Oral Presentation 22nd Annual Lorne Proteomics Symposium 2017

Evaluating the relationship between N-Glycosylation and protein stability in Campylobacter jejuni (#25)

Joel A Cain 1 2 , Nichollas E Scott 2 3 , Nestor Solis 2 3 , Melanie Y White 1 2 , Stuart J Cordwell 1 2 4 5
  1. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia
  2. School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia
  3. University of British Columbia, Vancouver, BC, Canada
  4. Mass Spectrometry Core Facility , University of Sydney, Sydney, NSW, Australia
  5. Discipline of Pathology, University of Sydney, Sydney, NSW, Australia

Campylobacter jejuni is a leading cause of acute gastroenteritis in developed countries, and was the first prokaryote demonstrated to extensively modify proteins by N-linked glycosylation. This highly conserved post-translational modification system is crucial for pathogenicity, albeit the mechanism remains to be elucidated. With over 120 modified sites identified to date, recent findings have suggested that N-glycosylation may be important for protein stability in various physiological and pathogenically relevant contexts. To explore this further, we employed iTRAQ-based labelling to determine the effect of either loss of the oligosaccharyltransferase (ΔpglB), or biosynthesis of the glycan (ΔpglDEF) on whole protein abundance in a relatively recent clinical isolate, C. jejuni JHH1. Of the 1077 C. jejuni proteins quantified, only 57 were deemed to have a significant change in abundance in either of the Δpgl strains relative to the wild-type isolate. A large proportion of known glycoproteins were quantified with ~17% displaying an altered abundance in the N-glycosylation negative strains. N-terminal amine isotopic labelling of substrates (N-TAILS) was also employed for pair wise comparisons of the N-degradome of wild-type JHH1 and individual pgl deletion strains to address the hypothesis that the N-linked glycan may provide protection from proteolytic degradation. We were able to identify and quantify 4122 unique N-termini from 766 C. jejuni proteins. From those derived from known N-linked glycoproteins, a number were found to be in close proximity to or contained the sites of N-linked glycosylation and in turn displayed a significant difference in their relative abundance in the Δpgl mutants. Further, we coupled N-TAILS to intact N-glycopeptide analyses to identify putative N-terminal N-glycopeptides. These proteomics-based approaches were complemented with various standard phenotypic tests to establish how the loss of N-glycosylation extended to broader changes in C. jejuni physiology.