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.

Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa

“…Individual values of each isoform were very close to this average. This confirms classification of these isoforms as bII class proteins (Sreerama and Woody, 2003), as these results are in agreement with the structural characteristics of other plant Kunitz-type inhibitors described in the literature (Araújo et al, 2005;Haq and Khan, 2003). In addition, static fluorescence spectra of the three isoforms measured after excitation at 280 and 295 nm, gave the same k max emission value at 341 nm; this value corresponds to the position of spectra of class II chromophores, in which tryptophan residues are exposed to the surface of the compact native protein molecule (Burstein et al, 1973).…”

Functional Analysis of the Kunitz Trypsin Inhibitor Family in Poplar Reveals Biochemical Diversity and Multiplicity in Defense against Herbivores

“…By contrast, TI3 and TI5 inhibited both trypsin and chymotrypsin with similar efficiency. TI6 was the only elastase inhibitor, a rare activity among plant KTIs (Valueva et al, 2000;Araujo et al, 2005;Sumikawa et al, 2006). These functional differences presumably reflect the sequence diversity of the KTI family.…”

“…The reactive site is located on a protruding reactive loop, which forms H-bonds with the active groove of the protease. Most KTI proteins have four conserved Cys residues that form two disulfide bridges, although examples with only one or no disulfide bridges are known (do Socorro et al, 2002;Araujo et al, 2005;Macedo et al, 2007). Evidence from several studies suggests that the first disulfide bridge, which surrounds the reactive loop, is necessary for the inhibitory activity of most KTIs (DiBella and Liener, 1969;do Socorro et al, 2002).…”

The effects of a plant proteinase inhibitor from Enterolobium contortisiliquum on human tumor cell lines

“…Interestingly, we found that EcTI efficiently inhibited the formation of both 66-kDa and 64-kDa MMP-2 under PMA/plasminogen-stimulating conditions, whereas aprotinin could only block transformation into 64-kDa MMP-2 but, similar to aprotinin EcTI, decreases the plasminogen-induced proMMP-9 activation approving direct inhibition of plasmin by the plant inhibitor. By contrast, neither the plant-derived elastase/cysteine proteinase inhibitor BbCI nor the kallikrein inhibitor BbKI Araujo et al, 2005) Bereitgestellt von | Universitaetsbibliothek der LMU Muenchen Angemeldet | 129.187.254.47 Heruntergeladen am | 19.12.13 12:38 showed any effect on the proMMP-2 and -9 activation, indicating that the respective target serine proteinases do not interact with the MMP system in our cell model.…”