Factor XIIIa Mediated Attachment of S. aureus Fibronectin-Binding Protein A (FnbA) to Fibrin:  Identification of Gln103 as a Major Cross-Linking Site

Abstract

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.

Ranking reactive glutamines in the fibrinogen αC region that are targeted by blood coagulant factor XIII

“…22,23,41 Curiously, there is no obvious consensus sequence for the Q-containing substrates of FXIIIa. [50][51][52] The reactive glutamines are often found in flexible regions of the substrates. It is important to point out that not all freely available glutamines are good FXIII substrates.…”

Examining the substrate specificity of factor XIIIa towards peptide substrate modelsaC fibrin domains.

“…The unlabeled control showed no peaks, while the FXIIIa-reacted sample showed the two sharp 15 N-amide proton peaks as anticipated. Similarly, α 2 AP(Q4P) (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) showed the sharp twin peaks in the spectrum of the FXIII-reacted sample, but the unlabeled control spectrum was not clear like that of K9 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). In the nonenzymatically reacted sample, there appeared a weak, broad peak at approximately 7 ppm, which was an unexpected result.…”

“…The crosslinking of fibrin monomers is achieved by FXIIIa's transglutaminase activity, which catalyzes an acyl transfer reaction between the side chain of a reactive glutamine (Q) and the side chain of normally a lysine (K) residue. 7 The γ-carboxyamide of the glutamine residue serves as the acyl donor while the lysine residue functions as the acyl acceptor. A Q-containing substrate is initially bound in the enzyme's catalytic thiol-containing site and is then followed by the nucleophilic attack of a K-containing substrate's sidechain amine.…”

“…This creates the isopeptide bond between the two residues and releases FXIII. 7 A diagram of the transglutamination between glutamine and lysine may be seen in Figure 5. A number of physiological substrates for FXIIIa have been identified; however, a clear consensus sequence for the glutamine-containing substrate is not yet known.…”

“…The Fnb A therefore allows the S. aureus to anchor on to its host cell. 7,8 Another effective FXIIIa substrate is α 2 -antiplasmin (α 2 AP), a protein that prevents the activation of plasmin from plasminogen. Plasmin is a protease that lyses existing clots.…”