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
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.
KeywordsReceptor for advanced glycation end products (RAGE) Protein glycoforms Released glycan profiling LC/mass spectrometry Thermal stability Protein-protein interactions Pichia pastoris
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.
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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.
- 7.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)CrossRefGoogle Scholar
- 8.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)CrossRefGoogle Scholar
- 13.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)CrossRefGoogle Scholar
- 14.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)CrossRefGoogle Scholar
- 17.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)CrossRefGoogle Scholar
- 19.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)CrossRefGoogle Scholar
- 21.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)CrossRefGoogle Scholar
- 26.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)Google Scholar
- 28.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)Google Scholar
- 30.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)CrossRefGoogle Scholar
- 35.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)CrossRefGoogle Scholar
- 36.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)CrossRefGoogle Scholar
- 37.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)CrossRefGoogle Scholar
- 38.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)CrossRefGoogle Scholar
- 41.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)CrossRefGoogle Scholar
- 42.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)CrossRefGoogle Scholar