Cloning, Expression And Functional Analysis Of Antithrombin Genes From Amphioxus Branchiostoma Belcheri Tsingtaunese

Coagulation is a complex process. In mammals the coagulation factors are highly conserved, and similar factors also exist in frog, turtle, salmon and ostrich. Recently, the jawless fish lampreys have been shown to possess a reduced set of the many clotting factors observed in higher vertebrates, while none of the principal clotting factors are found in the urochordate Ciona intestinalis. Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates, which is one of the most important animal model to research about the origin and evolution about vertebrates. Study on the project that if the coagulation factors exist in amphioxus is very important to develop the origin and evolution of the coagulation system.Antithrombin is a member of serpin superfamily. As a key protein working in the coagulation system, it is responsible for maintaining the balance of coagulation and anticoagulation in vivo.In this paper, we report the characterization, expression, and function of antithrombin from amphioxus (BbAT).Frist, we cloned the amphioxus antithrombin cDNA with an ORF coding for a protein of 338 amino acids. The ORF contains a conserved serpin domain between residues 1 to 336. A phylogenetic tree constructed shows that the BbAT belongs to the clade C (antithrombin) of serpin superfamily, to some extent also similar with the clade B (ancestor of the extracellular members). Further comparison of BbAT with antithrombins from the other vertebrates, revealed that BbAT shares about 40℅ identity with frog, ostrich and human antithrombins. The protein structure of BbAT predicated by SWISS-MODEL indicated the presence of 3β-sheets, 7α-helices and RCL, which are all similar to human antithrombin, and suggesting that they may perform a similar role.Antithrombin is mainly expressed in liver of vertebrate, and also at a low level in lung, spleen, kidney, heart etc. It is to be believed that the hepatic caecum in amphioxus is homologous to the vertebrate liver. In situ hybridization histochemistry demonstrated that BbAT transcript was most abundant in the hepatic caecum, and also presented in the hind-gut, gill and ovary, while it was absent in the muscle, testis, neural tube and notochord. This basically agrees with the notion that the hepatic caecum is the precursor of vertebrate liver.The recombinant protein of BbAT (rBbAT) were purified from P. pastoris X33 after inducing for the functional assay, both chromogenic assay and Western blot were used to examine the function of rBbAT, it was found that the activity of rBbAT was homologous with human antithrombin.In conclusion, BbAT has the feature of an ancestor of antithrombin in vertebrates, and the function is also similar to that of antithrombin in vertebrates.