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

Ding Liu,Carol H Miao,Weidong Xiao,Barbara A Konkle,Lei Li,Xiu-Feng Wan,Shuaishuai Wang,Junping Zhang

doi:10.3389/fchem.2021.691217

Abstract

Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the 18O-labeling method. O-glycopeptide enrichment and O-glycosite identification were achieved by an enzyme-assisted site-specific extraction method. An RP–LC–MS/MS system functionalized with collision-induced dissociation and stepped normalized collision energy (sNCE)-HCD tandem mass was applied to analyze the glycoforms of fibronectin. A total of 6 N-glycosites and 53 O-glycosites were identified, which were occupied by 38 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 77.31% of N-glycans were sialylated, and O-glycosylation was dominated by the sialyl-T antigen. These site-specific glycosylation patterns on human fibronectin can facilitate functional analyses of fibronectin and therapeutics development.

Highlights

As a major post-translational modification (PTM) of proteins, glycosylation has been reported to play critical roles in protein folding, stability, macromolecular interactions, functions, and activity (Mitra et al, 2003; Shental-Bechor and Levy, 2008)
The most common O-linked glycosylation refers to the attachment of an α-linked GalNAc residue to serine (Ser) or threonine (Thr) residues of protein by an O-glycosidic bond, whereas N-linked glycosylation refers to the attachment of a β-linked N-glycan to asparagine (Asn) residues within a consensus peptide sequence of Asn-Xxx-Ser/Thr via an N-glycosidic bond
Proteomic analysis of Glu-C-tryptic–digested fibronectin confirmed the identity of plasma fibronectin

Summary

Introduction

As a major post-translational modification (PTM) of proteins, glycosylation has been reported to play critical roles in protein folding, stability, macromolecular interactions, functions, and activity (Mitra et al, 2003; Shental-Bechor and Levy, 2008). N-linked glycosylation and O-linked glycosylation are two major types of protein glycosylation (Varki et al, 2015). Human plasma fibronectin is a large glycoprotein that plays a crucial role in wound healing (Furie and Furie, 2005; Patten and Wang, 2020). It is a dimer consisting of two nearly identical monomers

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Conclusion