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

Javier Garcia-Pardo, Sebastian Tanco, Lucía Díaz, Sayani Dasgupta, Juan Fernandez-Recio, Julia Lorenzo, Francesc X. Aviles, Lloyd D. Fricker

Research output: Contribution to journalArticle

2 Scopus citations

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.

Original languageEnglish (US)
Article numbere0187778
JournalPloS one
Volume12
Issue number11
DOIs
StatePublished - Nov 2017

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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    Garcia-Pardo, J., Tanco, S., Díaz, L., Dasgupta, S., Fernandez-Recio, J., Lorenzo, J., Aviles, F. X., & Fricker, L. D. (2017). Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains. PloS one, 12(11), [e0187778]. https://doi.org/10.1371/journal.pone.0187778