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

Detalhes bibliográficos
Autor(a) principal: Serveau, C.
Data de Publicação: 1999
Outros Autores: Lalmanach, G., Hirata, Izaura Yoshico [UNIFESP], Scharfstein, J., Juliano, Maria Aparecida [UNIFESP], Gauthier, F.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UNIFESP
Texto Completo: http://dx.doi.org/10.1046/j.1432-1327.1999.00032.x
http://repositorio.unifesp.br/handle/11600/25999
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.
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spelling 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 proteinasescysteine proteinasecruzipainenzyme specificityThe 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.Univ Tours, Enzymol & Prot Chem Lab, F-37032 Tours, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, BR-21941 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, São Paulo, BrazilWeb of ScienceBlackwell Science LtdUniv ToursUniversidade Federal de São Paulo (UNIFESP)Universidade Federal do Rio de Janeiro (UFRJ)Serveau, C.Lalmanach, G.Hirata, Izaura Yoshico [UNIFESP]Scharfstein, J.Juliano, Maria Aparecida [UNIFESP]Gauthier, F.2016-01-24T12:30:43Z2016-01-24T12:30:43Z1999-01-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersion275-280http://dx.doi.org/10.1046/j.1432-1327.1999.00032.xEuropean Journal of Biochemistry. Oxford: Blackwell Science Ltd, v. 259, n. 1-2, p. 275-280, 1999.10.1046/j.1432-1327.1999.00032.x0014-2956http://repositorio.unifesp.br/handle/11600/25999WOS:000077944300035engEuropean Journal of Biochemistryinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP2024-10-14T13:57:40Zoai:repositorio.unifesp.br/:11600/25999Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestbiblioteca.csp@unifesp.bropendoar:34652024-10-14T13:57:40Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false
dc.title.none.fl_str_mv 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
title 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
spellingShingle 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
Serveau, C.
cysteine proteinase
cruzipain
enzyme specificity
title_short 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_sort 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
author Serveau, C.
author_facet Serveau, C.
Lalmanach, G.
Hirata, Izaura Yoshico [UNIFESP]
Scharfstein, J.
Juliano, Maria Aparecida [UNIFESP]
Gauthier, F.
author_role author
author2 Lalmanach, G.
Hirata, Izaura Yoshico [UNIFESP]
Scharfstein, J.
Juliano, Maria Aparecida [UNIFESP]
Gauthier, F.
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Univ Tours
Universidade Federal de São Paulo (UNIFESP)
Universidade Federal do Rio de Janeiro (UFRJ)
dc.contributor.author.fl_str_mv Serveau, C.
Lalmanach, G.
Hirata, Izaura Yoshico [UNIFESP]
Scharfstein, J.
Juliano, Maria Aparecida [UNIFESP]
Gauthier, F.
dc.subject.por.fl_str_mv cysteine proteinase
cruzipain
enzyme specificity
topic cysteine proteinase
cruzipain
enzyme specificity
description 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.
publishDate 1999
dc.date.none.fl_str_mv 1999-01-01
2016-01-24T12:30:43Z
2016-01-24T12:30:43Z
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://dx.doi.org/10.1046/j.1432-1327.1999.00032.x
European Journal of Biochemistry. Oxford: Blackwell Science Ltd, v. 259, n. 1-2, p. 275-280, 1999.
10.1046/j.1432-1327.1999.00032.x
0014-2956
http://repositorio.unifesp.br/handle/11600/25999
WOS:000077944300035
url http://dx.doi.org/10.1046/j.1432-1327.1999.00032.x
http://repositorio.unifesp.br/handle/11600/25999
identifier_str_mv European Journal of Biochemistry. Oxford: Blackwell Science Ltd, v. 259, n. 1-2, p. 275-280, 1999.
10.1046/j.1432-1327.1999.00032.x
0014-2956
WOS:000077944300035
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv European Journal of Biochemistry
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 275-280
dc.publisher.none.fl_str_mv Blackwell Science Ltd
publisher.none.fl_str_mv Blackwell Science Ltd
dc.source.none.fl_str_mv reponame:Repositório Institucional da UNIFESP
instname:Universidade Federal de São Paulo (UNIFESP)
instacron:UNIFESP
instname_str Universidade Federal de São Paulo (UNIFESP)
instacron_str UNIFESP
institution UNIFESP
reponame_str Repositório Institucional da UNIFESP
collection Repositório Institucional da UNIFESP
repository.name.fl_str_mv Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)
repository.mail.fl_str_mv biblioteca.csp@unifesp.br
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