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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board Member: Buchner, Johannes / Ludwig, Stephan / Sies, Helmut / Turk, Boris / Wittinghofer, Alfred

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

Ana Paula Ulian Araújo1 / Daiane Hansen2 / Debora F. Vieira3 / Cleide de Oliveira4 / Lucimeire A. Santana5 / Leila M. Beltramini6 / Claudio A.M. Sampaio7 / Misako U. Sampaio8 / Maria Luiza V. Oliva9

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Corresponding author

Citation Information: Biological Chemistry. Volume 386, Issue 6, Pages 561–568, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: 10.1515/BC.2005.066, July 2005

Publication History

Received:
February 3, 2005
Accepted:
April 8, 2005
Published Online:
2005-07-07

Abstract

Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) 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 prepropeptide 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. BbCI 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 BbKI strongly inhibits plasma kallikrein (K i(app) 2.4 nM) and plasmin (K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the β-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.

Keywords: cathepsins; cruzipain; elastase; gene; kallikreins; proteinase inhibitors

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