title: O-Glycosylation of the N-terminal region of the serine-rich adhesin Srr1 of Streptococcus agalactiae explored by mass spectrometry.
abstract: Serine-rich (Srr) proteins exposed at the surface of Gram-positive bacteria are a family of adhesins that contribute to the virulence of pathogenic staphylococci and streptococci. Lectin-binding experiments have previously shown that Srr proteins are heavily glycosylated. We report here the first mass-spectrometry analysis of the glycosylation of Streptococcus agalactiae Srr1. After Srr1 enrichment and trypsin digestion, potential glycopeptides were identified in collision induced dissociation spectra using X! Tandem. The approach was then refined using higher energy collisional dissociation fragmentation which led to the simultaneous loss of sugar residues, production of diagnostic oxonium ions and backbone fragmentation for glycopeptides. This feature was exploited in a new open source software tool (SpectrumFinder) developed for this work. By combining these approaches, 27 glycopeptides corresponding to six different segments of the N-terminal region of Srr1 [93-639] were identified. Our data unambiguously indicate that the same protein residue can be modified with different glycan combinations including N-acetylhexosamine, hexose, and a novel modification that was identified as O-acetylated-N-acetylhexosamine. Lectin binding and monosaccharide composition analysis strongly suggested that HexNAc and Hex correspond to N-acetylglucosamine and glucose, respectively. The same protein segment can be modified with a variety of glycans generating a wide structural diversity of Srr1. Electron transfer dissociation was used to assign glycosylation sites leading to the unambiguous identification of six serines and one threonine residues. Analysis of purified Srr1 produced in mutant strains lacking accessory glycosyltransferase encoding genes demonstrates that O-GlcNAcylation is an initial step in Srr1 glycosylation that is likely required for subsequent decoration with Hex. In summary, our data obtained by a combination of fragmentation mass spectrometry techniques associated to a new software tool, demonstrate glycosylation heterogeneity of Srr1, characterize a new protein modification, and identify six glycosylation sites located in the N-terminal region of the protein.
authors.affiliation: From the ‡INRA, MICALIS UMR-1319, 78352 Jouy-en-Josas cedex, France; §AgroParisTech, MICALIS UMR-1319, 78352 Jouy-en-Josas cedex, France; ¶¶Institut Pasteur, Unité de Spectrométrie de Masse Structurale et Protéomique, 28 rue du Dr Roux, 75015 Paris, France;
authors.forename: Thibault
authors.initials: T
authors.lastname: Chaze
authors.affiliation: ¶INRA, PAPPSO, MICALIS UMR-1319, 78352 Jouy en Josas cedex, France;
authors.forename: Alain
authors.initials: A
authors.lastname: Guillot
authors.affiliation: ‖INRA, PAPPSO, Génétique végétale UMR-320, Ferme du Moulon, 91190 Gif sur Yvette, France;
authors.forename: Benoît
authors.initials: B
authors.lastname: Valot
authors.affiliation: ‖INRA, PAPPSO, Génétique végétale UMR-320, Ferme du Moulon, 91190 Gif sur Yvette, France;
authors.forename: Olivier
authors.initials: O
authors.lastname: Langella
authors.affiliation: ¶¶Institut Pasteur, Unité de Spectrométrie de Masse Structurale et Protéomique, 28 rue du Dr Roux, 75015 Paris, France; ‖‖CNRS UMR 3528, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France.
authors.forename: Julia
authors.initials: J
authors.lastname: Chamot-Rooke
authors.affiliation: **CEA, Institut de Biologie Structurale Jean-Pierre Ebel, F-38027 Grenoble, France;
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Di Guilmi
authors.affiliation: ‡‡Institut Pasteur, Unité de Biologie des Bactéries Pathogènes à Gram+, 28, rue du Dr Roux, 75015 Paris, France; §§Centre National de la Recherche Scientifique, CNRS ERL3526, Paris, France;
authors.forename: Patrick
authors.initials: P
authors.lastname: Trieu-Cuot
authors.affiliation: ‡‡Institut Pasteur, Unité de Biologie des Bactéries Pathogènes à Gram+, 28, rue du Dr Roux, 75015 Paris, France; §§Centre National de la Recherche Scientifique, CNRS ERL3526, Paris, France;
authors.forename: Shaynoor
authors.initials: S
authors.lastname: Dramsi
authors.affiliation: From the ‡INRA, MICALIS UMR-1319, 78352 Jouy-en-Josas cedex, France; §AgroParisTech, MICALIS UMR-1319, 78352 Jouy-en-Josas cedex, France; mistou@jouy.inra.fr.
authors.forename: Michel-Yves
authors.initials: MY
authors.lastname: Mistou
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D009005:Monosaccharides / Q000032:analysis; D012694:Serine; D012984:Software; D013292:Streptococcus agalactiae / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D018829:Adhesins, Bacterial; D006020:Glycopeptides; D009005:Monosaccharides; C524542:Srr-1 protein, Streptococcus agalactiae; D012694:Serine