| Abstract | Mass spectrometry investigations of partially purified C. jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan of mass 1406 Da and comprised of one hexose, five HexNAc and a species of mass 228 Da, consistent with a trideoxydiacetamidohexose. By means of soybean lectin affinity chromatography, a mixture of glycoproteins was obtained from a glycine extract, and 2D-gel proteomics analysis led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins. Glycopeptides were prepared from the glycoprotein mixture by pronase digestion and gel-filtration. The structure of the glycan was determined by using nano-NMR techniques to be GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,4-[Glcb1,3-]GalNAc-a1,4-GalNAc-a1,3-Bac- b1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing further evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the biosynthesis of bacillosamine |
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