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Characterization of thrombin cDNAs ranging from mammals to cyclostomes : structural analysis and evolution of prothrombin in vertebrates

University of British Columbia

The cDNA sequence of the B-chain of thrombin has been determined from nine vertebrate species (rat, mouse, rabbit, chicken, gekko, newt, rainbow trout, sturgeon, and hagfish). The amino acid sequence identities vary from 96.5% (rat vs. mouse) to 62.6% (newt vs hagfish). Of the 240 amino acids spanned in all the species compared, there is identity at 110 (45.8%) positions. When conservative changes are included, the amino acid similarity increases to 75%. The most conserved portions of the B-chain are the active-site residues and adjacent amino acids, the B-loop, and the primary substrate binding region. In addition, the Arg-Gly-Asp motif is conserved in 9 of the eleven species compared and the chemotactic/growth factor domain is well conserved in all of the eleven species compared. The least conserved regions of the B-chain correspond to surface loops including the thrombomodulin binding-site and one of the hirudin binding regions. The extent of the amino acid sequence similarity and the conservation of many of the functional / structural motifs suggests that in addition to their role in coagulation, vertebrate thrombins may also play an important role in the general mechanisms of wound repair. In addition, the complete cDNA sequences of chicken and hagfish prothrombin has been determined. The sequences predict that prothrombin from both species is synthesized as a prepro-protein consisting of a putative Gla domain, two kringles and a two chain protease domain. Like the mammalian prothrombin cDNAs, both chicken and hagfish prothrombin have either AGT or AGC encoding their active-site serine. Chicken and hagfish prothrombin share 51.6% amino acid sequence identity (313/627 residues) and 61.7% similarity. Hagfish prothrombin contains a 19 amino acid residue insertion between the Gla domain and the first kringle. This insertion in hagfish prothrombin may form a 16 amino acid residue disulphide loop. Both chicken and hagfish prothrombin are structurally very similar to human, bovine, rat, and mouse prothrombin and all six species share 41% amino acid sequence identity and 67.5% amino acid sequence similarity. Amino acid sequence alignments of human, bovine, rat, mouse, chicken, and hagfish prothrombin suggest that the thrombin B-chain and the Gla domain are the regions most essential for the common function of vertebrate prothrombins. The structural composition of hagfish prothrombin implies that vertebrate prothrombins acquired the propeptide, Gla domain, and both kringles prior to the divergence of cyclostomes from the main vertebrate lineage (400 MYA). Attempts to identify an invertebrate prothrombin homologue were unsuccessful, supporting the notion that the hemostatic processes in invertebrates and vertebrates may be quite dissimilar.

Text

2011-01-25

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http://hdl.handle.net/2429/30819

Biochemistry and Molecular Biology

University of British Columbia

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