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Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors.

ABSTRACT: A panel of intramolecularly quenched fluorogenic substrates containing the conserved QVVA and LVG inhibitory sequences of cystatin inhibitors was used to describe the specificity of the major cysteine proteinase of Trypanosoma cruzi (cruzipain or cruzain). This approach was based on the observations that: (1) cruzipain is strongly inhibited by chicken cystatin and rat T-kininogen, two representative members of cystatin families 2 and 3; (2) the QVVA- and LVG-containing substrates are specifically hydrolysed by papain-like proteinases; and (3) the cystatin-like motifs are similar to the proteolytically sensitive sequences in cruzipain that separate the pro-region and/or the C-terminal extension from the catalytic domain. Specificity constants (kcat/Km) were determined and compared with those of mammalian cathepsins B and L from rat liver lysosomes. Cruzipain and the mammalian proteinases cleaved cystatin-derived substrates at the same site, but their specificities differed significantly. Increased specificity for cruzipain was obtained by replacing amino acids at critical positions on both sides of the cleavage sites, especially at position P2'. The specificity constants (k(cat)/Km) obtained for the two substrates with a prolyl residue at P2' (O-aminobenzoyl-QVVAGP-ethylenediamine 2-4-dinitrophenyl and O-aminobenzoyl-VVGGP-ethylenediamine 2-4-dinitrophenyl) were about 50 times higher for cruzipain than for rat cathepsin L and about 100 times higher than for cathepsin B. Diazomethylketone derivatives, based on the non-prime sequence of cystatin-derived substrates, inhibited cruzipain irreversibly, but their inactivation rate constants were considerably lower than those for mammalian cathepsins B and L, confirming the importance of P' residues for cruzipain specificity.

SUBMITTER: Serveau C

PROVIDER: S-EPMC1217003 | biostudies-other | 1996 Feb

REPOSITORIES: biostudies-other

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Project description:Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin-peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.

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Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors.

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