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Flagellin Glycoproteomics of the Periodontitis Associated Pathogen Selenomonas sputigena Reveals Previously Not Described O-glycans and Rhamnose Fragment Rearrangement Occurring on the Glycopeptides*

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Flagellated, Gram-negative, anaerobic, crescent-shaped Selenomonas species are colonizers of the digestive system, where they act at the interface between health and disease. Selenomonas sputigena is also considered a potential human periodontal pathogen, but information on its virulence factors and underlying pathogenicity mechanisms is scarce. Here we provide the first report of a Selenomonas glycoprotein, showing that S. sputigena produces a diversely and heavily O-glycosylated flagellin C9LY14 as a major cellular protein, which carries various hitherto undescribed rhamnose- and N-acetylglucosamine linked O-glycans in the range from mono- to hexasaccharides. A comprehensive glycomic and glycoproteomic assessment revealed extensive glycan macro- and microheterogeneity identified from 22 unique glycopeptide species. From the multiple sites of glycosylation, five were unambiguously identified on the 437-amino acid C9LY14 protein (Thr149, Ser182, Thr199, Thr259, and Ser334), the only flagellin protein identified. The O-glycans additionally showed modifications by methylation and putative acetylation. Some O-glycans carried hitherto undescribed residues/modifications as determined by their respective m/z values, reflecting the high diversity of native S. sputigena flagellin. We also found that monosaccharide rearrangement occurred during collision-induced dissociation (CID) of protonated glycopeptide ions. This effect resulted in pseudo Y1-glycopeptide fragment ions that indicated the presence of additional glycosylation sites on a single glycopeptide. CID oxonium ions and electron transfer dissociation, however, confirmed that just a single site was glycosylated, showing that glycan-to-peptide rearrangement can occur on glycopeptides and that this effect is influenced by the molecular nature of the glycan moiety. This effect was most pronounced with disaccharides. This study is the first report on O-linked flagellin glycosylation in a Selenomonas species, revealing that C9LY14 is one of the most heavily glycosylated flagellins described to date. This study contributes to our understanding of the largely under-investigated surface properties of oral bacteria. The data have been deposited to the ProteomeXchange with identifier PXD005859.

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Author contributions: C.B.R. and F.S. designed and performed the substantive majority of the experiments and wrote the manuscript. R.F. performed the monosaccharide analyses. P.H.S. edited the manuscript. D.K. and C.S. supervised the project and provided overall guidance on study design, execution and manuscript preparation, and editing. All authors discussed the results and commented on the paper.

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This work was supported by the Austrian Science Fund FWF, projects P26836-B22 (to C.S.) and the PhD Programme “Biomolecular Technology of Proteins” W1224. F.S, P.H.S. and D.K. gratefully acknowledge generous funding by the Max Planck Society. D.K. acknowledges support by the European Union (Seventh Framework Programme “Glycoproteomics”, grant number PCIG09-GA-2011-293847 and IBD-BIOM project, grant number 305479). DK is the recipient of an Australian Research Council Future Fellowship (project number FT160100344) funded by the Australian Government.

This article contains supplemental material.

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These authors contributed equally to this work.