Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes

Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes

Author Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Brooks, Darren R. Google Scholar
Selzer, Paul M. Google Scholar
Pandolfo, Hector L. Google Scholar
Judice, Wagner AS Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Meldal, Morten Google Scholar
Sanderson, Sanya J. Google Scholar
Mottram, Jeremy C. Google Scholar
Coombs, Graham H. Google Scholar
Institution Univ Glasgow
Universidade Federal de São Paulo (UNIFESP)
Intervet Innovat GmbH
Carlsberg Res Lab
Abstract The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. in this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. the H84Y mutation mimics an additional change present in another isoenzyme, CPB18. the active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S-1-S-3 and S-1'-S-3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P-3 to P-3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. the kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S-3 to S-3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P-2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.
Keywords carboxydipeptidase
cysteine protease
fluorogenic peptides
Language English
Date 2004-09-01
Published in European Journal of Biochemistry. Oxford: Blackwell Publishing Ltd, v. 271, n. 18, p. 3704-3714, 2004.
ISSN 0014-2956 (Sherpa/Romeo, impact factor)
Publisher Blackwell Publishing Ltd
Extent 3704-3714
Origin http://dx.doi.org/10.1111/j.1432-1033.2004.04311.x
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000223711400010
URI http://repositorio.unifesp.br/handle/11600/27899

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