NDLI: Proteome-derived Peptide Libraries to Study the Substrate Specificity Profiles of Carboxypeptidases

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Tanco, Sebastian Lorenzo, Julia Garcia-Pardo, Javier Degroeve, Sven Martens, Lennart Aviles, Francesc Xavier Gevaert, Kris Van Damme, Petra
Description	Country affiliation: Spain Author Affiliation: Tanco S ( Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i de Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.)
Abstract	Through processing peptide and protein C termini, carboxypeptidases participate in the regulation of various biological processes. Few tools are however available to study the substrate specificity profiles of these enzymes. We developed a proteome-derived peptide library approach to study the substrate preferences of carboxypeptidases. Our COFRADIC-based approach takes advantage of the distinct chromatographic behavior of intact peptides and the proteolytic products generated by the action of carboxypeptidases, to enrich the latter and facilitate its MS-based identification. Two different peptide libraries, generated either by chymotrypsin or by metalloendopeptidase Lys-N, were used to determine the substrate preferences of human metallocarboxypeptidases A1 (hCPA1), A2 (hCPA2), and A4 (hCPA4). In addition, our approach allowed us to delineate the substrate specificity profile of mouse mast cell carboxypeptidase (MC-CPA or mCPA3), a carboxypeptidase suggested to function in innate immune responses regulation and mast cell granule homeostasis, but which thus far lacked a detailed analysis of its substrate preferences. mCPA3 was here shown to preferentially remove bulky aromatic amino acids, similar to hCPA2. This was also shown by a hierarchical cluster analysis, grouping hCPA1 close to hCPA4 in terms of its P1 primed substrate specificity, whereas hCPA2 and mCPA3 cluster separately. The specificity profile of mCPA3 may further aid to elucidate the function of this mast cell carboxypeptidase and its biological substrate repertoire. Finally, we used this approach to evaluate the substrate preferences of prolylcarboxypeptidase, a serine carboxypeptidase shown to cleave C-terminal amino acids linked to proline and alanine.
File Format	HTM / HTML
ISSN	15359476
e-ISSN	15359484
DOI	10.1074/mcp.M112.023234
Journal	Molecular & Cellular Proteomics
Issue Number	8
Volume Number	12
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2013-08-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Proteomics Carboxypeptidases Metabolism Peptide Library Animals Chemistry K562 Cells Mast Cells Enzymology Mice Models, Molecular Protein Conformation Proteome Substrate Specificity Research Support, Non-u.s. Gov't
Content Type	Text
Resource Type	Article
Subject	Medicine Analytical Chemistry Molecular Biology Biochemistry

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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