Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies

Bauhinia bauhinoides cruzipain inhibitor (BbCl) and Bauhinia bauhinioides kallikrein inhibitor (BbKl) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropepticle comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCl inhibits human neutrophil elastase (K-i(app) 5.3 nM), porcine pancreatic elastase (K-i(app) 40 nM), cathepsin G (K-i(app) 160 nM) and the cysteine proteinases cruzipain (K-i(app) 1.2 nM), cruzain (K-i(app) 0.3 nM) and cathepsin L (K-i(app) 2.2 nM), while BbKl strongly inhibits plasma kallikrein (K-i(app) 2.4 nM) and plasmin (K-i(app) 33 nM). Circular dichroism spectra of BbCl and BbKl were in agreement with the P-trefoil fold described for Kunitz inhibitors. the inhibitory potency of both BbCl- and BbKl-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulficle bridges.Universidade Federal de São Paulo, Escola Paulista Med, BR-04044020 São Paulo, BrazilUSP, Inst Fis Sao Carlos, BR-13560970 Sao Carlos, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, BR-04044020 São Paulo, BrazilWeb of Scienc