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Reactivity of the N-terminal region of fibronectin protein to transglutaminase 2 and factor XIIIA.

ABSTRACT: Transglutaminase 2 (TG2) is secreted by a non-classical pathway into the extracellular space, where it has several activities pertinent to fibronectin (FN), including binding to the gelatin-binding domain of FN and acting as an integrin co-receptor. Glutamines in the N-terminal tail of FN are known to be susceptible to transamidation by both TG2 and activated blood coagulation factor XIII (FXIIIa). We used immunoblotting, limited proteolysis, and mass spectrometry to localize glutamines within FN that are subject to TG2-catalyzed incorporation of dansylcadaverine in comparison to residues modified by FXIIIa. Such analysis of plasma FN indicated that Gln-3, Gln-7, and Gln-9 in the N-terminal tail and Gln-246 of the linker between fifth and sixth type I modules ((5)F1 and (6)F1) are transamidated by both enzymes. Only minor incorporation of dansylcadaverine was detected elsewhere. Labeling of C-terminally truncated FN constructs revealed efficient TG2- or FXIIIa-catalyzed dansylcadaverine incorporation into the N-terminal residues of constructs as small as the 29-kDa fragment that includes (1-5)F1 and lacks modules from the adjacent gelatin-binding domain. However, when only (1-3)F1 were present, dansylcadaverine incorporation into the N-terminal residues of FN was lost and instead was in the enzymes, near the active site of TG2 and terminal domains of FXIIIa. Thus, these results demonstrate that FXIIIa and TG2 act similarly on glutamines at either end of (1-5)F1 and transamidation specificity of both enzymes is achieved through interactions with the intact 29K fragment.

SUBMITTER: Hoffmann BR

PROVIDER: S-EPMC3173148 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Reactivity of the N-terminal region of fibronectin protein to transglutaminase 2 and factor XIIIA.

Hoffmann Brian R BR Annis Douglas S DS Mosher Deane F DF

The Journal of biological chemistry 20110711 37

Transglutaminase 2 (TG2) is secreted by a non-classical pathway into the extracellular space, where it has several activities pertinent to fibronectin (FN), including binding to the gelatin-binding domain of FN and acting as an integrin co-receptor. Glutamines in the N-terminal tail of FN are known to be susceptible to transamidation by both TG2 and activated blood coagulation factor XIII (FXIIIa). We used immunoblotting, limited proteolysis, and mass spectrometry to localize glutamines within F ...[more]

PMID: 21757696

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Project description:BackgroundEthacrynic acid (EA) is a loop diuretic that is approved orally and parenterally to manage edema-associated diseases. Nevertheless, it was earlier reported that it is also associated with bleeding upon its parenteral administration. In this report, we investigated the effects of EA on human factor XIIIa (FXIIIa) of the coagulation process using a variety of techniques.MethodsA series of biochemical and computational methods have been used in this study. The potency and efficacy of human FXIIIa inhibition by EA was evaluated using a bisubstrate-based fluorescence trans-glutamination assay under near physiological conditions. To establish the physiological relevance of FXIIIa inhibition by EA, the effect on FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) - α2-antiplasmin complex was evaluated using SDS-PAGE experiments. The selectivity profile of EA against other coagulation proteins was assessed by evaluating EA's effect on human clotting times in the activated partial thromboplastin time (APTT) and the prothrombin time (PT) assays. We also used molecular modeling studies to put forward a putative binding mode for EA in the active site of FXIIIa. Results involving EA were the average of at least three experiments and the standard error ± 1 was provided. In determining the inhibition parameters, we used non-linear regression analysis.ResultsFXIIIa is a transglutaminase that works at the end of the coagulation process to form an insoluble, rigid, and cross-linked fibrin rich blood clot. In fact, inhibition of FXIIIa-mediated biological processes has been reported to result in a bleeding diathesis. Inhibition of FXIIIa by EA was investigated given the nucleophilic nature of the thiol-containing active site of the enzyme and the Michael acceptor-based electrophilicity of EA. In a bisubstrate-based fluorescence trans-glutamination assay, EA inhibited FXIIIa with a moderate potency (IC50 ~ 105 µM) and efficacy (∆Y ~ 66%). In SDS-PAGE experiments, EA appears to significantly inhibit the FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) - α2-antiplasmin complex which indicates that EA affects the physiological functions of FXIIIa. Interestingly, EA did not affect the clotting times of human plasma in the APTT and the PT assays at the highest concentration tested of 2.5 mM suggesting the lack of effects on the coagulation serine proteases and potentially the functional selectivity of EA with respect to the clotting process. Molecular modeling studies demonstrated that the Michael acceptor of EA forms a covalent bond with catalytic residue of Cys314 in the active site of FXIIIa.ConclusionsOverall, our studies indicate that EA inhibits the physiological function of human FXIIIa in vitro which may potentially contribute to the bleeding complications that were reported with the association of the parenteral administration of EA.

Dataset Information

Reactivity of the N-terminal region of fibronectin protein to transglutaminase 2 and factor XIIIA.

Publications

Reactivity of the N-terminal region of fibronectin protein to transglutaminase 2 and factor XIIIA.

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