Cloning and characterization of a novel Kunitz-type inhibitor from scorpion with unique cysteine framework.

Kunitz-type proteins from animal venom are good tools for understanding structure-function relationships between serine proteases and their inhibitors. We used a cDNA library to clone and characterize the Buthus martensi Kunitz-type protease inhibitor (BmKPI) present in the venom gland of the scorpion B. martensi. The gene codes for a signal peptide of 19 residues and a mature peptide of 64 residues. The mature BmKPI peptide possesses a unique cysteine framework reticulated by four disulfide bridges, unlike many other Kunitz-type proteins with three disulfide bridges. The recombinant BmKPI peptide was functionally expressed and showed strong inhibitory activity toward trypsin (Ki 1.8 × 10⁻⁶ M), chymotrypsin (Ki 3.2 × 10⁻⁸ M), and elastase (Ki 1.6 × 10⁻⁷ M). Structure-functional relationship between elastase and BmKPI was further studied. Cysteine mutagenesis experiment showed that the unique disulfide bridge Cys53-Cys61 had little effect on its inhibiting elastase. Molecular dynamics simulation revealed that BmKPI possesses elastase inhibitory active sites similar to the classical Kunitz-type venom peptides, although their cysteine frameworks were different. These results showed that BmKPI is a new multifunctional serine protease inhibitor. To the best of our knowledge, BmKPI is the first functionally characterized Kunitz-type elastase inhibitor derived from scorpion venoms.