Glycoconjugate Journal

, Volume 36, Issue 1, pp 27–38 | Cite as

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

  • Genny Degani
  • Alberto Barbiroli
  • Paula Magnelli
  • Stefania Digiovanni
  • Alessandra Altomare
  • Giancarlo Aldini
  • Laura PopoloEmail author
Original Article


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.


Receptor 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.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10719_2018_9855_MOESM1_ESM.pdf (492 kb)
ESM 1 (PDF 491 kb)


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biosciences (DBS)University of MilanMilanItaly
  2. 2.Department of Food, Environmental and Nutritional Sciences (DeFENS)University of MilanMilanItaly
  3. 3.New England Biolabs, Inc.IpswichUSA
  4. 4.Department of Pharmaceutical Sciences (DISFARM)University of MilanMilanItaly

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