title: Cloning and characterization of a novel Kunitz-type inhibitor from scorpion with unique cysteine framework.
abstract: 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.
authors.affiliation: Institute of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China. zychen@whu.edu.cn
authors.forename: Zongyun
authors.initials: Z
authors.lastname: Chen
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Cao
authors.forename: Wenxin
authors.initials: W
authors.lastname: Li
authors.forename: Yingliang
authors.initials: Y
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D015723:Gene Library; D008958:Models, Molecular; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012604:Scorpion Venoms / Q000737:chemistry; D012605:Scorpions / Q000737:chemistry*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics
chemical_list: D012604:Scorpion Venoms; D015842:Serine Proteinase Inhibitors; D010196:Pancreatic Elastase; D003545:Cysteine
keywords: BmKPI; Elastase inhibitor; Kunitz-type; Scorpion; Unique cysteine framework