Exploiting pglB Oligosaccharyltransferase-Positive and -Negative Campylobacter jejuni and a Multiprotease Digestion Strategy to Identify Novel Sites Modified by N-Linked Protein Glycosylation

Abstract

Campylobacter jejuni is a bacterial pathogen encoding a unique N-linked glycosylation (pgl) system that mediates attachment of a heptasaccharide to N-sequon-containing membrane proteins by the PglB oligosaccharyltransferase (OST). Many targets of PglB are known, yet only a fraction of sequons are experimentally confirmed, and site occupancy remains elusive. We exploited pglB-positive (wild-type; WT) and -negative (ΔpglB) proteomes to identify potential glycosites. The nonglycosylated forms of known glycopeptides were typically increased in protein normalized abundance in ΔpglB relative to WT and restored by pglB reintroduction (ΔpglB::pglB). Sequon-containing peptide abundances were thus consistent with significant site occupancy in the presence of the OST. Peptides with novel sequons were either unaltered (likely not glycosylated) or showed abundance consistent with known glycopeptides. Topology analysis revealed that unaltered sequons often displayed cytoplasmic localization, despite originating from membrane proteins. Novel glycosites were confirmed using parallel multiprotease digestion, LC-MS/MS, and FAIMS-MS to define the glycoproteomes of WT and ΔpglB::pglB C. jejuni. We identified 142 glycosites, of which 32 were novel, and 83% of sites predicted by proteomics were validated. There are now 166 experimentally verified C. jejuni glycosites and evidence for occupancy or nonoccupancy of 31 additional sites. This study serves as a model for the use of OST-negative cells and proteomics for highlighting novel glycosites and determining occupancy in a range of organisms.

Multi-Omics of Campylobacter jejuni Growth in Chicken Exudate Reveals Molecular Remodelling Associated with Altered Virulence and Survival Phenotypes

“…We observed changes in proteins involved in lipid A, PGN, and N-linked glycosylation that suggest CE induced their rate of biosynthesis, and this aligned with a reduction in UDP-HexNAc levels, correlating with its usage (as UDP-GlcNAc) as the building block for these pathways. Increased abundances of Pgl proteins, including a moderate increase in the PglF rate-limiting step [90], correlated with increased abundances of many target glycoproteins, including some with high glycan site occupancy [91], yet no obvious functional relationships beyond those discussed (e.g., CmeABC efflux proteins) could be discerned. Increased N-glycan biosynthesis could also be associated with elevated free oligosaccharides and hence, improved defence against osmotic stress [92].…”

Integrating the analysis of human biopsies using post‐translational modifications proteomics

“…Glycosylation is prevalent PTM and defined by the addition of a glycan to an amino acid, namely asparagine (N‐linked) or serine and threonine (O‐linked; Cain et al, 2021 ; Girgis et al, 2024 ; Morelle et al, 2006 ). Glycosylation impacts many functions of a protein: its structure, protein–protein interactions and physical dynamics (Xu et al, 2023 ).…”