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

Christina Schäffer,Peter H Seeberger,Falko Schirmeister,Daniel Kolarich,Cornelia B Rath,Rudolf Figl

doi:10.1074/mcp.ra117.000394

Abstract

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.

Highlights

From the ‡Department of NanoBiotechnology, NanoGlycobiology unit, Universitat fur Bodenkultur Wien, 1190 Vienna, Austria; §Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany; ¶Department of Biology, Chemistry, Pharmacy, Institute of Chemistry and Biochemistry, Freie Universitat Berlin, 14195 Berlin, Germany; ʈDepartment of Chemistry, Division of Biochemistry, Universitat fur Bodenkultur Wien, 1190 Vienna, Austria; **Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, 4222, Australia
Native C9LY14 protein obtained from a crude S. sputigena cell extract traveled at 55– 60 kDa, a recombinant His6-tagged flagellin (C9LY14R) produced in E. coli was downshifted on the SDS-PAGE gel migrating as a periodic acid-Schiff (PAS)-negative band around the size calculated based on amino acid composition (46.1 kDa; Fig. 1A)
We provide the first clear evidence that S. sputigena ATCC 35185, a Gram-negative bacterium that has emerged as a potential periodontal pathogen, glycosylates its flagella

Summary

Introduction

Indications of Flagellin Glycosylation in S. sputigena ATCC 35185—The separation of a crude S. sputigena cell extract by SDS-PAGE and subsequent staining with PAS reagent revealed a single prominent carbohydrate-positive protein band with an apparent size of ϳ60 kDa (Fig. 1A). Native C9LY14 protein obtained from a crude S. sputigena cell extract traveled at 55– 60 kDa, a recombinant His6-tagged flagellin (C9LY14R) produced in E. coli was downshifted on the SDS-PAGE gel migrating as a PAS-negative band around the size calculated based on amino acid composition (46.1 kDa; Fig. 1A).

Results

Conclusion