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 involved in hemostasis. We synthesized 25 peptides from various regions of factor XIIIa and studied their effects on cross-linking fibrin, N,N'-dimethylcasein, or fibronectin. We found that two peptides, Asn72-Asp97 (peptide-4) and Asp190-Phe230 (peptide-7), inhibited factor XIIIa cross-linking of these substrates. The other peptides did not inhibit factor XIIIa activity. The inhibition of cross-linking was reversed by excess substrate, indicating that the peptides were interacting with fibrin 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 concentration did not reverse inhibition. Peptides-4 and -7 also had no effect on exposure of the active site of factor XIIIa and no synergistic inhibitory effects were detected. Peptides-4 and -7 had no effect on factor XIIIa binding to fibrin suggesting that the binding sites and the substrate 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-terminal (Lys221-Phe230) portions of peptide-7 were 20-60-fold less inhibitory compared to intact peptide-7. Peptides-4 and -7 also inhibited guinea pig liver tissue transglutaminase from cross-linking fibrinogen, N,N'-dimethylcasein, and fibronectin. In conclusion, we have identified two regions outside the active site pocket which are important for substrate recognition in factor XIIIa and tissue transglutaminase.
Achyuthan
K E
KE
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Slaughter
T F
TF
Santiago
M A
MA
Enghild
J J
JJ
Greenberg
C S
CS
D000588:Amines / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D002364:Caseins / Q000737:chemistry; D003432:Cross-Linking Reagents; D005337:Fibrin / Q000737:chemistry / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D008099:Liver / Q000201:enzymology; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
D000588:Amines; D002364:Caseins; D003432:Cross-Linking Reagents; D015335:Molecular Probes; C002087:N,N-dimethylcasein; D010446:Peptide Fragments; C076022:5-(biotinamido)pentylamine; D001710:Biotin; D005337:Fibrin; D011503:Transglutaminases