Substrate specificity of human metallocarboxypeptidase D : comparison of the two active carboxypeptidase domains
Garcia-Pardo, Javier (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Tanco, Sebastián Martín (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Díaz, Lucía (Barcelona Supercomputing Center)
Dasgupta, Sayani (Albert Einstein College of Medicine. Department of Molecular Pharmacology)
Fernández-Recio, Juan (Barcelona Supercomputing Center)
Lorenzo Rivera, Julia (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Avilés, Francesc X. (Francesc Xavier) (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Fricker, Lloyd D. (Albert Einstein College of Medicine. Department of Molecular Pharmacology)

Date:	2017
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.
Grants:	Ministerio de Economía y Competitividad BIO2016-79960-R Ministerio de Ciencia e Innovación BIO2016-78057-R Ministerio de Ciencia e Innovación BIO2013-44973-R
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Amino acid sequence ; Bortezomib ; Catalytic domain ; HEK293 cells ; Humans ; Hydrogen-ion concentration ; Kinetics ; Molecular docking simulation ; Peptides ; Point mutation ; Proteins ; Substrate specificity
Published in:	PloS one, Vol. 12 issue 11 (2017) , art. e0187778, ISSN 1932-6203

DOI: 10.1371/journal.pone.0187778
PMID: 29131831

29 p, 9.5 MB

The record appears in these collections:
Research literature > UAB research groups literature > Research Centres and Groups (research output) > Health sciences and biosciences > Institut de Biotecnologia i de Biomedicina (IBB)
Articles > Research articles
Articles > Published articles