Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains

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• dc.contributor.author García Pardo, Javierca
• dc.contributor.author Tanco, Sebastián Martínca
• dc.contributor.author Díaz, Lucíaca
• dc.contributor.author Dasgupta, Sayanica
• dc.contributor.author Fernández Recio, Juanca
• dc.contributor.author Lorenzo, Júliaca
• dc.contributor.author Avilés, Francesc Xavierca
• dc.contributor.author Fricker, Lloyd D.ca
• dc.date.accessioned 2018-07-13T07:53:49Z
• dc.date.available 2018-07-13T07:53:49Z
• dc.date.issued 2017
• dc.description.abstract Metallocarboxypeptidase D (CPD) is a membrane-bound component of the trans-Golgi network that cycles to the cell surface through exocytic and endocytic pathways. Unlike other members of the metallocarboxypeptidase family, CPD is a multicatalytic enzyme with three carboxypeptidase-like domains, although only the first two domains are predicted to be enzymatically active. To investigate the enzymatic properties of each domain in human CPD, a critical active site Glu in domain I and/or II was mutated to Gln and the protein expressed, purified, and assayed with a wide variety of peptide substrates. CPD with all three domains intact displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as does CPD with mutation of domain I. In contrast, the domain II mutant displayed >50% activity from pH 6.5-7.5. CPD with mutations in both domains I and II was completely inactive towards all substrates and at all pH values. A quantitative peptidomics approach was used to compare the activities of CPD domains I and II towards a large number of peptides. CPD cleaved C-terminal Lys or Arg from a subset of the peptides. Most of the identified substrates of domain I contained C-terminal Arg, whereas comparable numbers of Lys- and Arg-containing peptides were substrates of domain II. We also report that some peptides with C-terminal basic residues were not cleaved by either domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of domain I for C-terminal Arg was validated through molecular docking experiments. Together with the differences in pH optima, the different substrate specificities of CPD domains I and II allow the enzyme to perform distinct functions in the various locations within the cell.
• dc.description.sponsorship This work was funded by the Spanish Ministry of Innovation and Competitiveness grants BIO2013-44973-R and BIO2016-78057-R (to FXA), by Plan Estatal grant number BIO2016-79960-R from the Spanish Ministry of Economy and Competitiveness (to JFR), and by grant R01-DA004494 from the United States’ National Institute of Health (to LDF).
• dc.format.mimetype application/pdf
• dc.identifier.citation Garcia-Pardo J, Tanco S, Díaz L, Dasgupta S, Fernandez-Recio J, Lorenzo J et al. Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains. PLoS One. 2017 Nov 13;12(11):e0187778. DOI: 10.1371/journal.pone.0187778
• dc.identifier.doi http://dx.doi.org/10.1371/journal.pone.0187778
• dc.identifier.issn 1932-6203
• dc.identifier.uri http://hdl.handle.net/10230/35150
• dc.language.iso eng
• dc.publisher Public Library of Science (PLoS)ca
• dc.relation.ispartof PLoS One. 2017 Nov 13;12(11):e0187778
• dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/BIO2013-44973-R
• dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/BIO2016-78057-R
• dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/BIO2016-79960-R
• dc.rights © 2017 Garcia-Pardo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0/
• dc.subject.keyword Peptide libraries
• dc.subject.keyword Thyroglobulin
• dc.subject.keyword Sequence alignment
• dc.subject.keyword Enzymes
• dc.subject.keyword Recombinant proteins
• dc.subject.keyword Serum albumin
• dc.title Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domainsca
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion