Comprehensive N‐ and O‐glycosylation mapping of human coagulation factor V

Abstract

Background/Objective: Coagulation factor V (FV), a multidomain glycoprotein, is an essential cofactor in the blood clotting cascade. FV deficiency is a rare bleeding disorder that results in poor clotting after an injury or surgery. The only treatment for the disease is infusions of fresh frozen plasma and blood platelets. Glycosylation affects the biological activity, pharmacokinetics, immunogenicity, and in vivo clearance rate of proteins in the plasma. The glycan profile of FV, as well as how it affects the activity, stability, and immunogenicity, remains unknown. Methods:In this study, we comprehensively mapped the glycosylation patterns of human plasma-derived FV by combining multienzyme digestion, hydrophilic interaction chromatography enrichment of glycopeptides, and alternated fragmentation mass spectrometry analysis. | 1885 MA et Al.

Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila

“…The O -glycopeptidase OgpA has shown promise as a tool to map O -glycosylation sites since it generates a digestion pattern only where O -glycans are present. The enzymatic activity of OgpA combined with detailed analytical workflows, such as liquid chromatography coupled to mass spectrometry, have enabled detailed mapping of occupied O -glycosylation sites into therapeutic proteins such as human coagulation Factor V 80 . Specifically, using an OgpA-based analytical workflow, 18 O -glycosites were discovered in the B domain of Factor V compared to the 10 sites previously identified 81 .…”

Site-Specific N- and O-Glycosylation Analysis of Human Plasma Fibronectin

“…However, only 28 of those sites were precisely localized via a lectin enrichment method ( King et al, 2017 ). Here, we localized the exact O-glycosites by using the EXoO method ( Ma et al, 2020 ). O-glycoforms at each site were also identified and relatively quantified by using the modified EXoO method.…”

“…N-glycopeptides were enriched by an in-house packed ZIC-HILIC micro-tip as previously described ( Ma et al, 2020 ). The HILIC micro-tip was washed with 500 μL of ACN, water, and binding buffer (80% ACN/1% TFA) twice sequentially.…”

“…Moreover, an O-glycan–specific protease-assisted method named “site-specific extraction of O-linked glycopeptides” (EXoO) was recently developed, which allows for the comprehensive analysis of O-glycosites and O-glycans ( Yang et al, 2020 ). We have been using these advanced glycoproteomic techniques to elaborate glycosylation of key proteins in thrombosis, achieving site-specific glycan mapping of human factor FVIII ( Qu et al, 2020 ) and factor FV ( Ma et al, 2020 ).…”

“…In this study, we performed systematic glycan analysis of human plasma fibronectin by optimizing and applying an integrated approach we recently developed ( Ma et al, 2020 ). Briefly, Glu-C and trypsin were used for peptide mapping, N-glycopeptides were enriched by ZIC-HILIC, and O-glycopeptides were enriched through EXoO ( Yang et al, 2020 ).…”