Identification of factor XIIIA-reactive glutamine acceptor and lysine donor sites within fibronectin-binding protein (FnbA) from Staphylococcus aureus.
Staphylococcal fibronectin-binding protein (FnbA) is a surface-associated receptor responsible for the reversible binding of bacteria to human fibronectin and fibrin(ogen). Recently we have shown that FnbA serves as a substrate for coagulation factor XIIIa and undergoes covalent cross-linking to its ligands, resulting in the formation of heteropolymers (Matsuka, Y. V., Anderson, E. T., Milner-Fish, T., Ooi, P., and Baker, S. (2003) Staphylococcus aureus fibronectin-binding protein serves as a substrate for coagulation factor XIIIa: Evidence for factor XIIIa-catalyzed covalent cross-linking to fibronectin and fibrin, Biochemistry 42, 14643-14652). Factor XIIIa also catalyzes the incorporation in FnbA of fluorescent probes dansylcadaverine and glutamine-containing synthetic peptide patterned on the NH(2)-terminal segment of fibronectin. In this study, the above probes were utilized for site-specific labeling and identification of reactive Gln and Lys residues targeted by factor XIIIa in rFnbA. Probe-decorated rFnbA samples were subjected to trypsin or Glu-C digestion, followed by separation of labeled peptides using reversed phase HPLC. Sequencing and mass spectral analyses of isolated probe-modified peptides have been employed for the identification of factor XIIIa-reactive Gln and Lys residues. Analysis of dansylcadaverine-labeled peptides resulted in the identification of one major, Gln103, and three minor, Gln105, Gln783, and Gln830, amine acceptor sites. The labeling procedure with dansyl-PGGQQIV probe revealed that Lys157, Lys503, Lys620, and Lys762 serve as amine donor sites. The identified reactive glutamine acceptor and lysine donor sites of FnbA may participate in transglutaminase-mediated cross-linking reactions resulting in the covalent attachment of pathogenic Staphylococcus aureus to human host proteins.
Anderson
Elizabeth T
ET
Wyeth Research, Pearl River, New York 10965, USA.
Fletcher
Leah
L
Severin
Anatoly
A
Murphy
Ellen
E
Baker
Steven M
SM
Matsuka
Yury V
YV
D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026122:Factor XIIIa / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013211:Staphylococcus aureus / Q000378:metabolism*
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D009842:Oligopeptides; C078635:fibronectin-binding proteins, bacterial; D005973:Glutamine; D026122:Factor XIIIa; C008542:monodansylcadaverine; D008239:Lysine; D002103:Cadaverine