Title:
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Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains
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Author:
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García-Pardo, Javier; Tanco, Sebastian; Diaz, Lucia; Dasgupta, Sayani; Fernandez-Recio, Juan; Lorenzo, Julia; Aviles, Francesc X.; Fricker, Lloyd D.
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Other authors:
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Barcelona Supercomputing Center |
Abstract:
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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. |
Abstract:
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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). |
Abstract:
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Peer Reviewed |
Subject(s):
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-Àrees temàtiques de la UPC::Enginyeria biomèdica -Protein engineering -Metallocarboxypeptidase D (CPD) -Carboxypeptidase domains -Enginyeria de proteïnes |
Rights:
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Attribution-NonCommercial-NoDerivs 4.0 Spain
http://creativecommons.org/licenses/by-nc-nd/4.0/es/ |
Document type:
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Article - Published version Article |
Published by:
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Public Library of Science
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