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- Title
Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system.
- Authors
Jervis, Adrian J.; Wood, Alison G.; Cain, Joel A.; Butler, Jonathan A.; Frost, Helen; Lord, Elizabeth; Langdon, Rebecca; Cordwell, Stuart J.; Wren, Brendan W.; Linton, Dennis
- Abstract
N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1- dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system.
- Subjects
GLYCOSYLATION; GENE clusters; OLIGOSACCHARYLTRANSFERASE; GLYCANS; CAMPYLOBACTER
- Publication
Glycobiology, 2018, Vol 28, Issue 4, p233
- ISSN
0959-6658
- Publication type
Article
- DOI
10.1093/glycob/cwx110