title: Characterization of N-linked protein glycosylation in Helicobacter pullorum.
abstract: The first bacterial N-linked glycosylation system was discovered in Campylobacter jejuni, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologues are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria, including three Helicobacter species: H. pullorum, H. canadensis, and H. winghamensis. In contrast to C. jejuni, in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli. Analysis of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum, PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the -2 position of the N-glycosylation sequon, as for C. jejuni. Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.
authors.affiliation: Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester M13 9PT, United Kingdom.
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Jervis
authors.forename: Rebecca
authors.initials: R
authors.lastname: Langdon
authors.forename: Paul
authors.initials: P
authors.lastname: Hitchen
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Lawson
authors.forename: Alison
authors.initials: A
authors.lastname: Wood
authors.forename: Joanne L
authors.initials: JL
authors.lastname: Fothergill
authors.forename: Howard R
authors.initials: HR
authors.lastname: Morris
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.forename: Brendan
authors.initials: B
authors.lastname: Wren
authors.forename: Dennis
authors.initials: D
authors.lastname: Linton
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D016123:Campylobacter jejuni / Q000235:genetics / Q000378:metabolism; D020565:Epsilonproteobacteria / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D016998:Helicobacter / Q000235:genetics / Q000378:metabolism*; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase