Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man9GlcNAc2- to Man15GlcNAc2- with major Man10GlcNAc2- and Man11GlcNAc2- species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE- or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Vistoli, G., De Maddis, D., Cipak, A., Zarkovic, N., Carini, M., Aldini, G.: Advanced glycoxidation and lipoxidation end products (AGEs and ALEs): an overview of their mechanisms of formation. Free Radic. Res. 47(Suppl 1), 3–27 (2013)
Ott, C., Jacobs, K., Haucke, E., Navarrete Santos, A., Grune, T., Simm, A.: Role of advanced glycation end products in cellular signaling. Redox Biol. 2, 411–429 (2014)
Wang, Z.Q., Jing, L.L., Yan, J.C., Sun, Z., Bao, Z.Y., Shao, C., Pang, Q.W., Geng, Y., Zhang, L.L., Li, L.H.: Role of AGEs in the progression and regression of atherosclerotic plaques. Glycoconj. J. 35, 443–450 (2018)
Zhuang, A., Forbes, J.M.: Diabetic kidney disease: a role for advanced glycation end-product receptor 1 (AGE-R1)? Glycoconj. J. 33, 645–652 (2016)
Monnier, V.M., Taniguchi, N.: Advanced glycation in diabetes, aging and age-related diseases: conclusions. Glycoconj. J. 33, 691–692 (2016)
Neviere, R., Yu, Y., Wang, L., Tessier, F., Boulanger, E.: Implication of advanced glycation end products (Ages) and their receptor (rage) on myocardial contractile and mitochondrial functions. Glycoconj. J. 33, 607–617 (2016)
Raghavan, C.T., Smuda, M., Smith, A.J., Howell, S., Smith, D.G., Singh, A., Gupta, P., Glomb, M.A., Wormstone, I.M., Nagaraj, R.H.: AGEs in human lens capsule promote the TGFbeta2-mediated EMT of lens epithelial cells: implications for age-associated fibrosis. Aging Cell. 15, 465–476 (2016)
Verzijl, N., DeGroot, J., Ben, Z.C., Brau-Benjamin, O., Maroudas, A., R.A. Bank, Mizrahi, J., Schalkwijk, C.G., Thorpe, S.R., Baynes, J.W., Bijlsma, J.W., Lafeber, F.P., TeKoppele, J.M.: Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: a possible mechanism through which age is a risk factor for osteoarthritis. Arthritis Rheum. 46, 114–123 (2002)
Verzijl, N., DeGroot, J., Thorpe, S.R., Bank, R.A., Shaw, J.N., Lyons, T.J., Bijlsma, J.W., Lafeber, F.P., Baynes, J.W., TeKoppele, J.M.: Effect of collagen turnover on the accumulation of advanced glycation end products. J. Biol. Chem. 275, 39027–39031 (2000)
Yamagishi, S., Nakamura, N., Suematsu, M., Kaseda, K., Matsui, T.: Advanced glycation end products: a molecular target for vascular complications in diabetes. Mol. Med. 21(Suppl 1), S32–S40 (2015)
Fukami, K., Yamagishi, S., Okuda, S.: Role of AGEs-RAGE system in cardiovascular disease. Curr. Pharm. Des. 20, 2395–2402 (2014)
Bierhaus, A., Humpert, P.M., Morcos, M., Wendt, T., Chavakis, T., Arnold, B., Stern, D.M., Nawroth, P.P., Understanding, R.A.G.E.: The receptor for advanced glycation end products. J Mol Med (Berl). 83, 876–886 (2005)
Aldini, G., Vistoli, G., Stefek, M., Chondrogianni, N., Grune, T., Sereikaite, J., Sadowska-Bartosz, I., Bartosz, G.: Molecular strategies to prevent, inhibit, and degrade advanced glycoxidation and advanced lipoxidation end products. Free Radic. Res. 47(Suppl 1), 93–137 (2013)
Mizumoto, S., Takahashi, J., Sugahara, K.: Receptor for advanced glycation end products (RAGE) functions as receptor for specific sulfated glycosaminoglycans, and anti-RAGE antibody or sulfated glycosaminoglycans delivered in vivo inhibit pulmonary metastasis of tumor cells. J. Biol. Chem. 287, 18985–18994 (2012)
Xu, D., Young, J., Song, D., Esko, J.D.: Heparan sulfate is essential for high mobility group protein 1 (HMGB1) signaling by the receptor for advanced glycation end products (RAGE). J. Biol. Chem. 286, 41736–41744 (2011)
Rabbani, N., Ashour, A., Thornalley, P.J.: Mass spectrometric determination of early and advanced glycation in biology. Glycoconj. J. 33, 553–568 (2016)
Degani, G., Altomare, A.A., Colzani, M., Martino, C., Mazzolari, A., Fritz, G., Vistoli, G., Popolo, L., Aldini, G.: A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE). Redox Biol. 11, 275–285 (2017)
Koch, M., Chitayat, S., Dattilo, B.M., Schiefner, A., Diez, J., Chazin, W.J., Fritz, G.: Structural basis for ligand recognition and activation of RAGE. Structure. 18, 1342–1352 (2010)
Degani, G., Colzani, M., Tettamanzi, A., Sorrentino, L., Aliverti, A., Fritz, G., Aldini, G., Popolo, L.: An improved expression system for the VC1 ligand binding domain of the receptor for advanced glycation end products in Pichia pastoris. Protein Expr. Purif. 114, 48–57 (2015)
Dattilo, B.M., Fritz, G., Leclerc, E., Kooi, C.W., Heizmann, C.W., Chazin, W.J.: The extracellular region of the receptor for advanced glycation end products is composed of two independent structural units. Biochemistry. 46, 6957–6970 (2007)
Ostendorp, T., Weibel, M., Leclerc, E., Kleinert, P., Kroneck, P.M.H., Heizmann, C.W., Fritz, G.: Expression and purification of the soluble isoform of human receptor for advanced glycation end products (sRAGE) from Pichia pastoris. Biochem. Biophys. Res. Commun. 347, 4–11 (2006)
Park, S.J., Kleffmann, T., Hessian, P.A.: The G82S polymorphism promotes glycosylation of the receptor for advanced glycation end products (RAGE) at asparagine 81: comparison of wild-type rage with the G82S polymorphic variant. J. Biol. Chem. 286, 21384–21392 (2011)
Wright, H.T.: Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins. Crit. Rev. Biochem. Mol. Biol. 26, 1–52 (1991)
Behrens, A.J., Duke, R.M., Petralia, L.M., Harvey, D.J., Lehoux, S., Magnelli, P.E., Taron, C.H., Foster, J.M.: Glycosylation profiling of dog serum reveals differences compared to human serum. Glycobiology. 28, 825–831 (2018)
Park, H., Boyington, J.C.: The 1.5 a crystal structure of human receptor for advanced glycation Endproducts (RAGE) Ectodomains reveals unique features determining ligand binding. J. Biol. Chem. 285, 40762–40770 (2010)
Trimble, R.B., Atkinson, P.H., Tschopp, J.F., Townsend, R.R., Maley, F.: Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris. J. Biol. Chem. 266, 22807–22817 (1991)
Gemmill, T.R., Trimble, R.B.: Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta. 1426, 227–237 (1999)
Ziegler, F.D., Gemmill, T.R., Trimble, R.B.: Glycoprotein synthesis in yeast. Early events in N-linked oligosaccharide processing in Schizosaccharomyces pombe. J. Biol. Chem. 269, 12527–12535 (1994)
Vinogradov, E., Petersen, B.O., Duus, J.O.: Isolation and characterization of non-labeled and 13C-labeled mannans from Pichia pastoris yeast. Carbohydr. Res. 325, 216–221 (2000)
Mille, C., Bobrowicz, P., Trinel, P.A., Li, H., Maes, E., Guerardel, Y., Fradin, C., Martinez-Esparza, M., Davidson, R.C., Janbon, G., Poulain, D., Wildt, S.: Identification of a new family of genes involved in beta-1,2-mannosylation of glycans in Pichia pastoris and Candida albicans. J. Biol. Chem. 283, 9724–9736 (2008)
Santambrogio, C., Ricagno, S., Colombo, M., Barbiroli, A., Bonomi, F., Bellotti, V., Bolognesi, M., Grandori, R.: DE-loop mutations affect beta2 microglobulin stability, oligomerization, and the low-pH unfolded form. Protein Sci. 19, 1386–1394 (2010)
Robinson, P.J., Pringle, M.A., Woolhead, C.A., Bulleid, N.J.: Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation. J. Biol. Chem. 292, 6978–6986 (2017)
Breitling, J., Aebi, M.: N-linked protein glycosylation in the endoplasmic reticulum. Cold Spring Harb. Perspect. Biol. 5, a013359 (2013)
Xie, J., Reverdatto, S., Frolov, A., Hoffmann, R., Burz, D.S., Shekhtman, A.: Structural basis for pattern recognition by the receptor for advanced glycation end products (RAGE). J. Biol. Chem. 283, 27255–27269 (2008)
Matsumoto, S., Yoshida, T., Murata, H., Harada, S., Fujita, N., Nakamura, S., Yamamoto, Y., Watanabe, T., Yonekura, H., Yamamoto, H., Ohkubo, T., Kobayashi, Y.: Solution structure of the variable-type domain of the receptor for advanced glycation end products: new insight into AGE-RAGE interaction. Biochemistry. 47, 12299–12311 (2008)
Miller, S., Henry, A.P., Hodge, E., Kheirallah, A.K., Billington, C.K., Rimington, T.L., Bhaker, S.K., Obeidat, M., Melen, E., Merid, S.K., Swan, C., Gowland, C., Nelson, C.P., Stewart, C.E., Bolton, C.E., Kilty, I., Malarstig, A., Parker, S.G., Moffatt, M.F., Wardlaw, A.J., Hall, I.P., Sayers, I.: The Ser82 RAGE variant affects lung function and serum RAGE in smokers and sRAGE production in vitro. PLoS One. 11, e0164041 (2016)
Hofmann, M.A., Drury, S., Hudson, B.I., Gleason, M.R., Qu, W., Lu, Y., Lalla, E., Chitnis, S., Monteiro, J., Stickland, M.H., Bucciarelli, L.G., Moser, B., Moxley, G., Itescu, S., Grant, P.J., Gregersen, P.K., Stern, D.M., Schmidt, A.M.: RAGE and arthritis: the G82S polymorphism amplifies the inflammatory response. Genes Immun. 3, 123–135 (2002)
Osawa, M., Yamamoto, Y., Munesue, S., Murakami, N., Sakurai, S., Watanabe, T., Yonekura, H., Uchigata, Y., Iwamoto, Y., Yamamoto, H.: De-N-glycosylation or G82S mutation of RAGE sensitizes its interaction with advanced glycation endproducts. Biochim. Biophys. Acta. 1770, 1468–1474 (2007)
Kashiwagi, A., Araki, S.: Relation between polymorphisms G1704T and G82S of RAGE gene and diabetic retinopathy in Japanese type 2 diabetic patients. Intern. Med. 44, 397–398 (2005)
Liu, L., Xiang, K.: RAGE Gly82Ser polymorphism in diabetic microangiopathy. Diabetes Care. 22, 646 (1999)
Tae, H.J., Kim, J.M., Park, S., Tomiya, N., Li, G., Wei, W., Petrashevskaya, N., Ahmet, I., Pang, J., Cruschwitz, S., Riebe, R.A., Zhang, Y., Morrell, C.H., Browe, D., Lee, Y.C., Xiao, R.P., Talan, M.I., Lakatta, E.G., Lin, L.: The N-glycoform of sRAGE is the key determinant for its therapeutic efficacy to attenuate injury-elicited arterial inflammation and neointimal growth. J Mol Med (Berl). 91, 1369–1381 (2013)
Hamilton, S.R., Davidson, R.C., Sethuraman, N., Nett, J.H., Jiang, Y., Rios, S., Bobrowicz, P., Stadheim, T.A., Li, H., Choi, B.K., Hopkins, D., Wischnewski, H., Roser, J., Mitchell, T., Strawbridge, R.R., Hoopes, J., Wildt, S., Gerngross, T.U.: Humanization of yeast to produce complex terminally sialylated glycoproteins. Science. 313, 1441–1443 (2006)
Laukens, B., De Wachter, C., Callewaert, N.: Engineering the Pichia pastoris N-glycosylation pathway using the GlycoSwitch technology. Methods Mol. Biol. 1321, 103–122 (2015)
This work was partially supported by University of Milan. G.D. is the recipient of a Postdoc fellowship from University of Milano. The authors wish to thank Euroclone S.p.A., Via Figino 20/22, Pero (Milano, Italy) that, as a partner of the CBM consortium (Connecting bio-research and industry), supported this work with the grant Art. 13 DM 593 08/08/2000 and in particular we are grateful to Dr. Fabio Bolchi for helpful discussions and continuous support.
Conflicts of interest
The authors declare that they have no conflicts of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Degani, G., Barbiroli, A., Magnelli, P. et al. Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris. Glycoconj J 36, 27–38 (2019). https://doi.org/10.1007/s10719-018-09855-x
- Receptor for advanced glycation end products (RAGE)
- Protein glycoforms
- Released glycan profiling
- LC/mass spectrometry
- Thermal stability
- Protein-protein interactions
- Pichia pastoris