title: Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies.
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 beta-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.
authors: Instituto de Física de São Carlos, Universidade Federal de São Paulo, Avenida Trabalhador Sãocarlense 400, 13560-970 São Carlos, SP, Brazil.
authors: Ana Paula Ulian
authors: AP
authors: Araújo
authors: Daiane
authors: D
authors: Hansen
authors: Debora F
authors: DF
authors: Vieira
authors: Cleide
authors: C
authors: Oliveira
authors: Lucimeire A
authors: LA
authors: Santana
authors: Leila M
authors: LM
authors: Beltramini
authors: Claudio A M
authors: CA
authors: Sampaio
authors: Misako U
authors: MU
authors: Sampaio
authors: Maria Luiza V
authors: ML
authors: Oliva
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D036481:Bauhinia; D001665:Binding Sites; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D018076:DNA, Complementary / Q000302:isolation & purification; D018744:DNA, Plant / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015800:Protozoan Proteins; D018749:RNA, Plant / Q000032:analysis; D011994:Recombinant Proteins / Q000494:pharmacology; D012639:Seeds / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
chemical_list: C505411:BbCI protein, Bauhinia bauhinoides; C434623:BbKI protein, Bauhinia bauhinioides; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D018744:DNA, Plant; C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D015800:Protozoan Proteins; D018749:RNA, Plant; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C063214:cruzipain