Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes

Autores
Lufrano, Daniela; Cotabarren, Juliana; Garcia Pardo, Javier; Fernandez Alvarez, Roberto; Tort, Olivia; Tanco, Sebastián; Avilés, Francesc Xavier; Lorenzo, Julia; Obregon, Walter David
Año de publicación
2015
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8 Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B was demonstrated.
Fil: Lufrano, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina. Universitat Autònoma de Barcelona; España
Fil: Cotabarren, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina
Fil: Garcia Pardo, Javier. Universitat Autònoma de Barcelona; España
Fil: Fernandez Alvarez, Roberto. Universitat Autònoma de Barcelona; España
Fil: Tort, Olivia. Universitat Autònoma de Barcelona; España
Fil: Tanco, Sebastián. Universitat Autònoma de Barcelona; España. University of Ghent; Bélgica
Fil: Avilés, Francesc Xavier. Universitat Autònoma de Barcelona; España
Fil: Lorenzo, Julia. Universitat Autònoma de Barcelona; España
Fil: Obregon, Walter David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina
Materia
Andean Potatoes
Plant Protease Inhibitor
Potato Carboxypeptidase Inhibitor
Secondary Binding Site
Solanaceae
Solanum Tuberosum
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/50079
Acceder
id CONICETDig_5068880efc27e2e2efd87bfaee26d7cd
oai_identifier_str oai:ri.conicet.gov.ar:11336/50079
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoesLufrano, DanielaCotabarren, JulianaGarcia Pardo, JavierFernandez Alvarez, RobertoTort, OliviaTanco, SebastiánAvilés, Francesc XavierLorenzo, JuliaObregon, Walter DavidAndean PotatoesPlant Protease InhibitorPotato Carboxypeptidase InhibitorSecondary Binding SiteSolanaceaeSolanum Tuberosumhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8 Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B was demonstrated.Fil: Lufrano, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina. Universitat Autònoma de Barcelona; EspañaFil: Cotabarren, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; ArgentinaFil: Garcia Pardo, Javier. Universitat Autònoma de Barcelona; EspañaFil: Fernandez Alvarez, Roberto. Universitat Autònoma de Barcelona; EspañaFil: Tort, Olivia. Universitat Autònoma de Barcelona; EspañaFil: Tanco, Sebastián. Universitat Autònoma de Barcelona; España. University of Ghent; BélgicaFil: Avilés, Francesc Xavier. Universitat Autònoma de Barcelona; EspañaFil: Lorenzo, Julia. Universitat Autònoma de Barcelona; EspañaFil: Obregon, Walter David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; ArgentinaPergamon-Elsevier Science Ltd2015-12info: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/11336/50079Lufrano, Daniela; Cotabarren, Juliana; Garcia Pardo, Javier; Fernandez Alvarez, Roberto; Tort, Olivia; et al.; Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes; Pergamon-Elsevier Science Ltd; Phytochemistry; 120; 12-2015; 36-450031-9422CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0031942215300893info:eu-repo/semantics/altIdentifier/doi/10.1016/j.phytochem.2015.09.010info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:12:49Zoai:ri.conicet.gov.ar:11336/50079instacron: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 11:12:50.163CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes
title Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes
spellingShingle Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes
Lufrano, Daniela
Andean Potatoes
Plant Protease Inhibitor
Potato Carboxypeptidase Inhibitor
Secondary Binding Site
Solanaceae
Solanum Tuberosum
title_short Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes
title_full Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes
title_fullStr Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes
title_full_unstemmed Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes
title_sort Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes
dc.creator.none.fl_str_mv Lufrano, Daniela
Cotabarren, Juliana
Garcia Pardo, Javier
Fernandez Alvarez, Roberto
Tort, Olivia
Tanco, Sebastián
Avilés, Francesc Xavier
Lorenzo, Julia
Obregon, Walter David
author Lufrano, Daniela
author_facet Lufrano, Daniela
Cotabarren, Juliana
Garcia Pardo, Javier
Fernandez Alvarez, Roberto
Tort, Olivia
Tanco, Sebastián
Avilés, Francesc Xavier
Lorenzo, Julia
Obregon, Walter David
author_role author
author2 Cotabarren, Juliana
Garcia Pardo, Javier
Fernandez Alvarez, Roberto
Tort, Olivia
Tanco, Sebastián
Avilés, Francesc Xavier
Lorenzo, Julia
Obregon, Walter David
author2_role author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Andean Potatoes
Plant Protease Inhibitor
Potato Carboxypeptidase Inhibitor
Secondary Binding Site
Solanaceae
Solanum Tuberosum
topic Andean Potatoes
Plant Protease Inhibitor
Potato Carboxypeptidase Inhibitor
Secondary Binding Site
Solanaceae
Solanum Tuberosum
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8 Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B was demonstrated.
Fil: Lufrano, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina. Universitat Autònoma de Barcelona; España
Fil: Cotabarren, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina
Fil: Garcia Pardo, Javier. Universitat Autònoma de Barcelona; España
Fil: Fernandez Alvarez, Roberto. Universitat Autònoma de Barcelona; España
Fil: Tort, Olivia. Universitat Autònoma de Barcelona; España
Fil: Tanco, Sebastián. Universitat Autònoma de Barcelona; España. University of Ghent; Bélgica
Fil: Avilés, Francesc Xavier. Universitat Autònoma de Barcelona; España
Fil: Lorenzo, Julia. Universitat Autònoma de Barcelona; España
Fil: Obregon, Walter David. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; Argentina
description Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8 Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B was demonstrated.
publishDate 2015
dc.date.none.fl_str_mv 2015-12
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/11336/50079
Lufrano, Daniela; Cotabarren, Juliana; Garcia Pardo, Javier; Fernandez Alvarez, Roberto; Tort, Olivia; et al.; Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes; Pergamon-Elsevier Science Ltd; Phytochemistry; 120; 12-2015; 36-45
0031-9422
CONICET Digital
CONICET
url http://hdl.handle.net/11336/50079
identifier_str_mv Lufrano, Daniela; Cotabarren, Juliana; Garcia Pardo, Javier; Fernandez Alvarez, Roberto; Tort, Olivia; et al.; Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes; Pergamon-Elsevier Science Ltd; Phytochemistry; 120; 12-2015; 36-45
0031-9422
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0031942215300893
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.phytochem.2015.09.010
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Pergamon-Elsevier Science Ltd
publisher.none.fl_str_mv Pergamon-Elsevier Science Ltd
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
_version_ 1846781527581523968
score 12.982451

Publicaciones similares