The Type B Flagellin of Hypervirulent Clostridium difficile Is Modified with Novel Sulfonated Peptidylamido-glycans

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The Journal of Biological Chemistry - 2016 - The Type B Flagellin of Hypervirulent Clostridium difficile Is Modified with Novel Sulfonated Peptidylamido-glycans.pdf(1.21 MB)
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2016
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AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Abstract
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.
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bacteria, glycosylation, Gram-positive bacteria, mass spectrometry (MS), nuclear magnetic resonance (NMR), Clostridium difficile, flagellin, modification, sulfonated, TANDEM MASS-SPECTROMETRY, TYROBUTYRICUM ATCC 25755, PROTEIN GLYCOSYLATION, POSTTRANSLATIONAL MODIFICATION, IDENTIFICATION, MOTILITY, VIRULENCE, GENOME
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https://doi.org/10.1074/jbc.M116.749481
 https://repositorio.uc.cl/handle/11534/77664
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