Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies.
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.
Araújo
Ana Paula Ulian
AP
Instituto de Física de São Carlos, Universidade Federal de São Paulo, Avenida Trabalhador Sãocarlense 400, 13560-970 São Carlos, SP, Brazil.
Hansen
Daiane
D
Vieira
Debora F
DF
Oliveira
Cleide
C
Santana
Lucimeire A
LA
Beltramini
Leila M
LM
Sampaio
Claudio A M
CA
Sampaio
Misako U
MU
Oliva
Maria Luiza V
ML
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
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