A Novel Trypsin Inhibitor from Bungarus fasciatus Venom

ABSTRACTAIMTo purify and characterize a novel trypsin inhibitor named bungaruskunin 1 from the snake venom of Bungarus fasciatus, and to clone its cDNA from the cDNA library of B. fasciatus venomous glands.METHODSSephadex G-50, cation-exchange CM-Sephadex C-25, HPLC, and RP-HPLC (C4 column) methods were used to isolate bungaruskunin 1. The inhibition effects of the sample on the hydrolysis of synthetic chromogenic substrates by serine proteases were assayed in 50 mmol·L−1 Tris-HCl, pH 7.8 at room temperature. The total RNA of B.fasciatus venomous glands were extracted using TRIzol method and the cDNA library were constructed by a SMAR™ PCR cDNA synthesis kit (Clontech). Two primers (positive primer, based on the conserved cDNA fragment encoding signal peptides of beta-bungarotoxin B chains and Kunitz inhibitors from Bungarus snakes, and reverse primer based on the adopter sequence in the cDNA library) were used to screen Kunitz inhibitor and beta-bungarotoxin B chain encoding sequences. The cDNA products with about 700 bp in length were reclaimed from 1% agarose gel, and purified before ligation into the pMD18-T vector.RESULTSThe predicted precursor was composed of 83 amino acid (aa) residues including a 24 aa signal peptide and a 59 aa mature bungaruskunin 1. Bungaruskunin 1 showed maximal similarity (64%) with the predicted serine protease inibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin 1 is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin were also identified.CONCLUSIONThe overall structures of bungaruskunin 1 and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.