Factor XIIIa mediated attachment of S. aureus fibronectin-binding protein A (FnbA) to fibrin: identification of Gln103 as a major cross-linking site.
In the present study we investigated the role of factor XIIIa reactive Gln and Lys sites of staphylococcal FnbA receptor in cross-linking reaction with alpha chains of fibrin. For this purpose we produced two recombinant FnbA mutants in which either a single Gln103 site (1Q FnbA) or all identified reactive Gln103, 105, 783, 830 and Lys157, 503, 620, 762 sites (4Q4K FnbA) were substituted with Ala residues. The results of FXIIIa-catalyzed incorporation of dansylcadaverine and dansylated peptide patterned on the NH2-terminal segment of fibronectin revealed that the reactivity of Gln substrate sites was drastically reduced in 1Q FnbA and 4Q4K FnbA mutants, while the reactivity of Lys substrate sites was only moderately decreased in 4Q4K FnbA. When it was tested in the FXIIIa-mediated fibrin cross-linking reaction, the 1Q FnbA mutant exhibited about 70-85% reduction in reactivity compared to that of the wild-type FnbA. These results demonstrate that FnbA participates in cross-linking to alpha chains of fibrin predominantly via its Gln103 reactive site. Several minor sites, including residues replaced in 4Q4K FnbA mutant, contributed to an additional 15-30% of the total fibrin cross-linking reactivity of FnbA. Comparison of amino acid sequences that follow the major reactive Gln site in FnbA and several known substrate proteins revealed that FXIIIa displays a preference for the glutamine residue in an xQAxBxPx sequence, where Q represents reactive glutamine, x is any amino acid residue, A is a polar residue, B is either valine or leucine, and P is proline.
Severina
Elena
E
Department of Protein Chemistry, Wyeth Research, 401 North Middletown Road, Building 205/228, Pearl River, New York 10965, USA.
Nunez
Lorna
L
Baker
Steven
S
Matsuka
Yury V
YV
D018829:Adhesins, Bacterial / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D026122:Factor XIIIa / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism*; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D013211:Staphylococcus aureus / Q000737:chemistry*; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D018829:Adhesins, Bacterial; D003432:Cross-Linking Reagents; C078635:fibronectin-binding proteins, bacterial; D005973:Glutamine; D014508:Urea; D005337:Fibrin; D011392:Proline; D026122:Factor XIIIa; D007930:Leucine; D008239:Lysine