Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi

Autores
Frasch, Alejandra P.; Carmona, Raquel Adriana; Juliano, Luis; Cazzulo, Juan Jose; Niemirowicz, Gabriela Teresa
Año de publicación
2012
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Metallocarboxypeptidases (MCP) of the M32 family of peptidases have been identified in a number of prokaryotic organisms but they are absent from eukaryotic genomes with the remarkable exception of those of trypanosomatids. The genome of Trypanosoma brucei, the causative agent of Sleeping Sickness, encodes one such MCP which displays 72% identity to the characterized TcMCP-1 from Trypanosoma cruzi. As its orthologue, TcMCP-1, Trypanosoma brucei MCP is a cytosolic enzyme expressed in both major stages of the parasite. Purified recombinant TbMCP-1 exhibits a significant hydrolytic activity against the carboxypeptidase B substrate FA (furylacryloil)-Ala-Lys at pH 7.0-7.8 resembling the T. cruzi enzyme. Several divalent cations had little effect on TbMCP-1 activity but increasing amounts of Co 2+ inhibited the enzyme. Despite having similar tertiary structure, both protozoan MCPs display different substrate specificity with respect to P1 position. Thus, TcMCP-1 enzyme cleaved Abz-FVK-(Dnp)-OH substrate (where Abz: o-aminobenzoic acid and Dnp: 2,4-dinitrophenyl) whereas TbMCP-1 had no activity on this substrate. Comparative homology models and sequence alignments using TcMCP-1 as a template led us to map several residues that could explain this difference. To verify this hypothesis, site-directed mutagenesis was undertaken replacing the TbMCP-1 residues by those present in TcMCP-1. We found that the substitution A414 M led TbMCP-1 to gain activity on Abz-FVK-(Dnp)-OH, thus showing that this residue is involved in specificity determination, probably being part of the S1 sub-site. Moreover, the activity of both protozoan MCPs was explored on two vasoactive compounds such as bradykinin and angiotensin I resulting in two different hydrolysis patterns. © 2012 Elsevier B.V. All rights reserved.
Fil: Frasch, Alejandra P.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Carmona, Raquel Adriana. Universidade de Sao Paulo; Brasil
Fil: Juliano, Luis. Universidade de Sao Paulo; Brasil
Fil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Niemirowicz, Gabriela Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Materia
Carboxypeptidase
Fret Peptides
M32 Family
Peptidase
Trypanosoma Brucei
Trypanosoma Cruzi
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/75476
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/75476
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruziFrasch, Alejandra P.Carmona, Raquel AdrianaJuliano, LuisCazzulo, Juan JoseNiemirowicz, Gabriela TeresaCarboxypeptidaseFret PeptidesM32 FamilyPeptidaseTrypanosoma BruceiTrypanosoma Cruzihttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Metallocarboxypeptidases (MCP) of the M32 family of peptidases have been identified in a number of prokaryotic organisms but they are absent from eukaryotic genomes with the remarkable exception of those of trypanosomatids. The genome of Trypanosoma brucei, the causative agent of Sleeping Sickness, encodes one such MCP which displays 72% identity to the characterized TcMCP-1 from Trypanosoma cruzi. As its orthologue, TcMCP-1, Trypanosoma brucei MCP is a cytosolic enzyme expressed in both major stages of the parasite. Purified recombinant TbMCP-1 exhibits a significant hydrolytic activity against the carboxypeptidase B substrate FA (furylacryloil)-Ala-Lys at pH 7.0-7.8 resembling the T. cruzi enzyme. Several divalent cations had little effect on TbMCP-1 activity but increasing amounts of Co 2+ inhibited the enzyme. Despite having similar tertiary structure, both protozoan MCPs display different substrate specificity with respect to P1 position. Thus, TcMCP-1 enzyme cleaved Abz-FVK-(Dnp)-OH substrate (where Abz: o-aminobenzoic acid and Dnp: 2,4-dinitrophenyl) whereas TbMCP-1 had no activity on this substrate. Comparative homology models and sequence alignments using TcMCP-1 as a template led us to map several residues that could explain this difference. To verify this hypothesis, site-directed mutagenesis was undertaken replacing the TbMCP-1 residues by those present in TcMCP-1. We found that the substitution A414 M led TbMCP-1 to gain activity on Abz-FVK-(Dnp)-OH, thus showing that this residue is involved in specificity determination, probably being part of the S1 sub-site. Moreover, the activity of both protozoan MCPs was explored on two vasoactive compounds such as bradykinin and angiotensin I resulting in two different hydrolysis patterns. © 2012 Elsevier B.V. All rights reserved.Fil: Frasch, Alejandra P.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carmona, Raquel Adriana. Universidade de Sao Paulo; BrasilFil: Juliano, Luis. Universidade de Sao Paulo; BrasilFil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Niemirowicz, Gabriela Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaElsevier Science2012-08info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/75476Frasch, Alejandra P.; Carmona, Raquel Adriana; Juliano, Luis; Cazzulo, Juan Jose; Niemirowicz, Gabriela Teresa; Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi; Elsevier Science; Molecular and Biochemical Parasitology; 184; 2; 8-2012; 63-700166-6851CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.molbiopara.2012.04.008info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0166685112001016info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:42:02Zoai:ri.conicet.gov.ar:11336/75476instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:42:02.632CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi
title Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi
spellingShingle Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi
Frasch, Alejandra P.
Carboxypeptidase
Fret Peptides
M32 Family
Peptidase
Trypanosoma Brucei
Trypanosoma Cruzi
title_short Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi
title_full Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi
title_fullStr Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi
title_full_unstemmed Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi
title_sort Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi
dc.creator.none.fl_str_mv Frasch, Alejandra P.
Carmona, Raquel Adriana
Juliano, Luis
Cazzulo, Juan Jose
Niemirowicz, Gabriela Teresa
author Frasch, Alejandra P.
author_facet Frasch, Alejandra P.
Carmona, Raquel Adriana
Juliano, Luis
Cazzulo, Juan Jose
Niemirowicz, Gabriela Teresa
author_role author
author2 Carmona, Raquel Adriana
Juliano, Luis
Cazzulo, Juan Jose
Niemirowicz, Gabriela Teresa
author2_role author
author
author
author
dc.subject.none.fl_str_mv Carboxypeptidase
Fret Peptides
M32 Family
Peptidase
Trypanosoma Brucei
Trypanosoma Cruzi
topic Carboxypeptidase
Fret Peptides
M32 Family
Peptidase
Trypanosoma Brucei
Trypanosoma Cruzi
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Metallocarboxypeptidases (MCP) of the M32 family of peptidases have been identified in a number of prokaryotic organisms but they are absent from eukaryotic genomes with the remarkable exception of those of trypanosomatids. The genome of Trypanosoma brucei, the causative agent of Sleeping Sickness, encodes one such MCP which displays 72% identity to the characterized TcMCP-1 from Trypanosoma cruzi. As its orthologue, TcMCP-1, Trypanosoma brucei MCP is a cytosolic enzyme expressed in both major stages of the parasite. Purified recombinant TbMCP-1 exhibits a significant hydrolytic activity against the carboxypeptidase B substrate FA (furylacryloil)-Ala-Lys at pH 7.0-7.8 resembling the T. cruzi enzyme. Several divalent cations had little effect on TbMCP-1 activity but increasing amounts of Co 2+ inhibited the enzyme. Despite having similar tertiary structure, both protozoan MCPs display different substrate specificity with respect to P1 position. Thus, TcMCP-1 enzyme cleaved Abz-FVK-(Dnp)-OH substrate (where Abz: o-aminobenzoic acid and Dnp: 2,4-dinitrophenyl) whereas TbMCP-1 had no activity on this substrate. Comparative homology models and sequence alignments using TcMCP-1 as a template led us to map several residues that could explain this difference. To verify this hypothesis, site-directed mutagenesis was undertaken replacing the TbMCP-1 residues by those present in TcMCP-1. We found that the substitution A414 M led TbMCP-1 to gain activity on Abz-FVK-(Dnp)-OH, thus showing that this residue is involved in specificity determination, probably being part of the S1 sub-site. Moreover, the activity of both protozoan MCPs was explored on two vasoactive compounds such as bradykinin and angiotensin I resulting in two different hydrolysis patterns. © 2012 Elsevier B.V. All rights reserved.
Fil: Frasch, Alejandra P.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Carmona, Raquel Adriana. Universidade de Sao Paulo; Brasil
Fil: Juliano, Luis. Universidade de Sao Paulo; Brasil
Fil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fil: Niemirowicz, Gabriela Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
description Metallocarboxypeptidases (MCP) of the M32 family of peptidases have been identified in a number of prokaryotic organisms but they are absent from eukaryotic genomes with the remarkable exception of those of trypanosomatids. The genome of Trypanosoma brucei, the causative agent of Sleeping Sickness, encodes one such MCP which displays 72% identity to the characterized TcMCP-1 from Trypanosoma cruzi. As its orthologue, TcMCP-1, Trypanosoma brucei MCP is a cytosolic enzyme expressed in both major stages of the parasite. Purified recombinant TbMCP-1 exhibits a significant hydrolytic activity against the carboxypeptidase B substrate FA (furylacryloil)-Ala-Lys at pH 7.0-7.8 resembling the T. cruzi enzyme. Several divalent cations had little effect on TbMCP-1 activity but increasing amounts of Co 2+ inhibited the enzyme. Despite having similar tertiary structure, both protozoan MCPs display different substrate specificity with respect to P1 position. Thus, TcMCP-1 enzyme cleaved Abz-FVK-(Dnp)-OH substrate (where Abz: o-aminobenzoic acid and Dnp: 2,4-dinitrophenyl) whereas TbMCP-1 had no activity on this substrate. Comparative homology models and sequence alignments using TcMCP-1 as a template led us to map several residues that could explain this difference. To verify this hypothesis, site-directed mutagenesis was undertaken replacing the TbMCP-1 residues by those present in TcMCP-1. We found that the substitution A414 M led TbMCP-1 to gain activity on Abz-FVK-(Dnp)-OH, thus showing that this residue is involved in specificity determination, probably being part of the S1 sub-site. Moreover, the activity of both protozoan MCPs was explored on two vasoactive compounds such as bradykinin and angiotensin I resulting in two different hydrolysis patterns. © 2012 Elsevier B.V. All rights reserved.
publishDate 2012
dc.date.none.fl_str_mv 2012-08
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/75476
Frasch, Alejandra P.; Carmona, Raquel Adriana; Juliano, Luis; Cazzulo, Juan Jose; Niemirowicz, Gabriela Teresa; Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi; Elsevier Science; Molecular and Biochemical Parasitology; 184; 2; 8-2012; 63-70
0166-6851
CONICET Digital
CONICET
url http://hdl.handle.net/11336/75476
identifier_str_mv Frasch, Alejandra P.; Carmona, Raquel Adriana; Juliano, Luis; Cazzulo, Juan Jose; Niemirowicz, Gabriela Teresa; Characterization of the M32 metallocarboxypeptidase of Trypanosoma brucei: Differences and similarities with its orthologue in Trypanosoma cruzi; Elsevier Science; Molecular and Biochemical Parasitology; 184; 2; 8-2012; 63-70
0166-6851
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.molbiopara.2012.04.008
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0166685112001016
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science
publisher.none.fl_str_mv Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.982451

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