EVALUATING THE RELATIONSHIP BETWEEN <em>N</em>-GLYCOSYLATION AND PROTEIN STABILITY IN <em>CAMPYLOBACTER JEJUNI</em> — ASN Events
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  2. Lightning Talks One
  3. EVALUATING THE RELATIONSHIP BETWEEN N-GLYCOSYLATION AND PROTEIN STABILITY IN CAMPYLOBACTER JEJUNI

EVALUATING THE RELATIONSHIP BETWEEN N-GLYCOSYLATION AND PROTEIN STABILITY IN CAMPYLOBACTER JEJUNI (#102)

Joel JA Cain 1 2 , Nichollas NE Scott 1 2 3 , Nestor N Solis 1 2 3 , Melanie MY White 1 2 , Stuart SJ 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 one of the leading causes of acute gastroenteritis in the developed world, and a major antecedent for a number of debilitating autoimmune disorders. C. jejuni was the first prokaryotic organism found to possess an N-linked protein glycosylation system. Approximately 100 C. jejuni proteins to date have been shown to be targets of this post-translational modification1,2, and this system has been shown to be a crucial for pathogenicity albeit through an as yet undefined mechanism. Here, 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 strain 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-TAILS3) was also employed for pair wise comparisons of the N-degradome of wild-type JHH1 and individual pgl knock out strains to address the hypothesis that the addition of the N-linked glycan may provide protection from proteolytic degradation for the largely unstructured regions of the protein on which they’re commonly attached. 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.

These proteomics-based approaches were complemented with various standard phenotypic tests to establish if the observed effects of loss of N-glycosylation extended to broader changes in C. jejuni’s physiology.

  1. Scott et al., 2012. Modification of the Campylobacter jejuni N-Linked glycan by EptC protein-mediated addition of phosphoethanolamine. J. Biol. Chem.
  2. Scott et al., 2011. Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy collisional dissociation, and electron transfer dissociation MS applied to the N-Linked glycoproteome of Campylobacter jejuni. Mol. Cell. Proteomics.
  3. Kleifeld et al., 2010. Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat. Biotechnol.