Abstract
Serine protease inhibitors, or serpins, are a group of widely distributed proteins with similar structures that use conformational change to inhibit proteases. Antithrombin (AT) is a member of the serine protease inhibitor superfamily and a major coagulation inhibitor in all vertebrates, but its evolutionary origin remains elusive. In this study we isolated for the first time a cDNA encoding an antithrombin homolog, BjATl, from the protochordate Branchiostoma japonicum. The deduced protein BjATl consisted of 338 amino acids sharing 36.7% to 41.1% identity to known vertebrate ATs. BjATl contains a potential N-linked glycosylation site, two potential heparin binding sites and the reactive center loop with the absolutely conserved sequence Gly-Arg-Ser; all of these are features characteristic of ATs. All three phylogenetic trees constructed using Neighbor-Joining, Maximum-Likelihood and Bayesian-Inference methods also placed BjATl together with ATs. Moreover, BjATl expressed in yeast cells was able to inhibit bovine thrombin activity by forming a SDS-stable BjATl-thrombin complex. It also displays a concentration-dependent inhibition of thrombin that is accelerated by heparin. Furthermore, BjATl was predominantly expressed in the hepatic caecum and hind-gut, agreeing with the expression pattern of AT in mammalian species. All these data clearly demonstrate that BjATl is an ortholog of vertebrate ATs, suggesting that a primitive coagulation system emerged in the protochordate.
Highlights
Blood coagulation, or clotting, is of vital importance for the survival of both vertebrates and invertebrates-by preventing the leakage of blood from the sites of injury and impeding infection by the microbial invaders, the coagulation system of invertebrates is distinct from that of vertebrates [1,2]
The open reading frame (ORF) coded for a deduced protein of 338 amino acids
To explore the expression pattern of BjATl in adult B. japonicum, tissue section in situ hybridization was conducted and the results demonstrated that BjATl transcript was most abundant in the hepatic caecum and hind-gut, and at a lower level present in the gill and ovary, while it was absent in the epidermis, muscle, neural tube, notochord and testis (Fig. 9), implicating a tissue-specific expression pattern of BjATl in adult B. japonicum
Summary
Clotting, is of vital importance for the survival of both vertebrates and invertebrates-by preventing the leakage of blood from the sites of injury and impeding infection by the microbial invaders, the coagulation system of invertebrates is distinct from that of vertebrates [1,2]. The basal chordate, amphioxus, as the extant representative of subphylum Cephalochordata, has a heart homolog [7] and a circulation system with a fundamental organization found in all chordates [8,9], providing an ideal model for insights into the origin and evolution of vertebrate coagulation system. Bioinformatic approaches to inventory the presence or absence of genes involved in blood coagulation processes supports the view that these systems became progressively more complex during the period between the divergence of jawless fish and the appearance of mammals. The root of coagulation systems may extend back to protochordates. For this evolutionarily important organism, amphioxus, the coagulation system remains largely unclear
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