Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Determining the role of Campylobacter jejuni N-glycosylation in protein stability by N-terminomics (#113)

Joel Cain 1 2 , Nichollas Scott 1 , Nestor Solis 1 , Melanie White 1 2 3 , Stuart 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 the leading cause of gastroenteritis in the developed world, with human infections predominantly acquired via under-cooked or poorly prepared poultry, a host in which the organism is considered a commensal. C. jejuni encodes a protein N-glycosylation system (Pgl), which targets >100 membrane-associated proteins and is required for both human infection and chicken colonization. Despite this, the exact biological role of this post-translational modification system remains poorly understood. We employed iTRAQ-based labelling to show that loss of the PglB oligosaccharyltransferase (ΔpglB), or biosynthetic enzymes that generate the glycan (ΔpglDEF), have a limited effect on whole protein abundance relative to wild-type. Changes were largely associated with known glycoproteins, with ~17% of quantified N-glycoproteins displaying altered protein abundances. N-terminal amine isotopic labelling of substrates (N-TAILS) comparisons between wild-type and Δpgl mutants provided evidence for proteolytic processing in close proximity to known sites of N-linked glycosylation that was only identified in pgl mutant strains. We next performed intact glycopeptide analysis of N-glycopeptide enriched membrane protein fractions from wild-type C. jejuni, using protein level dimethylation to differentiate those N-glycopeptides localised at protein N-termini. Using this approach, we were able to identify 78 unique C. jejuni N-glycopeptides, including 2 novel sites. We also provide evidence that the proteolytic activity associated with sites of N-glycosylation is limited in the wild-type, further supporting the conclusion that N-glycosylation serves to protect N-glycoproteins from proteolytic degradation within the C. jejuni membrane.