Primary structure of blood coagulation factor XIIIa (fibrinoligase, transglutaminase) from human placenta.

Abstract

We have determined the primary structure of human placental factor XIJ1a, an enzyme [fibrinoligase, transglutaminase, fibrin-stabilizing factor, EC 2.3.2.13 (protein-glutamine:amine r-glutamyltransferase)] that forms intermolecular isopeptide bonds between fibrin molecules as the last step in blood coagulation. Placental factor XIa is an unglycosylated polypeptide chain of 730 amino acid residues (Mr = 83,005) that appears to be identical to the a subunit of the plasma zymogen factor XI. Ca2+-dependent activation of factor XHIa by thrombin removes a blocked amino-terminal peptide and unmasks a reactive thiol group at Cys-314. A second specific cleavage after Lys-513 by thrombin inactivates factor XIIa and produces an amino-terminal 56-kDa fragment and a 24-kDa fragment. The amino acid sequence of factor XIIa is unique and does not exhibit internal homology, but its active center is similar to that of the thiol proteases. The probable Ca2+-binding site of factor XIa has been identified by homology to the high-affmity sites in calmodulins. Knowledge of the primary structure of factor XMa will aid elucidation of the mechanism of its enzymatic action and that of the many tissue transglutaminases ofwhich it is the prototype. This will also facilitate production of factor XIa by recombinant DNA technology for use in treatment of congenital factor XIII deficiencies and in the postoperative healing of wounds.Factor XIIIa [FXIIIa; fibrinoligase, fibrin-stabilizing factor, EC 2.3.2.13 (protein-glutamine:amine y-glutamyltransferase)] is the last enzyme generated in the blood coagulation cascade (1, 2). It is a transglutaminase or transamidating enzyme that forms intermolecular y-glutamyl-e-lysine crosslinks between fibrin molecules, thereby stabilizing the fibrin clot mechanically and also conferring resistance to proteolysis. FXIIIa has a cysteine thiol active site; however, unlike thiol proteases such as cathepsins B and H that cleave peptide bonds, FXIIIa forms intermolecular isopeptide bridges in fibrin. Other substrates for FXIIIa include coagulation factor V, a2-macroglobulin, platelet myosin, actin, and fibronectin. Thus, in addition to its essential role in blood clotting, FXIIIa may function to stabilize cell surface molecules and membranes. FXIIIa is the prototype of a class of Ca2+-dependent transglutaminases with thiol active centers that are widespread in animal tissues and have been associated with cell proliferation, embryonic development, and growth (3-5). However, none except FXIIIa has been puri-, fled and characterized, and until now little has been published about the structure of FXIIIa except the sequence of the amino-terminal activation peptide of the human (6) and bovine (7) plasma FXIIIa. We report here the primary structure of human placental FXIIIa, including the sequence at the active site, and the sites of activation and inactivation by thrombin and the probable Ca2`-binding site.

Molecular cloning of human protein 4.2: a major component of the erythrocyte membrane.

“…5, large box), in which 20 of the 33 residues, or 60.6%, are identical for all three proteins. This highly conserved region contains the catalytic thiol sites of X111a and LTG where Cys-Trp is required for transglutaminase activity (24,26). P4.2, however, despite the high homology around the active site, has an alanine (residue 298 of P4.2; Fig.…”

“…The major band was excised and subcloned into pGEM 3zf plasmids (Promega). From >500 transformants, 24 Fig. 4, c).…”

Coagulation Factor XIIIa Undergoes a Conformational Change Evoked by Glutamine Substrate

“…The fibrinogen epitope was identified from CNBr and tryptic fragments to be located in the region of A␣ 529 -539, the only fragment from the combined digests that substantially inhibited (18%) binding of 5A2 to fibrinogen A␣-chains (20). Searches for similarities between the sequence of this peptide and the amino acid sequences (16,31) in XIIIA were made using both the PROPHET (Bolt Beranek and Newman, Inc., Cambridge MA) and the BLAST network service (NCBI). Both searches found a segment near the C terminus of Fig.…”

Identification of the Calcium Binding Site and a Novel Ytterbium Site in Blood Coagulation Factor XIII by X-ray Crystallography

“…Based on the amino acid sequence, the calcium binding site was predicted to be in a region (residues 468 -479) with high similarity to the EF-hand motif (17,18). The main calcium binding site, as seen in the preliminary crystallographic study (19), involves residues Asn-436, Asp-438, Ala-457, Glu-485, and Glu-490.…”