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

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

Author Serveau, Carole Google Scholar
Lalmanach, Gilles Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Scharfstein, Julio Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Gauthier, Francis Google Scholar
Institution UNIV TOURS
Universidade Federal de São Paulo (UNIFESP)
FED UNIV RIO DE JANEIRO
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 arl 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 (k(cat.)/K-m) 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.)/K-m) obtained for the two substrates with a prolyl residue at P2' (O-aminobenzoyl-QVVAGP-ethlylenediamine 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.
Language English
Date 1996-02-01
Published in Biochemical Journal. London: Portland Press Ltd, v. 313, n. 3, p. 951-956, 1996.
ISSN 0264-6021 (Sherpa/Romeo, impact factor)
Publisher Portland Press Ltd
Extent 951-956
Origin http://dx.doi.org/10.1042/bj3130951
Access rights Open access Open Access
Type Article
Web of Science ID WOS:A1996TU58500037
URI http://repositorio.unifesp.br/11600/42786

Show full item record




File

File Size Format View

There are no files associated with this item.

This item appears in the following Collection(s)

Search


Browse

Statistics

My Account