Abstract
Human alpha(2)-antiplasmin (alpha(2)AP), the main inhibitor of plasmin-mediated fibrinolysis, is a substrate for plasma transglutaminase, also termed activated factor XIII (FXIIIa). Of 452 amino acids in alpha(2)AP, only Gln(2) is believed to be a fibrin-cross-linking (or FXIIIa-reactive) site. Kinetic efficiencies (k(cat)/K(m)((app))) of FXIIIa and the guinea pig liver tissue transglutaminase (tTG) and reactivities of Gln substrate sites were compared for recombinant wild-type alpha(2)AP (WT-alpha(2)AP) and Q2A mutant alpha(2)AP (Q2A-alpha(2)AP). [(14)C]Methylamine incorporation showed the k(cat)/K(m)((app)) of FXIIIa to be 3-fold greater than that of tTG for WT-alpha(2)AP. With FXIIIa or tTG catalysis, [(14)C]methylamine was incorporated into Q2A-alpha(2)AP, indicating that WT-alpha(2)AP has more than one Gln cross-linking site. To identify transglutaminase-reactive sites in WT-alpha(2)AP or Q2A-alpha(2)AP, each was labeled with 5-(biotinamido)pentylamine by FXIIIa or tTG catalysis. After each labeled alpha(2)AP was digested by trypsin, sequence and mass analyses of each labeled peptide showed that 4 of 35 Gln residues were labeled with the following reactivities: Gln(2) > Gln(21) > Gln(419) > Gln(447). Q(2)A-alpha(2)AP was also labeled at Gln(21) > Gln(419) > Gln(447), but became cross-linked to fibrin by FXIIIa or tTG at approximately one-tenth the rate for WT-alpha(2)AP. These results show that alpha(2)AP is a better substrate for FXIIIa than for this particular tTG, but that either enzyme involves the same Gln substrate sites in alpha(2)AP and yields the same order of reactivities.
Highlights
Human ␣2-antiplasmin (␣2AP)1 contains 452 amino acid residues and has three functional domains that contribute to protecting fibrin clots from plasmin-mediated lysis [1,2,3]
Kinetic efficiencies (kcat/Km(app)) of FXIIIa and the guinea pig liver tissue transglutaminase and reactivities of Gln substrate sites were compared for recombinant wild-type ␣2AP (WT-␣2AP) and Q2A mutant ␣2AP (Q2A-␣2AP). [14C]Methylamine incorporation showed the kcat/Km(app) of FXIIIa to be 3-fold greater than that of tTG for WT-␣2AP
We compared the Gln cross-linking sites in ␣2AP for the tTG isolated from guinea pig liver versus FXIIIa using both wild-type ␣2AP (WT-␣2AP) and mutant ␣2AP in which Gln2 was replaced with Ala (Q2A-␣2AP)
Summary
Human ␣2-antiplasmin (␣2AP) contains 452 amino acid residues and has three functional domains that contribute to protecting fibrin clots from plasmin-mediated lysis [1,2,3]. Transglutaminases are Ca2ϩ-dependent enzymes that catalyze post-translational modification of proteins through the formation of ␥-glutamyl–⑀-lysine cross-links between polypeptide chains [6]. ␣2AP has been found in fibrin-rich matrix and granulation tissue of human skin wounds [14], and tTG has been suggested to promote wound healing [15]. Given these observations, we compared the Gln cross-linking sites in ␣2AP for the tTG isolated from guinea pig liver versus FXIIIa using both wild-type ␣2AP (WT-␣2AP) and mutant ␣2AP in which Gln was replaced with Ala (Q2A-␣2AP). We report here the expression and purification of WT-␣2AP and Q2A-␣2AP, the identification and reactivity of their respective Gln substrate sites, and the rates of FXIIIa- and tTG-mediated cross-linking for each ␣2AP species
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