FACTOR-XIIIA-DERIVED PEPTIDES INHIBIT TRANSGLUTAMINASE ACTIVITY - LOCALIZATION OF SUBSTRATE RECOGNITION SITES

Factor XIIIa is a transglutaminase that catalyzes intermolecular gamma -glutamyl-epsilon-lysyl bonds between fibrin and other proteins involv ed in hemostasis. We synthesized 25 peptides from various regions of f actor XIIIa and studied their effects on cross-linking fibrin, N,N'-di methylcasein, or fibronectin. We found that two peptides, Asn72-Asp97 (peptide-4) and Asp190-Phe230 (peptide- 7), inhibited factor XIIIa cro ss-linking of these substrates. The other peptides did not inhibit fac tor XIIIa activity. The inhibition of cross-linking was reversed by ex cess substrate, indicating that the peptides were interacting with fib rin and not factor XIIIa. The peptides were not pseudosubstrates since they were not cross-linked to fibrin. The peptides did not modify the primary amine binding site as increasing the primary amine concentrat ion did not reverse inhibition. Peptides-4 and -7 also had no effect o n exposure of the active site of factor XIIIa and no synergistic inhib itory effects were detected. Peptides-4 and -7 had no effect on factor XIIIa binding to fibrin suggesting that the binding sites and the sub strate recognition sites were distinct. Synthetic peptides containing shorter amino acid sequences of peptide-4 were inactive. In contrast, the amino-terminal (Asp190-Lys199, Tyr194-Tyr204) and the carboxyl-ter minal (Lys211-Phe230) portions of peptide-7 were 20-60-fold less inhib itory compared to intact peptide-7. Peptides-4 and -7 also inhibited g uinea pig liver tissue transglutaminase from cross-linking fibrinogen, N,N'-dimethylcasein, and fibronectin. In conclusion, we have identifi ed two regions outside the active site pocket which are important for substrate recognition in factor XIIIa and tissue transglutaminase.