation of CAMKII contributes directly to diabetic cardiomyopathy and to arrhythmias associated with diabetes. Prolonged elevation of O-GlcNAc, as occurs in diabetes, contributes directly to diabetic complications and is a major mechanism of glucose toxicity. Drugs that elevate O-GlcNAcylation in the brain, which prevents hyperphosphorylation, appear to be of benefit for the treatment of Alzheimer’s disease in animal models. To date, all cancers have elevated O-GlcNAc cycling, which may play a key role in the regulation of metabolism in cancer cells. Supported by NIH P01HL107153 and R01GM116891. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.

Lectures

101. Overexpressed X-linked inhibitor of apoptosis protein (XIAP) inhibits cancer cell growth through proteasomal degradation of OGT

Hyeon Gyu Seo 1,2 , Han Byeol Kim 1,2 , Jung Hwa Seo 1,2 ,Jin Won Cho 1,2; 1Yonsei University, Department of Integrated OMICS for Biomedical Science, 2Glycosylation Network Research Center, 03722, Seoul, Korea,

chojw311@yonsei.ac.kr

O-GlcNAc transferase (OGT) attaches a single GlcNAc to hydroxyl groups of serine and threonine residues on various nuclear and cytoplasmic proteins. Although the cellular protein levels of OGT are important to regulate cancer cell progression, the molecular mechanisms regulating the subcellular OGT protein levels are unclear. We report that X-linked inhibitor apoptosis protein (XIAP), a well-known caspase inhibitor, acts as an E3 ligase and promotes the proteasome-dependent degradation of OGT in vivo and in vitro. We shows that transiently overexpressing XIAP directly poly-ubiquitylated OGT and induced degradation of endogenous OGT levels in an independent of OGT mRNA levels. The HCT116 cells stably overexpressing XIAP show reduced OGT protein levels and decreased growth rate and colony formation compared to the control HCT116 cells. Our study suggests that a novel function of XIAP in the regulation of OGT, which is distinctly different from its well characterized anti-apoptotic properties.

102. Proteomic analysis of serum glycoproteins from colorectal cancer patients enriched by wheat germ aggrutinin

Voraratt Champattanachai 1 , Tanin-ek Sriwitool 2 , Pukkavadee Netsirisawan 1 , Parunya Chaiyawat 1 , Daranee Chokchaichamnankit 1 , Chantragan Srisomsap 1 , Jisnuson Svasti 1; 1Chulabhorn Research Institute, 2Chulabhorn Graduate Institute, Thailand

voraratt@cri.or.th

O-GlcNAcylation, a single attachment of N-acetylglucosamine to serine/threonine residues on cellular proteins, is emerging as a key mediator in pathophysiological processes of many diseases including cancer. Previously, we reported that O-GlcNAcylation level was elevated in primary colorectal cancer (CRC) tissues, CRC cell lines and their secretions. In this study, we aim to investigate and identify if (O)-GlcNAc modified proteins is present in serum of CRC patients. Wheat germ agglutinin (WGA), a lectin specifically binds to GlcNAc/sialic acid modified proteins, were used to enrich glycoproteins from serum of CRC patients and normal. Two dimensional (2D) gel based proteomics revealed alteration of WGA-enriched protein spots between two groups. In addition, 2D O-GlcNAc immunoblots detected by RL2 antibody showed that sera of CRC patients had intense in O-GlcNAc spots when compared to those in the normal sera. These O-GlcNAc protein spots were further identified using a nanoflow LC coupled with amaZon speed ion trap and validation by immune detections. The proteins identified may open up a new aspect of this glycosylation present in sera of cancer patients, especially in CRC.

Supported by the Chulabhorn Research Institute and the National Science and Technology

Development Agency, Thailand.

Session 22. Glycan Binding Proteins I

Keynote Lectures

103. Galectins: intra and extracellular functions

John Stegmayr 1 , Michael C. Carlsson 2 , Xiaoli Huang 3 , Fredrik Zetterberg 4 , Barbro Kahl-Knutson 1 , Hans Schambye 5 , Ulf J. Nilsson 6 , Stina Oredsson 3 , Hakon Leffler 1; 1Lund University, Department of Laboratory Medicine, 22184 Lund, Sweden, 2University of Copenhagen, Department of Cellular and Molecular Medicine, 2200 Copenhagen N, Denmark, 3Lund University, Department of Biology, 22362 Lund, Sweden, 4Galecto Biotech AB, Sahlgrenska Science Park, 41346 Gothenburg, Sweden, 5Galecto Biotech AB, DK-2200 Copenhagen N, Denmark, 6Lund University, Department of Chemistry, 22100 Lund, Sweden

hakon.leffler@med.lu.se

Galectins were discovered in a quest to find proteins binding cell surface carbohydrates and taking part in cell adhesion, and this is indeed one of the roles of galectins. However, they have many other roles. Galectins bind β-galactose containing glycans typically found at the cell surface and inside vesicles. Most studied galectin functions are due to this binding activity, and included formation of cell surface lattices, regulation of glycoprotein traffic and surface exposure of receptor, induction of endocytosis and others, that translate into higher level roles of galectins in immunity, inflammation and cancer. However, surprisingly, it was early discovered that galectins are synthesized as cytosolic proteins, without a signal peptide, and can also have functions in the cytosolic and nuclear compartments, such as interaction with RAS-proteins, ESCRT-complex, centrosomes and roles in transcription and RNA splicing. At the interface between the cytosolic and intravesicular compartments is the now well established carbohydrate-binding dependent rapid accumulation of galectin around disrupted vesicles and coupling of this to autophagy, and also to secretory autophagy. These multiple seemingly unrelated functional effects, suggest roles of galectins in feed back loops regulating membrane turnover and organization or similar.

To help study such galectin related phenomena we have developed binding assays to analyze galectin specificity and aggregation on encounter with natural and artificial ligands. We have generated mutants of galectins with altered fine specificity, permitting coupling of this property to cellular function. We have also developed potent small molecule galectin inhibitors (nM affinities) that can either be readily taken up by cells or not, permitting examination of galectin intracellular roles separate from extracellular roles. Such galectin inhibitors are also in development as therapeutics

Lectures

104. Development of potent inhibitors for targeting specific galectins and glycosidases

Chun-Hung Hans Lin 1; 1Institute of Biol Chem, Academia Sinica, Taiwan

chunhung@gate.sinica.edu.tw

We report the inhibitor development for two proteins, namely galectins and glycosidases. Human galectins are promising targets for cancer immunotherapeutic and fibrotic disease-related drugs. We report herein the binding interactions of three thio-digalactosides (TDGs) including TDG itself, TD139 (3,3’-deoxy-3,3’-bis-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside, recently approved for the treatment of idiopathic pulmonary fibrosis), and TAZTDG (3-deoxy-3-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside) with human galectins-1, -3 and -7 as assessed by X-ray crystallography, isothermal titration calorimetry and NMR spectroscopy. Five binding subsites (A–E) make up the carbohydrate-recognition domains of these galectins. We identified novel interactions between an arginine within subsite E of the galectins and an arene group in the ligands. In addition to the interactions contributed by the galactosyl sugar residues bound at subsites C and D, the fluorophenyl group of TAZTDG preferentially bound to subsite B in galectin-3, whereas the same group favored binding at subsite E in galectins-1 and -7. The characterized dual binding modes demonstrate how binding potency, reported as decreased Kd values of the TDG inhibitors from μM to nM, is improved and also offer insights to development of selective inhibitors for individual galectins.

Additionally we will discuss the structural basis of the complex structures of fucosidase/inhibitors. The inhibitors are iminocyclitols (also called azasugars) representing an important class of glycosidase inhibitors because they can be protonated at physiological pH to mimic the positive-charge feature of oxocarbenium-like transition state. According to our X-ray structures, the loop near the enzyme active site appears to be important for the potency and selectivity.

105. Galectin-3 competes with galectin-8 for regulation of antibacterial autophagy through differential recognition of host glycans on damaged phagosomes

I-Chun Weng 1 , Hung-Lin Chen 1 , Tzu-Han Lo 1 , Wei-Han Lin 1 , Huan-Yuan Chen 1 , Daniel K. Hsu 2 , Fu-Tong Liu 1; 1Academia Sinica, Institution of Biomedical Sciences, 11529, Taipei, Taiwan, 2University of California-Davis, Department of Dermatology, 95817, Davis, USA

ftliu@ibms.sinica.edu.tw

Glycans are generally displayed on the cell surface or confined in the lumen of organelles, but can become exposed to the cytosolic milieu when the organelle membrane integrity is compromised by various stress or pathogens. Galectins are a family of β-galactoside-binding animal lectins synthesized and predominantly localized in the cytosol. Recent research indicates that some galectins may act as “danger signal sensors” by detecting the unusual exposure of glycans to the cytosol. Galectin-8 has been shown to promote antibacterial autophagy by recognizing host glycans on ruptured vacuolar membrane and interacting with the autophagy adaptor protein NDP52. Galectin-3 also accumulates at damaged phagosomes containing bacteria, however its functional consequence remains obscure. By studying mouse macrophages infected with Listeria monocytogenes (LM), we showed that endogenous galectin-3 protects intracellular LM by suppressing autophagic response through a host N-glycan-dependent mechanism. Knock-out of galectin-3 gene expression resulted in enhanced LC3 recruitment to LM and decreased bacterial replication, a phenotype recapitulated when wild-type macrophages were depleted of host N-glycans. Moreover, since galectin-3 and galectin-8 antagonize each other in antibacterial autophagy and both exert their functions through binding to host glycans, the balance of autophagic response may be regulated by differential occurrence of preferred glycan ligands for galectin-3 and galectin-8. We explored the concept that alterations in cell surface glycosylation by extracellular factors can be “read” by galectins during the process of phagocytosis/endocytosis followed by rupture of phagosomal/endosomal membrane. We found that treatment of cells with sialidase, which removes sialic acid on glycans, increased galectin-3 binding and decreased galectin-8 binding, and led to a stronger anti-autophagic response. Our study demonstrated that cytosolic galectins may sense changes of glycosylation that occur on the cell surface and modulate cellular response through differential recognition of glycans on ruptured phagosomal membrane.

106. Identification of Siglec-15 ligands using proximity labeling method

Yi-Hsiu Chen 1 , Penk-Yeir Low 1 , Yi-Ju Chen 2 , Yu-Ju Chen 2 , Takashi Angata 1; 1Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, 2Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan

angata@gate.sinica.edu.tw

Siglecs are a family of receptor-type vertebrate lectins that recognize sialic acids and participate in self-nonself discrimination by the immune system. It is essential to understand the molecular mechanisms of the Siglec-ligand interaction to target this interaction for therapeutic purposes. However, limited information is available regarding Siglec ligands, because the affinity between Siglecs and their ligands tends to be low.

To overcome this problem, we developed a method to identify Siglec ligands by proximity labeling. In brief, the cells expressing Siglec ligands are first labeled with FLAG-tagged recombinant Siglec protein coupled with peroxidase-conjugated anti-FLAG antibody (Siglec-peroxidase complex). The cells are then incubated with hydrogen peroxide and biotin-tyramide, which is converted to a short-lived radical and covalently ligate to the protein in the vicinity of the Siglec-peroxidase complex. Biotinylated proteins are affinity-purified and identified by mass spectrometry. As a proof of concept, we used recombinant CD22/Siglec-2 to label its ligands on human B-lymphoma cell line, and identified several glycoproteins reported to be CD22 ligands, including CD45, IgM, and endogenous CD22 itself.

Siglec-15 is involved in osteoclast differentiation, but its ligand has not been identified. We applied the proximity labeling method to identify the potential ligands of Siglec-15 on RAW264.7 mouse macrophage cell line. We are investigating the contribution of these ligand candidates to osteoclast differentiation.

This study is supported by the Ministry of Science and Technology (MoST) of Taiwan (grant number: 104-2311-B-001-017-MY3).

107. GalNAc/Gal-Specific lectin from the sea mussel Crenomytilus grayanus induces tumor cells death and modulates immune response

Oleg Chernikov 2 , Kuo-Feng Hua 1 , Alexandra Kuzmich 2 , Irina Chikalovets 2 , Valentina Molchanova 2; 1G.B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS, 690022, Vladivostok, Russian Federation, 2National Ilan University, Department of Biotechnology and Animal Science, 260, Ilan, Taiwan

chernikov@piboc.dvo.ru

Marine biological resources are increasingly being used as sources of new physiologically active substances and objects for basic and applied biomedical research. The aim of the work was to study the biological properties of GalNAc/Gal-specific lectin (CGL) from the edible mussel Crenomytilus grayanus.

Glycan array assay revealed that CGL was able to bind both α and β anomer of galactose, but interaction with the αGal-terminated glycans was stronger. Analysis of most common glycan motifs for CGL showed high affinity to Galα1-4Galβ1-4GlcNAc motif similar to globotriose structure (Gb3: Galα1-4Galβ1-4Glc), the epitope of globotriaosylceramide. CGL recognized Gb3 on the surface of Burkitt`s lymphoma Raji cells (high Gb3 expression), leading to dose-dependent cytotoxic effect, G2/M phase cell cycle arrest and apoptosis. Lectin had no effect on erythroleukemia K562 cells (no Gb3 expression). The activity of CGL was specifically blocked by α-galactoside.

We demonstrated that CGL can activate immune responses in vitro and in vivo. In the in vitro cell models, CGL induced tumor necrosis factor-α and interleukin-6 (IL-6) secretion in mouse RAW264.7 macrophages, mouse bone marrow-derived macrophages and human THP-1 macrophages. The CGL-mediated cytokine production was regulated by reactive oxygen species, mitogen activated protein kinases, protein kinase C-α/δ and NF-κB. Interestingly, in LPS-activated macrophages, CGL induced endotoxin tolerance (characterized by the downregulation of nitric oxide, inducible nitric oxide synthase, IL-6 and cyclooxygenase II) via the downregulation of IRAK2 expression, JNK1/2 phosphorylation and the activation of NF-κB. CGL also increased the bactericidal activity of and induced cytokine production in the macrophages in the mouse model in vivo.

Overall, our data indicate that CGL has the potential to be used in cancer diagnosis and treatment and as a modulator or adjuvant in immunotherapy and vaccination.

The investigation was partially supported by the Program of FEB RAS “Far East”.

Session 23. O-GlcNAc Modification II

Keynote Lectures

108. O-GlcNAc: epigenetic modifier linked to human neurodegenerative disease

John Hanover 1; 1NIDDK, National Institutes of Health, USA

jah@helix.nih.gov

Proteostasis is essential in the mammalian brain where post-mitotic neurons must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites for proper function and is spared from metabolic deficits even during starvation. The nutrient sensitive nucleocytoplasmic posttranslational modification O-linked N-acetylglucosamine (O-GlcNAc) regulates protein homeostasis, is highly abundant in the mammalian brain, and has been linked to proteopathies, including neurodegenerative diseases such as such as Alzheimer’s, Parkinson’s, and Huntington’s. We suggest that O-GlcNAc cycling is essential to the brain proteostasis network and that its deregulation may have both developmental and pathological consequences. Findings in C. elegans, Drosophila and mouse model systems have demonstrated that the dynamic turnover of O-GlcNAc is critical for maintaining levels of key transcriptional regulators responsible for neurodevelopment fate decisions. In addition, pathways of autophagy and proteasomal degradation depend upon a transcriptional network dependent upon O-GlcNAc cycling. Like the quality control system in the endoplasmic reticulum which uses a “mannose-timer” to monitor protein folding, we propose that cytoplasmic proteostasis uses an “O-GlcNAc timer” that helps to regulate the lifetime and fate of cytosolic proteins. Worm, fly and mouse models harboring O-GlcNAc transferase and O-GlcNAcase knockout alleles have helped define the role O-GlcNAc plays in development and age-associated neurodegenerative disease. We anticipate that brain-selective knockout mouse models will be an important tool for understanding the role of O-GlcNAc in the physiology of the brain and its susceptibility to neurodegenerative injury. Blocking O-GlcNAc cycling is detrimental to mammalian brain development and interferes with neurogenesis, neural migration, and proteostasis. O-GlcNAc-dependent developmental alterations impact metabolism and growth of the developing mouse embryo and persist into adulthood. Thus, O-GlcNAcase is both a promising diagnostic and therapeutic target for human neurodegenerative disease.

Lectures

109. Calcium-dependent O-GlcNAc signaling drives liver autophagy in adaptation to starvation

Hai-Bin Ruan 3 , Yina Ma 1 , Sara Torres 3 , Bichen Zhang 1 , Colleen Feriod 2 , Kevin Qian 3 , Barbara Ehrlich 2 , Xiaoyong Yang 3; 1Yale University, Department of Comparative Medicine, 06520, New Haven, USA, 2Yale University, Department of Cellular and Molecular Physiology, 06520, New Haven, USA, 3Yale University, Department of Pharmacology, 06520, New Haven, USA

xiaoyong.yang@yale.edu

Starvation induces liver autophagy, which is thought to provide nutrients for the use of other organs and maintain whole-body homeostasis. Here we demonstrate that O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is required for glucagon-stimulated liver autophagy and metabolic adaptation to starvation. Genetic ablation of OGT in mouse liver reduces autophagy flux and production of glucose and ketone bodies. Upon glucagon-induced calcium signaling, calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates OGT, which in turn promotes O-GlcNAc modification and activation of Ulk proteins by potentiating AMPK-dependent phosphorylation. These findings uncover a signaling cascade by which starvation promotes autophagy through OGT phosphorylation and establish the importance of O-GlcNAc signaling in coupling liver autophagy to nutrient homeostasis.

110. Biological significance for the Cys S-nitrosylation of OGT

Su-Il Do 1; 1Ajou University, Korea

sido@ajou.ac.kr

Mechanistic link of protein hypo-O-GlcNAcylation to the pathogenesis of Alzheimer's disease (AD) remains unclear. The Cys S-nitrosylation of O-linked N-acetylglucosaminyltransferase (SNO-OGT) was induced by β-amyloid peptide (Aβ) exposure to human neuroblastoma cells. Aβ-induced SNO-OGT led to protein hypo-O-GlcNAcylation globally together with tau hypo-O-GlcNAcylation. Molecular basis for SNO-OGT induction comprises the concerted action of Aβ-triggered Ca2+ entry and nNOS-mediated NO production. It was intriguing that OGT was associated with nNOS and its association was enhanced during Aβ treatment. Early time-course Aβ-monitoring assay revealed that that SNO-OGT-mediated tau hypo-O-GlcNAcylation was paralled with SNO-Akt-mediated GSK3β activation for tau phosphorylation. These results suggest that tau hyperphosphorylation is proceeded by synergistic connection between SNO-OGT induction and GSK3β activation. Next, it was observed that Aβ-neurotoxicity including reactive oxygen species (ROS) production and cell death was amplified by DON treatment, whereas it was restored by PUGNAc treatment, GlcNH2 treatment or OGT overexpression. These results imply that hyper-O-GlcNAcylation state can aid to avoid Aβ-affected pathogenesis in neuronal system. Furthermore, Cys mapping demonstrates that cysteine-nitric oxide (Cys-NO) linkages in SNO-OGT occur at triple residues of Cys845, Cys921, and Cys965 in C-terminal catalytic domain (C-CAT) of OGT, resulting in null OGT activity. Finally, selectivity determinants of SNO and 3D-motifs for Cys-NO linkage triplet in SNO-OGT were investigated.

111. O-GlcNAc transferase (OGT) in hepatic stellate cells suppresses liver fibrogenesis

Yuanyuan Ruan 1 , Jianxin Gu 1; 1Fudan University, Key Laboratory of Glycoconjugate Research Ministry of Public Health, 200032, Shanghai, China

yuanyuanruan@fudan.edu.cn

Liver fibrosis represents the consequences of a sustained wound healing response to chronic liver injury, and remains a significant health problem estimated to affect over 100 million people worldwide. Hepatic stellate cells (HSCs) activation is considered to be the central event of liver fibrosis. In this study, we found that protein O-GlcNAcylation was increased in the septa of fibrotic liver in thioacetamide (TAA)-induced mouse model. HSCs-specific knock-out of O-GlcNAc transferase (OGT) promoted TAA-induced liver fibrosis, and administration of glucosamine therapeutically attenuated liver fibrogenesis in a HSC-derived OGT dependent manner. In vitro studies demonstrated that stimulation of protein O-GlcNAcylation repressed HSCs transdifferentiation and proliferation, but enhanced HSCs migration. Further studies revealed Smad3, a key modulator of type I collagen transcription, was O-GlcNAcylated. Increment in O-GlcNAcylation resulted in the inhibition of Smad3 activation and translocation into nucleus. In addition, the O-GlcNAc modification in the septa also positively correlated with the stage of liver fibrosis in clinical biopsy samples. Taken together, our data suggest OGT in HSCs as a novel suppressor of liver fibrogenesis, and protein O-GlcNAcylation may be a potential target for the treatment of liver fibrosis.

Session 24. Glycan Binding Proteins II

Lectures

112. In vivo tropism of Salmonella Typhi toxin to cells expressing a multiantennal glycan receptor

Yi-An Yang 1 , Sohyoung Lee 1 , Jun Zhao 2 , Ruth Nussinov 2,3 , Lingquan Deng 4 , Jeongmin Song 1; 1Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, 2Computational Structural Biology Section, Cancer and Inflammation Program, National Cancer Institute, NIH, Frederick, MD 21702, 3Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel, 4GlycoMimetics, Inc., 9708 Medical Center Drive, Rockville, MD 20850 USA

jeongmin.song@cornell.edu

Bacterial AB toxins exert very important roles in bacterial pathogenesis. Receptor binding “B” subunit binds specific glycan receptors on the cell surface to deliver enzymatic “A” subunit to target cells, as an essential part of bacterial virulence. For instance, typhoid toxin produced by the Gram-negative bacterium Salmonella Typhi contributes to the development of life-threatening typhoid fever. Typhoid toxin consists of two A subunits – CdtB (nuclease) and PltA (ADP-ribosyltransferase) – and a homopentamer of B subunit PltB. As PltB has five identical binding pockets, PltB preferentially binds its glycan receptor Neu5Ac displayed by multiantennary N-glycans, presumably with higher affinities than its binding to a single Neu5Ac. As a result of this multivalent binding, despite the ubiquity of Neu5Ac, typhoid toxin displays in vivo tropism to specific cells – brain endothelial cells of arterioles and immune cells – which associates with typhoid fever symptoms. Cumulatively, our results revealed remarkable features in the biology of a bacterial AB toxin in inducing virulence exclusively in specific cells at the organismal level.

113. Lectin domains in model plant species: evolutionary dynamics and physiological importance

Sofie Van Holle 2 , Kristof De Schutter 1 , Lore Eggermont 2 , Mariya Tsaneva 2 , Liuyi Dang 2 , Els J.M. Van Damme 2;1Ghent University, Department Molecular Biotechnology, 9000, Gent, Belgium, 2Ghent University, Department of Crop Protection, 9000, Gent, Belgium

ElsJM.VanDamme@ugent.be

Lectins are present throughout the plant kingdom. A comparative analysis was made of the lectin motifs in three important dicot species (Arabidopsis thaliana, Glycine max, Cucumis sativus) and the monocots Oryza sativa ssp. japonica and Oryza sativa ssp. indica. First, the genomes were screened for genes encoding lectin domains to examine the distribution of lectin domains. Subsequently, the phylogenetic relationships were analyzed by constructing maximum likelihood phylogenetic trees. Finally, functional analyses allowed investigating the importance of lectin domains for plant growth and development.

The results demonstrate that the majority of the lectin families are present in each of the species under study. Domain organization analysis showed that most identified lectins are multi-domain proteins, owing to the modular rearrangement of protein domains during evolution. A comparative and evolutionary genomics study was performed for the plant lectin domains and some other protein domains that are found in combination with the lectin domains. Several reoccurring protein domains (e.g. glycosidase, F-box, protein kinase) are known to be involved in plant defense, signaling and/or development. This observation agrees with the idea that lectin domains play a role in plant growth and defense. Some multi-domain proteins are widespread, indicating the retention of ancient fusions in a common ancestor. In contrast, the many species-specific domain arrangements can be explained by lineage-specific retention of a certain domain rearrangement or by a recent event of protein domain fusion or domain loss in a particular species.

It is clear that plants have a large variety of plant lectins at their disposal. During evolution, new lectin domain combinations have been created and used to the benefit of the plant to allow rapid adaption to changing environmental conditions. First experimental evidence show that lectins within one species exert complementary activities since they are present in different subcellular/tissue specific locations and recognize different carbohydrate structures, that are either present in the plant or part of pathogens.

114. Galectin-8 modulates autophagy induced by damaged vesicles corresponding to cell surface carbohydrate alterations

Ming-Hsiang Hong 2 , Wei-Han Lin 2 , I-Chun Weng 2 , Yu-Hsien Hung 1 , Hung-Lin Chen 2 , Huan-Yuan Chen 2 , Wei-Yuan Yang 1 , Fu-Tong Liu 2; 1Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China (R.O.C), 2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China (R.O.C)

ftliu@ibms.sinica.edu.tw

Galectin-8 is a β-galactoside-recognizing lectin with two homologous carbohydrate recognition domains (CRDs). These CRDs show differential affinities for glycoconjugates: the N-terminal CRD has a preference for sialylated or sulfated carbohydrates, while the C-terminal CRD has a preference for N-acetyllactosamine (LacNAc) structure. Galectin-8 is known to be recruited to damaged endocytic vesicles, but the precise molecular mechanism is not well understood. We applied a light-induced endocytic vesicle impairing scheme, which involves illumination of the endocytosed amphiphilic photosensitizer AlPcS2a (disulfonated aluminum phthalocyanine) with a red light. We provide evidence that internalized cell surface glycoconjugates contained in endocytic vesicles attracts galectin-8 accumulation in Chinese hamster ovary (CHO) cells. The amounts of accumulated galectin-8 were dependent on the composition and amount of glycans recognized by the protein, as demonstrated by using various glycosylation mutant CHO cell lines. Importantly, galectin-8 could sense cell surface glycoconjugate alterations, resulting in different amounts of the protein accumulating around damaged endocytic vesicles. In lung epithelial A549 cells, the amount of galectin-8 accumulated in damaged endocytic vesicles was significantly diminished when sialic acids were removed from cell surface. Moreover, accumulated galectin-8 was associated with autophagy adapter protein NDP52 and LC3; the amounts of these proteins accumulated were thus also significantly lower in cells with desialyated cell surface glycans. Our data indicate that galectin-8 is a novel intracellular sensor to decipher sophisticated messages from versatile cell surface carbohydrates and eventually convey the information into cells to modulate distinct cellular responses.

115. Galectins induce multicellularity in bacteria and increase bacterial adhesion to intestines

Chi-Shan Li 1 , Ting-Jui Tu 1 , Fu-Tong Liu 1; 1Academia Sinica, Institute of Biomedical Sciences, 11529, Taipei, Taiwan

ftliu@ibms.sinica.edu.tw

It is known that a number of different galectins are expressed by intestinal epithelial cells. We showed that galectins were present in the secretions from freshly isolated intestines of mice, especially the tandem-repeat type galectin-4 and galectin-9. We demonstrated that galectins had higher selectivity for intestinal bacteria compared to mammalian cells. In addition, galectin-4 was preferentially released from apical sides of polarized intestinal epithelial cells compared to basolateral sides. By flow cytometry, we found that human galectin-4 bound to Escherichia coli O19ab, Escherichia coli O86, Salmonella enteric Cubana and Salmonella enteric Worthington. The binding was through the carbohydrate-recognition domains. Galectin-4 did not affect bacterial survival, but induced chaining and clustering in bacteria. This was related to the divided cells failing to separate into individual daughter cells and galectins being highly enriched at cell-cell junctions and bridging the daughter cells. Moreover, Escherichia coli O19ab treated with tandem-repeat galectins increased their adhesion to intestinal epithelial cells. Finally, endogenous galectins enhanced bacterial colonization in vivo. Our data suggest that extracellular galectins can induce multicellularity in bacteria and increase their adhesion to intestines.

Session 25. Glyconeurobiology: Glycans in Neuroscience

Keynote Lectures

116. Genetic and environmental factors influence the expression of polysialic acid in brain

Chihiro Sato 1; 1Nagoya University, Bioscience and Biotechnology Center, 464-8601, Nagoya Japan

chi@agr.nagoya-u.ac.jp

Polysialic acid (polySia or PSA) is a homopolymer of sialic acid with the degree of polymerization 8-400 Sia residues. PolySia mainly modifies two N-linked glycans in the 5th immunoglobulin domain of neural cell adhesion molecules (NCAM) in embryonic brains. In adult brains, polySia expression is highly restricted to the plastic areas such as hippocampus and olfactory systems. In vertebrate brain, polySia is shown to be involved in memory, circadian rhythm, and social behaviors via varieties of neuro-cellular functions. The molecular mechanism underlying these phenomena is considered to be related not only with the bulky and hydrated properties, but also with reservoir functions as a scaffold for various neurological active molecules (1,2): the brain-derived neurotrophic factor (BDNF), catecholamine neurotransmitters, and the fibroblast growth factor 2 (FGF2). Recently it has been shown that polySia and a polysialyltransferase gene, ST8SIA2, have some relationship with psychiatric disorders such as schizophrenia and bipolar disorder. We investigated the effects of reported single nucleotide polymorphisms (SNPs) (cSNP, sSNP, iSNP and rSNP) of ST8SIA2/STX gene on their products and found that all the SNPs examined so far influenced on the structure and function of polySia (3-6). In addition, we have also been analyzing the effects of environmental factors that are known to affect the mental disorders on the polySia expression, showing that polySia expression is highly sensitive to the environmental factors at restricted area(s) of brain (7). Collectively, these data suggest that impairments of "quality and quantity" of the polySia structure, which is regulated by "genetic factors x environmental factors" in a brain region-specific manner, bring about the high risk of disorders, probably because polySia functions as a multi-molecule regulator.

References: 1) Sato, Kitajima (2013) Front Cell Neurosci. 7, 61 2) Sato, Kitajima (2013) J Biochem. 154, 115 3) Isomura et al. (2011) J Biol Chem. 286, 21535 4) Hane et al. (2012) Pure Appl Chem. 84, 1895 5) Hane et al. (2015) Glycobiology 10, 1112-1124 6) Hane et al. (2016) Biochem Biophys Res Commun. 478, 1123-1129 7) Abe et al. (2017) Int J Mol Sci. 18, 1123

Lectures

117. Elucidation of the mechanism underlying the trafficking of mucin-type O-glycans specifically to the proximal region of an axon in drosophila neuron

Takaaki Kinoshita 1 , Chikara Sato 2 , Shoko Nishihara 1; 1Department of Bioinformatics, Graduate School of Engineering, Soka University, 192-8577, Hachioji, Japan, 2Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 305-8566, Tsukuba, Japan

kiguri1988@gmail.com

T antigen (Galβ1-3GalNAcα1-Ser/Thr) is one of the most common structures of mucin-type O-glycans. In Drosophila and mammals, T antigen is expressed and functions in the nervous and blood systems (Yoshida H et al. 2008. Glycobiology. 18: 1094-1104; Xia L et al. 2004. J Cell Biol. 164: 451-459). Previously we have reported that T antigen is required for maintenance of hematopoietic stem cells and the localization of neuromuscular junction boutons in larval Drosophila (Fuwa TJ et al. 2015. Dev Biol. 401: 206-219; Itoh K et al. 2016. Dev Biol. 412: 114–127). However, detailed localization and function of T antigens in neurons are largely unknown. In this study, we found that T antigen is localized within the proximal axon segments of Drosophila primary cultured neuron (Kinoshita T et al. 2017. Sci Reports. 7: 41455). Ultrastructural analysis by atmospheric scanning electron microscopy (ASEM) showed that microtubule bundles cross one another at the intra-axonal boundary, and that F-actin are abundant there (Kinoshita T et al. 2014. Microscopy and Microanalysis. 20: 469-483). In addition, T antigens accumulated and formed circular pattern before the boundary. These characteristic structural features near the boundary might reflect the mechanism of their proximal localization. We then screened and identified Short stop (Shot), a crosslinker protein between F-actin and tubulin, as a key molecule for their proximal localization. Shot was localized mainly in the proximal axon segments like T antigen. In shot null mutant, the percentage of neurons with proximal localization of T antigens was significantly decreased. Moreover, the inhibition of their proximal localization was rescued by the expression of wild type Shot in shot mutant background. These results showed that Shot involves in trafficking of T antigens to proximal axon segments. The abnormal microtubule formation was observed by ASEM in shot null mutant axons but not in shotΔFBD mutant axons that express the Shot protein lacking only F-actin binding domain. However, preferential localization of T antigens was disturbed in shotΔFBD mutant neurons just like in the shot null mutant. Therefore, the F-actin binding domain of Shot is required for the specific trafficking to the proximal region of an axon (Kinoshita T et al. 2017. Sci Reports. 7: 41455).

118. Crosstalk between brain glycome and social behavior: brain glycome mapping of genetically modified mouse model

Jua Lee 1 , Jae Keung Yun 1 , Boyoung Lee 2 , Hee-Sup Shin 2 , Hyun Joo An 1; 1Graduate School of Analytical Science and Technology, Chungnam National University, 34134, Daejeon, South Korea, 2Center for Cognition and Sociality, 34133, Institute for Basic Science, Daejeon, South Korea

hjan@cnu.ac.kr

Glycan plays numerous roles in the nervous system during development, regeneration and synaptic plasticity. Despite the abundance of immunohistochemical evidence linking glycosylation with crucial brain functions, molecular investigation into the brain glycome has languished in relative obscurity due to the lack of effective analytical method. In this study, for the first time to determine regional specific glycome, we have analyzed 9 mouse brain regions (cerebral cortex, prefrontal cortex, striatum, hippocampus, olfactory bulb, diencephalon, midbrain, pons-medulla, cerebellum) using highly sensitive mass spectrometry-compatible method which is the pairing of tissue membrane extraction and structure-specific nano-LC/MS with microgram-level sensitivity. Structural information was further obtained by LC/MS/MS. Approximately one hundred N-glycans and gangliosides were identified in total with several unique “brain type” characters. Based on the profile of both glyco-conjugates, we proposed global ‘Brain Glycome Map’ covering biosynthetic pathways for the purpose of utilizing it as reference data to monitor alteration of glyco-conjugates in mouse brain whose candidate risk genes for neurodevelopmental disorders are deleted. For example, PLCβ1, whose decreased expression has been observed in the brains of schizophrenia patients in previous studies, yet still with weak evidence at molecular level was selected as the first target gene. We profiled glyco-conjugates in schizophrenia-associated PLCβ1-knockout mice brain. As a result, dramatic alteration of N-glycan profiles in knockout mouse were identified, uncovering disease-specific N-glycan synthesis trajectories. Changes in N-glycan signature related to disease context could lead to following insights into the glycosylation machinery, illustrating which glycosyltransferases or glycosidases may be involved in the etiology of schizophrenia. Glycosyltransferase candidates were listed for further investigation of expression and biological activity between KO and WT mouse lines. Complementing these findings with additional proteomics and biological tools would help gaining a broader picture, i.e., insights into linkage of glycosylation with the physiological and pathological function of the knockout mouse brain.

Session 26. Glycosylation disorder diseases

Keynote Lectures

119. Mutations in OGT are causual for X-linked intellectual disability

Nithya Selvan, Krithika Vaidyanathan, Stephan George, Peng Zhao, Charles Schwartz, Lance Wells 1; 1University of Georgia, USA

lwells@ccrc.uga.edu

O-GlcNAc transferase (OGT) appears to be the sole mammalian enzyme responsible for O-GlcNAc modification of serines and threonines on over two thousand nuclear and cytosolic proteins. Recently, six families with XLID have been discovered that all have different missense mutations in OGT. Interestingly, all of the variants are in the TPR domains of OGT that are thought to be involved in protein-protein interactions that likely help to target OGT to appropriate substrates. Here we present our data biochemically characterizing the variants with regards to stability, O-GlcNAc transferase activity, and HCF-1 cleavage activity. Further, we present our preliminary data using Cas9-engineered male hES cells, which contain the mutations. Finally, we will present our working model that the interactome of the OGT variants is disrupted in a cell-type specific manner.

Lectures

120. Accelerated aging and turnover of host anti-inflammatory enzymes contributes to the pathogenesis of gram-negative sepsis

Won Ho Yang 1 , Douglas M. Heithoff 2 , Peter V. Aziz 1 , Michael J. Mahan 2 , Victor Nizet 3 , Jamey D. Marth 1; 1Sanford Burnham Prebys Medical Discovery Institute, Center for Nanomedicine, 93106, Santa Barbara, United States, 2University of California Santa Barbara, Molecular Cellular and Developmental Biology, 93106, Santa Barbara, United States, 3University of California San Diego, Department of Pediatrics and Skaggs School of Pharmacy and Pharmaceutical Sciences, 92093, La Jolla, United States

wyang@sbpdiscovery.org

Recent discoveries by this laboratory identified a mechanism of secreted protein aging and turnover that is composed of glycosidase–mediated N-glycan remodeling (Yang et al., 2015). We now find that this mechanism is modulated in mouse models of sepsis caused by infection with the human bacterial pathogens Salmonella enterica serovar Typhimurium and Escherichia coli, and moreover differently from sepsis caused by the Gram-positive pathogens Streptococcus pneumoniae and Staphylococcal aureus. During the onset of sepsis, we have measured an increased rate of N-glycan remodeling with ensuing endocytic lectin ligand formation among secreted proteins in the blood plasma, resulting in rapid decreases in the abundance and function of two host anti-inflammatory enzymes, namely tissue non-specific and intestinal alkaline phosphatases (TNAP and IAP). Our findings demonstrate that both of these secreted glycoprotein enzymes are post-translationally regulated by sialylation as a means of determining their concentrations and activities in the blood. Our data further reveal that the endogenous sialyltransferase ST3Gal6 is responsible for TNAP and IAP sialylation, without which both alkaline phosphatase isozymes are rapidly cleared from circulation by the Ashwell-Morell receptor (AMR). This accelerated remodeling of TNAP and IAP in sepsis is due to the induction of host neuraminidase activity in the blood. This induction includes Neu1 and Neu3 and can be recapitulated by the lipopolysaccharide component of Gram-negative bacteria, thereby accelerating the aging and turnover of the anti-inflammatory

enzymes TNAP and IAP. We further show that the induction of Neu activity with the subsequent reduction of alkaline phosphatase activity are responsible for increased LPS-phosphate levels linked with inflammation and reduced survival of Gram-negative sepsis. These findings demonstrate unexpected features of pathogen and host interactions during sepsis that target an intrinsic host mechanism of secreted protein aging and turnover. The resulting rapid changes in protein abundance and function among the secreted proteome have significant impacts on the pathogenesis and survival of sepsis.

121. Application of lectin-based protein microarray in glyco-biomarker analysis of congenital disorder of glycosylation

Jaroslav Katrlík 1 , Martina Križáková 1 , Zuzana Pakanová 1 , Jana Ziburová 1 , Marek Nemčovič 1 , Peter Baráth 1 , Sergej Šesták 1 , Ján Mucha 1; 1Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38, Bratislava, Slovakia

katrlik@yahoo.com

Protein microarray based on optical detection enables effective high-throughput analysis of various biointeractions having large application potential in biomedicine, biology and biotechnology including determination of biomarkers. In the combination with lectins or other glycan binding probes is this platform powerful also in glyco-profiling of samples and analysis of glyco-biomarkers. Developed lectin-based microarray platform was used for the study of glycosylation in both serum and isolated transferrin samples from patient with congenital disorder of glycosylation (CDG) and from healthy controls. The samples were spotted and incubated with panel of biotinylated lectins. We have determined significant differences in lectin reactivity in patient samples vs. healthy controls for both kinds of samples, mostly in the case of mannose binding lectins. Analysis of isolated transferrin showed higher differences between patient vs. control samples than analysis of serum samples. Although interaction with lectins does not allow accurate identification of glycan structures, the microarray platform is suitable for rapid screening to detect changes or glycosylation abnormalities for a large number of samples making this method very useful in determining glyco-biomarkers. Identification of glycan structures on samples by MS methods confirmed glycosylation changes detected by microarray. MALDI TOF-MS and LC-MS/MS technologies were applied in the N- glycoprofiling of patient serum sample and purified serum transferrin and O- glycoprofiling of apolipoprotein CIII (apo-CIII). Other examples of analysis of glyco-biomarkers by lectin-based microarray are introduced as well.

Acknowledgement: This work was supported by the projects APVV-14-0753, VEGA 2/0162/14 and COST Action CA16113.

122. Carbohydrate-binding domain of the POMGnT1 stem region modulates O-mannosylation sites of α-dystroglycan

Naoyuki Kuwabara 1 , Hiroshi Manya 3 , Takeyuki Yamada 3 , Hiroaki Tateno 4 , Motoi Kanagawa 5 , Kazuhiro Kobayashi 5 , Keiko Akasaka-Manya 3 , Yuriko Hirose 6 , Mamoru Mizuno 6 , Mitsunori Ikeguchi 2 , Tatsushi Toda 5 , Jun Hirabayashi 4 , Toshiya Senda 1 , Tamao Endo 3 , Ryuichi Kato 1; 1High Energy Accelerator Research Organiztion (KEK), Institute of Materials Structure Science (IMSS), Structural Biology Research Center (SBRC), 300-3257, Tsukuba, Japan, 2Yokohama City University, Graduate School of Medical Life Science, 230-0045, Yokohama, Japan, 3Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, 173-0015, Itabashi-ku, Japan, 4National Institute of Advanced Industrial Science and Technology (AIST), Biotechnology Research Institute for Drug Discovery, 305-8568, Tsukuba, Japan, 5Kobe University, Graduate School of Medicine, 650-0017, Kobe, Japan, 6The Noguchi Institute, Laboratory of Glyco-organic Chemistry, 173-0003, Itabashi-ku, Japan

naoyuki.kuwabara@kek.jp

The dystrophin glycoprotein complex, which connects the cell membrane to the basement membrane, is essential for a variety of biological events, including maintenance of muscle integrity. An O-mannose-type GalNAc-β3-GlcNAc-β4-Man(-6-phosphate) (core M3) structure of α-dystroglycan (αDG), a subunit of the complex that is anchored to the cell membrane, interacts directly with laminin in the basement membrane. Hypo-glycosylation of αDG is linked to some types of inherited muscular dystrophy; consistent with this relationship, many disease-related mutations have been detected in genes involved in O-mannosyl glycan synthesis. Defects in protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), a glycosyltransferase that participates in the formation of GlcNAc-β2-Man glycan, are causally related to muscle-eye-brain disease (MEB), a congenital muscular dystrophy, although the role of POMGnT1 in post-phosphoryl modification of core M3 glycan remains elusive. We found that N-terminal domain of POMGnT1 (called stem domain) recognizes the β-linked GlcNAc of O-mannosyl glycan, an enzymatic product of POMGnT1. This interaction may recruit POMGnT1 to a specific site of α-DG to promote GlcNAc-β2-Man (core M1) clustering and also may recruit other enzymes that interact with POMGnT1, e.g., FKTN which is required for ribitol-phosphate modification of the core M3 glycan that is the first step of post-phosphoryl modification of core M3 glycan. These findings explain how POMGnT1 attaches GlcNAc-β to clustered O-mannose sites and influences post-phosphoryl modification of core M3. Our study provides important insight into how disease-associated mutations cause inherited muscular dystrophy pathogenesis.

123. N-Glycosylation profile of IgG in metabolic syndrome: a cross-sectional study in a real-life community-based chinese cohort

Youxin Wang 1 , Wei Wang 2 , Lucija Klarić 3 , Jing Dong 4 , Maja Pučić-Baković 3 , Di Liu 1 , Ivo Ugrina 3 , Manshu Song 1 , Manshu Song 1 , Xiuhua Guo 1; 1Beijing Municipal Key Laboratory of Clinical Epidemiology, School of Public Health, Capital Medical University, Beijing 100069, China, 2School of Medical and Health Sciences, Edith Cowan University, Perth WA 6027, Australia, 3Genos Glycobiology Research Laboratory, Zagreb 10000, Croatia, 4Center for Physical Examination, Xuanwu Hospital, Capital Medical University, Beijing 100050, China

wei.wang@ecu.edu.au

Background: Glycosylation is the most abundant, diverse and complex form of post-transcriptional modification and participates in multiple physiological processes. We previously demonstrated the association between plasma N-glycome and MetS. This study aims to further explore the association between MetS components and N-glycome of immunoglobulin G (IgG), a protein that is closer to biological inflammatory function.

Methods: A cross-sectional study including 891 adults from Xicheng community of Beijing, China was performed to explore the association between IgG glycosylation and MetS components.

Results: Among 77 IgG N-glycan traits, 5 digalactosylation traits (GP12, GP12n, GP17, GP20, GP13n) and 3 fucosylation traits (Fn total, FG2n total/G2n, and Bn/(Fn+FBn)) are associated with at least 3 of the 5 MetS components (waist circumference, blood pressure, total triglycerides, high density lipoprotein and fasting blood glucose) in Chinese Han population (gender and age adjusted correlation r: -0.145 to -0.086; P: 0.001 to 0.047). Furthermore, canonical correlation analysis (CCA) shows that 5 IgG galactosylation traits (GP6, GP6n, GP14, GP14n and G2n) prominently associate with all 5 MetS components (r=0.55, P<0.001).

Conclusion: The present findings demonstrate that variations in galactosylation and fucosylation of IgG associate with MetS. The abnormal glycosylation profile of IgG in MetS represents a potentially intermediate phenotype of inflammatory pathogenesis in MetS, might predicating high susceptibility to cardiovascular disease and other inflammatory diseases.

POSTER ABSTRACTS

Glycans in Signaling

124. The neurotrophic properties of GM1 oligosaccharide: a new promising story

Elena Chiricozzi 1 , Diego Yuri Pomè 1 , Margherita Maggioni 1 , Erika Di Biase 1 , Chiara Parravicini 2 , Ivano Eberini 2 , Sandro Sonnino 1; 1University of Milano, Department of Medical Biotechnology and Translational Medicine, 20090. Segrate, Italy, 2University of Milano, Department of Pharmacological and Biomolecular Sciences, 20133, Milano, Italy

elena.chiricozzi@unimi.it

Experimental evidence, both in vitro and in vivo, highlight the neurotrophic and neurodifferentiative effects of ganglioside GM1. So far, the molecular mechanism underlining the GM1 neuro activities is still unknown. In order to clarify the molecular mechanism by which GM1 exerts its neurotrophic action we investigated the role of its oligosaccharide portion. GM1 oligosaccharide was added to the culture medium of neuronal cells. To study the neurodifferentiation, cells were followed over the time to assess both morphological parameters and biochemical markers. In murine neuroblastoma cell lines we found that the oligosaccharide chain of GM1 is directly involved in the processes of neuronal differentiation by inducing TrkA-MAPK pathway activation. In this cell line, the GM1 oligosaccharide chain and TrkA receptor showed a direct interaction at the plasma membrane level. Using mouse primary neurons, we highlighted that GM1 oligosaccharide is able to influence the differentiation processes by accelerating the neuronal differentiation from both morphological and biochemical view. We surmise that the neurotrophic effect of GM1 is due to a direct interaction between the oligosaccharide chain and TrkA receptor. Trying to define the molecular mechanism of GM1, we have potentially found a new neurotrophic player, which in vitro is able to influence differentiation of mice primary neurons.

125. The wound healing effect of β-glucan

Gayoung Seo 1 , Hyeran Yu 1 , Karthika Muthuramalingam 1 , Yeungmi Kim 1 , Moonjae Cho 1; 1Jeju National University, Department of medicine, 63243, Jeju, Korea

moonjcho@jejunu.ac.kr

Wound healing is an intricate process whereby re-epithelialization and dermal contraction initiates after injury. We investigated the effect of β-glucan on the migration and viability of keratinocyte or fibroblast using the in vitro MTT assay, migration assay, invasion assay and western blotting as well as in vivo experiment. The migration of keratinocytes treated with 0.5% β-glucan was increased after 24 hours as well as proliferation. The FAK/Src pathway has well known to affect cell migration by forming lamellipodia. Β-glucan also promotes phosphorylation of FAK/Src in time-dependent. ROS is associated with fibroblast differentiation to contract dermal layer and synthesize collagens. We found that fibroblast was activated by increasing NOX expression.

We propose that β-glucan is capable of promoting keratinocyte migration via the induction of FAK/Src phosphorylation resulting acceleration of wound closure and activating dermal fibroblast differentiation through NADPH oxidase for matrix remodeling.

126. Mechanical cues-induced Skp2 expression mediates YAP-dependent cell cycle exit and oncogenic functions

Wonyul Jang 2 , Tackhoon Kim 2 , Ja Seung Koo 1 , Sang-Kyum Kim 1 , Dae-Sik Lim 2; 1KAIST, Department of Biological Science, Daejeon, Korea, 2Yonsei University College of Medicine, Department of Pathology, , Seoul, Korea

relago12@kaist.ac.kr

Mechanical tensions are usually generated during development at spatially defined regions within tissues. Such physical cues dictate the cellular decisions of proliferation or cell-cycle arrest. Yet, how mechanical stress controls cell cycle is not fully understood. Here, we report that mechanical cues function upstream of Skp2 transcription. We found that YAP, the mechano-responsive oncogenic Hippo signaling effector, directly promotes Skp2 transcription. YAP inactivation induces cell cycle exit (G0) by down-regulating Skp2, causing p21/p27 to accumulate. Both Skp2 reconstitution and p21/p27 depletion can rescue the observed defect in cell cycle progression. In context of a tissue-mimicking 3D culture system, Skp2 inactivation effectively suppresses YAP-driven oncogenesis and aberrant stiff 3D matrix-evoked epithelial tissue behaviors. Finally, we also found that the expression of Skp2 and YAP are positively correlated in breast cancer patients. Our results not only reveal the molecular mechanism by which mechanical cues induce Skp2 transcription, they also uncover a role for YAP-Skp2 oncogenic signaling in the relationship between tissue rigidity and cancer progression.

Glycans in Metabolism

127. Elucidation of the order of glycan acquisition by a phylogenetic profile analysis of human glycoside hydrolases

Takahiro Nakamura 1 , Jun Tanaka 1 , Hisao Kojima 1 , Masahiro Ito; 1Ritsumeikan University, Graduate of Life Science, Department of bioinformatics,525-8577,Shiga, Japan

sj0030hp@ed.ritsumei.ac.jp

Glycans with varying structures, which are related to their biological functions, are produced by glycosyltransferases and glycoside hydrolases. Recently, a large amount of functional data on glycoside hydrolases has been collected and has been stored in databases, but a genome-wide analysis of glycoside hydrolases has not yet been conducted. Herein, a phylogenetic profile analysis was performed on human glycoside hydrolases as the genome-wide approach in order to elucidate the in vivo function of human glycans. Data on the human glycoside hydrolases were obtained from three databases—UniProt, CAZy, and BRENDA. Orthologs of these glycoside hydrolases were confirmed in organisms with decoded genomes, and clustering was performed according to the presence or absence of the ortholog.

Consequently, the human glycoside hydrolases were classified into four classes: class 1, conserved only in chordates; class 2, conserved only in metazoans; class 3, conserved in metazoans/plants; class 4, widely conserved in eukaryotes. Focusing on the biological function of glycoside hydrolases of each class, the class 1/2 enzymes comprised secreted proteins involved in fertilization, protection against bacterial infection, and remodeling of extracellular matrix. This suggests that multicellular systems tuned by glycans, arisen by glycoside hydrolases, were acquired in the process of evolution into metazoans. With regard to glycoside hydrolases related to N-glycans, enzymes processing high mannose type glycans were in class 4, enzymes involved in the degradation of complex glycans were in class 2, and the endo-type degradation enzyme was in class 1. These results suggest that N-glycans have diversified among eukaryotes with the formation of more complex glycan structures as evolution progressed towards chordates. In glycosphingolipids, degradation of the glycan residues at the reducing end is catalyzed by a glycoside hydrolase that is widely conserved in eukaryotes and the non-reducing terminal glycan is degraded by an evolutionarily novel glycoside hydrolase; in other words, the glycosphingolipid glycan may have elongated through evolution. These results support the aforementioned hypotheses on N-glycan evolution and glycosphingolipid elongation.

128. Association between immunoglobulin G N-glycome and central obesity: a cross-sectional study in Chinese Han population

Di Liu 1 , Wei Wang 1 , Lucija Klarić 2 , Xi Chu 3 , Maja Pučić-Baković 2 , Zhongyao Zhao 1 , Ivo Ugrina 2 , Qi Sun 1 , Gordan Lauc 2 , Youxin Wang 1; 1Beijing Municipal Key Laboratory of Clinical Epidemiology, School of Public Health, Capital Medical University, Beijing 100069, China, 2Genos Glycobiology Research Laboratory, Zagreb 10000, Croatia, 3Center for Physical Examination, Xuanwu Hospital, Capital Medical University, Beijing 100050, China

wangy@ccmu.edu.cn

Objective: Glycosylation significantly affects structure and function of proteins, and thus participates in multiple physiology and pathologic process. N-Glycosylation of immunoglobulin G (IgG) has been observed to be associated with obesity determined by body mass index (BMI) in European populations. The study aims to investigate the association between IgG N-glycosylation and central obesity measured by waist-to-hip ratio (WHR) in a Chinese population. Method: We recruited 913 Chinese Han individuals from a community-based cohort in Xicheng District, Beijing, China. Plasma IgG N-glycome was determined by hydrophilic interaction liquid chromatography (HILIC). Results: FBG2n/G2n (fucosylation with bisecting GlcNAc of digalactosylated structures: r=0.113, P=0.007) and G1n (monogalactosylated structures: r=0.084, P=0.045) were positively correlated with WHR after controlling for gender and age. In addition, the IgG fucosylation was significantly higher in participants of central obesity than these of the normal WHR. Conclusions: The addition of fucose to IgG N-glycan is moderately correlated with increased WHR, might suggest that IgG N-glycosylation might be the link between obesity and increased cardiovascular risk.

Glycogene Expression and Regulation

129. Inhibitory effect of tumor necrosis factor-α on gene expression of renal neuraminidase, Klotho in radiation-induced senescence of renal epithelial cells

Eun Ju Kim 1 , Da Yeon Kim 1 , Minyoung Lee 2; 1Korea Institute of Radiological and Medical Sciences, Division of Basic Radiation Sciences, 01812, Seoul, Korea, 2Korea Institute of Radiological and Medical Sciences, Laboratory Animal Center, 01812, Seoul, Korea

mylee@kirams.re.kr

Radiation nephropathy could occur after accidental exposures of radiation. Moreover, radiotherapy for lymphoma, gynecologic cancers, and sarcomas of the upper abdomen may result in radiation-induced kidney injury. Radiation nephropathy generally progresses slowly, over a period of several years. However, the exact pathogenesis and/or mediators involved in radiation nephropathy remain under investigation. Ionizing radiation induces senescence, which involves various activities of senescence associated secretory phenotype. As the senescent cell fraction following irradiation is known to increase, we examined whether ionizing radiation causes the renal injury by inducing cellular senescence. We assessed the effects of ionizing radiation on canine renal epithelial cells (MDCK), which mostly used in in vitro renal cell models. We found that irradiation on MDCK cells induced cellular senescence. Remarkably, Klotho, a secretory renal neuraminidase that is essential for the function of fibroblast growth factor, was significantly decreased after irradiation in MDCK cells. Lectin affinity analysis determined that these reduction of klotho increased the sialylated form of epithelial calcium channel in MDCK cells. Furthermore, Klotho expression was decreased in kidney tissues of radiation-exposed mice. Tumor necrosis factor- α (TNF-α) production was increased in irradiated MDCK cells and also renal tissues of mice. We proposed that reduction of klotho gene expression could possibly associated with negative regulatory effect of TNF-α in radiation-exposed renal cells/or tissues. Therefore, our data suggest that increase of TNF-α and their inhibitory activity on Klotho gene expression following irradiation may contribute to the development of renal dysfunction through the acceleration of cellular senescence.

Glycotechnology

130. Intact glycopeptide analysis of targeted serum haptoglobin for biomarker discovery in gastric cancer

Seunghyup Jeong 1 , Unyong Kim 1 , Hyun Joo An 1; 1Chungnam National University, Graduate School of Analytical Science and Technology, 34134, Daejeon, Korea

hjan@cnu.ac.kr

Gastric cancer is one of the most common malignancy and leading cause of cancer death. Classically, CEA and CA19-9 have been used for gastric cancer detection and screening. However, CEA and CA19-9 insufficient for cancer detection due to low specificity and sensitivity. Glycosylation is the most common post-translational modification and plays an important role in various biological processes. Whole serum based glycan profiling has been already developed and widely used in cancer biomarker study. However, targeted glycoproteomic approach is needed in clinic for better sensitivity and specificity. In previous study, we targeted serum haptoglobin, a positive acute-phase protein with immunomodulatory properties, for gastric cancer. We found glycosylation changes of haptoglobin between gastric cancer patient and healthy control. Basically, released glycan analysis provide compositional and isomer information while cannot give actual site of glycosylation. On the other hand, intact glycopeptide analysis provide site-specific information. Based on this actual glycosylation site and glycan heterogeneity, we monitored more detailed glycan changes and aberrant glycosylation of each site for more sensitive and specific diagnosis. In this study, we have developed the method for intact glycopeptide analysis for biomarker discovery. First, haptoglobin was denatured and alkylated by DTT and IAA. Afterward, sample was treated by trypsin and analyzed by UHPLC Q-TOF MS without glycopeptide enrichment and purification. Haptoglobin has four glycosylation sites and three glycopeptides. We successfully separated glycopeptides and profiled the glycoform of GP1 and GP3. GP2 [NLFLNHSENATAK] was difficult to profile glycoform and cannot be obtained site-specific information according to two glycosylation sites. GP2 was fractionated and treated non-specific protease pronase E to generate site-specific glycopeptides. Further, cancer patient (n=40, stage IV) and healthy control (n=47) samples were applied for training set of biomarker study. A T-test based analysis was performed to identify potential biomarker (p<0.001). Ten potential glycopeptide biomarker and AUC to conclude patients and controls were 0.70 to 0.81. In further study, we will apply to large clinical sample to validate potential biomarker.

131. Two-component supramers strongly inhibiting influenza virus-to-cell adhesion in vivo

Alexander Chinarev 1 , Alexander Tuzikov 1 , Nicolai Bovin 1; 1Shemyakin&Ovchinnikov Institute of Bioorganic Chemistry (Russian Academy of Sciences), Carbohydrates Lab, 117997, Moscow, Russia

chinarev@carb.ibch.ru

We synthesized glycopeptides, [6'SLN-linker-Glyn-NHCH2]4C (6'SLN is Neu5Acα2-6Galβ1-4GlcNAcβ-; n≥7), which due to H-bonding spontaneously assemble in aqueous media into stable extended sheet-like supramers. Surfaces of the formed supramers are densely covered with multiple copies of 6'SLN residues; 6'SLN was recognized as receptor for human influenza viruses in the course of their adhesion to target cells. Tight multivalent binding occurs between the glycosupramers and the viruses, which leads to steric blockage of the virus particles and inhibition their interaction with the cells. Thus, antiviral activity of [6'SLN-linker-Gly7-9-NHCH2]4C measured in vitro was up to 100 times higher in comparison to 6'SLN. Also, we studied how the surface density of 6'SLN residues influence the binding of the virus with supramers. We synthesized succinylated peptide [Suc-Gly9-NHCH2]4C (Suc is HOOC(CH2)2CO-) and used it as "diluting" component upon formulation of blends with the glycopeptide [6'SLN-linker-Gly9-NHCH2]4C. Hydrophilic succinyl group does not disturb to the assembly and rise the supramers solubility in water. Passing from 1:1 to 1:20 molar ratios between the glycosylated and the succinylated components we noticed an additional tenfold rise of inhibitory activity of the resultant supramers. Finally, we estimated antiviral potency of the binary supra-molecular system, [6'SLN-linker-Gly9-NHCH2]4C/[Suc-Gly9-NHCH2]4C, 1:20, in vivo on ferret model with a contemporary human H1N1 strain. Intranasal inoculations of the infected animals (once a day, seven days, dosage 200-2000 μg/kg) allowed extenuating the clinical signs of the disease, and reducing the virus titers in nasal washings. Noteworthy, no signs of toxicity were observed; the described supramoluecular inhibitor was only slightly less potent than Zanamivir, widely accepted antiflu medication, used in the experiment as a reference.

132. Genetically engineered tissue to screen for glycan function in tissue formation

Maria Adamopoulou 1 , Emil M. Pallesen 2 , Asha Levann 2 , David Thein Aagaard 2 , Troels Boldt Rømer 2 , Irina N. Marinova 2 , Mathias Ingemann Nielsen 2 , Sally Dabelsteen 1 , Hans H. Wandall 2; 1University of Copenhagen, Department of Odontology, 2200, Copenhagen, Denmark, 2University of Copenhagen, Copenhagen Center for Glycomics, 2200, Copenhagen, Denmark

maad@sund.ku.dk

Glycans present a huge structural diversity with species and cell-type specificity that underlie specific biological functions. While, the glycome is a difficult entry point for discovery, the glycogenome is a feasible entry point, because most of the genes controlling glycosylation are now known. We use genetic engineering with CRISPR/Cas9 combined with 3D organotypic skin models to examine how distinct glycans influence epithelial formation. We have performed knockout and knockin of more than 100 select genes in the genome of human immortalized human keratinocytes, enabling a systematic analysis of the impact of specific glycans in the formation and transformation of the human skin. The genetic engineered human skin models (GlycoSkin) was designed with and without all major biosynthetic pathways in mammalian glycan biosynthesis, including GalNAc-O-glycans, O-fucosylation, O-mannosylation, with and without complex N-linked glycans, and with and without elongated glycosphingolipids. We believe that this is the first time tissue is developed presenting a repertoire of all human glycan structures in a combinatorial design presenting all possible glycoforms in their native environment. Such genetic engineered GlycoSkin tissue models can be used to study biological interactions involving glycan structure on lipids, or glycosaminoglycans. This engineering approach will allow us to investigate the functions of glycans in homeostasis and elucidate the role of glycans in normal epithelial formation, transformation and host pathogen interactions.

133. Luteolin reduces mucin-type O-Glycosylation through on-target inhibition of ppGalNAc-Ts

Feng Liu 1 , Kai Xu 1 , Zhijue Xu 1 , Yueyang Zou 1 , Xing Li 1 , Fang Wu 1 , Yan Zhang 1; 1Ministry of Education Key Laboratory of Systems Biomedicine,Shanghai Center for Systems Biomedicine (SCSB), Shanghai Jiao Tong University, Shanghai, China, 200240

liu89liu89liu@163.com

Mucin-type O-glycosylation is initiated by the members of polypeptide N-acetyl-α-galactosaminyltransferases (ppGalNAc-Ts) family and closely involved in physiological and pathological conditions. The lack of direct and selective inhibitors of ppGalNAc-Ts has largely impeded research progress regarding the molecular aspects of mucin-type O-glycosylation. Herein we identified eight ppGalNAc-T2 inhibitors with IC50 values ranging from 5 nM to 15 μM from ~5000 commercial compounds through an enzyme-coupled high-throughput screening assay. Among these, luteolin showed potent inhibitory effect in cells by lectin analyses. Kinetic analyses revealed that luteolin acted as a protein substrate-competitive inhibitor of ppGalNAc-T2 and exhibited selectivity within ppGalNAc-T family.

We first reported that luteolin could reduce mucin-type O-glycosylation by inhibiting ppGalNAc-Ts and further optimization based on luteolin may provide more potent and specific inhibitor for ppGalNAc-Ts.

134. Metallic element chelated tag labeling (MeCTL) for quantitation of N-Glycans in MALDI-MS

Lijun Yang 1 , Haojie Lu; 1Fudan University, Department of Chemistry and Institutes of Biomedical Sciences, 200032, Shanghai, P. R. China

luhaojie@fudan.edu.cn

N-glycosylation, as one of the most important and widespread PTMs of proteins, is involved in many physiological functions and biological pathways. Quantifying N-glycan is important since its alterations are related to a variety of human diseases, such as tumor progression and inflammation. In this study, we develop a novel N-glycan quantitative methods ( MeCTL) using metallic element chelated tag through reductive amination. MeCTL strategy not only provides accurate quantitation and help N-glycan ionized well in negative ion reflection mode, which provides more cross-ring fragmentation to distinguish N-glycan isomers. At least this strategy has been successfully used for analysis of N-glycan changes in human serum associated with CRC. The result showed this method has the potential application in clinical investigations in disease N-glycome profiling and relative quantitation.

135. Chemo-enzymatic synthesis of the sequence-regulated keratan sulfate oligomers by keratanase II-catalyzed transglycosylation

Yuji Yamazaki 1 , Shunsaku Kimura 1 , Masashi Ohmae; 1Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

ohmae@peptide.polym.kyoto-u.ac.jp

Keratan sulfate (KS) is a member of glycosaminoglycans, which consists of alternating repeats of (1,4)-β-Gal and (1,3)-β-GlcNAc 6-sulfate (GlcNAc6S); the Gal residue is partly sulfated at the C6, and the GlcNAc6S has sometimes an α-Fuc branch at the C3. The Gal residue at the non-reducing terminal of KS chain is capped by a variety of monosaccharides such as Neu5Ac, GlcNAc6S, etc. The repeating unit bearing (1,3)-α-Fuc is 6-O-sulfonato-Lewis X {Galβ(1,4)[Fucα(1,3)]GlcNAc6S; su-LeX}, which is found in selectin ligands and on cancer cell surfaces. In addition to these modifications, KS connects to the different types of the linkage oligosaccharides between the KS chain and the core protein, leading to higher structural diversity of KS. It is believed that physiological functions of KS depend on such structural diversity, which leads to difficulty in investigation of structure-activity relationships of KS. In order to unveil KS functions, it is essential to establish a facile and efficient method to prepare KS with well-defined structure.

In this study, we demonstrated the synthesis of the sequence-regulated KS derivatives having su-LeX units in appropriate orders by keratanase II (KSase II)-catalyzed transglycosylation. The oxazoline derivatives of 6-O-sulfonato-LacNAc [Galβ(1,4)GlcNAc6S; su-LacNAc] (1) and su-LeX (2) were effectively catalyzed by KSase II, affording the corresponding oligomers in a regio- and stereo-selective manner. The enzymatic glycosylations also proceeded effectively by using 1 and 2 as donors and the su-LacNAc and the su-LeX having an azido-oligoethyleneoxide linker (3 and 4, respectively) as acceptors, providing the corresponding glycosides (1+4 and 2+3). Notably, the su-LeX trimer derivative was obtained by the enzymatic glycosylation of 4 with the dimeric su-LeX (2+2) oxazoline donor. These results clearly indicate that sequence-regulated KS oligosaccharides can be synthesized by stepwise chain-elongation reaction catalyzed by KSase II.

Glycobiology in Animal Model System

136. The putative polypeptide N-Acetylgalactosaminyltransferase 18 attenuates endoplasmic reticulum stress during the LPS-induced neuroinflammation

Aidong Shan 1 , Jishun Lu, Zhijue Xu, Xing Li, Yingjiao Xu, Yan Zhang; 1Shanghai Jiao Tong University, China

aidong2006@sina.cn

UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase 18 (ppGalNAc-T18) is a distinct member of the vertebrate-specific Y subfamily of ppGalNAc-Ts. Unlike most of the other ppGalNAc-Ts localized in the Golgi apparatus, ppGalNAc-T18 is predominantly distributed on endoplasmic reticulum (ER). Furthermore, this enzyme is ubiquitously expressed in human tissues but lacks the catalytic activity. Previously, our study has shown that down-regulation of ppGalNAc-T18 expression in human lung adenocarcinoma epithelial A549 cell could alter cellular O-glycosylation, cell morphology and proliferation. Furthermore, ppGalNAc-T18 could modulate the catalytic activity of ppGalNAc-T2 and -T10 in vitro. In spite of these findings, the exact role of ppGalNAc-T18 in ER still remains obscure.

In this study, we found that concomitant with the enhanced expression of ER stress-associated proteins, ppGalNAc-T18 expression was specifically up-regulated in the neuroinflammation models including the mice induced by lipopolysaccharide (LPS) and the neuron-like PC12 cells treated with different pro-inflammatory stimuli. Overexpression of ppGalNAc-T18 in PC12 cells significantly inhibited the ER stress and cell apoptosis induced by pro-inflammatory stimuli. Conversely, silencing of ppGalNAc-T18 in PC12 clearly decreased the O-glycosylation level and led to the ER stress and cell apoptosis without any stimulation. Furthermore, loss of ppGalNAc-T18 in the nervous system of mice aggravated the ER stress induced by lipopolysaccharide injection. These results clearly demonstrate that ppGalNAc-T18 may attenuate the ER stress during neuroinflammation, which not only suggests the significance of this putative ppGalNAc-T isoform in cells, but provides new insights into the molecular mechanisms linking neuroinflammation to ER stress.

Glycoprotein Quality Control and Trafficking

137. Impaired autophagy and mitophagy in MPS II (Mucopolysaccharidosis Type II) disease models

Sungmi Kim 1 , Sungeun Kim 1 , Nanyoung Ko 1 , Sungho Hwang 1; 1MOGAM institute for Biomedical Research, Rare Disease Team, 16924, Yongin city, Korea

smkant@mogam.re.kr

Lysosomal storage disorders (LSDs) are caused by alterations of proteins involved in lysosomal degradation, and characterized by progressive accumulation of undigested cellular components within the cell due to lysosomal dysfunctions. The role of autophagy has been investigated in several LSDs, such as Multiple Sulfatase Deficiency (MSD), Mucopolysaccharidosis Type IIIA (MPS IIIA), GM1-Gangliosidosis, Pompe Disease (PD), Niemann-Pick Disease Type C (NPC), and Mucolipidosis Type IV. However, there has been no autophagic and cellular vesicular characterization study in Hunter Syndroms. MPS II is caused by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS, I2S), which leads to the accumulation of certain glycosaminoglycans (GAGs), dermatan sulfate and heparan sulfate. The purpose of this study is to address the cellular pathophysiology of MPS II by comparing with the patterns of matched controls. We characterized the cellular vesicilar phenotypes of IDS-deficient cells models of human MPS II. Biochemical analysis of MPS II-deficeitn cells revealed a progressive accumulation of cellular autophagic markers, Cox IV and mitofusin 1 (Mfn 1), also dramatically accumulated in IDS-deficient cells compared with normal cells. MPS II-deficient mice also showed similar results with increased levels of autophagic/mitophagic failure markers. Our characterization of IDS-deficient human cell line and -mouse model revealed an impaired cellular clearance pathway in MPS II, suggesting in view of the presently characterized cellular pathophysiology.

138. Identification of membrane topology of human DPY19L3 reveals that C-terminal region is essential for C-mannosyltransferase Activity

Yuki Niwa 2 , Yoshihiko Nakano 2 , Takehiro Suzuki 1 , Mizuo Yamagishi 2 , Kei Otani 2 , Naoshi Dohmae 1 , Siro Simizu 2; 1Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 223-8522, Yokohama, Japan, 2Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, 351-0198, Wako, Japan

niwa-yuki@keio.jp

C-mannosylation is a rare type of protein glycosylation in which α-mannose is enzymatically attached to the indole C2 carbon of a tryptophan residue via a C-C linkage in the endoplasmic reticulum (ER) lumen. Previously, we first identified human DPY19L3 as a C-mannosyltransferase for R-spondin1 at Trp156 (Mol. Biol. Cell, 27, 744-756, 2016). In the present study, we determined membrane topology of human DPY19L3, which is predicted to be multipass transmembrane protein and localizes to the ER membrane. To identify membrane topology of DPY19L3, we performed redox-sensitive luciferase assay using secretory luciferase (Gaussia luciferase) based on in silico analysis. As a result, we revealed that DPY19L3 comprises 11 transmembrane regions and 3 re-entrant loops with the N- and C-termini facing the cytoplasm and ER lumen, respectively. Human DPY19L3 has 4 N-glycosylation consensus sites at Asn118, Asn319, Asn439, and Asn704. We identified that DPY19L3 is N-glycosylated at Asn118 and Asn704 but not Asn319 and Asn439, supporting the accuracy of our topological model of DPY19L3; however, gain-of-function experiments using Drosophila S2 cells, which lack C-mannosyltransferase activity but are able to induce C-mannosylation by introducing DPY19L3, revealed that N-glycosylations at Asn118 and Asn704 are not required for its C-mannosyltransferase activity of DPY19L3. On the other hand, DPY19L3 has a splice variant, called isoform2, which lacks C-terminal luminal region of DPY19L3. We evaluated the C-mannosyltransferase activity of isoform2 by gain-of-function experiments and suggested that isoform2 has no C-mannosyltransferase activity. These results demonstrated that human DPY19L3 has C-terminal essential region for C-mannosyltransferase. The novel findings in this study provide the important insights for examining the mechanism of C-mannosylation.

139. Premelanosome protein (PMEL) is C-mannosylated at W104, W153 and W156

Ryosuke Komatsu 2 , Takehiro Suzuki 1 , Yuki Niwa 2 , Erina Shimizu 2 , Naoshi Dohmae 1 , Siro Simizu 2; 1Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 223-8522, Yokohama, Japan, 2Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, 351-0198, Wako, Japan

koma.ryoryo@keio.jp

In the melanosome, premelanosome protein (PMEL) is the functional protein as scaffolds for melanin synthesis and pigmentation. PMEL forms amyloid fibrils through aggregation of its fragments. In the previous studies, PMEL has several N-glycosylation and O-glycosylation sites and those are significant for fibril formation.

C-mannosylation is a unique type of protein glycosylation. The α-D-mannopyranose is attached to the first tryptophan residue in the consensus sequence, W-X-X-W/C (X represents any amino acid). PMEL has one predicted C-mannosylation consensus sequence, W153-K-T-W, in the N-terminal region (NTR) and W153 is probably C-mannosylated; however, the presence of C-mannosylation in PMEL has not been reported. In this study, we established a PMEL (NTR)-overexpressing CHO-K1 cell line and purified recombinant PMEL (NTR) protein from the cell cultured medium. Using mass spectrometry, we first demonstrated that PMEL is C-mannosylated at predicted W153. Moreover, PMEL is C-mannosylated at W104 and W156, which belong to W104-V-N-N and W156-G-Q-Y, respectively. To evaluate the role of C-mannosylation in PMEL on its function of fibril formation, we now establish full-length wild-type and C-mannosylation-defective mutants (W104F, W153F/W156F) PMEL-overexpressing melanoma cell lines. In conclusion, PMEL is C-mannosylated at W104, W153 and W156, and the C-mannosylation may associate with its processing and/or pigmentation.

Glycobiology in Plant

140. Expression and glycosylation of vaccine candidate for VHSV (Viral Hemorrhagic Septicemia Virus) recombinant protein in Nicotiana tabacum

Chun Ha Shin 1 , Kisung Ko 1; 1Therapeutic Protein Engineering Laboratory, Department of Medicine, College of Medicine, Chung-Ang University, 06974, Seoul, Korea

ksko@cau.ac.kr

Viral hemorrhagic septicemia (VHS) is world-wide important infectious disease caused by Viral hemorrhagic septicemia virus (VHSV). Viral hemorrhagic septicemia virus (VHSV) is the causative agent of serious systemic disease of fish, a disease that affects a number of teleost fish species including olive flounder (Paralichthys olivaceus). In this study, we focused on production of subunit vaccine, VHSV glycoprotein (G) including epitope by plant expression system. The recombinant gene, VHSV (G) FcK was fused to the immunoglobulin Fc fragment and extended with Endoplasmic Reticulum (ER) retention signal (KDEL) for oligomannose glycosylation. The recombinant expression vector was transformed into Agrobacterium tumefaciens (LBA4404) using electroporation method and plant transformation was conducted Agrobacterium-mediated plantlet method using tobacco plant (Nicotiana tabacum). Confirmation of gene insertion was performed by plant genomic DNA isolation and PCR analysis. VHSV (G) FcK protein expression was confirmed by western blot analysis. The Bioactivity of recombinant protein was analyzed through animal trials and ELISA. This study suggested to produce the fish vaccine using plant expression system.

This research was supported by agrant (Code#PJ011110) from the Korean Rural Development Administration, National Research Foundation of Korea Grant funded by the Korean Government (MEST)(NRF-2014R1A2A1A11052922).

141. Glycosylation and function of plant recycling for molecular biofarming to produce recombinant anti-cancer mAb

Deuk-Su Kim 1 , Ilchan Song 1 , Jinhee Kim 2 , Do-Sun Kim 2 , Kisung Ko 1; 1Therapeutic Protein Engineering Laboratory, Department of Medicine, College of Medicine, Chung-Ang University, 06974, Seoul, Korea, 2Vegetable Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju-gun, South Korea

ksko@cau.ac.kr

The expression and glycosylation patterns of anti-colorectal cancer therapeutic monoclonal antibody CO17-1A recognizing the tumor-associated antigen GA733-2, expressed in human colorectal carcinoma cells, were observed in the leaf and stem tissues of primary (0 cycle), secondary (1 cycle), and tertiary (2 cycle) growths of seedlings obtained from the stem cut of T2 plants. The bottom portion of the stem of T2 seedlings was cut to induce the 1 cycle shoot growth, which was again cut to induce the 2 cycle shoot growth. In the 1 and 2 cycle growths, the periods for floral organ formation (35 days) was shorter than that (100 days) for the 0 cycle growth. The levels of protein and glycosylation in the leaves and stem tissues from the 1 and 2 cycles were similar to those in the tissues from the 0 cycle. Surface plasmon resonance (SPR) confirmed that CO17-1A obtained from leaf and stem tissues of the 0, 1, and 2 cycles had similar binding affinity for the GA733-2 antigen. These data suggest that the shoot growth by bottom stem cutting is applicable to speed up the growth of plant biomass expressing CO17-1A without variation of expression, glycosylation, and functionality.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1A6A3A11930180), National Research Foundation of Korea Grant funded by the Korean Government (MEST) (NRF 2014R1A2A1A11052922).

142. New protein a agarose resins for purification of mAb from plants

Ilchan Song 1 , Su-lim Choi 2 , Hae Kyung Lee 3 , Kisung Ko 1; 1Therapeutic Protein Engineering Laboratory, Department of Medicine, College of Medicine, Chung-Ang University, 06974, Seoul, Korea, 2Biotech R&D center, Amicogen, Inc., Jinju 660-852, Korea, 3Division of zoonosis, Korea Centers for Disease Control and Prevention, Osong, Korea.

ksko@cau.ac.kr

New resins have been successfully developed for the purification of recombinant vaccines and monoclonal antibodies for immunotherapy. ProA™ (Amicogen) is a new protein A chromatography resin that is generally used for purification of the various antibody proteins including monoclonal antibodies, polyclonal antibodies and Fc fusion proteins. Protein A ligand of ProA™ is the native protein A originated from Staphylococcus aureus. Protein A is coupled to the epoxy activated 4% highly cross linked agarose bead. The epoxy group has a feature of attaching to hydroxyl group (-OH), amino group (-NH2), or sulfhydryl group (-SH) under different pH conditions. The binding capacity of protein A for plant-derived antibody proteins has been improved by coupling to agarose beads in the same direction with linearly erected, not zig-zag form, which enabled to capture more antibodies per protein A resin. The new resins were differenciated from other products in many points, showing high performance and durability and low price, respectively. These cost-effective mAb purification technology used Alkali tolerant protein A resins which could raise binding capacity and enable to reuse with no significant loss of binding capacity. And furthermore, this newly developed protein A resins can overcome high cost downstream process for purification of plant-derived recombinant antibody proteins.

This research was supported by agrant (Code#PJ011110) from the Korean Rural Development Administration, National Research Foundation of Korea Grant funded by the Korean Government (MEST)(NRF-2014R1A2A1A11052922).

143. Development of plant expression system to enhance efficacy of vaccine against colorectal cancer by modification of protein and glycan structures

Sohee Lim 1 , Kisung Ko 1; 1Therapeutic Protein Engineering Laboratory, Department of Medicine, College of Medicine, Chung-Ang University, 06974, Seoul, Korea

ksko@cau.ac.kr

The purpose of this study is to develop plant-based 'Clean Green Vaccine (CGV)' through optimization of vaccine protein and glycan structures for colorectal cancer and investigation of immunological mechanism of the optimized recombinant vaccine protein expressed in plant. The novel structures of the large quaternary GA733 (Gastrointestinal tumor-associated antigen) vaccine using IgA and IgM Fc were designed. The colorectal cancer antigen GA733 linked to the IgA or IgM Fc antibody fragment fused to KDEL, an ER retention motif. Since IgA and IgM are antibodies that are secreted primarily in the mucous membranes, the IgA and IgM Fc fusion vaccine can be applied to develop products that can be used for a variety of vaccines, such as the route of administration for edible, injectable, and patch vaccine products. The formation of large quaternary structure of the recombinant vaccine proteins using IgA or IgM type have not been attempted. The size of IgM fused vaccine is similar to 30 nm sizes of the virus. It is expected to have better vaccination effects than IgG fusion vaccine. This could be a new principle validation study about the effect of vaccination using the IgA and IgM. This study is applicable to the relevant knowledge to the next generation of new vaccine structure for a variety of cancer diseases, including other infectious diseases in plant expression system.

This research was supported by agrant (Code#PJ011110) from the Korean Rural Development Administration, National Research Foundation of Korea Grant funded by the Korean Government (MEST)(NRF-2014R1A2A1A11052922).

144. Expression and glycosylation of a human prostatic acid phosphatase (PAP)-IgM Fc fusion protein in plants using in vitro tissue subculture

Yang Joo Kang 1 , Kisung Ko 1; 1Department of Medicine, Therapeutic Protein Engineering Lab, College of Medicine, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, Korea

ksko@cau.ac.kr

In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1, SG2 and SG3 leaf section [top (T), middle (M), and base (BA)]. The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins.

This research was supported by agrant (Code#PJ011110) from the Korean Rural Development Administration, National Research Foundation of Korea Grant funded by the Korean Government (MEST)(NRF-2014R1A2A1A11052922).

145. Effect of Fc engineering to improve efficiency of plant-derived rabies virus monoclonal antibody

Solah Park 1 , Ilchan Song 1 , Dalmuri Han 2 , Hyegyeong Lee 2 , Kisung Ko 1; 1Therapeutic Protein Engineering Laboratory, Department of Medicine, College of Medicine, Chung-Ang University, 06974, Seoul, Korea, 2National Research Institute of Health, Cheongju, Korea

ksko@cau.ac.kr

Plant-derived monoclonal antibodies mAb SO57 with an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) (mAbpK SO57) have steadily studied. Production of therapeutic monoclonal antibodies in transgenic plants has several advantages such a large-scale production of mAb and absence of pathogenic animal contaminants. However, oligomannose (OM) type glycans structure of mAbpK SO57 in ER have showed a faster clearance of them compared to antibodies produced in animal cells. The neonatal Fc receptor (FcRn) regulates the persistence of immunoglobulin G (IgG) by the FcRn-mediated recycling pathway, which salvages IgG from lysosomal degradation within cells. In this study, Fc-engineering of mAbpK SO57 was performed to improve the efficiency of mAbpK SO57. Enzyme-Linked immunosorbent assay (ELISA) and Surface plasmon resonance (SPR) assay showed the difference of the binding affinity between the Fc region of mAbpK SO57 variants and hFcRn, respectively. N-glycan structure was also confirmed that all of mAbpK SO57 variants had OM type glycans structure similar to mAbpK SO57. In addition, after engineering of mAbpK SO57, the variants were effective as mAbpK SO57 in neutralizing the activity of the rabies virus CVS-11. These results told us that N-glycan structure and neutralization reaction of the variants were not affected. It suggests that their serum half-life will see the difference according to the binding affinity between mAbpK SO57 variants and hFcRn. This study shows that Fc-engineering could influence on the efficiency of plant-derived therapeutic monoclonal antibodies.

This research was supported by agrant (Code#PJ011110) from the Korean Rural Development Administration, National Research Foundation of Korea Grant funded by the Korean Government (MEST)(NRF-2014R1A2A1A11052922).

146. N-Glycan analysis of wheat germ exposed to high CO2 concentration

Risa Horiuchi 1 , Naoki Hirotsu 1 , Saman Seneweera 2 , Nobumitsu Miyanishi 1; 1Toyo University, Graduate School of Life Sciences, 374-0193, Gunma, Japan, 2University of Southern Queensland, Centre for Crop Health, 4350, QLD, Australia

miyanishi@toyo.jp

Wheat is one of the three major grains in the world and the staple diet because of nutritive value, handiness in processing and cultivation. Among wheat, durum and bread wheat are widely cultivated in the world. In the development and growth of wheat, it is unclear about the effect of high CO2 condition on wheat, and there are concerns about the effect of an increasing of global atmospheric CO2 concentrations accompanying global warming. Therefore, the assessment of high CO2 condition in wheat is important. On the other hand, N-glycans play important roles in various biological phenomena, and the structure and expression are sensitive to ambient environmental changes. In particular, N-glycans, which exist in wheat germ, are expected to have important roles in development and growth of wheat. Therefore, as a first model for assessment of the influence of CO2, we focused on the correlation of N-glycan structures and the effect of CO2 in wheat germs and analyzed the N-glycan structures in wheat germs exposed to elevated CO2 condition.

In this study, we analyzed typical cultivars, Tjikuri (durum wheat) and DART (bread wheat) grown under ambient or elevated CO2 conditions. Wheat germ N-glycans were prepared by hydrazinolysis, N-acetylation, and pyridylamination (PA). The resulting PA-N-glycans were separated by two kinds of HPLCs, and then structural determination of N-glycans was performed using MALDI-TOF mass spectrometry and glycosidase digestions. As a result of N-glycan analysis of wheat germs grown under ambient CO2 condition, N-glycans of Tjikuri and DART germ were composed of high-mannose, paucimannose, and minor plant complex type N-glycans. These results showed that the diversity and heterogeneity of N-glycan was very low in wheat germs. Furthermore, we also analyzed the N-glycans in wheat germs grown under elevated CO2 condition. The results of size-fractionation HPLC analysis, some peaks are significantly increased in elevated CO2 condition, suggesting that the N-glycan transition is expected to lead to a first model for assessment of the influence of CO2 on wheat.

147. N-Glycan core α1,3-fucose residue is required for basipetal auxin transport and gravitropic response in rice (Oryza sativa)

Ki Seong Ko 1 , Jae Yong Yoo 1 , Kyun Oh Lee 1; 1Gyeongsang National University, Division of Applied Life Science, 52828, Jinju, Korea

bug0@nate.com

In plants, α1,3-fucosyltransferase (FucT) catalyzes the transfer of fucose from GDP-fucose to asparagine-linked GlcNAc of the N-glycan core in the medial Golgi. To explore the physiological significance of this processing, we isolated two Oryza sativa (rice) mutants (fuct-1 and fuct-2) with loss of FucT function. Biochemical analyses of the N-glycan structure confirmed that α1,3-fucose is missing from the N-glycans of allelic fuct-1 and fuct-2. Compared with the wild-type cv Kitaake, fuct-1 displayed a larger tiller angle, shorter internode and panicle lengths, and decreased grain filling as well as an increase in chalky grains with abnormal shape. The mutant allele fuct-2 gave rise to similar developmental abnormalities, although they were milder than those of fuct-1. Restoration of a normal tiller angle in fuct-1 by complementation demonstrated that the phenotype is caused by the loss of FucT function. Both fuct-1 and fuct-2 plants exhibited reduced gravitropic responses. Expression of the genes involved in tiller and leaf angle control was also affected in the mutants. We demonstrate that reduced basipetal auxin transport and low auxin accumulation at the base of the shoot in fuct-1 account for both the reduced gravitropic response and the increased tiller angle.

148. Facilitated formation of the largest N-glycan by limited addition of the 6-arm β1,2-linked N-acetylglucosamine (GlcNAc) residue in plants

Jae Yong Yoo 1 , Ki Seong Ko 1 , Kyun Oh Lee 1; 1Gyeongsang National University, Division of Applied Life Science, 52828, Jinju, Korea

jayong77@nate.com

The most abundant N-glycan in plants is the paucimannosidic N-glycan with core β1,2-xylose and α1,3-fucose residues (XylMan3FucGlcNAc2). Here, we report a mechanism in Arabidopsis thaliana that efficiently produces the largest N-glycan in plants. Genetic and biochemical evidence indicates that the addition of the 6-arm β1,2-GlcNAc residue by N-acetylgluco-saminyltransferase II (GnTII) is less effective than additions of the core β1,2-xylose and α1,3-fucose residues by XylT, FucTA, and FucTB in Arabidopsis. Furthermore, analysis of gnt2 mutant and 35S:GnTII transgenic plants shows that the addition of the 6-arm non-reducing GlcNAc residue to the common N-glycan acceptor GlcNAcMan3GlcNAc2 inhibits additions of the core β1,2-xylose and α1,3-fucose residues. Our findings indicate that plants limit the rate of the addition of the 6-arm GlcNAc residue to the common N-glycan acceptor as a mechanism to facilitate formation of the prevalent N-glycans with XylMan3FucGlcNAc2 and GlcNAc2Xyl-Man3FucGlcNAc2 structures.

149. N-Glycosylation is an issue for the use of Chlorella as protein expression host

Reka Tunde Mocsai 2 , Elisabeth Svehla 2 , Rudolf Figl 2 , Richard Strasser 1 , Friedrich Altmann 2; 1University of Natural Resources and Life Sciences, Department of Chemistry, 1190, Vienna, Austria, 2University of Natural Resources and Life Sciences, Department of Applied Genetics and Cell Biology, 1190, Vienna, Austria

reka.mocsai@boku.ac.at

O-methylated carbohydrate structures have been found in various organisms like bacteria, fungi, nematodes and gastropods but not in mammals [1]. Non-vascular plants, like algae and mosses are a prominent example containing O-methylated N-glycans. Green microalga species are considered as potential hosts for genetic engineering and expression of heterologous proteins. There are many advantages of unicellular plant protein expression systems like rapid growth, low production costs and low risk of contamination. They can be a cost-effective alternative to mammalian cell systems, provided the glycosylation of product proteins could be adapted. Therefore, product quality and safety would require the clarification of the glycosylation pathway of certain Chlorella strains.

In this work we aimed at the analysis of N-glycan structures of different Chlorella strains. LC-ESI-MS/MS, MALDI-TOF-MS/MS and GC-MS instruments were used to access the structural differences between Chlorella pyrenoidosa, Chlorella vulgaris and Chlorella sorokiniana N-glycosylation.

Recently, we found a 3-O-methyl-arabinose in the main N-glycan of Chlorella pyrenoidosa, meanwhile in Chlorella vulgaris all the oligomannosidic structures were methylated, mainly in the form of 3-O-methyl-mannose. Chlorella Sorokiniana on the other hand contains a heavily methylated and perfectly new glycan with a monoisotopic mass of 1318.455 Da and a possible structure of HexNAc2Hex4Pent2. Interestingly, these closely related species showed very different glycan profiles that could imply a wider genetic diversity than previously thought.

Reference:

1 Staudacher, Erika. "Methylation - an uncommon modification of glycans." Biological Chemistry 393.8 (2012): 675-685.

Glycolipid

150. Differential regulation of cell phenotypes by tumor associated glycosphingolipids

Keiko Furukawa 1 , Mariko Kambe 1 , Yuki Ohkawa 1 , Yuhsuke Ohmi 1 , Rika Takeuchi 1 , Orie Tajima 1 , Koichi Furukawa 1; 1Dept. Biomed. Sci., Chubu Univ. 1200 Matsumoto-cho, Kasugai, Aichi, 487-8501, Japan

keikofu@isc.chubu.ac.jp

Ganglioside GD3 is widely expressed in human malignant melanoma cell lines and tumors. Previously, we reported that GD3-expressing human melanoma cells show increased cell proliferation and invasion.

In this study, we analyzed cell proliferation, adhesion to type I collagen and relative migration velocity to clarify the effects of sugar chain structure of gangliosides to these cell phenotypes using GM3-, GM2-, GM1-, GD3- or GD2-expressing human melanoma cells. They are stable transfectants of glycosyltransferase cDNAs. The cell proliferation of GD3+ cells and GD2+ cells increased comparing with the control cells (GM3+ cells), GM2+ cells and GM1+ cells. The cell adhesion to type I collagen of GD3+ and GD2+ cells as analyzed by real time cell electron sensor system (RT-CES) were increased, and adhesion of GD2+ cells was particularly enhanced. Relative migration velocity of cells during adhesion to type I collagen was measured by time-lapse imaging. Relative migration velocity of GM3+ cells and GM2+ cells were faster than GD3+ cells and GD2+ cells. In particular, GD2+ cells showed the slowest velocity among all cells.

These results suggest that cell adhesion intensity and migration activity are inversely correlated. Although GD2 has been considered to be highly expressed especially in metastatic stage of melanomas, migration activity of GD2+ cells was very weak among all cells, suggesting that GD2 might contribute in the solid fixation of melanoma cells at the metastasized sites, although proliferation of GD3+ cells and GD2+ cells increased almost equally.

151. Metabolic labeling of cholesteryl glucosides in helicobacter pylori reveals how uptake of human lipids enhances bacterial virulence

Sasikala Muthusamy 1 , Yi Chen Chan, Hau-Ming Jan, Chun-Hung Hans Lin; 1Academia Sinica, Taiwan

ss.kala4@gmail.com

Helicobacter pylori infects approximately half of the human population and is the main cause of various gastric diseases. This pathogen is auxotrophic for cholesterol, which it converts upon uptake to various cholesteryl α-glucoside derivatives, including cholesteryl 6α-acyl and 6α-phosphatidyl α-glucosides (CAGs and CPGs). Owing to a lack of sensitive analytical methods, it is not known if CAGs and CPGs play distinct physiological roles or how the acyl chain component affects function. Herein we established a metabolite-labeling method for characterizing these derivatives qualitatively and quantitatively with a femtomolar detection limit. By using this labeling method, we provide a series of MS/MS database for CGds and reveal overall lipid profiles of CGds in H. pylori. Through these profiling, we found that these bacteria acquire phospholipids from the membrane of epithelial cells for CAG biosynthesis. The resulting increase in longer or/and unsaturated CAG acyl chains helped to promote lipid raft formation and thus delivery of the virulence factor CagA into the host cell, supporting the idea that the host/pathogen interplay enhances bacterial virulence.

152. Lysosomal dysfunction leads to the alteration of plasma membrane glycosphingolipid composition with the onset of cell damage

Maura Samarani 1 , Giulia Lunghi 1 , Nicoletta Loberto 1 , Paola Giussani 1 , Rosaria Bassi 1 , Alessandro Prinetti 1 , Sandro Sonnino 1 , Massimo Aureli 1; 1University of Milano, Department of Medical Biotechnology and Translational Medicine, 20090, Segrate (Milano), Italy

maura.samarani@unimi.it

Increasing evidence implicates lysosomal dysfunction in the etiopathology of lysosomal storage disorders. Nevertheless, the molecular mechanisms by which the perturbation of lysosomal homeostasis induced by undegraded metabolites may affect the cell function are still unknown. In this context, altered metabolism of glycosphingolipids (GSLs) could play an active role in the onset of cell damage. To explore this issue, we used an artificial in vitro model of lysosomal impairment represented by human fibroblasts subjected to sucrose loading.

Interestingly, in sucrose-loaded cells we observed a significant slowdown in cell proliferation and autophagy activation. Moreover, after sucrose loading we found an increased content of GSLs (glucosylceramide, lactosylceramide, gangliosides) both intracellularly and at the plasma membrane level (PM). As a result of an augmented lysosomal exocytosis, in sucrose-loaded fibroblasts we observed an increased activity of the main GSLs catabolic enzymes associated with the cell surface. Interestingly, we demonstrated that the increased activity of glycohydrolases at the PM leads to the ectopic production of ceramide through the in-situ hydrolysis of complex GSLs. Taken together, these data suggest a new molecular mechanism, triggered by lysosomal storage, responsible for altered GSLs composition as well as increased glycohydrolytic activities at the PM level. The relationship between these events and the production of ceramide at the PM unveils a new role of GSLs metabolism in the onset of cell damage.

153. β-glucocerebrosidase GBA2: a new promising target to reduce the cystic fibrosis lung inflammation

Domitilla Schiumarini 2 , Nicoletta Loberto 2 , Paola Brocca 2 , Silvia Munari 1 , Giulia Mancini 2 , Rosaria Bassi 2 , Maria Cristina Dechecchi 1 , Massimo Aureli 2 , Sandro Sonnino 2; 1University of Milano, Department of Medical Biotechnology and Translational Medicine, 20090, Segrate, Italy, 2University Hospital of Verona, Laboratory of Molecular Pathology, 37126, Verona, Italy

domitilla.schiumarini@unimi.it

Cystic fibrosis (CF) is characterized by progressive chronic infection and inflammation of the airways, which cause the obstructive lung disease in CF patients. Resultant progressive remodeling leads to irreversible damage and fibroses, which is a major cause of mortality in patients.

Several evidences highlight the role of sphingolipids (SLs) in CF. Interestingly, an accumulation of ceramide has been demonstrated in lower airway of CF patients. Moreover, the infection of bronchial epithelial cells with Pseudomonas aeruginosa (PAO) activated host acid sphingomyelinase leading to ceramide generation at the cell surface. This ceramide regulates the release of inflammatory cytokines, such as IL-8. Ceramide production at the plasma membrane level could be triggered also by the action of the non-lysosomal glycohydrolase β-glucosylceramidase GBA2. Here, we report the importance of GBA2 in regulating the pro-inflammatory state of CF cells as well as the inflammatory response after PAO infection. In particular, we examined the impact of lowering the expression of GBA2 in CF cells exposed to PAO by siRNA and we found that the IL-8 expression is reduced in both uninfected and infected cells.

For these experiments, we developed nanoparticles (NP) for the systemic delivery of siRNA targeting GBA2 as a possible promising new anti-inflammatory therapy for CF lung disease.

This project was supported by the Italian Cystic Fibrosis Research Foundation, FFC#24/2014 to S.S.

154. Role of glycosphingolipids in CFTR plasma membrane stability

Nicoletta Loberto 2 , Giulia Mancini 2 , Domitilla Schiumarini 2 , Paola Giussani 2 , Silvia Munari 1 , Maria Cristina Dechecchi 1 , Sandro Sonnino 2 , Anna Tamanini 1 , Massimo Aureli 2; 1University of Milano, Department of Medical Biotechnology and Translational Medicine, 20090, Segrate, Italy, 2University Hospital of Verona, Laboratory of Molecular Patology, 37126, Verona, Italy

nicoletta.loberto@unimi.it

Human lung tissue is characterized by a complex sphingolipids (SLs) pattern composed by neutral glycolipids and fourteen different types of gangliosides, including the most abundant GM3 and GM1. Interestingly, in Cystic Fibrosis (CF) patients, which are characterized by mutation loss of function in the gene coding for CF transmembrane conductance regulator (CFTR), alterations in the SLs pattern of lungs have been observed.

In particular, it has been found a direct correlation between plasma membrane (PM) levels of GM1 and CFTR expression: lack of CFTR is responsible for a decrease of the GM1 content in bronchial epithelial cells.

Even if promising, the current therapeutic options based on the use of CFTR potentiators and correctors fail in restoring the CFTR activity for the most common mutation delF508. Interestingly, we found that in delF508 CF bronchial epithelial cells the use of corrector and potentiator did not change the GM1 cellular content. Conversely, in CF cells loaded with exogenous GM1, the treatment with the drugs results in a significative increase of CFTR at the cell surface, indicating a role of this ganglioside in the regulation of CFTR PM-stability.

In the next future, we plan to test the effect of GM1 in a more physiological cell model, represented by primary bronchial epithelial cells obtained from both CF patients and normal subjects. Ongoing experiments are aimed to characterize the lipid pattern of these cells during the differentiation to the air-liquid interface epithelium.

This project was supported by the Italian Cystic Fibrosis Research Foundation, FFC#24/2014 to S.S. and FFC#9/2015 to A.T.

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

Sara Grassi 2 , Livia Cabitta 2 , Simona Prioni 2 , Yana Zorina 1 , Laura Mauri 2 , Maria Grazia Ciampa 2 , Sandro Sonnino 2 , Alessandro Prinetti 2; 1University of Milan, Dept. of Medical Biotechnology and Translational Medicine, 20090, Segrate (MI), Italy, 2Acorda Therapeutics Inc., 10502, Ardsley (NY), USA

sara.grassi@unimi.it

Recombinant human IgM22 (rHIgM22) binds to myelin and oligodendrocytes (OLs), and promotes remyelination in mouse models of multiple sclerosis. rHIgM22 preferentially reacts with sulfatide-positive (O4+) OLs, and binding of rHIgM22 is abolished in CNS tissue slices from Cst(-/-) mice, suggesting that its binding requires the presence of a product of cerebroside sulfotransferase, possibly sulfatide, highly expressed in OLs and myelin. However 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 mouse brain, CNS myelin, mixed glial cells, and O4+ OLs using TLC immunostaining and SPR with lipid monolayers.

Our preliminary results show that IgM22 binds to sulfatide in vitro, while it does not bind to other myelin sphingolipids suggesting that sulfatide at the OLs surface might be important for the binding of rHIgM22 to these cells and to myelin. However, IgM22 does not bind structures expressing sulfatide outside the nervous system, so additional factors are likely relevant for the immunoreactivity of IgM22 in CNS. Indeed, in lipid extracts from different sources we found another lipid antigen selectively recognized by rHIgM22, whose identity is under investigation. This lipid is also present in the extracts from mixed glial cultures, which do not contain mature O4+ OLs, suggesting that other glial cells in addition to OLs might be important in the response to rHIgM22.

Proteoglycan

156. Biochemical and mutational analysis of α-L-iduronidase in glycosaminoglycan storage disorder of Thai patients

Lukana Ngiwsara 1 , Rattana Charoenwattanasatien 1 , Supattra Sukcharoen 3 , Siriporn Keeratichamroen 1 , Somporn Liammongkolkul 4 , Pornswan Wasant 4 , Thipwimol Tim-Aroon 5 , Dangrurdee Wattanasirichaigoon 5 , Chulaluck Kuptanon 2 , Suthipong Pangkanon 2 , Voraratt Champattanachai 1 , James R. Ketudat-Cairns 1,6, , Jisnuson Svasti 1,3; 1Biochemistry Laboratory, Chulabhorn Research Institute, Bangkok, 10210, Thailand, 2Queen Sirikit National Institute of Child Health, Bangkok, Thailand, 3Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 4Department of Pediatrics, Siriraj Hospital, Mahidol University, Bangkok, 5Division of Medical Genetics, 6School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand

Lukana@cri.or.th

The most abundant heteropolysaccharides in the body are the glycosaminoglycans (GAGs) or mucopolysaccharides. Impaired degradation of GAGs with consequent intralysosomal accumulation of undegraded products causes a group of lysosomal storage disorders known as mucopolysaccharidoses (MPSs). MPSs are recognized by increased excretion in urine of partially degraded GAGs which ultimately result in progressive cells, tissues, and organ dysfunctions. There are eleven different enzymes involved in the stepwise degradation of GAGs. Deficiencies of those enzymes result in seven different MPSs, all sharing a series of clinical features, though in variable degrees. Our works focus on mucopolysaccharidosis type I (MPS I) which is an autosomal recessive disorder resulting from the deficiency of α-L-iduronidase (IDUA; E.C. 3.2.1.76). MPS I presents the wide variation of clinical manifestation from attenuated to severe forms. More than 100 different IDUA mutations have been reported in Human Gene Mutation Database (http://www.hgmd.org). Here we present six Thai unrelated patients with MPS I who exhibited low-to-absent levels of leukocyte IDUA activity. Genetic analysis reveals eight different mutant alleles in IDUA gene. Three novel nonsense mutations are c.983G>T (p.E299X), c.935G>A (p.W312X) and c.1138 C>T (p.Q380X) while other known pathogenic mutations include three missense mutations c.311G>A (p.A75T), c.1986C>T (p.S633L) and c.345G>A (p.R89Q), one stop read through mutation c.*1T>C (p.X654R) and a frameshift mutation 252insertC. These molecular finding expand the diverse set of defective IDUA genes in the population that it will be useful for molecular diagnosis and treatment of disease.

This research was supported by the Chulabhorn Research Institute.

157. Accumulation of heparan sulfate in MPS II cells

Sung Eun Kim 1 , Hyunju Kang 1 , Sungho Hwang 1; 1MOGAM institute, Rard Disease team, 16924, Youngin, Korea

sekim@mogam.re.kr

Mucopolysaccharides or glycosaminoglycans (GAGs) are long polysaccharides consisting of a repeating disaccharide unit and occur as a proteoglycan form in cellular membranes or as a free form inside or outside cells. GAGs are essential for many cell functions, but too much GAG accumulation can cause diseases. For example, mucopolysaccharidosis type II (MPS II) is caused by a lack of functional iduronate-2-sulfatase (IDS) which plays a key role in the first step of heparan sulfate (HS) degradation. Although the MPS II physiology in patients or animal models has been widely studied, relatively little is known at the cellular level. Hence, we determined to characterize the pathophysiology in IDS-KO cells, a cellular model of MPS II, compared with wild type cells. First, we quantified the amount of cellular HS to examine whether more HS is accumulated in MPS II cells than in wild-type cells. For this, we extracted and purified GAGs from either wild-type or IDS-KO cells. Then, GAGs were treated with heparinases to generate HS disaccharide mixtures. The HS disaccharides were tagged by a fluorescent molecule, 2-AB, and quantified using high-performance liquid chromatography (HPLC). As a result, IDS-KO cells expressed two times higher HS than wild-type cells. In particular, the increase of 2-O-sulfated disaccharides was bigger than that of 2-O-nonsulfated ones. This result correlated to the fact that IDS removes 2-O-sulfate group from 2-sulfoglucuronates in HS disaccharides. Next, to test whether HS accumulates in lysosomes, we performed immunocytochemistry using a lysosome marker, LAMP1 and a monoclonal anti-HS antibody, 10E4. Unexpectedly, we did not observe HS in lysosomes in IDS-KO cells. Collectively, our results suggest altered lysosomal homeostasis in MPS II cells by HS over-accumulation. In conclusion, for the first time, we have shown the HS accumulation in IDS-KO cells and our observations will contribute to the further understanding of MPS II pathophysiology at the cellular level.

Sialic Acid

158. Enzymatic synthesis of lactosylated and sialylated derivatives of epothilone A

Prakash Parajuli 1 , Ramesh Prasad Pandey 1 , Jae Kyung Sohng 1; 1SunMoon University

sohng@sunmoon.ac.kr

Epothilone A is a derivative of 16-membered polyketide natural product, which has comparable chemotherapeutic effect like taxol. Introduction of sialic acids to these chemotherapeutic agents could generate interesting therapeutic glycoconjugates with significant effects in clinical studies. Since, most of the organisms biosynthesize sialic acids in their cell surface, they are key mediators in cellular events (cell-cell recognition, cell-matrix interactions). Interaction between such therapeutic sugar parts and cellular polysaccharides could generate interesting result in drugs like epothilone A. Based on this hypothesis, epothilone A glucoside (epothilone A 6-O-β-D-glucoside) was further decorated by conjugating enzymatically galactose followed by sialic acids to generate epothilone A 7-O-β-D-glucopyranosyl, 4'-O-α-D-galactoside i.e., lactosyl epothilone A (lac epoA) and two sialosides of epothilone A namely epothilone A 7-O-β-D-glucopyranosyl, 4'-O-α-D-galactopyranosyl 3″-O-α-N-acetyl neuraminic acid and epothilone A 7-O-β-D-glucopyranosyl, 4'-O-α-D-galactopyranosyl 6″-O-α-N-acetylneuraminic acid i.e., 3'sialyllactosyl epothilone A: 3'SL-epoA, and 6'sialyllactosyl epothilone A: 6'SL-epoA, respectively. These synthesized analogs were spectroscopically analyzed and elucidated, and biologically validated using HUVEC and HCT116 cancer cell lines.

159. Analysis of relationship between polysialyltransferases and malignant phenotype of the B16 mouse melanoma cell

Erino Araki 1 , Kana Tsuda 1 , Ken Kitajima 1 , Chihiro Sato 1; 1Nagoya University, Bioscience and Biotechnology Center, 464-8601, Nagoya, Japan

takahashi.erino@j.mbox.nagoya-u.ac.jp

Polysialic acid (polySia) is a polymerized structure of sialic acid with degree of polymerization ranging from 2 to 400. PolySia functions not only as a negative regulator of cell-cell/extracellular matrix adhesion due to its hydration effect and steric hindrance, but also as a reservoir for biologically active molecules due to its molecule-binding property. For example, polySia can bind FGF2, and the cell surface polySia has the ability to inhibit the FGF2-dependent heparan sulfate-mediated cell proliferation. PolySia is known to be present in some tumors and its expression is demonstrated to be positively correlated with metastasis. However, it largely remains unclarified how polySia expression is involved in the malignant phenotypes of cancerous cells. Interestingly, we have recently demonstrated that molecule-binding properties are different between those polySia structures synthesized by two polysialyltransferases, STX (ST8SIA2) and PST (ST8SIA4). In this study, we thus investigated how polySia structures synthesized by STX and PST affected malignant phenotypes of the B16 mouse melanoma cell.

The plasmid encoding the mouse STX or PST gene, or empty plasmid was transfected into B16 cells which express NCAM, but not STX or PST, and each stable cell line was established as B16-STX, B16-PST, or Mock, respectively. These cells were analyzed for the cell surface oligo/polySia structure by flow cytometry using specific anti-oligo/polySia antibodies. Cell surface polySia for both B16-STX and B16-PST were significantly increased compared with that for Mock. These cells were also analyzed for proliferation rates, and B16-PST cells showed dramatically higher proliferation rate than B16-STX or Mock cells. Furthermore, to see whether the expression of STX and PST genes enhanced malignancy of B16 cells, anchorage-independent growth was examined by soft-agar colony formation assay. The results showed that the highest number and the largest size of the colonies were observed for B16-PST cells. Taken together, it is suggested that the expression of PST gene enhances abnormal growth in B16 cells, providing additional evidence for the importance of PST in tumor malignancy.

160. Monitoring of non-human glycan epitope on biotherapeutic glycoproteins with advanced technologies

Myung Jin Oh 1 , Youngsuk Seo 1 , Yi-Nae Jeon 1 , Hyun Joo An 1; 1Chungnam National University, GRAST, 34134, Daejeon, Korea

hjan@cnu.ac.kr

Therapeutic glycoproteins represent a great accomplishment for treatment of various diseases. Biotherapeutics are frequently manufactured in mammalian expression systems to achieve appropriate glycosylation, and the glycosylation pattern is often critical for the drug’s stability, immunogenicity, PK/PD, and bioactivity. Unlike other mammalian except chicken, human cannot biosynthesize the sialic acid NeuGc because the human gene CMAH, encoding CMP-N-acetylneuraminic acid hydroxylase, the enzyme responsible for producing CMP-Neu5Gc from CMP-N-acetylneuraminic acid (CMP-Neu5Ac), is irreversibly mutated. Human express anti-NeuGc antibodies, which leads to immune response against biotherapeutics carrying NeuGc glycan. Therefore, analysis of the non-human glycan is critical to regulate of potential immunogenicity for process development and manufacturing procedures of therapeutic glycoproteins. Here, we propose LC/MS and MS/MS screening as a method for rapid identification and structural elucidation of biopharmaceutical glycosylation containing NeuGc. Biotherapeutic glycoproteins produced by various cell-based expression systems were prepared and analyzed. After removing salts and detergents, N-glycans were enzymatically released and purified by graphitized carbon solid phase extraction. PGC chip nano-LC/Q-TOF provided chromatographic isomer separation profiling and structural characterization. Numerous NeuGc-sialylated glycans were discovered in mAbs produced by mouse Sp2/0 cells. Isobaric glycans carrying NeuAc and NeuGc glycan were separated by PGC column and NeuGc-layted glycans was confirmed by LC/MS/MS. Strong signals for fragment ions at m/z 308 (NeuGc) and m/z 673 (NeuGc attached to galactose and N-acetylglucosamine) are represent presence of NeuGc glycan. The approach is fast, does not need for labeling or derivatization and is applicable for highly accurate identification of NeuGc glycosylation.

161. Occurrence and biosynthesis of sulfated Sialic acids in mammals

Nursah Ertunc 1 , Thanyaluck Phitak 1 , Hiroshi Fujita 1 , Chihiro Sato 1 , Ken Kitajima 1; 1Nagoya University, Bioscience and Biotechnology Center under Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

nursahertunc@gmail.com

Sialic acid (Sia), an essential monosaccharide for survival of mammals, modifies glycoconjugates on cell surface and extracellular matrix, and mediates and regulates cellular recognition and signaling events. Sia shows an extremely large structural diversity that emerges from modification by acetylation, methylation, sulfation and so on. However, metabolism and biological functions of those modified Sias have not been clarified, except for a limited knowledge of acetylated Sia. Sulfation of Sia residues is known to occur in various animals including mammals and sea urchin. It has been shown that sulfated Sia (SiaS) is involved in sperm-egg interaction and sperm motility in sea urchin. In contrast, nothing is known about significance of SiaS in mammals. Thus, our objective is to understand the biological significance of SiaS in mammals, and here we report our recent results on occurrence and biosynthesis of SiaS. First, we performed immunohistochemical observations of SiaS using monoclonal antibody (mAb).3G9 that recognizes 8-O-sulfated Neu5Ac residues (Yamakawa et al. 2007), and demonstrated ubiquitous occurrence of SiaS in normal and cancer cells and tissues from mouse and human. Second, we examined the expression level of SiaS in various mammalian cells by immunochemical methods, such as Western blotting and flow cytometry, using mAb.3G9, as well as by the DMB derivatization-fluorometric HPLC analysis. Some mouse cell lines and tissues were found to show dynamic changes of SiaS expression. Third, we searched for a sulfotransferase (SULT) responsible for sulfation of Sia. After various trials and errors, we finally found a candidate sulfotransferase gene (SULT-X), and cloned. When we transfected some mammalian cells with the cDNA for SULT-X, flow cytometry and the fluorometric HPLC analysis showed that the amount of SiaS on the cell surface was increased. These results suggest that SULT-X is responsible for expression of 8-O-sulfated Neu5Ac.

162. The enhanced bacterial clearance by α2,3- and α2,6-sialyllactose in the PAK-induced pneumonia model

Jimin Kim 1 , Jae Wha Kim 1 , Yong-Jae Kim 1 , Su-Hyun Shin 1 , Young-Eun Ko 1 , Jinseon Jeong 1 , Kwanghoon Yang 1 , Solji Choi 1 , Daehee Kim 2 , Jinsuk Woo 2; 1Korea Research Institute of Bioscience and Biotechnology, Division of Systems Biology and Bioengineering, KS015, Daejeon, Republic of Korea, 2GeneChem, KS015, Daejeon, Republic of Korea

wjkim@kribb.re.kr

The biological efficacy of sialyllactose for the protection from invading pathogen has been well documented for long time. Improvement of innate immunity by sialyllactose enforces bacterial clearance. Pneumonia model was constructed by introduction of pseudomonas aeruginosa K (PAK) via intranasal administration. Our data showed that orally administered sialyllactose has prominent effects on bacterial clearance with significance in the PAK-induced pneumonia model. In the mouse model, inoculated pathogen was promptly eliminated in the sialyllactose added group within 4 hours after bacteria insemination and chemokine macrophage inflammatory protein-2 (MIP-2), was increased when sialyllactose was added. The result suggests that increased MIP-2 might yield neutrophil activation including neutrophil recruitment and phagocytosis and eventually result in bacterial clearance and return to homeostasis. Modulation of chemokines by sialyllactose was further examined in the cultured monocyte derived cell line, THP-1. Co-stimulation of PAK and sialyllactose promotes C-X-C motif chemokine ligand 8 (CXCL8) in the cultured monocytes and thus neutrophil recruitment to the chemokine gradient following its activation of phagocytosis. Our results demonstrate that sialyllactose treatment significantly promotes neutrophil activity like as chemotactic activity and phagocytosis by increasing chemokines. These findings will provide new insights for developing a therapeutic for pneumonia with sialyllactose.

163. Mechanism of uptake of deaminoneuraminic acid (KDN) in mammalian cells

Yuya Iwaki 1 , Chihiro Sato 1 , Ken Kitajima 1; 1Bioscience and Biotechnology Center, Nagoya University, 464-8601, Nagoya, Japan

iwaki.yuuya@e.mbox.nagoya-u.ac.jp

Deaminoneuraminic acid (KDN) residues have unique properties that are absent in N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). KDN residues are resistant against commonly used sialidases, and terminate the elongation of α2,8-linked oligo-polysialic acid chains. Mammalian cells de novo synthesize a very little KDN and abundant Neu5Ac and Neu5Gc, while they also utilize those free sialic acids that are incorporated from culture medium. It has been reported that free Neu5Ac and Neu5Gc are taken up into mammalian cells by clathrin-independent endocytic pathways mainly through a macropinocytosis. However, no study has ever been performed on uptake mechanism of free KDN in mammalian cells. In this study, to reveal the uptake mechanism of free KDN, mouse melanoma B16 cells and human embryonic kidney HEK293 cells were cultured in the presence of free KDN under various inhibitors for membrane transport for 12 hours. After washing, the cells were quickly frozen on liquid nitrogen, and fractionated into the cytosol and membrane fractions, followed by quantitation of KDN. In both B16 and HEK293 cells, uptake of free KDN was diminished by inhibitors for caveolae-mediated endocytic and clathrin-mediated endocytic pathways, but not by a macropinocytosis inhibitor. Interestingly, inhibitors for monocarboxylate transporters reduced the KDN uptake in B16 cells, but not in HEK293 cells. In addition, no KDN uptake was inhibited by co-incubation with high concentrations of Neu5Ac. These results suggest that mammalian cells have different uptake mechanisms for KDN than those for Neu5Ac and Neu5Gc, and that they have a specific machinery for incorporation of free KDN, because caveolae- and clathrin-mediated endocytic pathways are known to be triggered by recognition of a target to be taken up.

164. Unique properties of polyvalent disialic acid-linked dextran toward the sialic acid-binding lectin Siglec-7

Atsushi Yoshimura 2 , Yu Yasuda 2 , Airi Mori 2 , Hiroshi Tanaka 1 , Ken Kitajima 2 , Chihiro Sato 2; 1Nagoya University, Bioscience Biotechnology Center, 4648601, Nagoya, Japan, 2Tokyo Institute of Technology, Department of Chemical Science and Engineering, 152-8552, Meguro Tokyo, Japan

yoshimura.atsushi@j.mbox.nagoya-u.ac.jp

Sialic acid (Sia) is present as monosialyl residues at non-reducing termini of glycan chains on glycoproteins and glycolipids in vertebrates. Sia plays important roles in various biological phenomena including immune tolerance and infection. Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs) are mainly expressed in blood cells in a cell-type-specific manner and regulate immune signaling via recognition of sialylated glycans. Siglec-7 is expressed on NK cells and monocytes, and is known to bind to α2,8-linked disialic acid (diSia) and branched α2,6-linked Sia structures. The inhibitor of Siglec-7 is considered to be useful not only as an immunomodulatory reagent, but also as a functional probe for elucidation of the regulatory mechanism of the Siglec-7-mediated events. In this study we prepared diSia-Dex, a new Sia-containing glycopolymer that is fluorescent probe-conjugated dextran with high-density diSia, and examined its effect on the Sia recognition of Siglec-7.

Recombinant protein of the extracellular domain of Siglec-7 fused with a Fc region of human Ig (Siglec7EcFc) was purified from the culture medium of CHO cells stably expressing Siglec7EcFc. The binding abilities to gangliosides were examined by an ELISA-based method with Siglec7EcFc and Siglec7EcFc (R124A) that was mutated at the essential amino acid residue (R124) in the canonical Sia binding site. Siglec-7 specifically bound to the diSia structure of GD3, not to the monoSia structure of GM3 through the R124 residue. Siglec7EcFc was then found to bind strongly to diSia-Dex and its dissociation rate was very slow as examined through the kinetic analysis using the bio-layer interferometry (BLI) method. Furthermore, the inhibition analysis toward the ligand (GD3) binding of Siglec7EcFc demonstrated that diSia-Dex was the best compound. On the cell surface of the CHO cells stably expressing the full-length Siglec-7, diSia-Dex was specifically interacted with Siglec-7 when evaluated by a flow cytometric method. All these data suggest that diSia-Dex is a key molecule to modulate the Siglec-7 ligand binding.

Glycopathology: Glycans in Cancer

165. WNT signaling accelerates gastric tumorigenesis through galectn-3 dependent STAT3 activation

Seok-Jun Kim 1 , Hyeok-Gu Kang 1 , Kyung-Hee Chun 1; 1Yonsei University, College of Medicine, Department of Biochemistry & Molecular Biology, 03722, Seoul,Korea

khchun@yuhs.ac

Aberrant activation of WNT signaling is known to play a critical role in gastric tumorigenesis. Interestingly, we detected activated STAT3 with WNT1 overexpression, suggesting that these two pathways crosstalk during gastric tumorigenesis. Using clinicopathological analysis of gastric cancer(GC) patient tissues and genetically engineered(K19-Wnt1/C2mE and K19-WNT1) mice, increased β-catenin expression correlated with increased phosphorylation of STAT3. However, STAT3 phosphorylation was diminished in stomach tissues of K19-WNT1/lgals3-/- mice and lgals3-/- MEFs. WNT1 overexpression increased the phosphorylation and nuclear-localization of STAT3, whereas galectin-3 depletion reversed these effects. The T45-Q48 domain of galectin-3 interacted directly with STAT3 and increased its phosphorylation by interaction with JAK2. Galectin-3 moved together with STAT3 into the nucleus and increased its transcriptional activity. We detected the high expression and co-localization of β-catenin, phosphorylated STAT3, and galectin-3 in malignant GC patient tissues. Therefore, we propose that WNT1 activates STAT3 through galectin-3, which explains the crosstalk between WNT and STAT3 pathways.

166. Synergistic regulation of lung cancer cells growth with human lactoferrin N-lobe and sulfated glycosaminoglycans

Akira Shiga 1 , Takumi Matsuzaki 1 , Atsushi Sato 1 , Masao Nakamura 1; 1Tokyo University of Technology, Graduate School of Bionics, Computer and Media Sciences, 1920982, Japan

nakamuramo@stf.teu.ac.jp

Lactoferrin (Lf) is a potential seed for drug discovery because of its antitumor activity and anti-inflammatory action. Lf is a multifunctional lectin contained in exocrine fluids such as breast milk and tears. This lectin is a glycoprotein belonging to the transferrin family, and is composed of two globular domains (N-lobe and C-lobe). Thus far, the N-terminal region of N-lobe has been identified as a binding site of sulfated glycosaminoglycan (sGAG). Recently, it was reported that sGAG forming the tumor microenvironment affected the proliferative and adhesion abilities of lung cancer cells, and contributed to the enhancement of metastatic potential. However, the contribution of sGAG in the regulation of lung cancer cell proliferation by Lf is unknown. In this study, using the growth inhibitory activity of the three human lung cancer cell lines (poorly differentiated adenocarcinoma PC-14, moderately differentiated adenocarcinoma PC-3, and differentiated adenocarcinoma PC-9) as an index, the impact of sGAG on the activity of Lf N-lobe was evaluated. The results indicated that: i) the growth inhibitions of the three lung cancer cell lines by Lf N-lobe differed depending on their degree of differentiation, ii) the level of activity of Lf N-lobe was controlled by the presence of sGAG, and iii) Lf N-lobe had high affinity for a specific sGAG, uptake of Lf N-lobe into lung cancer cells was inhibited, and the growth inhibitory effect on cancer cells was attenuated. These results suggested that the presence and structure of sGAG were important for growth control of lung cancer cells by Lf N-lobe.

167. Enzymatic stable isotope labelling of small O-glycans for improved tumor biomarker analysis

Johannes Führer 1 , Friedrich Altmann 1; 1University of Natural Resources and Life Sciences, Department of Chemistry, 1190, Vienna, Austria

johannes.fuehrer@boku.ac.at

The epithelium of organs and inner cavities is coated with a carbohydrate-rich, protective barrier consisting mainly of heavily O-glycosylated mucin proteins. Especially in early development of metastatic cancer the usually complex O-glycans are found to be truncated, which makes them interesting as biomarkers. [1] These mono- and disaccharidic structures, known as Tn- and T-antigen respectively are, due to their diminutive size, notoriously difficult to assess with both lectin-based and instrumental analysis methods. The latter consists of chromatographic analysis of the released O-glycans using porous graphitic carbon coupled to electrospray mass spectrometry (PGC-LC-ESI-MS). [2][3]

This work aims to improve the detection of these biomarkers using the isomer specific analysis method PGC-LC-ESI-MS. Crucial for this approach is the enzymatic elongation of the Tn-antigen found on mucins and other secreted proteins using recombinantly expressed core 1 β-1,3-galactosyltransferase (C1GalT) together with α-2,3-sialyltransferase to create a trisaccharide. The incorporation of 13C6-labelled galactose creates a glycan that is easily discernable from native structures by a characteristic mass difference of six dalton.

For this purpose we expressed soluble C1GalTA from Drosophila melanogaster in Sf9 cells and used it to add 13C6-galactose to peptide-bond Tn-antigen. Further application in the O-glycan profiling of bovine submaxillary mucin also yielded promising results. As the substrate specificity of the insect GalT may be sub-optimal for human mucins we are also expressing C1GalT1 from Mus musculus together with its chaperone Cosmc.

Our labelling strategy could serve as a useful tool once it is integrated into workflows in research and routine diagnosis of small tumor related O-glycans – especially as an orthogonal substantiation of lectin-based results.

[1] Ju, T., Wang, Y., Aryal, R.P., Lehoux, S.D., Ding, X., Kudelka, M.R., Cutler, C., Zeng, J., Wang, J., Sun, X., Heimburg-Molinaro, J., Smith, D.F., Cummings, R.D., Proteomics Clin. Appl. 7 (2013) 618–631

[2] Dotz, Viktoria, Haselberg R., Shubhakar A., Kozak R.P, Falck D., Rombouts Y, Reusch D., Somsen G.W., Fernandes D.L., Wuhrer M., Trends Anal. Chem. 73 (2015): 1–9

[3] David C., Mao L., Dis. Markers 33, no. 1 (2012): 1–10

168. Differential expression of ST6GAL1 during CRC progression

Sen Zhang 1 , Jishun Lu 3 , Bo Feng 2 , Yan Zhang 3; 1Ruijin Hospital, 197, Ruijin Er Road, Shanghai 200025, China, 2Shanghai Jiao Tong University, Shanghai Center for Systems Biomedicine (SCSB), 20024, Shanghai, China, 3Department of General Surgery,Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Minimally Invasive Surgery Center, 200025, Shanghai China

zhangsen6886@163.com

Altered expression of ST6GAL1 has been observed in multiple types of carcinomas. However, the mechanistic roles of this sialyltransferase in colorectal cancer(CRC) are still obscure. Here, we found that ST6GAL1 was significantly higher in the colorectal tumors at stage I and II (no metastasis) but not those at stage III and IV (metastasized) when compared with their pair-matched uninvolved tissues. Consistently, overexpression of ST6GAL1 in colon cancer cell line SW480 clearly inhibited cell migration. Metabolic labeling of the sialylated membrane proteins followed by mass spectrometry analysis identified 318 membrane proteins differentially regulated after ST6GAL1 overexpression. Bioinformatic analysis revealed a list of potential substrates that might mediate the function of ST6GAL1 in tumor metastasis. Among these, we showed that ST6GAL1 overexpression decreased the phosphorylation levels of EGFR and Erk1/2 and increased the expression of ABCB1, providing a possible explanation for the downregulation of ST6GAL1 at the stage III and IV of CRC. Taken together, our results indicate a dynamic change in the expression of ST6GAL1 during the CRC progression and provide numerous sialylated proteins potentially relevant to the different functions of ST6GAL1 in colon cancer cells.

169. Investigation of the mechanistic roles of ST6GAL1 in HCC progression

Jishun Lu 1 , Feijie Lu 1 , Hao Shen 1 , Jianguo Gu 2 , Yan Zhang 1; 1Shanghai Jiao Tong University, Shanghai Center for Systems Biomedicine (SCSB), 20024, Shanghai, China, 2Tohoku Medical and Pharmaceutical University, Institute of Molecular Biomembrane and Glycobiology, 981-8558, Sendai, Miyagi, Japan

lujishun@sjtu.edu.cn

Altered glycosylation is a common feature of a tumor cell. Despite the long-standing observations, our understanding of the molecular mechanisms linking altered glycosylation to tumor cell behavior still remains obscure. Here, we combined the high through-put technologies of lectin array and proteomic analysis to investigate the mechanistic roles of the glycosylation in HCC metastasis by using two HCC cell lines MHCC97L and HCCLM3 with similar genetic background but different metastatic potentials (MHCC97L<< HCCLM3). Lectin array and FACS analysis showed that the reactivity of MHCC97L against SNA lectin, which specifically recognizes the α2,6 linked sialic acids on N-glycans, was greatly higher than that of HCCLM3. Further data suggested that this was mainly due to the high expression of ST6GAL1 (primarily responsible for α2,6 sialylation on N-glycans) in MHCC97L cells as compared with HCCLM3. Forced expression of ST6GAL1 in HCCLM3 cells inhibited not only cell migration but also cell invasion, indicating that ST6GAL1 may negatively controls the HCC metastasis. To explore the cell surface sialylated proteins that mediate these functions, we metabolically labeled cell surface sialic acids with ManNAz and pulled down the sialylated proteins for MASS analysis. The results revealed a list of proteins whose expression was significantly affected between these two cells. Taken together, our study indicates that ST6GAL1 may suppress the HCC metastasis by regulating the expression or sialylation status of some important proteins on cell surface.

170. Lectin from Sambucus sieboldiana abrogates the anoikis resistance of colon cancer cells conferred by N-acetylglucosaminyltransferase V during hematogenous metastasis

Jeong-Hwa Lee 1 , Dae In Ha 2 , Su Bin Moon 2 , Jeong Mi Lee 2 , Yong-Sam Kim 2; 1Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea, 2Genome Editing Research Center, KRIBB, Daejeon, Republic of Korea

omsys1@kribb.re.kr

Anoikis is a form of anchorage-dependent apoptosis, and cancer cells adopt anokis-resistance molecular machinery to conduct metastasis. Here, we report that N-acetylglucosaminyltransferase V gene expression confers anoikis resistance during cancer progression. Overexpression of N-acetylglucosaminyltransferase V protected detached cancer cells from apoptotic death, and suppression or knockout of the gene sensitized cancer cells to the apoptotic death. The gene expression also stimulated anchorage-dependent as well as anchorage-independent colony formation of cancer cells following anoikis stress treatments. Importantly, treatment with the lectin from Sambucus sieboldiana significantly sensitized anoikis-induced cancer cell deaths in vitro as well as in vivo. We propose that the lectin alone or an engineered form could offer a new therapeutic treatment option for cancer patients with advanced tumors.

171. C-Type lectin-like receptor-1 promotes the progress of gastric cancer via activating PI3K/mTOR/Akt signaling pathway

Shushu Song 1; 1Fudan University,Key Laboratory of Glycoconjugate Research Ministry of Public Health, 200032, Shanghai, China

13111010022@fudan.edu.cn

The C-type lectins are classical molecules containing at least one C-type lectin domain that have been classified into different groups by their ability to bind carbohydrates in a Ca2+-dependent manner. Among the vast C-type lectin superfamily, the C-type lectin-like proteins are a subgroup of receptors that possess the C-type lectin domain but lack Ca2+-binding elements. Although DC-associated C-type lectin-1 (DECTIN-1), oxidized low-density lipoprotein receptor-1 (LOX-1) and C-type lectin-like receptor-1 (CLEC-2) which belong to dectin-1 cluster have been studied extensively, only very limited information is available for C-type lectin-like receptor-1 (CLEC-1). In present study, we found that the expression of CLEC-1 was remarkably up-regulated in gastric tumor tissues compared to non-tumor tissues. High CLEC-1 staining intensity was positively correlated with tumor size, tumor invasion depth, lymph mode metastasis, TNM stage, and reduced patients’ survival. Multivariate analysis identified CLEC-1 as an independent prognostic factor, which could improve the predictive accuracy for overall survival when incorporated into TNM staging system. Over-expression of CLEC-1 promoted viability, colony formation and cell cycle of gastric cancer cells in vitro, and it facilitated the growth of subcutaneous tumor xenografts in vivo. MGC80-3 cells with CLEC-1 over-expression had increased phosphorylation levels of mTOR, and S6 kinase, 4E-BP1 and AKT at Ser473 the downstream targets of mTOR. And all these changes were prevented by the mTOR inhibitor rapamycin or PI3K inhibitor LY294002. Furthermore, over-expression of CLEC-1 in MGC80-3 cells increased protein and messenger RNA levels of PI3K subunits p85 and p110. In conclusion, CLEC-1 promotes growth of gastric cancer cells by inducing the activation of PI3K-mTOR-Akt signaling pathway. And CLEC-1 was proposed as a novel independent prognostic factor and potential therapeutic target for gastric cancer.

172. Differential expression profiles of ST3Gal4 and ST3Gal6 influence the progression of bladder cancer

Han Zhang, Bo Fan, Yujie Zhao, Liping Wang, Qingmn Yuan, Xiao Yu, Shujing Wang 1; 1Dalian Medical University, China

wangshujing@dmu.edu.cn

It has been reported that the abnormal expression of sialyltransferases is closely related to the tumorigenesis and tumor progression. Bladder cancer (Bca) is a common malignancy with high morbidity and mortality. However, the role and potential mechanism of ST3Gal4 and ST3Gal6 in bladder cancer remain unknown. In this study, we found that expression of ST3Gal4 and ST3Gal6 was dramatically down-regulated in BCa tissues and cells compared with that in normal urothelial epithelium tissues. Low levels of ST3Gal4 and ST3Gal6 in Bca were associated with aggressive phenotype and poor prognosis. Furthermore, we examined the roles and mechanisms of ST3Gal4 and ST3Gal6 in BCa tumorigenesis and metastasis in vitro and in vivo. ST3Gal4 and ST3Gal6 overexpression inhibited the proliferation, migration, and invasion of Bca T24 and 5637 cells. Additionally, T24 cells that overexpressed ST3Gal4 and ST3Gal6 formed significantly smaller tumors in mouse xenograft model, implying that ST3Gal4 and ST3Gal6 overexpression can suppress BCa cell malignant transformation. Importantly, ST3Gal4 and ST3Gal6 may be affected the development of BCa by regulating PI3K-AKT signaling pathway. Taken together, our study indicates that ST3Gal4 and ST3Gal6 could inhibit growth and invasion of BCa cells through PI3K-AKT signaling pathway, and they might be a potential markers for prognosis and therapy of BCa. Supported by the National Natural Science Foundation of China (NO.31470799, 31570953).

Glycoimmunology (Immunity & Sugar Chain)

173. Polysaccharides from Pseudopterogorgia americana modulates immune response in macrophages

Lee-Chiang Lo 1 , Oleg Chernikov 2 , Hsiao-Wen Chiu 3 , Kuo-Feng Hua 3; 1National Taiwan University, Department of Chemistry, Taipei, Taiwan, 2G.B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS, Vladivostok, Russia, 3National Ilan University, Department of Biotechnology and Animal Science, 260 Ilan, Taiwan

kuofenghua@gmail.com

The marine polysaccharides have a profound impact on the regulation of immune response. However, the mechanism of immune modulation by polysaccharides isolated from the coral Pseudopterogorgia americana (PPA) in mammalian cells remains unclear. Here, we demonstrated that PPA can activate immune responses in macrophages. PPA induced secretion tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and cyclooxygenase-2 in mouse RAW264.7 macrophages. The PPA-mediated cytokine production was regulated by reactive oxygen species (ROS), mitogen-activated protein kinases (MAPK), protein kinase C-α (PKC-α) and NF-κB. Interestingly, in lipopolysaccharide-activated macrophages, PPA induced endotoxin tolerance (characterized by the downregulation of TNF-α and IL-6) via the downregulation of IRAK2 expression, MAPK phosphorylation and NF-κB activation. Overall, our data indicate that PPA has the potential to be used as an immune modulator in mammals.

174. Synthesis of structural diverse muramyl dipeptide derivatives and their use in a study of human NOD2 stimulation activity

Zhen Zhuo Teo 1 , Huang-Yi Lee 2 , Kuo-Ting Chen 2 , Pi-Hui Liang 1 , Wei-Chieh Cheng 2; 1National Taiwan University, Department of Pharmacy, 10055, Taipei city, Taiwan (R.O.C.), 2Academia Sinica, Genomics Research Center, 11529, Nankang, Taiwan (R.O.C.)

wcheng@gate.sinica.edu.tw

N-Acetyl muramyl dipeptide (N-acetyl MurNAc-L-Ala-D-iso-Gln) is the minimal structure of bacteria’s peptidoglycan for the NOD2 activation and recently it has been demonstrated to directly bind to NOD2, stimulating NF-κB production, triggering innate immune responses.1

We provide a flexible strategy toward the preparation of diverse N-substituted muramyl dipeptides (N-substituted MDPs) from different protected monosaccharides. Diverted MDPs were synthesized including N-acetyl MDP and N-glycolyl MDP, and this methodology allows for structural variation at six positions, including the muramic acid, peptide, and N-substituted moieties.2 On the other hand, sugar part of MDP were modified in order to generate a simplified structure to induce human NOD2 activation. Those compounds of synthesized for NOD2 activation are study now.

Referrences

1. C. L. Grimes, L. de Ariyananda, J. E. Melnyk, E. K. OShea, J. Am. Chem. Soc. 2012, 134, 13535

2. K. T. Chen, D. Y. Huang, C. H. Chiu, W. W. Lin, P. H. Liang, W. C. Cheng, Chem. Eur. J. 2015, 21, 11984

175. Inhibition of allergic rhinitis by anti-sulfated-glycan monoclonal antibodies

Jotaro Hirakawa 1 , Hiroto Kawashima 1; 1Chiba University, Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, 260-8675, Chiba, Japan

jotaro@chiba-u.jp

The novel anti-sulfated-glycan monoclonal antibody S2 which recognizes 6-sulfo sialyl Lewis X (sLeX) expressed on high endothelial velunes (HEV) in the nasal-associated lymphoid tissue (NALT). NALT is a mucosal lymphoid tissue that functions as a border of defense against inhaled antigens. Lymphocyte recruitment to NALT is mediated by the interaction of L-selectin on lymphocytes and sulfated-glycans on HEV in NALT. HEV are specialized post capillary venules regulating lymphocyte entry form blood into parenchyma of lymphoid organs. It has been previously reported that lymphocyte recruitment to NALT was markedly blocked in HEV-expressed N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) -1 and -2 double-deficient (DKO) mice. In the DKO mice, immune responses against nasally administered ovalbumin (OVA) including OVA-specific IgE, sneezes and nose scratches were significantly diminished.

S2 was established by immunizing GlcNAc6ST-1/2 DKO mice with CHO-K1 cells stably expressing 6-sulfo sLeX on the cell surfaces. S2 administration inhibited lymphocyte recruitment to NALT and allergic rhinitis induced by intranasal administration of OVA. S2 administration to OVA-immunized mice resulted in the reduction of OVA-specific serum IgE, and counts of sneezes and nose scratches after nasal immunization. Consistently, total cell numbers in NALT and expression of Th2-related cytokines such as IL-5 and IL-13 were diminished after S2 administration. Thus, these results suggest that S2 would serve as a novel therapeutic agent useful for the treatment of allergic rhinitis.

176. Glucosamine inhibits inflammation in LPS-induced acute lung injury mice

Kyoung-Hong Kim 1 , Ji-Sun Hwang 1 , Yunkyoung Lee 1 , Jiwon Park 1 , Sang-Min Kim 1 , Jaehee Lee 1 , Jinwook Shin 2 , Inn-Oc Han 1; 1Department of Physiology and Biophysics, College of Medicine, Inha University, 100 inharo, Nam-gu Incheon 22212, Korea, 2Department of Microbiology, College of Medicine, Inha University, 100 inharo, Nam-gu Incheon 22212, Korea

iohan@inha.ac.kr

Acute lung injury (ALI) is a common complication of sepsis in patients with impaired immune function. Previous studies have shown that glucosamine inhibits LPS-induced inflammation by inhibiting NF-κB activation in vitro. In this study, we aimed to investigate suppressive effects of glucosamine on the inflammatory response in the lungs of LPS-induced sepsis mice. Mice were intraperitoneally (i.p.) injected with lipopolysaccharide (LPS, 5 mg/kg) to induce sepsis-induced acute lung injury. One group mice were i.p. injected with glucosamine (200 mg/kg) at 2hr before LPS injection. Our data show that glucosamine inhibited LPS-induced nitric oxide (NO) and lactate production in serum as well as attenuated LPS-induced elevation of NO synthase (iNOS) in lung homogenates. Expression of interleukin-1β, interleukin-6 and tumor necrosis factor-α by LPS was inhibited in the glucosamine-pretreated LPS group. In addition, immunohistochemistry and western blot analysis show that glucosamine-pretreated LPS group significantly increased O-GlcNAcylation compared to LPS group. In particular, O-GlcNAcylation of c-Rel, a member of NF-kB, increased in lung homogenates of LPS-induced sepsis mice, while glucosamine attenuated these effects. These results demonstrate that glucosamine inhibits inflammations LPS-induced acute lung injury by inhibiting NF-kB activity.

177. Distribution of two medically relevant polysaccharides in Acinetobacter baumannii population by CPS antibody, its passive protection and bifurcated pathway study for new targets as vaccine candidates

Feng-Ling Yang 1 , I-Ming Lee 1 , Shu-Chen Kuo 2 , Wan-Ling Wu 1 , Jeffy Chern 1 , Shui-Tsung Chen 1 , Nien-Tsung Lin 3 , Shih-Hsiung Wu 1; 1Academia Sinica, Institute of Biological Chemistry, 11529, Taipei, Taiwan, 2National Health Research Institutes, 35053, Miaoli County, Taiwan, 3Tzu Chi University, Institute of Microbiology, 907, Hualien, Taiwan

shwu@gate.sinica.edu.tw

Treating drug-resistant A. baumannii with high mortality is a challenge in this post-antibiotic era due limitation of available drug choices. Although the treatments of tigecycline, colistin, and sulbactam have favorable clinical outcomes, the low serum level and bacteriostatic nature of tigecycline, nephrotoxicity of colistin and emerging resistance against sulbactam limit their use in critically ill patients. Therefore, it is an urgent need for a novel therapeutic modality.

Bacterial surface polysaccharides elicit antibodies during infection and are essential for resistance to complement killing. Capsular polysaccharide (CPS) (K antigen) localized in the outer layer of the bacteria is immunogenic and plays an important role in the pathogenesis. Until now, there are more than 20 A. baumannii CPS structures have been determined. The chemical structures of the capsular polysaccharide from two clinic A. baumannii SK44 and 54149 in Taiwan were determined by the cleavage of specific phage enzymes, GC-MS, NMR, and mass spectrometry.

Our polyclonal antibodies were generated from purified capsular polysaccharides. The polyclonal antibody of SK44 CPS and 54149 CPS reacted with 60% and 25% of Taiwan clinical isolates (250 strains) respectively. Furthermore, twenty-two clinic strains isolated from America were also assayed. Out of them, 19 strains were recognized by SK44 CPS antibody, and 3 strains were recognized by 54149 CPS antibody.

We found that SK44 CPS-induced antibody provided 55% protection in 32 μg per mice against challenge in an animal model. Also, this antibody reduced the lung bacterial infection significantly even at low dosage (4 μg). The high antigenic specificity exists in the SK44 CPS antibody.

A. baumannii was discovered to have a bifurcated pathway for O-glycosylation and capsule synthesis. Therefore, we used immunoprecipitation (IP) to identify possible CPS-anchored outer membrane proteins. From MS/MS and bio-informatics analysis, we identified 12 outer membrane proteins as the possible CPS-anchored candidates, including OmpA/motB, putative outer membrane protein W, TonB-dependent receptor, and Omp38, along with their corresponding proposed glycosylation sites. These findings are consistent with a recent A. baumannii O-glycosylation study that OmpA/motB belongs to an O-linked glycoprotein.

178. Evaluation of plasma N-Glycome as potential biomarker for neurocysticercosis

Pedro Bonay 1 , Isabel Quintana 1 , Agnes Fleury 2 , Andrea Toledo 2 , Maria Jesus Pertegeuer 3 , Teresa Garate 3; 1Universidad Autonoma de Madrid-Centro de Biologia Molecular "Severo Ochoa", 28049, Madrid, Spain, 2Unidad Periférica del Instituto de Investigaciones Biomédicas en el Instituto Nacional de Neurología y Neurocirugía, Universidad Nacional Autónoma de México, México D.F., México., 3Instituto de Salud Carlos III, Centro Nacional de Microbiología, Servicio de Parasitología, 28220 Majadahonda, Madrid, Spain.

pbonay@cbm.csic.es

Human neurocysticercosis (NCC) is a heterogeneous pathology. Clinical manifestations (from asymptomatic to a severe intracranial hypertension) vary according to number, size, stage and location of cysts, intensity of host immune response and parasite genotype. Although diagnosis tools and treatments have improved during the last decades, the prognostic of the patients is highly variable; some of them will completely respond to the first therapeutical cycle while in others, parasites will persist in spite of numerous treatment courses and severe complications can be developed. Therefore, the availability of new markers, e.g. related to disease prognosis, different NCC pathological forms, response to cysticidal drugs and to anti-inflammatory treatments, etc, would help clinician in the patient management and its follow-up.

In recent years, plasma N-glycans have emerged as biomarkers for health and disease. During the development of disease, changes in cellular glycosylation are observed, indicating that alterations of the glycome occur in extracellular fluids as well as in plasma/serum and could therefore serve as disease progression biomarkers.

The aim of this work was to identify new potential glycan biomarkers in NCC.

Plasma was collected from 80 NCC patients diagnosed with active or inactive cerebral cisticercosis (40 HP10 positive and 40 HP10 negative), 12 patients with other noninfectious neuropathologies and 60 spanish age-matched healthy controls. Total plasma N-glycome was measured by comprehensive and quantitative analysis by UPLC after deglycosylation and labeling with 2-AB.

From each plasma sample, 38 individual glycans and 17 different traits (antennae, sialylation, galactosylation, core and outer fucosylation, high-mannosylation) were evaluated.

We demonstrated, for first time whole plasma glycan profiling of inactive and active CC patients in comparison to healthy controls and showed that some individual glycans exhibit differential variation in their level in both groups when compared among each other and with the control population.

179. Synthesis of capsular polysaccharide of streptococcus pneumonia serotype 6A in various lengths for vaccine development

Mettu Ravinder 1 , Vulupala Hanmanth Reddy 1 , Hong-Jay Lo 1 , Chung-Yi Wu 1; 11Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nanakang, Taipei 115, Taiwan.

ravichemphd@gate.sinica.edu.tw

The objective of the current study was to develop a synthetic vaccine candidate against Streptococcus pneumonia which causes invasive pneumococcal diseases (IPD) in humans. Although, there are about 90 different serotypes of this bacterium were identified, few serotypes are virulent and responsible for IPD and those are current vaccine targets. Native polysaccharide vaccine PPV-23 and glycoconjugate vaccines such as PCV13, PCV 10, and PCV 7 are currently available pneumococcal vaccines and both of which have its own impact on controlling IPD. However, the effectiveness of both type of vaccines decreases with age and time since its vaccination, therefore revaccination is the only option to maintain the antibody titers for a long time. In recent days sequential PCV13 and PPV-23 vaccination are suggested which is a more economic burden. Both the vaccines afford protection against serotypes that included in the vaccine but not protective from non-vaccine serotypes. For instance, serogroup 6 consist of 6A, 6B, 6C, 6D serotypes out of which 6A and 6B are common and pathogenic, therefore targeted in all pneumococcal vaccines. But, rising of pneumococcal diseases caused by non-vaccine serotype 6C has been observed worldwide. Furthermore, it is very difficult to incorporate all the serotypes into the vaccine. However, there is the chance of cross-reactivity due to the structural similarity of these serotypes and the antibodies induced by one serotype are able to recognize remaining serotypes of the same group. Therefore the development of common vaccine candidate in the same group is preferable.

The development of such vaccine candidate is a challenging task and a detailed structure and length of the saccharide that suitable for immunogenic is required. We believe that synthetic vaccine candidate can solve those problems. Herein, we described the synthesis of various lengths of capsular polysaccharide of serotype 6A which is structurally similar to 6B and 6C.

180. Detection and functional characterization of Sialyl Lewis X antigen in mice using novel anti-carbohydrate monoclonal antibodies

Ryuji Matsumura 3 , Jotaro Hirakawa 1 , Minoru Fukuda 2 , Yasuyuki Imai 3 , Hiroto Kawashima 1; 1University of Shizuoka, Graduate School of Pharmaceutical Sciences, 4228526, Shizuoka, Japan, 2Chiba University, Graduate School of Pharmaceutical Sciences, 2608675, Chiba, Japan, 3Sanford Burnham Prebys Medical Discovery Institute, 97037, La Jolla, USA

h-kawashima@chiba-u.jp

Sialyl Lewis X (sLex) antigen functions as a common carbohydrate determinant recognized by all three members of the selectin family. However, its expression and function in mice remain undefined due to the poor reactivity of conventional anti-sLex monoclonal antibodies (mAbs) with mouse tissues. In the present study, we developed novel anti-sLex mAbs, termed F1 and F2, which react well with both human and mouse sLex, by immunizing fucosyltransferase (FucT)-IV and FucT-VII doubly deficient mice with 6-sulfo sLex-expressing cells transiently transfected with an expression vector encoding CMP-N-acetylneuraminic acid hydroxylase. F1 and F2 specifically bound both the N-acetyl and the N-glycolyl forms of sLex as well as 6-sulfo sLex, a major ligand for L-selectin expressed in high endothelial venules, and efficiently blocked physiological lymphocyte homing to lymph nodes in mice. Importantly, both of the mAbs inhibited contact hypersensitivity responses not only when administered in the L-selectin-dependent sensitization phase but also when administered in the elicitation phase in mice. When administered in the latter phase, F1 and F2 efficiently blocked rolling of mouse leukocytes along blood vessels expressing P- and E-selectin in the auricular skin in vivo. Consistent with these findings, the mAbs blocked P- and E-selectin-dependent leukocyte rolling in a flow chamber assay. Taken together, these results indicate that novel anti-sLex mAbs reactive with both human and mouse tissues with the blocking ability against leukocyte trafficking mediated by all three selectins have been established. These mAbs should be useful to determine the role of sLex antigen under physiological and pathological conditions.

181. Marker antibodies recognizing carbohydrate structures on glycoproteins and glycolipids on human iPS/ES cells

Toshisuke Kawasaki 3 , Hiromi Nakao 3 , Yuko Nagai 1 , Hidenao Toyoda 1 , Tomoya Akama 2 , Nobuko Kawasaki 3; 1Ritsumeikan University, Research Center for Glycobiotechnology, 525-8577, Kusatsu, Japan, 2Ritsumeikan University, College of Pharmaceutical Sciences, Laboratory of Bio-analytical Chemistry, 525-8577, Kusatsu, Japan, 3Kansai Medical University, Department of Pharmacology, 573-1010, Hirakata, Japan

tkawasak@fc.ritsumei.ac.jp

Carbohydrate-recognizing antibodies are useful tools for monitoring cell surface glycan structures with high sensitivity and strict specificity. Most of the marker antibodies to human embryonic stem (hES) and human-induced pluripotent stem (hiPS) cells are carbohydrate-recognizing antibodies, which include stage specific embryonic antigen (SSEA)-3 and SSEA-4, recognizing globosides, and tumor rejection antigen (TRA)-1-60 and TRA-1-81, recognizing glycoproteins. However, these antibodies also recognize human embryonal carcinoma (hEC) cells. Accordingly, we generated monoclonal antibodies, R-10G (1) and R-17F (2), which bind strongly to hiPS/hES cells but exhibit little or no binding to hEC cells. R-10G epitope is characterized as a type of keratan sulfate and the core protein was identified as podocalyxin (1). In contrast, the predominant part of the R-17F epitope was identified as a glycolipid, lacto-N-fucopentaose I (LNFP I), Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc, although it was found later that R-17F epitope is expressed significantly on podocalyxin and other glycoproteins as well. Interestingly, R-17F, when added to hiPS/ES cell suspensions, exhibited potent dose-dependent cytotoxicity. Cell death was not mediated by apoptosis but by necrosis. An ELISA test with a series of N-acetyllactosamine tetrasaccharides indicated that the minimum epitope structure of R-10G is Galβ1-4GlcNAc(6S)β1-3Galβ1-4GlcNAc(6S) β1 (type 2-type 2, 2S) and that of TRA-1-60 is Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1 (type 1-type 2, 0S), being in agreement with the previous report of Natunen et al., (3). In addition, it was shown for the first time that TRA-1-60/81 recognizes sulfated type 1-type 2 N-acetyllactosamine as well as non-sulfated one. Furthermore, keratanase II was shown to degrade not only type 2-type 2 glycans but also type 1-type 2 glycans slightly. These observations might be interesting in considering controversies about whether the type 1-type 2 N-acetyllactosamine epitopes recognized by TRA-1-60/81 on hiPS/ES cells are modifications of keratan sulfate or mucin-type O-glycans.

(1) Kawabe, K., et al., Glycobiology, 23, 322–336 (2013)

(2) Matsumoto, S., et al., J Biol Chem, 290, 20071–20085 (2015),

(3) Natunen, S., et al., Glycobiology, 21,1125–1130 (2011)

Glyco(proteo)mics analysis

182. Highly fucosylated N-Glycan as a hallmark of human saliva

Hantae Moon 1 , Bum Jin Kim 1 , Hyun Joo An 1; 1Graduate School of Analytical Science and Technology, Chungnam National University, 34134, Daejeon, Korea

hjan@cnu.ac.kr

In forensics, saliva encountered at a crime scene is one of the important evidences for crime investigation and thus, identification of human saliva from other body fluids such as blood and sperm and other mammalian saliva is an essential prerequisite for further investigation. However, conventional methods such as biochemical amylase test and UV-based ALS to identify saliva still have a limitation by cross-reactivity with other body fluids. Therefore, new method is necessary to definitely determine human saliva from other body fluids. Glycosylation is highly sensitive to biological environment and numerous studies have been linked to changes in glycosylation to human fluids. In particular, saliva is highly rich in glycosylation containing mucins and glycoproteins such as IgA and PRG. Here, we explored saliva glycosylation closely using nano-LC/MS-based glycomic approach and identified human saliva-specific glyco-signatures compared with other human body fluids (serum and milk) and animal saliva (rat and bovine), respectively. Briefly, N-glycans in human saliva were enzymatically released by PNGase F and enriched by PGC-SPE. N-glycans were characterized by nano-LC-PGC chip/Q-TOF MS and structural information was obtained by nano-LC/MS/MS. We identified about 100 glycans including neutral and acidic glycans in human saliva. Most of them were decorated with a fucose, and the content of fucosylated glycans was more than 50%. In order to examine specific features of human saliva, we compared glycan compositions and amount with other glycans found in human fluids (serum and milk) and animal saliva (rat and bovine). We conclusively determined that highly fucosylated N-glycans containing from tetra to octa fucose (Hex5-7HexNAc4-6Fuc4-8) were saliva-specific molecules found in only human saliva. Among them, tetra fucosylated bi-antennary (Hex5HexNAc4Fuc4) and tri-antennary (Hex6HexNAc5Fuc4) glycans were present in all human saliva. Individual variation and stability of saliva glycans were also examined.

183. Quantitative analysis of glycan in yeast using metabolic isotope labeling of polysaccharides with isotopic glucose (MILPIG)

Ji-Yeon Kim 1 , Soo-Hyun Choi 1 , Jae-Min Lim 1 , Yeo-Jin Park 2 , Hye-Jung Choi 2 , Woo-Hong Joo 2 , Seong-Hun Kim 3; 1Changwon National University, Department of Chemistry, 51140, Changwon, South Korea, 2Changwon National University, Department of Biology and Chemistry, 51140, Changwon, South Korea, 3Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejeon 305-333, South Korea

jmlim@changwon.ac.kr

Glycosylation is one of the most common protein post-translational modifications (PTMs). Glycans are the glycosylated carbohydrate moieties on proteins. Glycans are involved in many biological functions such as cell-cell recognition, cell differentiation, cell development, tumorigenesis and metastasis, and inflammation etc. Because changes in the expression level of these glycans affect many physiological functions, it is important to analyze changes in glycans expression levels by quantitative analysis. Therefore, mass spectrometry-based quantitative analysis of glycans has been developed. Many quantitative analysis of glycan by mass spectrometry typically have been used by labeling methods such as isotopic permethylation, reductive stable isotope labeling, IDAWG, and so on.

In this study, we applied metabolic isotope labeling strategy for glycan quantitation in yeast (Saccharomyces cerevisiae). We used the Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose (MILPIG) method to label the light (12C) or heavy (13C1) glucose on glycans of yeast. As a result, we quantitatively analyzed the alteration of glycan in yeast under the conditions.

184. Quantitative analysis of N-linked glycan in rice plants using metabolic labeling with isotopic glucose by mass spectrometry

Soo-Hyun Choi 1 , Ji-Yeon Kim 1 , Jae-Min Lim 1 , Jae-Yong Yoo 2 , Kyun-Oh Lee 2; 1Changwon National University, Department of Chemistry, 51140, Changwon, South Korea, 2Gyeongsang National University, Department of Life Science, 52828, Gyeongsang, South Korea

jmlim@changwon.ac.kr

N-glycosylation is one of the most important post-translational modifications occurring in living organisms. In the case of animals, N-linked glycans have heterogeneous structures and have specific functions involved in cell-cell recognition, cell division, cell development, cell transport, cell differentiation, immune response, protein stability, and other many important biological phenomena depending on their structures. On the other hand, in the case of plants, N-linked glycan-mediated biological functions remain obscure.

Herein, we quantitatively analyzed the N-linked glycan of rice plant (Oryza sativa) using metabolic labeling with isotopic glucose by a mass spectrometry. For quantitative analysis, isotopic glucose is used for metabolic labeling of the glycan with a heavy condition (13C1-glucose), which can be achieved by replacing the carbon source needed to grow the plants in dark condition. We obtained the glycan from the shoot portion of rice plants grown from seed to adult in the environment of isotopic glucose containing Murashige and Skoog (MS) medium.

185. Comparison of membrane with cytoplasmic glycan profile changing with cellular senescence and human aging

Yoko Itakura 1 , Norihiko Sasaki, Masashi Toyoda; 1Tokyo Metropolitan Institute of Gerontology, Japan

yitakura@tmig.or.jp

Cells have various glycan-conjugated proteins. The glycans play essential roles in biological functions such as differentiation and immune response. Moreover, they have been used as a sign of disease or stem cell markers. Therefore, the glycans are expected to be biomarkers for physiological condition. However, the details of the specific glycans involved in aging are limited. 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 α2-6 and α2-3sialylated N- and O-glycans on the surface of fetus-derived cells more existence those of elderly-derived cells at early passage. Moreover, both cell types exhibited sequentially decreased α2-3sialylated O-glycan structures during the cellular senescence process (ref. 1). On the other hand, it was shown that intracellular glycans hardly changed with cellular senescence. Although cell surface glycans were changed with cellular senescence clearly within much younger cells, intracellular glycans were going to maintain without remarkable changes.

We consider that the glycan changes with aging in cell surface were significant and the keeping intracellular glycans was maintained for biological system in cells. Therefore, glycan profiling might be useful for the characterization of aging biomarkers.

Reference:

1) Itakura Y, et al., Cell Biosci., 2016, 18;6:14 DOI 10.1186/s13578-016-0079-5

186. Determination of O-glycosylation heterogeneity in recombinant coagulation factor IX using LC/MS/MS

Youngsuk Seo 1 , Gyeong Mi Park 1 , Myung Jin Oh 1 , Hyun Joo An 1; 1Chungnam National University, Graduate School of Analytical Science and Technology, 34134, Daejeon, Korea

ysseo910@gmail.com

Recombinant coagulation factor IX (rFIX) is an extremely heterogeneous glycoprotein having multiple N-and O-glycosylation which influence on pharmacological functions including biological activity, safety, and in vivo stability. The study of glycosylation on rFIX has mainly focused on N-glycans due to an inherent complexity of O-glycosylation and the absence of analytical platform. Here, we comprehensively characterized the O-glycosylation on recombinant coagulation factor IX (rFIX) using MS-based glycomic and glycoproteomic approach. Briefly, O-glycans were chemically released using β-elimination while O-glycopeptides were prepared by pronase digestion to determine site-specific O-glycosylation distribution. Successfully we could identify unique O-glycosylation modifications such as O-fucosylation and O-glucosylation as well as a typical O-glycosylation on rFIX using nanoLC separation, mass profiling, and diagnostic fragment ions by CID MS/MS. Particularly, through O-glycopeptide analysis, we demonstrated that O-glucosylation and O-fucosylation were uniformly displayed on O-glycosylation sites Serine 53 and 61 of epidermal growth factor domain, respectively. While core 1-type O-glycosylations were overwhelmingly distributed across other O-glycosylation sites (Threonine 159, 169, 172, and 179) in activation peptide of rFIX. Site-specific profiling including micro-and macro-heterogeneity could provide macroscopic aspect to figure out O-glycosylation pattern decorated on rFIX. Our analytical strategy based on O-glycomics and O-glycoproteomics would be a new paradigm to assess the quality and bioequivalence of biosimilars, particularly for therapeutic glycoproteins bearing multiple O-glycosylations.

187. One-fraction nanoLC-MS2/MS3 analysis for high throughput glycome-wide precision mapping of glycotopes

Yen-Ying Chen 2 , Cheng-Te Hsiao 2 , Po-Wei Wang 2 , Hua-Chien Chang 2 , Mei-Chun Yang 1 , Tong-Hsuan Chang 1 , Kay-Hooi Khoo 2; 1Institute of Biological Chemistry, Academia Sinica, 115, Taipei, Taiwan, 2GlycoNex Inc., 221, New Taipei City, Taiwan

biocanto@gmail.com

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. Since the same glycotopes can be equally or preferentially distributed over glycoproteins and glycolipids, a holistic view of their regulated expression necessitates a comprehensive mapping of N-, O-glycans, and glycolipids. Taking advantage of the increasing speed and performance of latest MS instruments, we have recently established a one-fraction, one-shot glycotope-centric glycomic workflow that would shorten analytical time without compromising the attainable glycomic resolution and precision. The GSL-derived glycan, N- and O-glycans from cells/tissues were released sequentially into one fraction, permethylated with or without additional separation into sulfated vs non-sulfated pool, and directly subjected to RP C18 nanoLC-MS2/MS3 analysis on an Orbitrap Fusion Tribrid MS system in both positive and negative ion modes. Productive MS2 containing diagnostic ions of glycotopes measured at the high mass accuracy and resolution are then filtered out by in-house developed glycomic data mining tool, GlyPick, along with any associated MS3 for both linkage confirmation and relative quantification. We now further append to this primary workflow several iterations that would collectively extend the achievable glycomic depth without increasing the total sample amount needed. These novel nanoLC-MS/MS approaches were optimized and validated against glycomic samples derived from gastrointestinal adenocarcinoma cells, tumor and adjacent non-cancerous tissues, which presented a rich source of Lewis a/x, sialyl Lewis a/x, H antigen type 1/2, and sulfated fucosylated glycotopes, to be quantitatively mapped at high throughput and precision.

188. Site-specific N-glycosylation in the endoplasmic reticulum and golgi of mammalian cells

Corina Mathew 1 , Marie Estelle Losfeld 1 , Chia-wei Lin 1 , Markus Aebi 1; 1ETH Zürich, Institute of Microbiology, 8093, Zürich, Switzerland

mathewc@ethz.ch

N-glycosylation is a common post-translational modifications that can be found on proteins. The process starts in the ER, where a preassembled glycan is transferred onto polypeptides and then subsequently modified by hydrolases and glycosyltransferases. Remodeling of the N-linked glycan takes places both in the ER and the Golgi compartment.

N-glycan processing results in a heterogeneous population of structures and the composition of the glycans varies depending of the cell type, protein and position of the N-glycan on a given protein.

We hypothesize, that the protein surface and its interaction with the covalently attached glycans are a major determinant of the N-glycan processing and hence responsible for these differences in glycan composition.

To investigate glycan structures on individual sites on model proteins we perform a quantitative, MS-based analysis and combine it with in vivo and in vitro approaches.

For the in vivo studies, yeast PDI containing five glycosylation sites was expressed in CHO cells and the glycan composition of analyzed in a site-specific manner. One of the sites (site 4) showed less processed glycans than the other sites. To evaluate the role of protein surface -glycan interaction, amino acids in the proximity of this glycosylation-site were mutated. We were able to alter site-specific N-glycan processing by altering the primary sequence of the model protein.

To characterize the role of protein surface-glycan interaction more precisely, we turned to in vitro analysis of glycoprotein processing. Different ER and Golgi hydrolases and glycosyltransferase were purified from overexpressing insect cells. Site-specific processing on protein-bound N-glycans of recombinant yeast PDI was monitored using quantitative MS analysis. We showed that the glycans from site 4 were processed significantly slower by enzymes such as α-1,2-mannosidases or α-1,3- β-N-acetylglucosaminyltransferases.

This site-specific effect is due to the tertiary structure of the protein because reducing and alkylating the model protein before the in vitro assay prevented site-specific processing.

189. Efficient LC-MS2/MS3-based glycotope-centric in depth spatial glycomic analysis of single mouse brain

Pao-Yuan Wang 1 , Hua-Chien Chang 1 , Cheng-Te Hsiao 1 , Chien-Yu Lin 2 , Hsing-Lin Lai 2 , Yijuang Chern 2 , Kay-Hooi Khoo 1; 1Academia Sinica, Institute of Biological Chemistry, 11529, Taipei, Taiwan, 2Academia Sinica, Institute of Biomedical Sciences, 11529, Taipei, Taiwan

kkhoo@gate.sinica.edu.tw

The characteristic complex type N-glycans of mouse brain are known to comprise truncated structures with 2 to 3 non-galactosylated GlcNAc termini, and bi- to multi-antennary structures elaborated with a combination of ±sialyl LacNAc and Lewis X termini. Other less abundant glycotopes including disialylated LacNAc and various sulfated structures have also been reported on specific isolated glycoproteins but not in the context of global glycomic analyses on single mouse brain. Taking advantages of recent advances in mass spectrometry (MS), we have undertaken in depth glycomic analyses of tissues derived from different mouse brain regions. Our unique nanoLC-MS2/MS3-based glycotope-centric glycomic workflow relies on i) permethylation to allow facile enrichment of sulfated glycans for separate analyses in negative ion mode; ii) glycotope MS2 diagnostic ion-dependent MS3; and iii) in house developed software tool for automated data processing and analysis. We not only identified all previously reported glycotopes but additionally resolved the relative amount of isomeric i) fucosylated LacNAc glycotopes (LeX, LeA, H1, H2); ii) terminal disialyl unit (NeuAc-NeuAc-Hex-HexNAc) versus disialylated type 1 structure (NeuAc-Hex-3(NeuAc6)HexNAc; and iii) Gal-3-O-, Gal-6-O- versus GlcNAc-6-O-sulfated glycotopes. Remarkably, we found a high abundance of terminal sulfated GlcNAc on the truncated structures, and both mono- and di-sulfated LacNAc, LeX, and sialyl LacNAc, not previously reported. Our glycomic analyses further demonstrated substantial difference in the expressed sulfo-glycotopes among distinct brain regions, whereas any difference for the non-sulfated glycans are not apparent. The rapid, efficient and highly sensitive data acquisition and analysis pipeline now allows us to meaningfully looking into brain glycomic changes induced by genetical manipulations and/or disease onsets.

190. Rapid chemical deglycosylation and derivatization for analysis of glycoprotein N-Linked glycans

Akihiko Kameyama 1 , Santha K. Dissanayake 1 , Wai Wai Thet Tin 1; 1AIST, Biotechnology Research Institute for Drug Discovery, 305-8568, Tsukuba, Japan

aki-kameyama@aist.go.jp

Glycan analysis inevitably comes with exploitation of glycan biomarkers and evaluation of the glycosylation heterogeneity of biopharmaceuticals. With increasing attention on such research, an easy and rapid method for glycan analysis is of great interest. For N-linked glycan analysis by high performance liquid chromatography with fluorescent detection, burdensome pretreatments including protein denaturation, protease digestion, PNGase F or A digestion, glycan purification, and labeling with fluorescent tags are usually required. This procedure often takes over 24 hours and consumes expensive enzymes. Hydrazinolysis has also been used to prepare N-linked glycans from glycoproteins. However, this reaction requires completely anhydrous conditions that take a long time to prepare, as well as hydrazine that must be handled with meticulous care because it is extremely toxic and hazardous. Furthermore, hydrazinolysis is incompatible with glycans containing N-glycolylneuraminic acid (NeuGc). We investigated alkaline hydrolysis of the asparagine glycosyl carboxamide of glycoproteins as a deglycosylation reaction. By adding hydroxylamine into the alkaline de-N-glycosylation, we could suppress degradation of the released glycans. The reaction could be finished within 1 h, and the obtained glycan derivatives could be easily tagged with 2-aminobenzamide (2-AB) by reductive amination. Here we demonstrated N-linked glycan analysis using this method for a monoclonal antibody, bovine apo-transferrin, and horse radish peroxidase, and showed that the method can be applied to variety of glycoproteins regardless of the presence of NeuGc and Fuc α1-3 GlcNAc which are unsuited for hydrazinolysis and PNGase F, respectively.

191. Mass spectrometry-based sequencing and identification of multiply O- and N-glycosylated peptides from receptor protein tyrosine phosphatases

Chu-Wei Kuo 1 , Yu-Chun Chien 1 , Deepa Sridharan 1 , Takashi Angata 1 , Tzu-Ching Meng 1 , Kay-Hooi Khoo 1; 1Academia Sinica, Institute of Biological Chemistry, 11529, Taipei, Taiwan

kkhoo@gate.sinica.edu.tw

Recent advances in mass spectrometry (MS) has enabled increasingly meaningful glycoproteomic venture but such global undertaking often lacks in specific details and misses the key glycoforms from key protein sites. Many false positives and mis-assignments of glycan compositions exists in the reported positive spectrum matches produced from searching the large MS2 dataset against protein database and glycan library using software tools such as Byonic. MS2-sequencing of N- and O-glycopeptides each presents its own problems and is particularly daunting when the peptides are not only heavily O-glycosylated but further carrying N-glycans. Such is the case for many cell surface receptors containing mucin-like domains, including members of the receptor protein tyrosine phosphatase (RPTP) family. Focusing on PTPRA, which contains as many as 7 N-glycosylation sites on a 120 amino acid-ectodomain that is Ser/Thr/Pro rich with only few tryptic sites, we show here how different strategies in genetic and enzymatic manipulations coupled with advanced HCD and EThcD MS/MS on Orbitrap Fusion enabled us to confirm the presence of densely-spaced O-glycans in between the N-glycosylations. In the case of PTPRE, its entire ectodomain is only 25 amino acid long but carries 2 N-glycans and multiple O-glycans, which could be detected and analyzed intact. However, distributing the deduced overall glycan composition into individual N- and O-glycans on specific sites necessitate good quality EThcD to delineate the huge combinatorial possibilities. A prior glycomic knowledge of the host cells, particularly those carried on the target glycoproteins, is also essential not only to construct sensible glycan library for MS2 spectral matching searches but also to ensure not missing out subsets of glycoforms less favorable to MS detection and sequencing.

192. Semi-quantitative measurement of a specific glycoform using a DNA-tagged antibody and lectin affinity chromatography for glyco-biomarker development

Nan-Ee Lee 1 , Do Yon Kim 1 , Kwang Sun Yoo 1 , Yong-Sam Kim 1; 1Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea,

omsys1@kribb.re.kr

Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising α-fetoprotein, hemopexin, and α-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the α-fetoprotein level or fuco-αfetoprotein test alone. The developed method is expected.

193. N-linked glycan imaging of tissue microarray using MALDI FT-ICR for early biomarker detection in hepatocellular carcinoma

Connor West 1 , Richard Drake 1 , Anand Mehta 1; 1Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA.

westco@musc.edu

Hepatocellular carcinoma (HCC) remains as the fifth most common cancer in the world and accounting for more than 700,000 deaths annually. Due to the relationship of survival rates and the time of detection, the need for early detection is critical. Our laboratory has developed a method of tissue based N-linked glycan imaging. This method bypasses the need for microdissection and solubilization of tissue prior to analysis. When matrix is applied across the tissue section, desorption can be targeted to specific “points” in a pattern and the data rasterized. The resulting spectra can then be used to generate two-dimensional maps of hundreds of glycans directly from the surface of a tissue section. These molecular maps display the relative abundance and spatial distribution of these molecules. Thus, MALDI tissue profiling has the power to link the molecular detail of mass spectrometry with molecular histology, generating mass spectra correlated to locations within a thin tissue section. We used this methodology in the analysis of over 180 HCC samples and compared the N-linked glycans in cancer tissue with either adjacent tissue or tissue from patients with just liver cirrhosis and no cancer. MALDI-Glycan imaging has identified over 5 glycans that were elevated in >85% of all HCC samples and many of these are found in serum. In addition, our data suggest that specific glycan alterations may be associated with specific genetic lesions that drive cancer development. This work represents the first complete characterization of the N-linked glycans in HCC tissue in a manner that retains tissue morphology and pathological information.

194. Analysis of fucosylated glycopeptide in alpha-fetoprotein immunoprecipitated from hepatocellular carcinoma serum by parallel reaction monitoring

Kwang Hoe Kim 1,2, Heeyoun Hwang 1, Ju Yeon Lee 1 , Ji Yeong Park 1,2 , Eun Sun Ji 1 , Hyun Joo An 2 , Soo-Youn Lee 3 , Jin Young Kim 1 , Jong Shin Yoo 1,2; 1 Biomedical Omics Group, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongju, 28119, Republic of Korea, 2Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea, 3 Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Republic of Korea

kkh2907@kbsi.re.kr

Fucosylation is one of the most common glycosylation and has been associated with the development of hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is clinically used as serological marker to diagnose HCC. Although the level of AFP increased in HCC patients, its sensitivity for diagnosis of HCC is poor because AFP levels are also increased in liver diseases, such as liver cirrhosis and chronic hepatitis. Recently, fucosylated fraction of AFP have been developed as a serological marker for HCC to increase the diagnostic sensitivity. Here, we introduced detection of AFP glycopeptides by parallel reaction monitoring (PRM) mass spectrometry (MS) combined with immunoprecipitation. Also, de-sialylation of AFP glycopeptides was used to improve MS detection limit (LOD < 2 ng/mL) and to obtain reliable signal (CV < 20%) from sub μL serum. Finally, we compared serum samples from HCC with liver disease patients using relative percentage of fucosylated glycopeptide in AFP (AFP-fuc%). AFP-fuc% showed an area under the ROC curve (AUC = 0.949, p value < 0.0001) to discriminate between HCC and liver disease patients. These results suggest the potential of PRM-based AFP-fuc% in early diagnosis of HCC using serum samples.

195. Characterization of site-specific N-Glycopeptides of α-1-acid glycoprotein from human plasma by an interlaboratory study using LC-MS/MS

Ju Yeon Lee 1 , Hyun Kyoung Lee 1, 2 , Gun Wook Park 1, 2 , Heeyoun Hwang 1 , Hoi Keun Jeong 1, 2 , Ki Na Yun 1, 3 , Eun Sun Ji 1, 4 , Kwang Hoe Kim 1, 2 , Jun Seok Kim 5 , Jong Won Kim 5 , Sung Ho Yun 6 , Chi-Won Choi 6 , Seung Il Kim 6 ; Jong-Sun Lim 7 , Seul-Ki Jeong 7 , Young-Ki Paik 7 , Soo-Youn Lee 8, 9 , Jisook Park 10 , Su Yeon Kim 9 , Young-Jin Choi 11 , Yong-In Kim 11 , Jawon Seo 11 , Je-Yoel Cho 11 , Myoung Jin Oh 2 , Nari Seo 2 , Hyun Joo An 2 , Jin Young Kim 1 , Jong Shin Yoo 1, 2; 1Korea Basic Science Institute, Cheongju-Si, Republic of Korea, 2Chungnam National University, Daejeon, Republic of Korea, 3Sogang University, Seoul, Republic of Korea, 4Hannam University, Daejeon, Republic of Korea, 5Osong Medical Innovation Foundation, Cheongju, Republic of Korea, 6Korea Basic Science Institute, Daejeon, Republic of Korea, 7Yonsei University, Seoul, Republic of Korea, 8Sungkyunkwan University, Seoul, Republic of Korea, 9Samsung Medical Center, Seoul, Republic of Korea, 10Samsung Biomedical Research Institute, Seoul, Republic of Korea, 11Seoul National University, Seoul, Republic of Korea

jylee@kbsi.re.kr

Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using α-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC-MS/MS platforms. We used three test and one blind samples to evaluate instrument performance with entire sample preparation workflow. These samples were provided to seven laboratories along with trypsin, glycopeptide enrichment kit, the recommended LC-MS/MS conditions and protocols (protein digestion and glycopeptides enrichmen). All raw data were processed by IGPA (Integrated GlycoProteome Analyzer)1 in our laboratory. Total 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Differences of LC-MS performance and laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Therefore, this analytical method could be used as the standard protocol to characterize site-specific N-glycopeptide isoforms of glycoproteins. The protocol was successfully applied for the relative quantification of the 10 major N-glycopeptides from AGP in normal and HCC human plasma samples using various types of MS analyzers.

196. Comparison of site-specific N-glycosylation between human and mouse plasma using LC-MS/MS and GPA system

Hyun Kyoung Lee 1,2 , Ju Yeon Lee 1 , Gun Wook Park 1 , Jin Young Kim 1 , Jong Shin Yoo 1,2; 1Korea Basic Science Institute, O-chang Cheongju, Korea, 2Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea

hklee@kbsi.re.kr

Mouse has been used as an animal model for scientific research owing to its physiological similarity to human. However, their N-glycosylation of protein showed many differences between mouse and human. Usually N-glycosylation is directly involved in various biological process and plays crucial role in human diseases due to their unusual biological sensitivity. Therefore, the site-specific characterization of N-glycosylation in model mouse is necessary in order to investigate progression of disease.

This study provided comparison of the site-specific N-glycosylation between human and mouse plasma. We compared site-specific N-glycosylation in human and mouse plasma samples using LC-MS/MS with GPA (GlycoProteome Analyzer) system1. The site-specific N-glycosylation between human and mouse has three differences in plasma. First, as reported previously, the sialic acid of N-glycopeptides was almost entirely Neu5Gc in mouse plasma, while in human plasma was Neu5Ac. Second, O-acetylated sialylation of N-glycopeptide was identified in mouse plasma only at the NeuGc form of sialic acids. Third, a unique trisialylated biantennary N-glycopeptide (peptide_Hex2HexNAc2NeuGc3+Man3GlcNAc2) was specifically identified from several glycoproteins in mouse plasma. The trisialylated biantennary glycoform was recently reported in mouse serum, although it could not determine the binding position of the third Neu5Gc. We found that it is directly linked to GlcNAc with evidence of specific oxonium and glycopeptide fragment ions in MS/MS spectra. This clearly proves that the new structure of trisialylated biantennary N-glycopeptide found only in mouse plasma. In conclusion, our study has provided an overview of the site-specific N-glycosylation in plasma proteins of human and mouse.

Reference

1. Gun Wook Park and Jin Young Kim et al., Scientific Reports, 6:21175 (2016)

197. Classification of core and outer fucosylation in N-glycoproteins by mass spectrometry with machine learning

Hoi Keun Jeong 1,2 , Heeyoun Hwang 1 , Gun Wook Park 1 , Ju Yeon Lee 1 , Hyun Kyoung Lee 1,2 , Jin Young Kim 1 , Jong Shin Yoo 1,2; 1Biomedical Omics Group, Division of Bioconvergence Analysis, KBSI, Ochang, Korea, 2Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea

jhg12@kbsi.re.kr

Fucosylation of protein N-glycosylation is known to be highly involved during the progress of various cancers. Recently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been emerged as a powerful technique for identification of N-glycoproteins. However, it is still remained challenge to classify the fucosylation types of N-glycopeptides using collision-induced dissociation (CID) MS/MS spectrum. Here, we constructed training and test sets of more than 800 N-glycopeptides from MS/MS analysis of human immunoglobulin gamma (IgG), and alpha 1-acid-glycoprotein (AGP) standards identified by I-GPA software. We selected fourteen features based on their manual assignment of fragments such as B/Y fragment ions in CID MS/MS spectra. Then, for classifying fucosylation type of N-glycopeptides, we performed Support Vector Machine (SVM) and Deep Learning (DL) analysis using relative abundance of those fourteen features. Their accuracies for classification of fucosylation types of IgG and AGP were 80.9% and 85.9% compared to manual validation, respectively. For application, we classified the fucosylation types of N-glycoproteins from human plasma of normal and hepatocellular carcinoma, whereas more than 90% equality was shown between SVM and DL results. Two machine learning results indicated that the N-glycoproteins without fucosylation was decreased but those with core and outer fucosylation was increased in HCC.

Reference

1 GW Park et al., Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation. Sci Rep. (2016)6, 21175

198. Mass spectrometric characterizaton of fucosylation in liver-secreted N-glycoproteins from human plasma of hepatocellular carcinoma patients

Eun Sun Ji 1 , Heeyoun Hwang 1 , Gun Wook Park 1,2 , Ju Yeon Lee 1 , Hyun Kyoung Lee 1,2 , Hoi Keun Jeong 1,2 , Kwang Hoe Kim 1,2 , Jin Young Kim 1 , Jong Shin Yoo 1,2; 1Biomedical Omics Group, Korea Basic Science Institute, Ochang, Republic of Korea, 2Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Republic of Korea

heeyounh@kbsi.re.kr

Aberrant N-glycoproteins play important roles in biological processes of various diseases. Especially fucosylated N-glycoproteins have been related to hepatocellular carcinoma (HCC). However, few studies have been reported for the site-specific fucosylation of various liver-secreted N-glycoproteins. In this study, we characterized the fucosylation patterns of major plasma glycoproteins using a workflow to identify and quantify site-specific N-glycoprotein. We selected 13 major liver-secreted glycoproteins in plasma. A total of 189 N-glycopeptides from 13 target glycoproteins in human plasma was manually identified by nanoRPLC-MS/MS with HCD/CID fragmentation, where 71 fucosylated N-glycopeptides were found. Of the fucosylated N-glycopeptides, bi- and tri-antennary glycoforms were the most common ones identified in liver-secreted proteins from HCC plasma. Therefore, we suggest that this site-specific analysis of N-glycoproteins is an effective method for characterizing fucosylation in proteins secreted from liver.

199. Direct analysis of site-specific N-glycopeptides of serological proteins in dried blood spot samples

Heeyoun Hwang 1 , Na Young Choi 2 , Eun Sun Ji 1 , Gun Wook Park 1 , Ju Yeon Lee 1 , Hyun Kyoung Lee 1,3 , Jin Young Kim 1 , Jong Shin Yoo 1,3; 1Biomedical Omics Group, Korea Basic Science Institute, Ochang, Korea, 2Lab Solution Sales, Agilent Technologies, Seoul, Korea, 3Graduated School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea

heeyounh@kbsi.re.kr

Dried blood spot (DBS) samples have a number of advantages, especially with respect to ease of collection, transportation, and storage, and to reduce biohazard risk. N-glycosylation is a major post-translational modification of proteins in human blood that is related to a variety of biological functions, including metastasis, cell-cell interactions, inflammation, and immunization. Here, we directly analyzed tryptic N-glycopeptides from glycoproteins in DBS sample using liquid chromatography tandem mass spectrometry (LC-MS/MS) without centrifugation of blood sample, depletion of major proteins, desalting of tryptic peptides, and enrichment of N-glycopeptides. Using this simple method and I-GPA program [1], we identified a total of 41 site-specific N-glycopeptides from 16 glycoproteins in the DBS sample, from immunoglobulin gamma 1 (IgG-1, 10 mg/mL) down to complement component C7 (50 μg/mL). Of these, 32 N-glycopeptides from 14 glycoproteins were consistently quantified over 180 days stored at room temperature. The major abundant glycoproteins in the DBS sample were IgG-1 and -2, which contain 9 asialo-fucosylated complex types of 16 different N-glycopeptide isoforms. Sialo-non-fucosylated complex types were primarily detected in the other glycoproteins such as alpha-1-acid glycoprotein 1, 2, α-1-antitypsin, α-2-macroglobulin, haptoglobin, hemopexin, Ig α 1, 2 chain C region, kininogen-1, prothrombin, and serotransferrin. We first report the characterization of site-specific N-glycoproteins in DBS sample by LC-MS/MS with minimal sample preparation.

Reference

Part et al. Scientific Reports (2016) 6, 21175

200. Automatic identification and quantification of site-specific N- and O-glycoproteins using glycoproteomeanalyzer

Gun Wook Park 1 , Jin Young Kim 1 , Ju Yeon Lee 1 , Young-Mook Kang 1 , Heeyoun Hwang 1 , Hyun Kyoung Lee 1,2 , Eun Sun Ji 1,3 , Kwang Hoe Kim 1,2 , Hoi Keun Jeong 1,2 , Ki Na Yun 1,4 , Yong-Sam Kim 5 , Jeong-Heon Ko 5 , and Jong Shin Yoo 1,2; 1Department of Mass Spectrometry, Korea Basic Science Institute, Ochang, Republic of Korea, 2Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Republic of Korea, 3Department of Chemistry, Hannam University, Daejeon, Republic of Korea, 4Department of Chemistry, Sogang University, Seoul, Republic of Korea, 5Cancer Biomarkers Development Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea

cancun@kbsi.re.kr

We have developed Integrated GlycoProteomeAnalyzer (GPA) for high throughput analysis of N-and O-glycoproteome, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. We created novel scoring algorithms with calculation of false discovery rate (FDR) and label-free quantification method using the combined intensities of top three isotope peaks at three highest MS spectral points (3TIQ). All glycoprotein samples were digested with trypsin. Then, those site-specific N-glycopeptides were enriched by hydrophobic interaction liquid chromatography and analyzed by nano-reversed-phase liquid chromatography coupled to tandem mass spectrometry with both HCD and CID-MS/MS fragmentation. The resultant LC/MS/MS data were then automatically analyzed using GPA: tryptic N-glycopeptides were identified against the database from Uniprot, quantified using the 3TIQ, and finally compared between multiple samples. In GPA, all amino acid sequence and N-and O-glycosylation site information were obtained from the Uniprot database. From the Swiss-Prot accession number of human protein, GPA program automatically construct N-glycopeptide database for human plasma sample. GPA has been designed to easily handle high-throughput glycoproteomic data with a graphical user interface. GPA software is demonstrated on website (https://www.igpa.kr/). It can also be integrated with cloud computing service that eliminates the need for local clusters and increases throughput of data analysis. Additionally, O-GPA, newly developed search engine, allows direct analysis of site-specific O-glycopeptides from glycoprotein mixtures using the O-GPA-DB from Uniprot with estimated FDR ≤ 1%. O-GPA is capable of automatic identification and quantification of O-glycopeptides using HCD and ETD-MS/MS spectra.

201. In-depth strucutral characterization of EPO (Erythropoietin)

Myung Sin Lim 1; 1Osong medical innovation foundation, Korea

sinistemcells@kbio.kr

Recombinant erythropoietins (EPOs) are an important class of biotherapeutics that stimulate red blood cell production. Pharmacokinetic parameters of EPOs are known to be importantly affected by their glycosylation including sialic acid and its O-acetylation. Therefore, in-depth characterization of glycosylation is critical step in development and manufacturing processes to ensure efficacy, safety, and pharmacokinetic properties of EPOs. Recently, mass spectrometry (MS) has been emerged as premier tool for glycomic analysis. Recently, it provides rapid and sensitive detection of sample preparation and analysis can be used as a precise tool for structural elucidation. Therefore, Mass has contributed significantly to recent progress towards understanding the role of the glycome in biotherapeutics glycoproteins including mAbs, recombinant proteins, therapeutic enzymes, and fusion proteins. In this study, we provide of glycan analysis platform for glycomic characterization of EPO biotherapeutics.

Structural glycobiology

202. Mass spectrometry characterization of the human endosulfatase HSulf-2

Ilham Seffouh 1 , Cédric Przybylski 1 , Amel Seffouh 2 , Rana Masri 2 , Dian Lebeau 1 , Romain Vivès 2 , Florence Gonnet 1 , Régis Danial 1; 1Université Paris-Saclay, CNRS, CEA, Univ. Evry, Laboratoire Analyse et Modélisation pour la Biologie et l’Environnement, F-91025 Evry, France, 2Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, F- 38000 Grenoble, France

regis.daniel@univ-evry.fr

The human heparan sulfate 6-O-endosulfatases (HSulf-1 and HSulf-2) catalyse the regioselective hydrolysis the 6-O- sulfate groups within heparan sulfate chains present on cell surface proteoglycans. Hsulfs exhibit two unique features among human sulfatases, as they are the only known sulfatases to be secreted and active in the extra-cellular medium, and to be active at the polymer level. By specifically modifying the 6-O-sulfation patterns of heparan sulfate (HS), HSulfs affect the polysaccharide modulatory properties towards a large number of growth factors, morphogens and chemokines.

Governing an unprecedented post-biosynthetic edition of the sulfation HS, HSulfs are involved in important developmental and tumor processes. Despite their key roles, the Hsulfs enzymes remain elusive protein objects as regard their structure and functions. Much effort has been devoted to the understanding the enzyme reaction specificities, while much less is know about the structural organization. HSulf-2 is a heterodimer made of a 75 kDa and 50 kDa subunits likely joined by disulfide bonds, and comprising several potential N-glycosylation sites. While HSulfs are indirectly detected in most reported studies by western-blotting and through the monitoring of their catalytic activity, our goal was to detect and characterize Hsulf-2 by MALDI and electrospray mass spectrometry.

In the present study, we report the first mass spectrometry characterization of HSulf-2, allowing its direct detection at the protein level. We obtained the coverage of the full protein sequence by using a combination of several proteases in a bottom-up proteomics analysis.

203. Molecular insights of arginine glycosylation by crystal structure of neisseria meningitidis EarP, an arginine glycosyltransferase

Junbae Park 1 , Youngki Yoo 1 , Ong Xiang Yu Belinda 1 , Hyun-Soo Cho 1; 1Yonsei University, Department of Systems Biology, 03722, Seoul, Korea

hscho8@gmail.com

Elongation factor P (EF-P) is one of the component of the protein translation machinery and this protein is required for efficient peptide bond synthesis in ribosome. Post-translational modification of EF-P is important and essential for its function. Previous research demonstrated that some bacterial EF-P is rhamnosylated by the EarP enzyme, which is glycosyltransfease. Surprisingly, the glycosylation target is arginine residue and the molecular mechanism of arginine glycosylation is unknown. Here, we report the crystal structure of EarP from Neisseria meningitides to gain an insight to the chemical mechanism. From the structure, the putative arginine binding pocket has negatively charged residues and they seem to interact with guanidine of arginine via ionic interactions. Furthermore, as compared to the other arginine modifiable enzyme such as arginine kinase or arginine methyltransferase, EarP seems to have a similar chemical mechanism for arginine modification. The strongly negative charged pocket in EarP is able to increase the nucleophilicity of guanidine and the rhamnosyl group from TDP-rhamnose may possibly form a covalent linkage with the activated guanidine. Here, we propose the enzyme mechanism about how arginine can be glycosylated based on the structure. Also, this structural information suggests that EarP protein can be a good target for drug against meningococcal bacteria.

204. Crystal structure of heparan sulfate bounded human RAGE

Junbae Park 1 , Youngki Yoo 1 , Ong Xiang Yu Belinda 1, Hyun-Soo Cho; 1Yonsei University, Department of Systems Biology, 03722, Seoul, Korea

hscho8@gmail.com

Receptor for advanced glycation end products (RAGE) is a single transmembrane receptor which causes various inflammatory diseases. RAGE is composed of three consecutive IgG-like domains (V-C1-C2 domain) which interact with various ligands at the extracellular space. Such ligands are able to induce the different oligomeric states of RAGE for ligand-specific signal transduction. Through the X-ray crystallographic approach, we report the heparan sulfate-bounded RAGE structure in 3.6Å resolution. In the structure, positively charged dimeric V-C1 domain pocket interacts with heparan sulfate via ionic interactions. Compared to the DNA bounded RAGE structure, the overall structure is similar but the ligand binding pocket of V-C1 domain is smaller to accommodate the heparin sulfate. Thus, this suggests that the V-C1 domain pocket is flexible and changeable depending on the specific ligands. Interestingly, the β-strand swapping dimer is found in asymmetric unit, which a β-strand of one protomer is exchanged with the other protomer to form a dimer structure. Exchanged β-strand is located in distinct position from the ligand binding pocket independently with heparin. This suggests an additional oligomeric state is possible under acidic condition (pH 4.6) indicating RAGE dynamics in physiological conditions.

Microbial glycobiology

205. Functional analysis of N-glycans assembled on surface mannoproteins in the interaction of cryptococcus neoformans with host cells

Jungho Kim 1 , Eun Jung Thak 1 , Seung Yeon Chung 1 , Dong-Jik Lee 1 , Hyun Ah Kang 1; 1Chung-Ang University, Department of Life Science, 06974, Seoul, Korea

hyunkang@cau.ac.kr

Cryptococcus neoformans is the opportunistic pathogenic fungus causing life-threatening meningoencephalitis in immunocompromised individuals, such as AIDS patients. Several C. neoformans mannoproteins (MPs), such as MP98, MP88, and MP84, are reported as a key antigens stimulating host CD4(+) T-cell response. In this study, we investigated the effect of truncated N-glycans on the interaction of Cryptococcal surface MPs with host cells. The expression pattern analysis of the histidine (His)-tagged and GPI-anchorless MPs in the wild-type (WT) and various N-/O-glycosylation mutant strains indicated that MP98 is modified mainly by N-glycosylation, while MP88 and MP84 are subject to both N-glycosylation and O-mannosylation. His tagged MPs were secreted from the WT and a N-glycosylation defective Cnalg3Δ mutant strain in the cap59Δ mutant background to facilitate purification of secretory proteins by inhibiting capsule biosynthesis. The in vitro adhesion assay using the purified MPs showed that all of the MPs adhered much more efficiently to epithelial lung cells than to macrophage cells. The alg3Δ-secreted MP88(H) bearing shortened N-glycans showed slightly decreased adhesion to lung cells compared to the WT-secreted MP88(H). However, the alg3Δ-secreted MP98(H) and MP84(H) showed rather apparently increased adherence to lung cells than the WT-secreted MPs. These results indicate that the effect of truncated N-glycans on the adhesion efficiency of C. neoformans is protein-specific. The purified MPs with altered glycosylation would be used for further investigation on the interaction with dendritic cells to elucidate the structures of cryptococcal glycans responsible for stimulating host immune response.

206. Metabolic engineering of Escherichia coli for the biosynthesis of amino deoxy-sugar-conjugated flavonoids

Sumangala Darsandhari 1 , Ramesh Prasad Pandey 2 , , Prakash Parajuli 1 , Biplav Shrestha 1 , Jae Kyung Sohng 1; 1Sun Moon University, Department of Life Science and Biochemical Engineering,31460,Tangjeong-myeon,Korea, 2Sun Moon University, Department of BT-Convergent Pharmaceutical Engineering,31460,Tangjeong-myeon,Korea

dsumanng@gmail.com

Escherichia coli strains was constructed for the biosynthesis of amino deoxy-sugar by disrupting three genes; glucose phosphate isomerase (pgi), glucose-6-phosphate dehydrogenase (zwf), and uridylyltransferase (galU) genes from the genome and overexpressing two genses; dTDP-D-glucose synthase (tgs) and dTDP-Dglucose 4,6-dehydratase (dh) to divert the flow of carbon flux from glucose-1-phosphate to thymidine diphosphate 4-keto 4,6-dideoxy-D-glucose (dTKDG). We successfully synthesized the 4-amino 4,6-dideoxy-D-galactose and 3-amino 3,6-dideoxy-D-galactose sugars conjugated flavonoids by biotransformation in E.coli.

207. Innate immune responses to improve anti-carbohydrate vaccines against bacterial pneumonia

Paulina Kaplonek 2 , Felix Bröcker 2 , Adam Calow 2 , Ling Yao 1 , Claney L. Pereira 2 , Leif Sander 1 , Peter H. Seeberger 2; 1Max-Planck-Institute of Colloids and Interfaces, Department of Biomolecular Systems, Am Mühlenberg 1, 14476 Potsdam, Germany, 2Medizinische Klinik m.S. Infektiologie und Pneumologie, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany

Paulina.Kaplonek@mpikg.mpg.de

High mortality rates of bacterial pneumonia and increase of antibiotic resistance are the major reasons to develop novel vaccine strategies against Streptococcus pneumonia. Ideal vaccines should be affordable, broadly available, easy to administer and induce long-lived protective immunity which requires activation of both innate and adaptive immune responses. Our project leads to develop a new generation of vaccines against bacterial pneumonia based on the latest discoveries both in the field of innate immunity and pathogen recognition, and novel techniques in carbohydrate antigen design and production.

Preliminary results from our group showed protective effects of an SP3-tetra (the synthetic repeating unit tetrasaccharide of the S. pneumonia serotype 3 (SP3) capsular polysaccharide) CRM197 conjugate vaccine in vivo, but long-term protection was limited. New adjuvants, such as prokaryotic mRNA and other vita-PAMPs (viability-associated pathogen-associated molecular patterns), have been shown to promote long-lasting humoral immunity in mice.

We design PRR (pattern recognition receptor)-ligand based adjuvant formulations to selectively induce innate signals that promote Th1 and TFH differentiation and humoral immunity. Encapsulated purified PAMPs (e.g. bacterial mRNA), or synthetic ligands (e.g. Imiquimod) in poly(lactic-co-glycolic acid particles (PLGA) are used to improve the immunoprotective effect of SP3-tetra in conjugation to both CRM197 and conserved pneumococcal proteins (single amino acid substitution mutants of pneumolysin). We use an in vivo mouse model (C57BL/6 mice) immunized with SP3-tetra conjugate.

208. Inspired from naturally nucleoside antibiotics to develop an expeditious synthesis of bacterial translocase MraY inhibitors

Wan-Ju Liu 1 , Gong-Siang Hu 2 , Tzu-Yuan Lee 3 , Chia-Ming Hu 2 , Chu-Ruil Li 4 , Wei-Chieh Cheng 2 , Lee-Chiang Lo 1; 1National Taiwan University, Department of Chemistry, 10617, Taipei, Taiwan (R.O.C.), 2Academia Sinica, Genomics Research Center, 11529, Taipei, Taiwan (R.O.C.), 3National Chung Hsing University, Department of Chemistry, 40249, Taichung, Taiwan (R.O.C.), 4National Cheng-Kung University, Department of Chemistry, 70101, Tainan, (R.O.C.)

lclo@ntu.edu.tw

The rise of antibiotic-resistant bacteria has posed a serious public health problem and has stimulated the activity toward development of new antibiotics. Bacterial cell wall MraY, also called translocase, is one of essential enzymes in peptidoglycan biosynthesis. Therefore, development of MraY inhibitors will provide a new direction for synthesis of antibiotics.

Inspired from naturally occurring but structurally complicate nucleoside antibiotics, we would like to develop a general and flexible synthetic approach to not only prepare highly diverse nucleoside derivatives but also simplify chemical structures of synthetic molecules. To assembly fragments together efficiently, several accessible synthetic strategies such as an amide formation, click chemistry and urea are applied to carry out our bio-conjugation purpose.

Several structural modifications including the lipid diversity, uracil modification, and studying the possibility to replace the aminoribose moiety with other accessible groups were systematically investigated. With assistance of the synthesizer, semi-automated liquid handler and solid-phase extraction technique, our nucleoside-based library possesses a substituent diversity, chiral center diversity, and scaffold diversity.

Currently, our diverse library containing 50 members will be applied to study antibacterial activity and translocase MraY inhibition activity. More detailed SAR (structure activity relationship) is ongoing.

209. Defective N-glycan assembly by ALG3 deletion in cryptococcus neoformans impacts on macrophage lysis during host infection

Eun Jung Thak 1 , Dong-Jik Lee 1 , Hyun Ah Kang 1; 1Chung-Ang University, Department of Life Science, 06974, Seoul, Korea

hyunkang@cau.ac.kr

Cryptococcus neoformans is a basidiomycetous human fungal pathogen, causing over one million cases of lung and brain infection each year. To investigate the roles of N-glycans in cryptococcal pathogenesis, we have identified and functionally analyzed a C. neoformans ALG3 homolog (CnALG3), coding for a dolichyl-phosphate-mannose dependent α-1,3-mannosyltransferase. A null mutant strain of CnALG3 (Cnalg3Δ) was constructed and subjected to N-glycan structure analysis by HPLC, exoglycosidase treatment, and MALDI-TOF. It was shown that Cnalg3Δ had a defect in the conversion of Man5GlcNAc2-Dol-PP to Man6GlcNAc2-Dol-PP, the earliest step to attach a mannose residue to the lipid-linked N-oligosaccharide in the ER. Interestingly, most of the truncated N-glycans of Cnalg3Δ appeared to be devoid of a xylose residue. The mannoproteins produced in Cnalg3Δ, such as phospholipase PLB1, aiding fungal traversal across the blood-brain barrier, and T-cell antigen MP98, were shown to have truncated N-glycans. Cnalg3Δ displayed a moderate defect in capsule biosynthesis and in resistance to oxidative and cell wall stresses compared to the wild-type (WT) strain. However, noticeably, the Cnalg3Δ mutant strain showed fully attenuated virulence in a mouse model of cryptococcosis, suggesting that the defect in lipid-linked N-glycan assembly affect considerably the pathogenicity of C. neoformans. However, the attachment to macrophage and the non-opsonic phagocytosis of Cnalg3Δ was shown to be comparable to that of WT during cryptococcal pathogenesis, indicating that the truncated N-linked glycans may not affect the early steps in interaction with macrophages. The capacity to drive macrophage lysis (pyroptosis) after phagocytosis was greatly decreased in Cnalg3Δ, indicating a critical role of N-glycans in inducing host cell lysis as a mechanism of host cell escape.

210. Glycoside hydrolases encoded by the Methyloferula stellata genome

Daniil G. Naumoff 1; 1Winogradsky Institute of Microbiology, Laboratory of Wetland Microbiology, 117312, Moscow, Russia

daniil_naumoff@yahoo.com

Methyloferula stellata AR4 is an aerobic acidophilic methanotroph of the Alphaproteobacteria family Beijerinckiaceae. It is unable to grow on any multicarbon substrates, including carbohydrates. So, it is reasonable to conclude that all of its glycoside hydrolases use substrates synthesized intracellularly. The M. stellata draft genome sequence has been generated at the Joint Genome Institute (GenBank, ARWA00000000.1).

Homology search of M. stellata genome allowed us to reveal 30 genes encoding putative glycoside hydrolases. Based on the catalytic domains, they belong to 14 different families: GH2, GH3, GH10, GH13, GH15, GH23, GH25, GH39, GH65, GH77, GH94, GH102, GH103, and PF06202. The PF06202 family is closely related to families GH63 and GH133 of glycoside hydrolases. We used each obtained glycoside hydrolase catalytic domain as a query for screening the protein database by blastp program. As a result, we found that in the vast majority of cases the closest homologue belongs to Alphaproteobacteria. The only exceptions are two M. stellata proteins representing GH39 and GH65 families of glycoside hydrolases. Eleven closest homologues of the GH39-family putative β-galactosidase (GenPept, WP_020173696.1) belong to bacteria from phyla Actinobacteria and Firmicutes (23%–31% of sequence identity). Five closest homologues of the GH65-family putative carbohydrate phosphorylase (WP_020177329.1) belong to Betaproteobacteria (60%–64%).

The data obtained suggest a low input of distant lateral transfer into evolution of glycoside hydrolase genes in the obligately methanotrophic bacteria. This is unusual for other bacteria and most probably is caused by the special role of their glycoside hydrolases not involved in environmental carbohydrate utilization.

211. Chemical features matters: molecular mass impacts the immunomodulatory properties of Lactobacilli polysaccharides

Sabina Gorska 1 , Ewa Brzozowska 1 , Andrzej Gamian 1; 1Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland

sabina.gorska@iitd.pan.wroc.pl

Lactobacillus strains have been mostly characterized as a probiotic bacteria due to its beneficial effect to the host. The activity of probiotics depend on the strain. Accumulating evidence demonstrates that probiotics interact with host immune cells due to production of specific compounds. This compounds include the bacterial surface-located molecules, such as polysaccharides. Lactobacilli polysaccharides (PS) have been attracted intensive attentions in food research fields due to their rheological properties. Their structural diversity is largely due to the presence of different sugar monomers, glycosidic linkages, sequence of sugar units, degrees of polymerization, and branching point. Many polysaccharides with unique chemical composition have been found to be applicable for potential applications [1, 2, 3]. The aim of the present work was compare the chemical properties and the immunomodulatory effects of PS isolated from probiotic bacteria Lactobacillus rhamnosus LOCK 0908 to those of Lactobacillus rhamnosus LOCK 0900.

Methods

Strains were cultured in MRS medium under microaerophilic conditions. PS were isolated and purificated according to methods used in our laboratory [3]. The structures were determined using component analysis and NMR spectroscopy. We examined their ability to modulate immune response of the mouse bone marrow-derived dendritic cells (BD-DC) to Lactobacillus plantarum WCFS1.

Results

In combination with component analysis, NMR spectroscopy shows that LOCK 0908 strain produced two structurally different polysaccharide L908/1 and L908/2. However, the structure of L908/1 and L908/2 is identical to that of L900/2 and L900/3 isolated from L. rhamnosus LOCK 0900, respectively [3]. However, the polymers differ in that the molecular mass of L908/1 was calculated to be 2,2 x 10^6 Da, whereas that of L900/2 was reported to be 0,84 x 10^6 Da; the molecular mass of L908/2 was calculated to be 19,4 x 10^3 Da, whereas that of L900/3 was reported to be 18 x 10^3 Da. First, we observed that both polymers of L. rhamnosus LOCK 0908 induced BM-DC to produce IL-10 and IL-12 when applied alone, while PS from L. rhamnosus LOCK 0900 did not [3]. Second, exposure to L908/2 didn’t enhanced IL-12p70 production induced by L. plantarum WCFS1, while in contrast L900/3 did.

Conclusion

It can be deduced that the bioactivities of polysaccharides are restricted by their molecular mass.

This work was supported by grant co-founded by the National Science Centre of Poland under a grant decision no UMO-2012/05/D/NZ7/02494 to S. Górska

O-GlcNAc modification

212. O-GlcNAcylation of TRIM31 controls intestinal autophagy

Taeyun A. Lee 1 , Hyunbin D. Huh 1 , Eun A. Ra 1 , Areum Park 1 , Hyun Jin Choi 1 , Sungwook Lee 2 , Boyoun Park 1; 1Yonsei University, Department of Systems Biology, 03722, Seoul, Korea, 2National Cancer Center, Research Institute, 10408, Goyang, Korea

bypark@yonsei.ac.kr

Autophagy is a critical process for the recognition and elimination of intracellular pathogens, thus acting as an important defensive barrier against bacterial invasion. We have previously reported that human TRIM31 (tripartite motif), an intestine-specific protein localized in mitochondria, is essential for promoting bacteria-induced alternative autophagy. Here, we show that TRIM31 is O-GlcNAcylated by O-GlcNAc transferase at its C-terminal region. In particular, deletion of the C-terminal portion of TRIM31 (TRIM31Δ294-425) significantly increases TRIM31-mediated cytosolic autolysosomal formation in intestinal cells, when compared with wild-type TRIM31. These results indicate that O-GlcNAcylated TRIM31 tightly controls autophagy through a negative autoregulatory mechanism, which enables intestinal homeostasis. Thus, our findings provide new insights into the mechanism of O-GlcNAcylation in TRIM31-mediated intestinal autophagy, which may protect excessive autophagy activation to maintain intestinal homeostasis.

213. O-GlcNAcylation is impairing regeneration capacity during osteoarthritis pathogenesis

Sunyoung Lee 1 , Jin-Hong Kim 1; 1Seoul National University, school of biological sciences, 08826, Seoul, Korea

christy0925@naver.com

Osteoarthritis (OA) is a prevalent degenerative disease, which involves progressive and irreversible destruction of articular cartilage. We aimed to promote cartilage repair through stimulating anabolism pathway to synthesize chondrocyte ECM proteins. We identified a key regulator that is negatively correlated with cartilage anabolism axis. The inhibitor of this factor in mouse chondrocytes stimulated synthesis of ECM proteins. In addition, in vivo surgery-induced cartilage destruction mouse model, intra-articular injection of pharmacological inhibitors targeting this factor effectively enhanced cartilage regeneration. Recent studies reported an increase of global O-GlcNAcylation in OA cartilage of human patients. From this, we have hypothesized that functional changes of the key regulator are from the cross-talk between O-GlcNAcylation and phosphorylation. Through YingOYang in silico analysis, both O-GlcNAcylation and phosphorylation sites of this factor appeared very densely at the N-terminal domains. This result provides a strong evidence of O-GlcNAcylation in key regulators of OA pathogenesis. Further studies on the O-GlcNAcylation of the various OA regulator will contribute to understanding of OA pathogenesis.

214. The role of O-GlcNAcylation in osteoarthritic cartilage homeostasis

Jungkwon Shin 1 , Jin-Hong Kim 1; 1Seoul National University, School of biological sciences, 08826, Seoul, Korea

sjk0104@snu.ac.kr

O-GlcNAc modification is one of the dynamic post-translational modifications that regulate fundamental cellular processes. This modification is regulated by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), in a nutrient responsive manner. The alteration in protein O-GlcNAcylation has linked to the pathogenesis of various diseases such as Alzheimer, cancer and diabetes. In osteoarthritic cartilage, it has been recently reported that the global levels of O-GlcNAcylations are elevated. However, the role of O-GlcNAcylated proteins in osteoarthritis (OA) remains largely unknown. Our aim was to study whether and how O-GlcNAcylation affects the progression of OA. The effect of alterations in cellular O-GlcNAcylation level on chondrocyte catabolism and anabolism, which balances cartilage homeostasis was tested. In addition, we studied how O-GlcNAcylation is regulated in OA cartilage.

215. An insight into nuclear translocation mechanism of O-GlcNAcase

Yeon Jung Kim 1,2 , Eunah Kim 1,2 , Jin Won Cho 1,2; 1Yonsei University, Department of Integrated OMICS for Biomedical Science, 2Glycosylation Network Research Center, 03722, Seoul, Korea,

chojw311@yonsei.ac.kr

O-GlcNAc is a carbohydrate post-translational modification occurs on hydroxyl groups of serine, threonine residues of cytosolic and nuclear proteins. O-GlcNAcase (OGA) is the enzyme that catalyzes the removal of O-GlcNAc moiety. It has two isoforms, full OGA and variant OGA, and in early reports it is known that full OGA exists both in cytosol and nucleus, primarily to the cytoplasm. However, there are two opposite reports about the subcellular localization of two OGA isoforms by different research groups. So these two findings motivate our laboratory to commence this work as to nucleocytoplasmic shuttling mechanism of two OGA isoforms. We also focus on the various post-translational modifications on OGAs. Full OGA should weigh 103 kDa, but it weighs approximately 130kDa in reality. So we speculate that various modifications might be related to OGA’s subcellular distribution. To get the answers to several issues, we overexpressed FLAG tagged-OGA isoforms in HeLa cells to trace the subcellular localization. Consequentially we can get schematic drawing as to OGA’s nucleocytoplasmic shuttling, and we expect that this research makes it easy to understand O-GlcNAc modification.

Acknowledgment

This research was supported by the NRF funded by the Ministry of Education, Science and Technology (Grants NRF-2016R1A5A1010764 and NRF-2015M3A9B6073840 to J.W.C.).

216. Protein O-linked N-acetylglucosamine glycosylation aggravated NLRP3 inflammasome in human macrophages

Ji Young Kim 1 , Soo Hyun Kim 1 , Jin Hee Kim 1 , Hyo Seong Jeon 1 , Byung-Wan Lee 2 , Eun Seok Kang 2 , Bong-Soo Cha 2 , Yong-Ho Lee 2; 1Department of Internal Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea, 2Severance Biomedical Science Institute, Yonsei Biomedical Research Institute, Yonsei University College of Medicine, Seoul 03722, Republic of Korea

YHOLEE@yuhs.ac

O-linked b-N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification that affects numerous intracellular pathways including energy metabolism and inflammation. Nod-like receptor family protein 3 (NLRP3) inflammasome has known as one of the major contributor in sterile inflammation in obesity, atherosclerosis and diabetes. However, there is no report regarding the relationship between O-GlcNAc and NLRP3 inflammasome. Here, we investigated the role of O-GlcNAc glycosylation (O-GlcNAcylation) in the regulation of NLRP3 inflammasome using human primary macrophages. Peripheral blood mononuclear cells (PBMCs) were isolated from blood in subjects with type 2 diabetes and differentiated into macrophages after incubation with M-CSF for 7 days. Low and high concentration of glucose, followed by synthetic ThiaMet-G (10 μM), an O-GlcNAcase (OGA) inhibitor were pre-treated in PBMCs and then lipopolysaccharide (LPS) / ATP were used for activation of NLRP3 inflammasome. IL-1β secretion from PBMCs was significantly increased by glucose concentration in dose-dependent manner. Western blot revealed that O-GlcNAcylation of proteins was increased in macrophages under high glucose or ThiaMet-G treated condition. Increased O-GlcNAcylation by ThiaMet-G further aggravated the IL-1β release from PBMCs. However, protein O-linked N-acetylglucosamine glycosylation can regulate the activity of NLRP3 inflammasome in human macrophages.

217. O-GlcNAcylation modulates physiological properties of neurons in the hippocampus

Hongik Hwang 1 , Ghilsoo Nam 2 , Pann-Ghill Suh 3 , Hyewhon Rhim 1; 1Center for Neuroscience, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, South Korea, 2Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, South Korea, 3School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan 44919, South Korea

hrhim@kist.re.kr

Post-translational modification (PTM) plays a critical role in diversifying protein functions. Along with phosphorylation, another highly dynamic PTM is O-GlcNAcylation, and the cycling of O-GlcNAc is entirely mediated by the two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Notably, the expression levels of OGT and OGA are high in the brain, and many neuron-specific proteins undergo O-GlcNAcylation. Despite its prevalence, however, the role of O-GlcNAcylation in regulating neuronal properties is relatively unknown. We employed a transgenic mouse with heterozygous loss of Oga (Oga+/-), and examined how the chronically elevated O-GlcNAcylation influences neuronal properties and animal behaviors. While the elevated O-GlcNAc modification did not affect intrinsic properties and basal synaptic transmission, Oga+/- mice showed impaired synaptic plasticity, a cellular basis of learning and memory, and hippocampus-dependent memory formation. Given that the phosphorylation of GluA1 (AMPA receptor subunit) is required for synaptic plasticity, we examined the phosphorylation of GluA1 in Oga+/- mice under chemically-induced LTP and LTD, and showed that it led to aberrant phosphorylation of GluA1. This result suggests that elevated O-GlcNAcylation interferes with trafficking of AMPARs during neuronal activity, which may underlie the impaired memory in Oga+/- mice. In addition, we also examined how acutely elevated O-GlcNAcylation modulates neuronal functions by pharmacologically inhibiting OGA, and found that it significantly decreased the intrinsic excitability. This decrease was due to the facilitation of afterhyperpolarization, and was rescued by inhibiting Ca2+-activated K+ channels. Blockade of OGA is also known to cause NMDAR-independent LTD, and we found that this increased O-GlcNAcylation of GluA2, which inhibits its association with transmembrane AMPA receptor regulatory protein. This may lead to the endocytosis of AMPARs, which was evidenced by the decrease in the amplitude of miniature excitatory postsynaptic currents. Taken together, hyper-O-GlcNAcylation of neuronal proteins influences ion channel conductance and protein-protein interaction, and thereby modulates the physiological properties of neurons.

218. O-GlcNAcylation regulates PPARγ2 via gain of protein stability during adipogenesis

Junkyu Song 1 , Jung-Hwan Baek 1 , Kyung-Hee Chun 1; 1Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul, Republic of Korea

junkyusong@yahoo.com

O-GlcNAylation is regulates diverse cellular processes which extensively modified by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties in Nuclear, cytoplasmic, and mitochondrial proteins at serine or threonine residues. O-GlcNActransferase (OGT) and O-linked β-N-acetylglucosaminase (OGA) participate in addition and removal of O-GlcNAc, respectively. Perturbations in protein O-GlcNAcylation are associated with a wide range of human diseases and conditions.

The nuclear peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily of ligand-inducible transcription factors. PPARs control the expression of networks of genes involved in adipogenesis, lipid metabolism, inflammation and maintenance of metabolic homeostasis. Among the PPARs, PPARγ is a master regulator of gene expression for metabolism, inflammation, and other pathways in many cell types, especially adipocytes.

Recently, many reports have suggested that PPARγ could be modulated by post-translational modifications (PTMs), and modulation of PTM has been considered as novel approaches for treating metabolic disorders. Although, O-GlcNAylation studied arise a global PTMs. but, O-GlcNAcylation of PPARγ were poorly understood. Here, we report that PPARγ O-GlcNAcylated during adipogenesis. O-glcNAcylation site prediction and mutant studies revealed that novel O-GlcNAcylation residue of PPARγ2 which close in acetylation sites. This O-GlcNAcylation has inverse correlation with acetylation status in PPARγ2. Moreover, inhibitions of O-GlcNAcylation led to attenuation of PPARγ2 in protein level. Our results suggest that the O-GlcNAc state of PPARγ2 influences its stability and is involved in adipocyte differentiation.

219. Alloxan inhibited the enhancing effect of lipopolysaccharide (LPS)-mediated iNOS/NO production by GlcN in RAW264.7 cells

Ji-Sun Hwang 1 , Kyung-Hong Kim 1 , Ji-Won Park, Sang-Min Kim 1 , Jea-Hee Lee 1 , Yunkyoung Lee 1 , Inn-Oc Han 1; 1Inha University, Korea

iohan@inha.ac.kr

Previously, we demonstrated that glucosamine (GlcN) increased LPS-induced expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. In the present study, we investigated that Alloxan (Alx) regulates the enhancing effect of LPS-mediated production of iNOS/NO by GlcN in macrophage cells. Alx suppressed the production of NO in LPS plus GlcN treated cell culture media but not LPS-mediated NO production. Moreover, LPS-induced iNOS, COX-2, and TNF-α mRNAs expression were enhanced by GlcN. Treatment with Alx, inhibited this enhancing effect. In addition, we found that GlcN enhanced the LPS-induced increase in O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) as well as DNA binding of c-Rel and p65 to the iNOS promoter, which was reversed by Alx. These results suggesting that inhibitory effect of Alx on GlcN-mediated enhancing of iNOS/NO expression in response to LPS is mediated by NF-κB inhibition.

220. Elevation of O-GlcNAcylation enhances progression of cholangiocarcinoma cells

Chatchai Phoomak 2 , Atit Silsirivanit 2 , Dayoung Park 1 , Kanlayanee Sawanyawisuth 2 , Kulthida Vaeteewoottacharn 2 , Chaisiri Wongkham 2 , Carlito B. Lebrilla 1 , Sopit Wongkham 2; 1Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand, 2Department of Chemistry, University of California, Davis, CA, 95616, US

chatchai_phoo@hotmail.com

O-GlcNAcylation is a post-translational modification of nucleocytoplasmic proteins with a single N-acetylglucosamine (GlcNAc). This process is controlled by two enzymes; the O-GlcNAc transferase (OGT) which transfers GlcNAc to serine or threonine of protein yielding the O-GlcNAcylated proteins (OGPs); and O-GlcNAcase (OGA) which catalyzes the reversed reaction. The OGPs are important in many cellular processes including tumor progression. Over-expression of OGT in cholangiocarcinoma (CCA) tissues and its correlation with shorter survival of CCA patients have been reported recently. In addition, knockdown of OGT alleviated the migration/invasion of CCA cell lines. This study, we further elucidate the novel OGPs that modulate the progression of CCA cells. To enhance the OGPs in CCA cells, suppression of OGA using PUGNAc, an OGA inhibitor, was used as the model of study. Increase of O-GlcNAcylation via PUGNAc treatment significantly increased CCA cell migration and invasion in two CCA cell lines. The OGPs related to the progression of CCA were further determined using Click-iT™ O-GlcNAc Enzymatic Labeling System to label O-GlcNAcylated peptides and identified by Q-Exactive Orbitrap mass spectrometer. Majority of OGPs found in KKU-213 and KKU-214 cells were nucleocytoplasmic proteins. There were 21 OGPs commonly found in both cell lines with almost similar intensity, of which 12 have not yet been reported for their O-GlcNAcylated forms, e.g., the solute carrier family 3 member 2, meprin A subunit alpha, heterogeneous nuclear ribonucleoprotein K, and calnexin, etc. The novel OGP candidates were selected and confirmed for the O-GlcNAcylated forms using immunoprecipitation and sWGA pull-down assay. Further studies are required to demonstrate the O-GlcNAcylated site of the novel OGP candidate and its role related to cancer progression.

221. Change of localization of the O-GlcNAcylated actin accompanied with diabetic nephropathy

Yoshihiro Akimoto 1 , Hayato Kawakami 1 , KunimasaYan 2 , Yuri Miura 6 , Tosifusa Toda 3 , Toshiyuki Fukutomi 4 , Daisuke Sugahara 1 , Akihiko Kudo 1 , Gerald W. Hart 5 , Tamao Endo 6; 1Kyorin University School of Medicine, Department of Anatomy, 181-8611, Tokyo, Japan, 2Kyorin University School of Medicine, Department of Pediatrics, 181-8611, Tokyo, Japan, 3Tokyo Metropolitan Institute of Gerontology, Research Team for Mechanism of Aging, 173-0015, Tokyo, Japan, 4Yokohama City University, Advanced Medical Research Center, 36-0004, Yokohama, Japan, 5Kyorin University School of Medicine, Department of Pharmacology and Toxicology, 181-8611, Tokyo, Japan, 6Johns Hopkins University School of Medicine, Department of Biological Chemistry, 21205, Baltimore, USA

yakimoto@ks.kyorin-u.ac.jp

In the kidney of the non-insulin dependent diabetes model Goto-Kakizaki (GK) rat, an elevation of O-GlcNAcylation occurs in cytoskeletal proteins such as actin, α-actinin 4, tubulin, myosin and mitochondrial proteins such as ATP synthase and pyruvate carboxylase (Akimoto et al. Clin Proteomics 2011, 8: 15).

In order to elucidate the role of O-GlcNAcylated actin in diabetic nephropathy, the changes of the localization of O-GlcNAcylated actin in the kidney of the diabetic GK rats were examined immunohistochemically. There are five O-GlcNAcylation sites in the amino acid sequence of actin. We chose one site from them in which not only O-GlcNAcylation but also phosphorylation occurs, which is important for the extension of actin filament. A specific anti-O-GlcNAcylated actin antibody was produce by using synthetic O-GlcNAcylated peptide as antigen.

The antibody against O-GlcNAcylated actin reacted only with G-actin, but did not react with F-actin. It stained moderately both cytoplasm and nucleus. Compared with control normal kidney, O-GlcNAcylated actin was increased in the foot process, cytoplasm and nucleus of podocyte in the glomerulus of diabetic kidney. In the proximal tubule O-GlcNAcylated actin localized in the base of microvilli. O-GlcNAcylated actin increased in the proximal tubule of diabetic kidney in comparison with control normal one.

From these results, it was suggested that O-GlcNAcylation of actin regulates the extension of actin fiber by inhibiting phosphorylation and elevation of O-GlcNAcylation might cause the morphological changes in the foot process of podocyte and microvilli of proximal tubule by inhibiting the extension of actin fiber. The role of O-GlcNAcylated actin in the nucleus remains to be elucidated. This study was supported in part by Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (C-24590260 to Y.A.) and from Japan Diabetes Foundation (to Y.A).

Glycan Binding Proteins

222. Variable lymphocyte receptors (VLRs) of hagfish (Eptatretus burgeri) against glycans of avian influenza virus H9N2

Se Pyeong Im 1 , Jung Seok Lee 1 , Jaesung Kim 1 , Tae Sung Jung 1; 1College of Veterinary Medicine, Gyeongsang National University, 660-70, Jinju, South Korea

arcayze@gmail.com

The adaptive immune system consists of highly efficient cellular mechanisms enables the host to eliminate the pathogens by evoking a more rapid and specific response against their repeated attacks. With respect to invertebrates possessing only the innate immune system, recent studies revealed that jawless vertebrates possess a unique immune receptor system. They possess particular molecules referred to as variable lymphocyte receptors (VLRs) such as VLR-A, VLA-C which are believed to be counterparts of T- cell receptors (TCRs) while VLRB functions as B- cell receptors (BCRs or Ig) of higher vertebrates.

To gain a better understanding of the adaptive immune responses of hagfish, we investigated the transcription profiles of VLRB at various time points after immunized with avian influenza virus (LPAIV) subtype H9N2. Moreover, we isolated and expressed monoclonal VLR-B proteins specific for the target virus confirmed by using a monoclonal antibody against VLR-B. These virus-binding clones showed high affinity and selectivity in ELISA and immunoblotting. Progeny virus from intentionally infected Madin Darby Canine Kidney (MDCK) cell line and chicken tissue were stained by our VLR-B clones, thus indicating specific recognition of antigens in immunocytochemistry (ICC) and immunohistochemistry (IHC) and flow cytometric analysis. To define antigenic determinants of the virus which are recognized by VLR-B clones in terms of glycocalyx, we assessed the selectivity using printed glycan microarray which are featured with 100 synthetic glycans. β-Glc-Sp, GlcNAc-β-1,3-Gal-β-1,4-Glc-β-Sp, Gal-α-1,3-Gal-β-1,3-GlcNAc-β-Sp, Tobramycin were the main reactive structures. However, additional studies will be required to more fully characterize the primary epitopes.

The unique phylogenetic position of VLRs in the evolution of adaptive immunity provides many potential advantages in VLR research. Previous studies have shown that VLR system can be used as specific binding scaffold for hen egg lysozyme, Bacillius anthracis spore surface protein (BclA), especially TFα (tumor-associated carbohydrate antigen). Here we applied VLR system in avian influenza virus which is one of the most deadly pathogens. The ability of hagfish immunity to produce mature VLRs that can rearrange to respond to specific antigens make it a good candidate for antibody therapies and may be beneficial in treating significant diseases.

223. Development of safe and effective Rotavirus vaccine by conjugating VP8, Rotavirus antigen, to Vi polysaccharide

Wookjin Park 1 , Sudeep Kothari 1 , Yeon-Kyung Yoon 1 , Viliam Pavliak 1; 1International Vaccine Institute, Department of Vaccine Process Development, 151-742, Seoul, Korea

viliam.pavliak@ivi.int

Rotavirus (RV) and Salmonella enterica serotype typhi (S. typhi) are important pathogens that transmit through a common fecal-oral route, causing gastroenteritis and typhoid fever, respectively in humans.

RV infection causes symptoms of severe diarrhea and dehydration among infants and young children. A worldwide evaluation in 2008 showed that RV infection led to approximately 453,000 deaths in young children. It accounts for 37% of deaths caused by diarrhea and 5% of all deaths in children younger than 5 years. Typhoid fever remains an important public health problem in developing countries where water quality is poor and sanitation is inadequate. WHO estimates the global incidence of typhoid fever to be 21 million cases, with a fatality rate ranging between 1% to 4%.

The two currently available live oral rotavirus vaccines, Rotarix® and RotaTeq®, are being introduced into immunization programs in many countries. These vaccines are highly efficacious in the high-income countries. However, the efficacy of the vaccines in low-income countries such as Africa and Southeast Asia is low. In addition, the two modified live-attenuated vaccines increase the risk of serious intestinal disorder, intussusception, in infants.

In this study, we developed and evaluated a conjugated vaccine candidate that consisted of the spike protein VP8 of RV and Vi polysaccharide of S. typhi. This vaccine will be against rotavirus and has the potential to provide broad protection from typhoid fever.

VP8 recombinant protein is easily made via E. coli expression system. BMPH, a linker, is used for conjugating the VP8 to Vi polysaccharide. Among various possible delivery routes, VP8-Vi polysaccharide conjugated vaccines are administered either subcutaneously or by microneedle injection.

224. In silico Analysis and development of a novel core-fucose binding Aleuria aurantia lectin

Harmin Herrera 1 , Sandhya Kortagere 1 , Anand Mehta 2; 1Drexel University, Department of Microbiology and Immunology, 19102, Philadelphia, PA, USA, 2Medical University of South Carolina, Department of Cell and Molecular Pharmacology, 29425, Charleston, SC, USA

herrera@musc.edu

Increased levels of core α-1,6 linked fucosylation have been associated with cancer development and progression. Currently, no commercially available lectin will bind specifically to only core α-1,6-linked fucose. In our recent molecular dynamics (MD) simulations of two recombinant AAL mutants, rAAL N224Q and rAAL N129Q, we observed that a single amino acid mutation in the fucose-binding site, altered the overall dynamics of the whole lectin. Interestingly, this altered both the specificity and affinity to different fucose-linkage. Based on the results from MD simulations, X-ray crystallography, surface plasmon resonance (SPR), glycan array and lectin blotting of the N224Q mutant, we proposed the design of another AAL mutant, rAAL Q101N. This mutant is localized to the second fucose binding site that primarily binds fucose in non α-1,6-linkages. We hypothesize that this mutant would have reduced binding to outer arm fucosylated structures while maintaining superior core-fucose binding. Indeed, our preliminary results show that the rAAL Q101N mutant has higher specificity to α-1,6-fucose, and completely abrogates binding to the H antigen (α-1,2-), Lewis X (α-1,3) and Lewis A( α-1,4). The design of linkage-specific lectin using MD simulations and biochemical analysis will be useful to specifically examine the role of specific fucose linkages in cancer development. We believe that this method can be easily applied to another lectin for discoveries of linkage-specific lectins.

225. Glycan-nanoparticle conjugates for detection of Helicobacter pylori

Ji Young Hyun 1 , Seong-Hyun Park 1 , Injae Shin 1; 1Yonsei University, Department of Chemistry, 03722, Seoul, Korea

injae@yonsei.ac.kr

Various pathogens express lectins on their surface for infection of host cells through interactions of the pathogenic lectins (adhesins) with the host cell surface glycans. H. pylori is one of the most popular infectious pathogens that affect nearly one-third of the world's population. This pathogen is known to cause chronic gastritis, which may lead to peptic ulcer disease and gastric cancer. H. pylori recognize specific host cell glycans, as exemplified by the well-characterized blood group antigen-binding adhesin (BabA). To detect lectins expressed on H. pylori cell surfaces, we prepared and characterized Lewis antigen conjugated, dual-modal fluorescent magnetic nanoparticles (FMNPs). When H. pylori J99 strain expressing BabA was incubated with glycoNPs, Leb and H1 conjugated but not Lea conjugated NPs bound to this strain. However, these glycoNPs did not interact with H. pylori strains lacking BabA. GlycoNPs were also used to isolate BabA expressing H. pylori by using a magnet. Furthermore, the glycoNPs have the ability to block adhesion of H. pylori to mammalian cells. The present study show that glycan-conjugated FMNPs can serve as useful tools to sensitively detect lectins expressed on pathogen cell surfaces, enrich lectin expressing pathogens and prevent pathogen infection. It is anticipated that this study provides opportunities for developing finely tuned multifunctional nanoparticle-based drugs and diagnostics.

226. Glycan microarrays for simultaneous screening of glycans whose binding to the cell-surface lectin stimulates the cellular response

Ji Young Hyun 1 , Injae Shin 1; 1Yonsei University, Department of Chemistry, 03722, Seoul, Korea

injae@yonsei.ac.kr

Interactions of immune cell surface lectins with glycans expressed on the exterior of pathogens elicit the cellular responses. Mouse SIGN-R1 (SIGN-related 1) is known to recognize mannose-rich and fucose containing glycans in a Ca2+-dependent manner. When SIGN-R1 binds to glycans on host cells, bacteria or viruses, glycan antigens enter cells via lectin-mediated endocytosis to elicit cellular responses. We applied carbohydrate microarrays containing various glycans to rapidly screening functional glycans that promote cell surface lectin-associated cellular responses. Because binding of glycan ligands to SIGN-R1 on the cell surface triggers a cellular response that leads to production of reactive oxygen species (ROS), the functional glycans on the microarrays are readily identified by using a ROS fluorescent probe (PF1). Glycan microarrays were constructed by immobilizing 31 unmodified glycans on hydrazide-modified glass slides. SIGN-R1 expressing cells pre-treated with PF1 were applied to the glycan microarrays and the fluorescence intensity of the cells was then measured by using a confocal fluorescence microscopy or a microarray scanner. The results of microarray experiments revealed that Man and Fuc bearing glycans stimulate ROS production, a phenomenon that was abrogated in the presence of a ROS scavenger or an NADPH oxidase inhibitor. The present study demonstrated that carbohydrate microarrays can be employed for rapid screening of functional glycans that enhance the cell-surface lectin-associated cellular response.

227. Intracellular activity of small molecule galectin-3 inhibitors with high or low cell membrane permeability

John Stegmayr 2 , Hakon Leffler 2 , Michael C. Carlsson 1 , Gunjan Sharma 2 , Xiaoli Huang 5 , Fredrik Zetterberg 3 , Barbro Kahl-Knutson 2 , Hans Schambye 3 , Ulf J. Nilsson 4 , Stina Oredsson 5; 1Lund University, Department of Laboratory Medicine, Division of Microbiology, Immunology, and Glycobiology (MIG), SE-22184, Lund, Sweden, 2University of Copenhagen, Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, DK-2200, Copenhagen N, Denmark, 3Lund University, Department of Biology, SE-22100, Lund, Sweden, 4Galecto Biotech AB, DK-2200, Copenhagen N, Denmark, 5Lund University, Department of Chemistry, Center for Analysis and Synthesis, SE-22100, Lund, Sweden

john.stegmayr@med.lu.se

Background and aims: The carbohydrate binding protein family of galectins are known to have important regulatory functions both inside and outside the plasma membrane of cells. These functions, including regulation of autophagy, modulation of RNA-splicing, cell migration and formation of membrane associated microdomains, are in the majority of cases believed to be dependent on galectin-glycan interactions. Several galectin inhibitors with high affinity for the galectin carbohydrate binding site have been developed and as these inhibitors have both academically and clinically relevant applications it has become of increasing interest to test them in biological systems (i.e. cell cultures and animal models).

Different assays for extracellular inhibition of galectins exists, but intracellular evaluation of galectin antagonists are less clear and reliable assays for this purpose do not yet exist. To fill this void, we utilized the phenomenon that galectins accumulate around damaged vesicles, in a carbohydrate binding dependent fashion, which could be observed by fluorescence microscopy as puncta in the cytosolic space of cells. We then postulated that this galectin characteristic would be a measurable readout for intracellular inhibition of galectins and a suitable assay for determination of intracellular activity of galectin inhibitors.

Results and conclusions: Results show that the galectin-3 accumulation observed, following treatment with a lysosomotropic agent, was reduced when cells were pre-treated with small-molecule galectin inhibitors. Furthermore, the assay was shown to be highly dependent on the cellular uptake of the inhibitor and the duration of pre-treatment, as expected for an intracellular assay and not only on the affinity between the galectin and the inhibitor. In addition to a reliable assay for assessment of intracellular activity of galectin inhibitors, we also present three high-affinity galectin-3 inhibitors with large differences in intracellular activity. These inhibitors could prove to be useful tools in research where e.g. extracellular versus intracellular roles of galectins are to be investigated in cell culture or animal models.

228. Conjugation strategies using glycopeptides containing mannose-6-phosphate glycans derived from glyco-engineered yeast

JiYeon Kang 1 , Keun Koo Shin 1 , Su Yeon Kim 1 , Eun Bi Cha 1 , Ohsuck Kwon 1 , Doo-Byoung Oh 1; 1KRIBB, 34141, Daejeon, Korea

jykang@kribb.re.kr

Many therapeutic enzymes used for treatment of lysosomal storage diseases require high content of mannose-6-phosphate (M6P) glycan, which is a key factor for lysosomal targeting and therapeutic efficacy. We constructed the glyco-engineered yeast harboring high content of mannosylphosphorylated glycans, which can be converted M6P glycans by uncapping of the outer mannose residue. In this study, the cell wall of this yeast was employed as a natural source of M6P glycans for conjugation to therapeutic enzymes. The extracted cell wall mannoproteins were digested by pronase to generate short glycopeptides, which were further elaborated by uncapping and α(1,2)-mannosidase digestion process. The resulting glycopeptides containing M6P glycans (M6PgPs) showed proper cellular uptake and lysosome targeting, which were greatly enganced by M6P-selective purification using titanium dioxide resin. The purified M6PgPs were successfully conjugated to a therapeutic enzyme, recombinant acid a-glucosidase(rGAA) used for treatment of Pompe disease, by two-step reactions using two hetero-bifunctional corsslinkers. After each amine reactive group of the crosslinkers was conjugated to primary amine(s) of M6PgPs or rGAA, the resulting crosslinker-attached M6PgPs and rGAA were linked together in the second reaction by using copper-free click chemistry without the loss of enzyme activity. The M6PgP-conjugated rGAA had 18-fold higher content of M6P glycan than rGAA, which results in greatly increased cellular uptake by pompe disease patient fibroblasts.

229. Analysis of possible role of S-nitrosylation in Galectin-2 protein function

Mayumi Tamura 2 , Tomoharu Takeuchi 2 , Tomomi Hatanaka 2 , Kazuo Ohtake 2 , Hiroaki Tateno 1 , Jun Hirabayashi 1 , Jun Kobayashi 2 , Yoichiro Arata 3; 1Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, Saitama 350-0295, Japan, 2Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, Ibaraki 305-8568, Japan, 3Faculty of Pharma-Science, Teikyo University, Tokyo 173-8605, Japan

arata@pharm.teikyo-u.ac.jp

Galectins are a group of animal lectins characterized by their specificity for β-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. A proteomic analysis identified Gal-2 as a protein that was S-nitrosylated when mouse gastric mucosal lysates were reacted with S-nitrosoglutathione, a physiologically relevant S-nitrosylating agent. Recombinant mouse Gal-2 was S-nitrosylated using nitrosocysteine (CysNO), which had no effect on the sugar-binding specificity and dimerization capacity of the protein. On the other hand, Gal-2 oxidation by hydrogen peroxide resulted in the loss of sugar-binding ability, while S-nitrosylation prevented hydrogen peroxide-inducted inactivation, presumably by protecting the Cys residue(s) in the protein. To study which of the two highly conserved Cys residues in Gal-2 (Cys57 and Cys75) must be S-nitrosylated for protection against oxidative inactivation, we used point-mutated recombinant Gal-2 proteins. Mutation of Cys57 to a Met residue (C57M) did not result in lectin inactivation following hydrogen peroxide treatment, whereas Cys75 mutation to Ser (C75S) led to significantly reduced lectin activity, as is the case for wild-type Gal-2. However, pretreatment of the C75S mutant with CysNO protected the protein from hydrogen peroxide-induced inactivation. Therefore, Cys57 is suggested to be responsible for oxidative inactivation of the Gal-2 protein, and protection of the sulfhydryl group of the Cys57 in Gal-2 by S-nitrosylation is likely important for maintaining Gal-2 protein function from oxidative stress in the gastrointestinal tract.

230. Probing the role of Galectin-3 in cell migration using unique small inhibitors

Xiaoli Huang 1 , John Stegmayr, Kristoffer Peterson, Fredrik Zetterberg, Barbro Kahl Knutson, Ulf Nilsson, Hakon Leffler, Stina Oredsson; 1Lund University. Sweden

xiaoli.huang@biol.lu.se

Background: Breast cancer is the most common cancer seen in women worldwide. The five-year survival rate for early-diagnosed breast cancer is 99%. However, only 23 women out of every 100 survive if the cancer is detected at the metastatic stage. Galecin-3, a member of the β-galactoside-binding lectin family, is highly expressed in several cancer types including breast cancer. The aim of this study is to use novel galectin-3 inhibitors to determine the function of galectin-3 in breast cancer cell metastasis.

Methods: JIMT-1 breast cancer cells were treated with galectin-3 inhibitors. The viability of the cells after treatment was examined with an MTT assay. The effect of the inhibitors on cell migration was investigated using wound-healing, and Boyden chamber assays. The binding affinities between the inhibitors and galectin-3 were determined with a fluorescence anisotropy assay. The ability of the inhibitors to cross biological membranes was previously explored with a Caco-2-based permeability assay.

Results: All tested galectin-3 inhibitors were non-toxic against breast cancer cells at concentrations ranging from 0 to 40 μM. Compounds with high affinity to galectin-3 and good cell permeability showed inhibitory effects on cell migration. The data indicate that the inhibition of cell migration is related to galectin-3.

Conclusions: By using novel galectin-3 inhibitors, we found that galectin-3 activity associates with breast cancer cell migration.

231. Roles of polyvalency of glycotopes in the mechanism of protein-glycan interactions. One of the important directions for the future research in glycosciences

Albert M. Wu 1 , Yung Liang Chen 2; 1Glyco-immunochemistry Res, Lab., College of Medicine, Chang-Gung University, Kwei-san, Taoyuan 333, Taiwan, 2Graduate Institute of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu City 300, Taiwan.

yunliang@mail2000.com.tw

Lectins are an important class of proteins or glycoproteins that specifically or selectively bind to carbohydrates and play many critical roles in life processes. In order to characterize recognition roles of lectins, the following aspects have been taken into consideration: i) lectins affinity to monosaccharides; ii) expression of reactivities toward oligosaccharides (mammalian structural units/recognition units) and iii) the most active ligand. Furthermore most lectins with the same mono- or oligosaccharide specificity may demonstrate different interaction, intensity and specificities in reaction with polyvalent forms ¬– it has shown a shift of binding specificiy of lectin from one type of carbohydrate ligand to another when the density of the carbohydrate ligand changed. Therefore, characterization of lectin specificity has been extended to: iv) simple oligovalent or cluster effect; and v) complex multivalent or cluster effects. Simple oligovalent effect concerns the reactivity of lectins with oligomeric glycoconjugtes (e.g. branched oligosaccharides carring several active disaccharides, glycopeptides with several Tα or Tn glycotopes). A complex multivalent effect applies to interaction with high-molecular or aggregated molecules carrying multiple glycotopes recognition factors recognized by a lectin. In this presentation, we are focusing on the resulting intensities of three basic recognition factors – (iii) essential mammalian structural units, (iv) their clusters and (v) polyvalency in the recognition processes.

Glyconeurobiology: Glycans in Neuroscience

232. GlcN ameliorates hypoxia-induced inflammatory response and learning-memory impairments in adult zebrafish

Yunkyoung Lee 2 , Jiwon Park 2 , Soo-Jung Lee 1 , Jaehee Lee 2 , Kyoung-Hong Kim 2 , Sang-Min Kim 2 , Ji-Sun Hwang 2 , Inn-Oc Han 2; 1Department of Physiology and Biophysics, College of Medicine, Inha University, Incheon, Korea, 2Department of Biological Sciences, Inha University, Incheon, Korea

iohan@inha.ac.kr

Normal brain function requires constant oxygen flow and nutrient supply, and brain damage due to oxygen deficiency causes inflammation, as well as the reduced exploratory activity and impaired cognitive ability. Recent studies have demonstrated that glucosamine (GlcN) exerts anti-inflammatory, antioxidative, and antiapoptotic effects as well as immunosuppressive effects in vitro and in vivo. However, the effect of GlcN on the hypoxic brain injury model is unknown. The aim of this study is to investigate the effects of GlcN on inflammatory response, learning-memory, and behavior in an adult zebrafish model exposed to hypoxia. The mortality of zebrafish exposed to hypoxia was increased, and was inhibited by 1 g/L GlcN pre-treatment. Moreover, GlcN suppressed hypoxia-induced upregulation of neuroglobin (Ngb), a small monomeric heme protein with oxygen-binding properties, and NOS2α, an isoform of iNOS in zebrafish, mRNAs. Locomotion and exploratory activity of zebrafish exposed to hypoxia were decreased, and GlcN only restored the locomotion activity. In passive avoidance test, GlcN also restored the impaired learning and memory abilities impaired by hypoxia. In addition, the phosphorylation of ERK and Akt were decreased after hypoxia exposure, and these were reversed by GlcN. Our findings suggest that GlcN may be useful as a potential drug to protect hypoxia-induced the neuroinflammation, and learning and memory impairments.

233. Different properties of polysialic acids synthesized by either or both of the polysialyltransferases ST8SIA2 and ST8SIA4

Airi Mori 1 , Masaya Hane 1 , Ken Kitajima 1 , Chihiro Sato 1; 1Nagoya Univ., Biosci. Biotech. Center, 4648601, Nagoya, Japan

mori.airi@d.mbox.nagoya-u.ac.jp

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 (1). PolySia is synthesized by polysialyltransferases (polySTs), ST8SIA2/STX and/or ST8SIA4/PST. PolySia has also been speculated to be involved in the development of several human psychiatric disorders. STX (SNP-7) was found in a schizophrenic patient. We demonstrated that the enzymatic activity was much lower than STX (Wild Type), and that the reservoir function of polySia synthesized by STX (SNP-7) was weaker than that synthesized by STX (Wild Type) (2-4). Based on the results, polySia expression is considered to be highly regulated for normal neurological functions, and the related biosynthetic enzymes are involved in the regulated expression of polySia structure. Our objective is, thus, to elucidate the quality of polySia-NCAM depending on polySTs and the relationship between the quality and the function of polySia-NCAM. First, we analyzed the structures of polySia-NCAM. Secondly, the function of polySia-NCAM was analyzed with SPR-based method. We found polySia-NCAM synthesized by STX displayed both different attractive and repulsive fields compared with that by PST, although the difference could not be clearly detected using conventional immunological and chemical methods. All these data suggest that polySia displays attractive and repulsive fields at the same time and the functional fields of polySia are highly regulated at least by polySTs (5).

References

(1) Sato and Kitajima (2013) J. Biochem. 154, 115-136

(2) Isomura et al. (2011) J. Biol. Chem. 286, 21535-21545

(3) Hane M. et al. (2012) Pure Appl. Chem. 84, 1895-1906

(4) Hane M. et al. (2015) Glycobiology. 25, 1112-1124

(5) Mori et al. (2017) Glycobiology. Submitted

234. Effects of chlorpromazine, an anti-schizophrenia reagent, on the expression of the polysialic acid in human neuroblastoma cells and mouse brains

Chikara Abe 1 , Saki Nishimura 1 , Airi Mori 1 , Yuki Niimi 1 , Yi Yang 1 , Masaya Hane 1 , Ken Kitajima 1 , Chihiro Sato 1; 1Bioscience Biotechnology Center, Nagoya University, Chikusa, Nagoya, 464-8601, Japan

abe.chikara@f.mbox.nagoya-u.ac.jp

Polysialic acid (polySia) is a linear homopolymer of sialic acid with the degree of polymerization (DP) 8-400 Sia residues. PolySia is mainly expressed on the neural cell adhesion molecule (NCAM) and changes the cell adhesion molecule to an anti-adhesion molecule in several restricted areas, such as hippocampus. PolySia is shown to be involved in cell migration, plasticity, memory, and social interaction. Recently, relationships between polySia or polysialyltransferase ST8SIA2 and psychiatric disorders such as schizophrenia and bipolar disorder have been reported. For example, the cell number of polySia-expressing cells at prefrontal cortex was decreased in schizophrenic brains and several significant SNPs on the ST8SIA2 change the quantity and quality of polySia. Therefore, we hypothesize that quantity and quality of polySia are important for normal brain functions. It is well-known that environmental factors are also the cause of psychiatric disorders; however, it is still unknown whether and how environmental factors influence the expression of polySia. In this study, we focused on an anti-schizophrenia reagent, chlorpromazine (CPZ) as an environmental factor, and analyzed it's effects on the expression of polySia in neural cells and mouse brains.

We analyzed the effects of the CPZ addition on the expression of polySia-NCAM in human neuroblastoma cell line, IMR-32 cells, by immunochemical and chemical methods. We found that polySia-related gene expressions remain unchanged. Interestingly, polySia on the cell surface was increased after CPZ addition but whole polySia expression was unchanged. We also found that Brefeldin A, an inhibitor of endocytosis, suppressed the CPZ-induced cell surface polySia expression. Then, we analyzed the effects of the CPZ on polySia-expression at five brain regions in adult mice and found that the CPZ only influenced the total amounts of polySia-NCAM at prefrontal cortex where polySia expression has been reported to be reduced in schizophrenia brains. These results suggest that the CPZ administration regulated the total polySia amounts in a brain region-specific manner. Based on the celluar and animal level studies, CPZ up-regulates the surface expression of polySia, consistent with a counter effect for the sympton of schizophrenic brains.

ETC

235. Effects of glycosylation of natural flavonoids with various sugars on anti-diabetes in vitro

Jeong Yeon Seo 1 , Hyeon Jeong Kim 1 , Ramesh Prasad Pandey 2 , Jae Kyung Sohng 2 , Yong Il Park 1; 1Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi-do, 14662, Republic of Korea, 2Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Asansi, Chungnam, 31460, Republic of Korea

yongil382@catholic.ac.kr

Even with accumulating evidence showing that various beneficial effects of many flavonoids against a broad range of human diseases including cardiovascular diseases, autoimmune disease, ulcer, inflammation and tumor, some limitations on natural flavonoids, such as extremely poor water-solubility, chemical instability and toxicity, have been reported. Therefore, attempts to resolve these limits have been made in many directions. One of these trials is modification of natural flavonoids by glycosylation with various sugars such as glucose, rhamnose, galactose, and xylose, which alter their physicochemical and pharmacokinetic properties, such as increased water solubility and stability, reduced toxicity, and sometimes enhanced or even new pharmacological activities. In this regard, the diabetes mellitus has been spreading continuously and its prevalence rate in Korean reached 13.7% in 2014. Thus, extensive and aggressive therapies to treat and ameliorate the diabetic complication are urgently needed. A plant natural flavonoid (NF), and its glycosylated derivatives with rhamnose (F-3-R), glucose (F-3-G), or xylose (F-3-X) were prepared using system biology-based techniques, evaluated and compared for their anti-diabetic effects in 3T3-L1 mature adipocyte. The results showed that NF, F-3-R, F-3-G, and F-3-X did not alter viabilities of 3T3-L1 cells with 6 h-exposure compared with untreated control. As representative index for anti-diabetic effects, activities of glucose uptake were assayed using fluorescent glucose analog, 2-NBDG (2-[N-(7- nitrobemz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose) in fully differentiated 3T3-L1 adipocyte. The results showed that F-3-R and F-3-X considerably improved glucose uptake compared to NF. These results suggested that the F-3-R and F-3-X may be promising candidates for the development of health beneficial agents that can prevent or treat diabetes mellitus.

236. Therapeutic roles of autophagy induction and NLRP3 inflammasome inhibition on non-alcoholic steatohepatitis

Soo Hyun Kim, Yong-Ho Lee 1 , Gyuri Kim, Milim Lee, Jin Hee Kim, Hyo Seong Jeon, Ji Young Kim, Byung-Wan Lee, Bong-Soo Cha, Myung-Shik Lee; 1Yonsei University College of Medicine, Korea

YHOLEE@yuhs.ac

Impairment in autophagy flux and inflammasome activation are common characteristics of nonalcoholic steatohepatitis (NASH). Considering the lack of approved agents for treating NASH, drugs that can enhance autophagy and modulate inflammasome pathways may be beneficial. Here, we investigated the novel mechanism of ezetimibe, a widely prescribed drug for hypercholesterolemia, as a therapeutic option for ameliorating NASH. Human liver samples with steatosis and NASH were analyzed. For in vitro studies of autophagy and inflammasomes, primary mouse hepatocytes, human hepatoma cells, mouse embryonic fibroblasts with AMPK or Tuberous sclerosis 2 knockout, and human or primary mouse macrophages were treated with ezetimibe and palmitate. Steatohepatitis and fibrosis were induced by feeding Atg7 wild-type, haploinsufficient, and knockout mice a methionine- and choline-deficient diet with ezetimibe (10 mg/kg) for 4 weeks. Human livers with steatosis or NASH presented impaired autophagy with decreased nuclear TFEB and increased SQSTM1, Light Chain 3-II, and NLRP3 expression. Ezetimibe increased autophagy flux and concomitantly ameliorated lipid accumulation and apoptosis in palmitate-exposed hepatocytes. Ezetimibe induced AMPK phosphorylation and subsequent TFEB nuclear translocation, related to MAPK/ERK. In macrophages, ezetimibe blocked the NLRP3 inflammasome/IL1B pathway in an autophagy-dependent manner and modulated hepatocyte-macrophage interaction via extracellular vesicles. Ezetimibe attenuated lipid accumulation, inflammation, and fibrosis in liver-specific Atg7 wild-type and haploinsufficient mice, but not in knockout mice. Ezetimibe ameliorates steatohepatitis by autophagy induction through AMPK activation and TFEB nuclear translocation, related to independent MTOR ameliorative effect and MAPK/ERK pathway. Ezetimibe dampens NLRP3 inflammasome activation in macrophages by modulating autophagy and a hepatocyte-driven exosome pathway.

237. Aloe emodin-O-glucoside induces G1 phase cell cycle arrest and apoptosis in human lung cancer cells

Ji Won Choi 1 , Jun Il Kim 1 , Thi Huyen Trang Nguyen 2 , Jae Kyung Sohng 2 , Yong Il Park 1; 1Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi-do 14662, Korea, 2Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Asansi, Chungnam 31460, Republic of Korea

yongil382@catholic.ac.kr

Anthraquinones, a common phenolic family of naturally occurring yellow, orange and red pigments, have been reported to possess diverse health beneficial effects including anti-cancer, anti-oxidant and anti-microbial activities. Recently, it was demonstrated that glycosylation of phenolic compounds enhances their aqueous solubility while retaining biological activities. Thus, we synthesized a glucosylated derivatives (AEG) of aloe emodin (AE) by enzymatic modification and studied its in vitro anti-cancer effects against human lung cancer using A549 cells. While treatment of cells with 5 μM AE did not affect the viability of A549 cells, AEG reduced cell viability, with a 40% reduction at the same concentration. Western blot analysis showed that AEG significantly induces the activation of caspase 9 and poly (ADP-ribose) polymerase (PARP) by 3.4 and 2.1-folds, respectively, compared to those in AE-treated cells, suggesting that the cytotoxicity of AEG is associated with apoptotic events. We further investigated the molecular mechanisms involved in the anti-cancer effect of AEG by western blot analysis. AEG (5 μM) decreases phosphorylation of ERK, p38 and Akt, by 35, 44, and 57%, respectively, compared to untreated control cells. Also we confirmed that AEG also suppressed the expression of cylin D which is known to responsible for G1-S phase transition in cell cycle. Taken collectively, these results suggested that AEG exerts more effective anti-cancer activity in A549 cells than AE via apoptotic cell death, and this AEG-induced apoptosis is associated with the arrest of G1 phase in cell cycle through down-regulating Akt and MAPKs signaling pathway. Thus, aloe emodin-O-glucoside can be a potent candidate for the development of anti-cancer agents against human lung cancer.

238. Inter-species analysis of O-GlcNAcylated proteins

Jun Tanaka 1 , Masaoki Fujii 1 , Hisao Kojima 1 , Masahiro Ito 1; 1Ritsumeikan University, Graduate school of Life Science, 525-8577, Shiga, Japan

sj0022ip@ed.ritsumei.ac.jp

O-GlcNAcylation, the attachment of one GlcNAc to serine/threonine residues, is essential for protein function in eukaryotes. Unlike typical glycosylation, this modification occurs mainly in the nucleus and cytoplasm. O-GlcNAcylation is regulated by only two enzymes—O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Despite this simple regulation, O-GlcNAcylated proteins are involved in cellular functions such as transcription and translation. We aim to predict the consensus sequence and function of O-GlcNAcylated proteins using sequence analysis. Additionally, we are analyzing their involvement with proteins possessing intrinsically disordered regions (IDRs), which are also mainly localized in the nucleus. However, no clear relationship between sequences and their functions has been clarified yet.

In this study, we clustered O-GlcNAcylated proteins to investigate the relation between their evolutionary conservation and function. First, a homology search was conducted among eukaryotes with completely sequenced genomes using human O-GlcNAcylated proteins as queries. Second, O-GlcNAcylated residues in human proteins were compared with orthologous proteins using amino acid sequence alignment. Finally, O-GlcNAcylated proteins were clustered according to the degree of the conservation. The conservation of the OGA/OGT domains was also investigated, and a similar evolutionary analysis of A. thaliana O-GlcNAcylated proteins was conducted.

Consequently, O-GlcNAcylated proteins were clustered into nine clusters. Both, ratios of O-GlcNAcylation within IDRs and co-occurrence of O-GlcNAcylation/phosphorylation at an identical region were high in evolutionarily young clusters. Evolutionarily, OGT was widely conserved in eukaryotes, but OGA was conserved only in animals. Additionally, in plants, fungi, and protists, OGT was conserved only in the catalytic domain. Therefore it was suggested that orthologous OGT modified targets different from those modified by human OGT, because O-GlcNAc could not be removed owing to absence of paired OGA. The analysis of A. thaliana proteins showed that over 80 % of O-GlcNAcylated A. thaliana proteins were not conserved in animals. This result supports the hypothesis that the target proteins of O-GlcNAcylation are different in plants and animals.

Late Submissions

239. The proof-of-principle of substrate reducion therapy in an MPS II disease model

Hyun-Ja Nam 1 , Sung Eun Kim 1 , Nanyoung Go 1 , Sungho Hwang 1; 1Rare disease team, MOGAM Institute for Biomedical Research, 16924, Yongin-si, Republic of Korea

hjnam098@mogam.re.kr

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by the deficiency of the lysosomal enzyme iduronate-2-sulfatase, which is required for hydrolysis heparan sulfate and dermatan sulfate, leading to progressive accumulation of glycosaminoglycans (GAG) in tissues and organs. Enzyme replacement therapy (ERT) has been used to treat MPS II disease. However, a major drawback of this approach is the inability of an exogenous enzyme to traverse the blood brain barrier and treat neurological symptoms. An alternative approach to ERT is substrate reduction therapy (SRT), a treatment strategy that employs small molecule inhibitors to reduce the biosynthesis of storage metabolites. To investigate whether substrate reduction might be feasible for treating MPS II disease, we developed in vitro and in vivo SRT models in which GAG biosynthesis is inhibited by rhodamine B. Treatment of MPS II patient-derived fibroblasts with rhodamine B led to decreased GAG levels and induced autophagy flux. In addition, administration of rhodamine B to MPS II mice resulted in a decrease in GAG levels in urine and tissues compared to untreated MPS II mice. These results provide proof-of-principle for inhibition of GAG biosynthesis as a target of SRT, which may prove beneficial for treating MPS II disease.

240. Synthetic glycolipids as inhibitors of mycobacterial biofilms and as supplements to the effect of isoniazid

Krishnagopal Maiti 1 , Kirtimaan Syal 2 , Dipankar Chatteri 2, N. Jayaraman 1; 1Department of Organic Chemistry, 2Molecular Biophysics Unit, Indian Institute of Science, Bangalore – 560 012, India

jayaraman@orgchem.iisc.ernet.in

Adherence to a surface and colonization of planktonic mycobacterial cells promote the formation of biofilm matrix. Biofilm-formed mycobacteria encounter sessile growth phase, where metabolic processes are different when compared to planktonic cells. Designed glycolipids exhibit an ability to interfere enzymatic assembly of cell wall components and phenotype functions. In a query, we undertook to study the effects of designed synthetic glycolipids on mycobacterial growth and biofilm-formed mycobacterium. Arabinofuranoside and mannopyranoside-containing oligosaccharides and their corresponding glycolipids were synthesized. These oligosaccharide glycolipids exert an influence on the mycobacterial growth and biofilm matrix. M. smegmatis was chosen to study the effect the synthetic glycolipids. We observe a significant reduction of the mycobacterial growth in the presence of synthetic glycolipids (50 – 100 mg/mL). Synthetic glycolipids are also found to inhibit biofilm growth. Reduction of biofilm matrix is found to as much as 85 – 90%, in a concentration-dependent manner of the glycolipids. When analyzing the extracellular matrix of biofilms, we observe a significant downregulation of mycolic acids, acylated lipids, mannosylated phospholipids, among few other components, for the glycolipid-treated biofilms, in comparison to untreated biofilms. Advancing the studies, we also find that synthetic glycolipids supplement the action of isoniazid, an important cocktail drug to treat pathogenic M. tuberculosis, on biofilm-formed mycobacterial cells. Features of studies leading to the establishment of synthetic glycolipids as inhibitors of mycobacterial growth, biofilm matrix and their synergism on the inhibition activities of isoniazid will be presented.

References

(a) K. Syal, K. Maiti, K. Naresh, P. G. Avaji, D. Chatterji, N. Jayaraman, Glycoconj. J. 2016, 33, 763-777.

(b) K. Syal, K. Maiti, K. Naresh, P. G. Avaji, D. Chatterji, N. Jayaraman, Revised manuscript submitted 2017.

241. The analysis of N-glycosylation for the biotherapeutic drugs

Min-Kyung So 1 , Myung-Sin Lim 1 , Byoung-Joon Ko 1; 1Division of Drug Screening and Evaluation,New Drug Development Center,Osong Medical Innovation Foundation Osong Saengmyung-Ro 123, Heungdeok-go, Cheongju-si, Chungbok,363-951

mks@kbio.kr

The N-glycan profile of a biologics, especially therapeutic antibody is commonly defined as a critical quality attribute (CQA) in bioprocess development. Fc glycosylation is reported to be important in the antibody-dependnet cell-mediated cytotoxicity (ADCC) and for complement-dependent cytotoxicity (CDC) functions through modulating the binding to Fcy receptors and C1q respectively. Removal of core fucose can also increase the ADCC. Therefore the analysis of N-glycan is very important and critical in the function of therapeutic antibodies. Recently, most of the analytical techniques in N-glycan analysis are using PNGase and Fluorescent labeling such as 2-AB. Resultant released glycans are analysed in the HPLC coupled with fluorescent detector and Mass. As the methods take 2~3 day to get the results it cannot be good ones in bioprocess development of therapeutic antibodies that they are requiring rapid analysis. Here, we tested an N-glycan labeling method that could provide rapid and enhanced fluorescence response/Mass sensitivity in the detection of N-glycan. Furthermore, this method has simplicity and high throughput capacity.

242. Brain storagte of heparan sulfate in Sanfilippo C syndrome causes synaptic dysfunction

Camila de Britto Pará de Aragão 1,2, Luigi Bruno 1,2, Graziella DiCristo 1, Jean-Claude Lacaille 3, Peter McPherson 4, Erika Freemantle 3, Carlos R. Morales 2, Alexey V. Pshezhetsky 1,2; 1CHU Sainte-Justine Research Center, University of Montreal, Montreal, QC, CA., 2Department of Anatomy and Cell Biology, McGill University, Montreal, QC, CA., 3Department of Neurosciences, Faculty of Medicine, University of Montreal, QC, CA., 4Montreal Neurological Institute, McGill University, Montréal, QC, Canada

alexei.pchejetski@umontreal.ca

Sanfilippo syndrome type C, also known as mucopolysaccharidosis type IIIC (MPSIIIC) is a rare genetic disorder triggered by mutations in the HGSNAT gene that leads to the lysosomal storage of heparan sulfate. In humans, MPSIIIC causes rapid neurological decline and death before adulthood, however the mechanisms underlying the neuropathophysiology are still not well understood. In mice, inactivation of the Hgsnat gene leads to hyperactivity, cognitive and memory decline, neuroinflammation and neuronal death. We hypothesise that prior neuronal death, the cognitive dysfunction in MPSIIIC mice is caused by defects in neurotransmission.

We studied synaptic structure in hippocampal neurons of MPSIIIC Hgsnat KO mice. We also established cultures of hippocampal neurons from E16 embryos and analysed them by transmission electron microscopy, immunocytochemistry and whole cell patch clamp to assess the electrophysiological patterns.

Our data demonstrate deficiency in synaptic vesicle trafficking in hippocampal neurons of MPSIIIC mice directly affecting pre- and postsynaptic neurons. The MPSIIIC neurons have shown a decrease or abnormal distribution of several synaptic markers including vGLUT1 and PSD-95, associated to the glutamatergic neurotransmission. The markers associated to the GABAergic pathway (Gephyrin and vGAT1) were not affected. The formation and maturation of dendritic spines in the MPSIIIC neurons were deficient both in vivo and in vitro. Cultured neurons revealed massive lysosomal storage, disorganized microtubules network and reduction of the postsynaptic densities, whereas their synaptic terminals presented sparse synaptic vesicles. MPSIIIC neurons showed a four-fold increase in the frequency of miniature excitatory postsynaptic currents (mEPSC’s) while the miniature inhibitory postsynaptic currents (mIPSC’s) were comparable to those in the WT cells. Together our data demonstrate that the heparan sulfate storage in MPSIIIC causes several abnormalities hippocampal neurons primarily affecting the excitatory neurotransmission pathways.

243. CLEC5A is a critical innate immunity receptor in host defense against Listeria infection

Shie-Liang Hsieh 1, 1Genomics Research Center, Academia Sinica, Taipei, Taiwan

slhsieh@gate.sinica.edu.tw

The DAP12-assoicated myeloid C-type lectin member 5A (CLEC5A/MDL-1) is a pattern recognition receptor to members of Flavivirus and plays critical roles in dengue virus (DV) and Japanese encephalitis virus (JEV)-induced lethal diseases. In this work, we further find that CLEC5A plays more important roles than TLR2 against L. monocytogenes infection. CLEC5A is responsible for bacteria-induced neutrophil extracellular trap (NETs) formation and the production of ROS and proinflammatory cytokine. Rapid bacteria spreading, increased bacterial loads in blood and liver, and severe liver necrosis were noted in Clec5A-/- mice after inoculation of sublethal dose of Listeria monocytogenes. Dramatic downregulation of IL-1b, IL-17A, and TNF-alpha accompanied with upregulation of CCL2 and massive infiltration of CD11b+Ly6chiCCR2hiCX3CR1low inflammatory monocytes were observed in Clec5A-/- mice liver. In addition, reduction of IL-17A-secreting gd T cells accompanied with severe liver necrosis and high mortality were found in Clec5A-/- mice at day 5 post Listeria infection. Interestingly, CLEC5A and TLR2 were upregulated and co-localized on the cell surface after incubation with bacteria, and Clec5A-/-Tlr2-/- mice were more susceptible than Clec5A-/- mice to L. monocytogenes infection. Thus, CLEC5A plays a pivotal role in the activation of multiple aspects of innate immunity, and co-activation of CLEC5A and TLR2 enhances host immunity against bacteria invasion.

244. Roles of galectin-1 in erythropoiesis and megakaryocyte differentiation

Ching-Yuan Cheng, Wan-Wan Lin 1, 1Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan

wwllaura1119@ntu.edu.tw

Galectin-1 (Gal-1) is a soluble beta-galactoside binding animal lectin, and can exert cellular functions through intracellular and extracellular (extrinsic) pathway. In our previous study, we have demonstrated non-receptor tyrosine kinase Syk as a novel upstream signaling molecule of erythropoietin receptor (EPOR) and GM-CSF receptors in TF-1 erythroleukemia cells, and mediate downstream signaling pathways like ERK, STAT5 and Akt for erythropoiesis. In this study, we found Gal-1 is expressed in plasma membrane, cytosol and nucleus of TF-1 cells, and can associate with Syk. Moreover, DB21, an inhibitor of extracellular Gal-1, can reduce TF-1 cell viability, suggesting the potential role of Gal-1 in positive regulation of cell proliferation and survival. We also used K562 cells as a cell model to understand the roles of Gal-1 in megakaryocyte and erythrocyte differentiation. We found PMA-induced CD61 and GATA-2 (markers of megakaryocytes) and hemin-induced hemoglobin-g (Hb-g) were reduced in K562 cells with Gal-1 silencing or treatment with DB21. Extracellular Gal-1 treatment can enhance CD61 gene expression. The inhibition effect of shRNA-Gal-1 on CD61 expression is not reversed by exogenous Gal-1, implying the existence of multifaceted actions of Gal-1 through intracellular and extracellular actions. Taken together, Gal-1 is required for cell viability and acquisition for cell differentiation into megakaryocytes and erythrocytes.

245. Intelectins (X-type lectins) from human and Xenopus laevis display different pathogen glycan binding specificities

Jin Kyu Lee 1, Jonathan Viola 1, J. Michael Pierce 1, 1Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30605

jinkyu@uga.edu

The intelectin first studied was the Xenopus laevis oocyte cortical granule lectin (XL35) in oocytes and embryos. Since XL35 was first identified, members of the family of intelectins have been reported in all deuterostomes studied, including ascidians, fishes, frogs and humans1. Several independent studies on this family of lectins strongly suggest that some members may be released upon infection by pathogens2. In addition, some family members have been shown to bind to cell surface glycans of bacterial pathogens. Two human XL-35 homologs, Intelectin-1(Int-1) and Intelectin-2 (Int-2) were identified; Int-1showed selective expression in heart, small intestine, colon, and thymus, with lower levels in ovary, testis, and spleen. Int-2, however, was expressed only in small intestine and was localized by immunostaining to the Paneth cells, specialized secretory cells whose main function is in pathogen surveillance. Therefore, this family of lectins participates in pathogen surveillance as part of the innate immune system. Infection by several types of pathogens induces a dramatic increase in the expression of intelectins in intestinal and bronchial epithelia, mediated by IL-13. This result strongly suggests that intelectins are involved in T helper type 2 (Th2) cell-mediated immunity in allergic inflammation and infections. Glycan binding array analysis by Core H of the Consortium for Funtional Glycomics showed no binding of recombinant human Int-1 to any of 600 synthetic glycans. By contrast, the binding specificity of XL35 on the same array was clearly glycans expressing Gala1-3GalNAc. Using the CFG Microbial Glycan Array, however, which contained 313 pathogen’s outer polysaccharides, human Int-1 and Int-2 showed somewhat unique binding profiles, binding to several polysaccharides with high affinity, and confirming their innate immune function in pathogen surveillance. Int-2 bound to a smaller set of pathogen surface polysaccharides, however. XL-35 showed a distinct pathogen polysaccharide binding pattern in comparison to to that of the human intelectins.

1. Lee et al. Glycoconjugate J. 21, 443-450, 2004

2. Wesener et al. Nature Structural & Molecular Biology 22, 603-610, 2016

Author Index

Aagaard, D.T. 132

Abe, C. 234

Adachi, Y. 90

Adamopoulou, M. 132

Aebi, M. 86, 188

Affolter, I. 86

Ahn, J.K. 79

Ai, X. 94

Akama, T. 181

Akasaka-Manya, K. 122

Akimoto, Y. 221

Akmacic, I.T. 19

Altmann, F. 149, 167

An, H. 79

An, H.J. 21, 40, 60, 118, 130, 160, 182, 186, 194, 195

Angata, T. 22, 29, 106, 191

Annese, V. 19

Araki, E. 159

Arata, Y. 229

Argunov, D. 81

Aureli, M. 152, 153, 154

Aziz, P.V. 37, 120

Baek, J.-H. 218

Bagdonaite 78

Bajzert, J. 80

Baksi, K. 15

Balmaña, M. 58

Bamforth, S. 13

Bandyopadhyay, G. 18

Banerjee, A. 15

Banerjee, D. 15

Bang, M. 82

Baráth, P. 121

Bard, F. 32

Bassi, R. 152, 153

Bellis, S.L. 56

Bello-Gil, D. 71

Bennett, E.P. 78

Bhide, G. 55

Bhuiyan, R.H. 14

Biase, E.D. 124

Bilan, M.I. 62

Blennow, K. 76

Bonay, P. 178

Borodina, E.Y. 62

Bouché, L. 73

Bovin, N. 71, 131

Brenner, M. 18

Brinkmalm, G. 76

Brocca, P. 153

Bröcker, F. 207

Bruno, L. 242

Brzozowska, E. 211

Buckley, C. 73

Cabitta, L. 49, 155

Calow, A. 207

Campos, D. 58

Carlsson, M.C. 103, 227

Casabuono, A. 77

Cavallero, G. 77

Cha, B.-S. 216, 236

Cha, E.B. 228

Cha, H.-S. 79

Chaisiri 24

Chaiyawat, P. 102

Champattanachai, V. 102, 156

Chan, Y.C. 151

Chang, H.-C. 187, 189

Chang, T.-H. 187

Charoenwattanasatien, R. 156

Chatteri, D. 240

Chen, C.-C. 72

Chen, G. 28

Chen, H.-L. 105, 114

Chen, H.-Y. 105, 114

Chen, K.-T. 174

Chen, R. 34

Chen, S.-T. 177

Chen, Y. 54

Chen, Y.-H. 106

Chen, Y.-J. 106

Chen, Y.-J. 29, 106

Chen, Y.-Y. 187

Chen, Y.L. 231

Cheng, C.-Y. 244

Cheng, W-C. 174, 208

Chern, J. 177

Chern, Y. 189

Chernikov, O. 107, 173

Chi, X. 94

Chiba, Y. 31

Chien, Y.-C. 22, 191

Chikalovets, I. 107

Chinarev, A. 131

Chiricozzi, E. 124

Chiu, H.-W. 173

Cho, B. 82

Cho, H.-S. 203, 204

Cho, J.-Y. 195

Cho, J.W. 101, 215

Cho, M. 125

Cho, S. 25

Choi, C.-W. 195

Choi, H.-J. 183

Choi, H.J. 212

Choi, J.W. 25, 237

Choi, N.Y. 199

Choi, S. 162

Choi, S.-H. 183, 184

Choi, S.-l 142

Choi, Y. 53

Choi, Y.-J. 195

Chokchaichamnankit, D. 102

Chu, X. 128

Chun, K.-H. 165, 218

Chung, S.Y. 205

Ciampa, M.G. 155

Colley, K. 55

Couchman, J.R. 53

Couto, A. 77

Cramer, J.T. 9

Cui, B. 18

Culebro, A. 97

Cummings, R.D. 6

Dabelsteen, S. 78, 132

Dang, L. 113

Danial, R. 202

Darsandhari, S. 206

de Britto Pará de Aragão, C. 242

De Schutter, K. 113

Dechecchi, M.C. 153, 154

Delannoy, C. 85

Delannoy, P. 52

Dell, A. 73

Deng, L. 112

DiCristo, G. 242

Dissanayake, S.K. 190

Do, S.-I. 110

Dobrinić, P. 20

Dohmae, N. 138, 139

Dong, J. 123

Drake, R. 193

Du, Y. 96

Duarte, H. 58

Dulal, H. 90

Dzijan, S. 19

Eberini, I. 124

Edwards, N. 84

Eggermont, L. 113

Ehrlich, B. 109

Elass, E. 85

Endo, T. 8, 122, 221

Ertunc, N. 161

Fahey, R. 11

Fan, B. 172

Fan, W. 54

Fang, J. 75

Fedorov, R. 9

Feng, B. 168

Feriod, C. 109

Figl, R. 149

Filer, A. 73

Fleury, A. 178

Foley, D. 55

Freemantle, E. 242

Freitas, D. 58

Führer, J. 167

Führing, J.I. 9

Fujdiarova, E. 89

Fujii, M. 238

Fujita, H. 161

Fukuda, M. 180

Fukutomi, T. 221

Funayama, S. 30

Furukawa, K. 14, 150

Furukawa, K. 14, 150

Gamian, A. 211

Garate, T. 178

Ge, Y. 57

George, S. 119

Gerardy-Schahn, R. 9

Gilborne, L. 76

Giussani, P. 152, 154

Go, N. 137, 239

Goldman, R. 84

Gomes, C. 58

Gomes, J. 58

Gonnet, F. 202

Gorska, S. 211

Grady, R. 13

Grassi, S. 49, 155

Greville, G. 11

Gu, J. 169

Gu, J. 59, 68, 111

Guan, N. 34

Guerardel, Y. 85, 95

Guo, H.-b. 66

Guo, X. 123

Gusthart, J. 98

Ha, D.I. 170

Hafkenscheid, L. 12

Hallinan, S. 11

Haltiwanger, R. 13

Han, D. 145

Han, I.-O. 176, 219, 232

Han, J. 59

Hane, M. 233, 234

Hanover, J. 108

Hansen, L. 78

Harazono, A. 36

Harnett, M. 73

Hart, G. 100

Hart, G.W. 221

Hashimoto, H. 57

Hashimoto, N. 14

Haslam, S. 73, 74

Hatanaka, T. 229

Heithoff, D.M. 37, 120

Herrera, H. 61, 224

Hibi, M. 57

Hidaka, T. 36

Hilliard, M. 11

Hirabayashi, J. 44, 87, 122, 229

Hirakawa, J. 175, 180

Hirose, Y. 122

Hirotsu, N. 146

Holdener, B. 13

Hong, M.-H. 114

Horiuchi, R. 146

Houser, J. 89

Hsiao, C.-T. 187, 189

Hsieh, S.-L. 243

Hsu, D.K. 105

Hsu, P.-H. 29

Hsu, S.-T.D 92

Hsu, T.-L. 30

Hu, C.-M. 208

Hu, G.-S. 208

Hua, K.-F. 107, 173

Huang, H.-W. 72

Huang, X. 103, 227, 230

Huang, X. 76

Huebner, J. 81

Huh, H.D. 212

Huizinga, T.W. 12

Hung, Y.-H. 114

Hwang, H. 194, 195, 197, 198, 199, 200

Hwang, H. 217

Hwang, J.-S. 176, 219, 232

Hwang, S. 137, 157, 239

Hyun, J.Y. 225, 226

Ikeguchi, M. 122

Im, S.P. 222

Imai, Y. 180

Imamura, T. 18

Imberty, A. 2, 89

Inamori, K.-i. 48

Inokuchi, J.-i. 45, 48

Ishii, A. 36

Itakura, Y. 44, 185

Ito, M. 50

Ito, M. 127, 238

Ito, Y. 38

Itoh, K. 36, 50

Iwabuchi, K. 46

Iwaki, Y. 163

Jachymek, W. 80

Jan, H.-M. 151

Jancarikova, G. 89

Jang, B. 53

Jang, H. 82

Jang, W. 126

Jayaraman, N. 240

Jennings, M. 64

Jeon, H.S. 216, 236

Jeon, Y.-N. 160

Jeong, H.K. 195, 197, 198, 200

Jeong, J. 162

Jeong, S. 130

Jeong, S.-K. 195

Ji, E.S. 194, 195, 198, 199, 200

Jia, X. 35, 43

Joo, W-H. 183

Joshi, H.J. 78

Josipović, G. 20

Ju, J. 35

Jung, J. 82

Jung, T.S. 222

Kahl-Knutson, B. 230, 103, 227

Kaji, H. 31

Kambe, M. 150

Kameyama, A. 190

Kanagawa, M. 122

Kaneko, K. 14

Kang, E.S. 216

Kang, H. 157

Kang, H.-G. 165

Kang, H.A. 205, 209

Kang, J. 228

Kang, J.G. 40, 67

Kang, M. 82

Kang, Y.-M. 200

Kang, Y.J. 144

Kanoh, H. 45

Kaplonek, P. 207

Kaszuba, K. 93

Kato, K. 39

Kato, R. 122

Katrlík, J. 121

Kawakami, H. 221

Kawasaki, N. 181

Kawasaki, T. 181

Kawashima, H. 175, 180

Keeratichamroen, S. 156

Ketudat-Cairns, J.R. 156

Khoo, K.-H. 22, 187, 189, 191

Kim, B.J. 182

Kim, D. 162

Kim, D.-S. 141

Kim, D.-S. 141

Kim, D.Y. 129

Kim, D.Y. 192

Kim, E. 215

Kim, E.J. 129

Kim, G. 236

Kim, H.B. 101

Kim, H.J. 235

Kim, J. 79

Kim, J. 222

Kim, J. 162

Kim, J. 141

Kim, J. 205

Kim, J.-H. 60

Kim, J.-H. 213, 214

Kim, J.-Y. 183, 184

Kim, J.H. 216, 236

Kim, J.H. 60

Kim, J.I. 237

Kim, J.S. 195

Kim, J.W. 162

Kim, J.W. 195

Kim, J.Y. 216, 236

Kim, J.Y. 194, 195, 196, 197, 198, 199, 200

Kim, K.-H 176, 232

Kim, K.-H. 219

Kim, K.H. 194, 195, 198, 200

Kim, K.H. 79

Kim, K.K. 98

Kim, N.-S. 67

Kim, S. 40

Kim, S. 137

Kim, S. 137

Kim, S.-H. 183

Kim, S.-J. 165

Kim, S.-K. 126

Kim, S.-M. 176, 219, 232

Kim, S.C. 25

Kim, S.E. 157, 239

Kim, S.H. 216, 236

Kim, S.I. 195

Kim, S.Y. 195, 228

Kim, T. 126

Kim, U. 130

Kim, W.J. 25

Kim, Y. 125

Kim, Y.-I. 195

Kim, Y.-J. 162

Kim, Y.-S. 67, 170, 192, 200

Kim, Y.J. 215

Kimura, S. 135

Kinoshita, T. 117

Kitajima, K. 57, 159, 161, 163, 164, 233, 234

Kitazume, S. 30

Kizuka, Y. 30

Klarić, L. 123, 128

Klasic, M. 19, 20

Ko, B.-J. 241

Ko, J.-H 40, 200

Ko, J.H. 67

Ko, K. 41, 140, 141, 142, 143, 144, 145

Ko, K.S. 147, 148

Ko, Y.-E. 162

Kobayashi, I. 36

Kobayashi, J. 229

Kobayashi, K. 122

Koca, J. 91

Kojima, H. 127, 238

Komatsu, D. 13

Komatsu, R. 139

Kominami, J. 44

Koo, J.S. 126

Kortagere, S. 224

Kothari, S. 223

Križáková, M. 121

Krylov, V. 81

Kudo, A. 221

Kumar, A. 89

Kuno, A. 31

Kuo, C.-W. 22, 191

Kuo, S.-C. 177

Kuptanon, C. 156

Kuwabara, N. 122

Kuzmich, A. 107

Kwon, O. 228

Lacaille, J.-C. 242

Lai, H.-L. 189

Larson, G. 76

Lauc, G. 19, 27, 128

Lavrsen, K. 78

Lebeau, D. 202

Lebrilla, C.B. 88, 220

Lee, B. 118

Lee, B.-W. 216, 236

Lee, C.W. 25

Lee, D.-J. 205, 209

Lee, H. 145

Lee, H.-Y. 174

Lee, H.K. 142

Lee, H.K. 195, 196, 197, 198, 199, 200

Lee, I.-M. 177

Lee, J. 176, 232

Lee, J. 25

Lee, J. 118

Lee, J. 196

Lee, J.-H. 219

Lee, J.-H. 67, 170

Lee, J.K. 245

Lee, J.M. 170

Lee, J.S. 222

Lee, J.Y. 194, 195, 197, 198, 199, 200

Lee, K.-O. 184

Lee, K.O. 42, 147, 148

Lee, M. 236

Lee, M. 129

Lee, M.-S. 236

Lee, N.-E. 192

Lee, S. 25

Lee, S. 112

Lee, S. 212

Lee, S. 213

Lee, S.-J. 232

Lee, S.-Y. 194, 195

Lee, S.H. 60

Lee, S.S. 98

Lee, T.-Y. 208

Lee, T.A. 212

Lee, W. 53

Lee, Y. 176, 219, 232

Lee, Y.-H. 216, 236

Leffler, H. 103, 227, 230

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Li, C.-S. 115

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Li, R. 96

Li, X. 133, 136

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Liang, P.-H. 174

Liang, X. 94

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Lim, J.-M. 183, 184

Lim, J.-S. 195

Lim, M.-S. 241

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Lim, S. 143

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Lin, C.-w. 86, 188

Lin, C.-Y. 189

Lin, H.-Y. 92

Lin, K.-I. 29, 72

Lin, N.-T. 177

Lin, W-H. 105, 114

Lin, W.-W. 244

Liu, D. 123, 128

Liu, F. 133

Liu, F.-T. 105, 114, 115

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Liu, W.-J. 208

Liu, X. 43

Liu, Y. 94

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