Oral Presentation 26th Annual Lorne Proteomics Symposium 2021

Exploiting pglB negative and positive Campylobacter jejuni to identify occupied and unoccupied sites of N-linked protein glycosylation (#28)

Joel A Cain 1 2 , Ashleigh L Dale 1 2 , Stuart J Cordwell 1 2 3 4
  1. School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia
  2. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia
  3. Discipline of Pathology, University of Sydney, Sydney, NSW, Australia
  4. Sydney Mass Spectrometry, University of Sydney, Sydney, NSW, Australia

Campylobacter jejuni is a Gram negative human pathogen that contains a unique N-linked protein glycosylation system that is encoded by the pgl gene cluster. The heptasaccharide N-glycan is attached to membrane-associated proteins in the periplasm by the PglB oligosaccharyltransferase at the consensus sequon Asp/Glu-X-Asn-X-Ser/Thr. Over 500 sequons are contained in the C. jejuni proteome and >130 sites have been experimentally verified. We performed quantitative proteomics of C. jejuni NCTC11168 compared with a pglB deletion mutant (ΔpglB) and a pglB restored strain (ΔpglB::pglB) with the aim of identifying differentially regulated sequon-associated peptides.  We quantified 164 sequon-associated peptides corresponding to 120 unique sequons, of which 73 contained previously experimentally verified glycosites. Analysis of differential abundances for these peptides allowed us to make inferences regarding site-level occupancy by the N-glycan in vivo. By referencing the subcellular topology of modified proteins we were able to show that these predictions were in agreement with the presentation of sequons relative to the N-glycosylation machinery. Finally we utilised a series of proteases (trypsin, Glu-C, Asp-N, thermolysin, pepsin and chymotrypsin) to define the glycoproteome of wild-type and pglB restored C. jejuni. We identified 1915 glycopeptides corresponding to 140 glycosylation sites, including the identification of 31 sites not previously observed and 17 highlighted by the ΔpglB proteomic analysis.