title: O-linked glycosylation of the PilA pilin protein of Francisella tularensis: identification of the endogenous protein-targeting oligosaccharyltransferase and characterization of the native oligosaccharide.
abstract: Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genus Francisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation in Francisella tularensis and its sufficiency for PilA glycosylation in Escherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica and demonstrated that the protein undergoes multisite, O-linked glycosylation with a pentasaccharide of the structure HexNac-Hex-Hex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains of Francisella subspecies thus express an O-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved in Francisella species, O-linked protein glycosylation may be a feature common to members of this genus.
authors.affiliation: Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway. w.m.egge-jacobsen@imbv.uio.no
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Egge-Jacobsen
authors.forename: Emelie Näslund
authors.initials: EN
authors.lastname: Salomonsson
authors.forename: Finn Erik
authors.initials: FE
authors.lastname: Aas
authors.forename: Anna-Lena
authors.initials: AL
authors.lastname: Forslund
authors.forename: Hanne C
authors.initials: HC
authors.lastname: Winther-Larsen
authors.forename: Josef
authors.initials: J
authors.lastname: Maier
authors.forename: Anna
authors.initials: A
authors.lastname: Macellaro
authors.forename: Kerstin
authors.initials: K
authors.lastname: Kuoppa
authors.forename: Petra C F
authors.initials: PC
authors.lastname: Oyston
authors.forename: Richard W
authors.initials: RW
authors.lastname: Titball
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Thomas
authors.forename: Åke
authors.initials: Å
authors.lastname: Forsberg
authors.forename: Joann L
authors.initials: JL
authors.lastname: Prior
authors.forename: Michael
authors.initials: M
authors.lastname: Koomey
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000378:metabolism*; D005604:Francisella tularensis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000378:metabolism*; D015151:Immunoblotting; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006020:Glycopeptides; D008565:Membrane Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D036781:Fimbriae Proteins; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase