Cloning and characterization of a novel Kunitz-type inhibitor from scorpion with unique cysteine framework
2013
Chen, Zongyun | Cao, Zhijian | Li, Wenxin | Wu, Yingliang
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.
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