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.

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 / G. Degani, A. Barbiroli, P. Magnelli, S. Digiovanni, A. Altomare, G. Aldini, L. Popolo. - In: GLYCOCONJUGATE JOURNAL. - ISSN 0282-0080. - 36:1(2019 Feb), pp. 27-38. [10.1007/s10719-018-09855-x]

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

G. Degani
Primo
;
A. Barbiroli
Secondo
;
S. Digiovanni;A. Altomare;G. Aldini
Penultimo
;
L. Popolo
Ultimo
2019-02

Abstract

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.
LC/mass spectrometry; Pichia pastoris; Protein glycoforms; Protein-protein interactions; Receptor for advanced glycation end products (RAGE); Released glycan profiling; Thermal stability; Biochemistry; Molecular Biology; Cell Biology
Settore BIO/11 - Biologia Molecolare
Settore BIO/10 - Biochimica
Settore CHIM/08 - Chimica Farmaceutica
Settore BIO/13 - Biologia Applicata
5-gen-2019
Article (author)
File in questo prodotto:
File Dimensione Formato  
Revised manuscript.pdf

embargo fino al 07/02/2020

Tipologia: Post-print, accepted manuscript ecc. (versione accettata dall'editore)
Dimensione 165.19 kB
Formato Adobe PDF
165.19 kB Adobe PDF Visualizza/Apri
Degani2019_Article_InsightsIntoTheEffectsOfN-glyc.pdf

accesso riservato

Tipologia: Publisher's version/PDF
Dimensione 2.51 MB
Formato Adobe PDF
2.51 MB Adobe PDF   Visualizza/Apri   Richiedi una copia
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/619265
Citazioni
  • ???jsp.display-item.citation.pmc??? 1
  • Scopus 5
  • ???jsp.display-item.citation.isi??? 5
social impact