Discrimination of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, and mammalian cathepsins B and L, by a pH-inducible fluorogenic substrate of trypanosomal cysteine proteinases

Discrimination of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, and mammalian cathepsins B and L, by a pH-inducible fluorogenic substrate of trypanosomal cysteine proteinases

Autor Serveau, C. Google Scholar
Lalmanach, G. Google Scholar
Hirata, Izaura Yoshico Autor UNIFESP Google Scholar
Scharfstein, J. Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Gauthier, F. Google Scholar
Instituição Univ Tours
Universidade Federal de São Paulo (UNIFESP)
Universidade Federal do Rio de Janeiro (UFRJ)
Resumo The substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, was investigated using a series of dansyl-peptides based on the putative autoproteolytic sequence of the proteinase (VVG-GP) located at the hinge region between the catalytic domain and the C-terminal extension. Replacing Val with Pro at P2 in this sequence greatly improved the rate of cleavage by cruzipain. Tyr and Val residues are preferred at P3 by all cysteine proteinases whatever their origin, whereas only cruzipain and cathepsin L cleaved substrate with a His at that position. the combination of a Pro at P2 and His at P3 abolished cleavage by cathepsin L, so that only cruzipain was able to cleave the HPGGP peptide at the GG bond. A substrate with intramolecularly quenched fluorescence was raised on this sequence (Abz-HPGGPQ-EDDnp) which was also specifically cleaved by cruzipain (k(cat)/K-m of 157 000 (M-1.s-1)) and by a homologous proteinase from Trypanosoma congolense. the pH activity profile of cruzipain on Abz-HPGGPQ-EDDnp showed a narrow peak with a maximum at pH 5.5 and no cleavage above pH 6.8, although trypanosomal cysteine proteinases remain active at basic pH. the lack of activity at neutral and basic pH was due to a decrease in k(cat), while the K-m remained essentially unchanged, demonstrating that the substrate still binds to the enzyme and therefore behaves as an inhibitor. Changing the substrate into an inhibitor depended on the deprotonation of the His residue in the substrate, as deduced from a comparison of the pH activity profile with that of a related, but uncharged, substrate. Abz-HPGGPQ-EDDnp also inhibited mammalian cathepsins B and L but was not cleaved by these proteinases at any pH. the importance of the His residue at P3 for cleavage by cruzipain was confirmed by substituting Lys for His at that position. the resulting peptide was not cleaved by cruzipain in spite of the presence of a positively charged group at P3, but still interacted with the enzyme. It was concluded that the presence of an imidazolium group at P3 was essential to endow the HPGGPQ sequence with the properties of a cruzipain substrate.
Palavra-chave cysteine proteinase
cruzipain
enzyme specificity
Idioma Inglês
Data de publicação 1999-01-01
Publicado em European Journal of Biochemistry. Oxford: Blackwell Science Ltd, v. 259, n. 1-2, p. 275-280, 1999.
ISSN 0014-2956 (Sherpa/Romeo, fator de impacto)
Publicador Blackwell Science Ltd
Extensão 275-280
Fonte http://dx.doi.org/10.1046/j.1432-1327.1999.00032.x
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000077944300035
Endereço permanente http://repositorio.unifesp.br/handle/11600/25999

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