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(1): 27-38, 2019 02.
Article
em En
| MEDLINE
| ID: mdl-30612271
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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polissacarídeos
/
Simulação de Dinâmica Molecular
/
Receptor para Produtos Finais de Glicação Avançada
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article