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

García Pardo, Javier; Tanco, Sebastián Martín; Díaz, Lucía; Dasgupta, Sayani; Fernández Recio, Juan; Lorenzo, Júlia; Avilés, Francesc Xavier; Fricker, Lloyd D.

doi:http://dx.doi.org/10.1371/journal.pone.0187778

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

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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

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.