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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/50079
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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 Técnicas2025-09-29T09:44:05Zoai: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:34982025-09-29 09:44:05.751CONICET 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 |
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1844613387953111040 |
score |
13.070432 |