Deciphering Protein O-Glycosylation: Solid-Phase Chemoenzymatic Cleavage and Enrichment

Abstract

Glycosylation plays a critical role in the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Over 50% of mammalian cellular proteins are typically glycosylated; this modification is involved in a wide range of biological functions such as barrier formation against intestinal microbes and serves as signaling molecules for selectins and galectins in the innate immune system. N-linked glycosylation analysis has been greatly facilitated owing to a range of specific enzymes available for their release. However, system-wide analysis on O-linked glycosylation remains a challenge due to the lack of equivalent enzymes and the inherent structural heterogeneity of O-glycans. Although O-glycosidase can catalyze the removal of core 1 and core 3 O-linked disaccharides from glycoproteins, analysis of other types of O-glycans remains difficult, particularly when residing on glycopeptides. Here, we describe a novel chemoenzymatic approach driven by a newly available O-protease and solid phase platform. This method enables the assignment of O-glycosylated peptides, N-glycan profile, sialyl O-glycopeptides linkage, and mapping of heterogeneous O-glycosylation. For the first time, we can analyze intact O-glycopeptides generated by O-protease and enriched using a solid-phase platform. We establish the method on standard glycoproteins, confirming known O-glycosites with high accuracy and confidence, and reveal up to 8-fold more glycosites than previously reported with concomitant increased heterogeneity. This technique is further applied for analysis of Zika virus recombinant glycoproteins, revealing their dominant O-glycosites and setting a basis set of O-glycosylation tracts in these important viral antigens. Our approach can serve as a benchmark for the investigation of protein O-glycosylation in diseases and other biomedical contexts. This method should become an indispensable tool for investigations where O-glycosylation is central.

Mapping the O‐glycoproteome using site‐specific extraction of O‐linked glycopeptides (EXoO)

“…OpeRATOR, identified from the mucin degrading human intestinal bacterium Akkermansia muciniphila , recognizes O‐linked glycans and cleaves O‐linked glycopeptides at the N‐termini of O‐linked glycan‐occupied Ser or Thr to release site‐specific O‐linked glycopeptides with their conjugated O‐linked glycans. During the peer‐review period of our study, a manuscript by Yang et al (), described the analysis of O‐linked glycosylation sites from several simple glycoproteins including fetuin, mucin, and Zika viral proteins. This study took use of the enzyme, OpeRATOR, with cleavage of peptide sequences at the N‐termini of the O‐linked glycosylation sites (Yang et al , ).…”

“…During the peer‐review period of our study, a manuscript by Yang et al (), described the analysis of O‐linked glycosylation sites from several simple glycoproteins including fetuin, mucin, and Zika viral proteins. This study took use of the enzyme, OpeRATOR, with cleavage of peptide sequences at the N‐termini of the O‐linked glycosylation sites (Yang et al , ). However, the glycan specificity and cleavage specificity for the O‐linked glycosylation sites by OpeRATOR enzyme are not clearly defined.…”

The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins

“…Systems with truncated forms of glycosylation, such as engineered "SimpleCells" lacking O-glycan elaboration machinery can simplify mucin analysis and glycosite identification, but functionally important glycan structures beyond the initiating O-GalNAc are lost (26). Recently, an "O-protease" was reported that cleaves N-terminally to glycosylated serine and threonine residues (27,28). However, this enzyme requires substrate desialylation for optimal activity, and is not specific for mucins.…”

Glycomics and glycoproteomics of viruses: Mass spectrometry applications and insights toward structure–function relationships

“…The enzyme, OpeRATOR, appears to be broadly active on O‐glycosylation sites occupied by multiple core types. It generates a new N‐terminus by cleavage of the peptide bond between the occupied Ser or Thr and its N‐terminal amino acid residue (Yang et al, 2018a, c). The enzyme has been used successfully to investigate Zika virus envelope E and NS‐1 proteins (Yang et al, 2018a).…”