Browsing by Author "Bouche, Laura"

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    The Type B Flagellin of Hypervirulent Clostridium difficile Is Modified with Novel Sulfonated Peptidylamido-glycans
    (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2016) Bouche, Laura; Panico, Maria; Hitchen, Paul; Binet, Daniel; Sastre, Federico; Faulds Pain, Alexandra; Valiente, Esmeralda; Vinogradov, Evgeny; Aubry, Annie; Fulton, Kelly; Twine, Susan; Logan, Susan M.; Wren, Brendan W.; Dell, Anne; Morris, Howard R.
    Glycosylation of flagellins is a well recognized property of many bacterial species. In this study, we describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile. We used mass spectrometry (nano-LC-MS and MS/MS analysis) to identify a number of putative glycopeptides that carried a variety of glycoform substitutions, each of which was linked through an initial N-acetylhexosamine residue to Ser or Thr. Detailed analysis of a LLDGSSTEIR glycopeptide released by tryptic digestion, which carried two variant structures, revealed that the glycopeptide contained, in addition to carbohydrate moieties, a novel structural entity. A variety of electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive and negative ion collisionally activated decomposition MS/MS, which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a taurine-containing peptidylamido-glycan structure. Finally, NMR analysis of flagellin glycopeptides provided complementary information. The glycan portion of the modification was assigned as -Fuc3N-(13)--Rha-(12)--Rha3OMe-(13)--GlcNAc-(1)Ser, and the novel capping moiety was shown to be comprised of taurine, alanine, and glycine. This is the first report of a novel O-linked sulfonated peptidylamido-glycan moiety decorating a flagellin protein.