Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies
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2005-06-01
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Araujo, APU
Hansen, D.
Vieira, D. F.
Oliveira, C. de
Santana, L. A.
Beltramini, L. M.
Sampaio, CAM
Sampaio, M. U.
Oliva, MLV
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Abstract
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.
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Biological Chemistry. Berlin: Walter de Gruyter & Co, v. 386, n. 6, p. 561-568, 2005.