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Proteome-derived peptide libraries to study the substrate specificity profiles of carboxypeptidases

(2013) MOLECULAR & CELLULAR PROTEOMICS. 12(8). p.2096-2110
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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.
Keywords
IDENTIFICATION, METALLOCARBOXYPEPTIDASE, GENE FAMILY, I-TASSER, PROTEASE, SEQUENCE-ANALYSIS, PROTEOLYTIC-ENZYMES, PROCARBOXYPEPTIDASE A2, DIAGONAL CHROMATOGRAPHY, MAST-CELL CARBOXYPEPTIDASE

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Citation

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Chicago
Tanco, Sebastian, Julia Lorenzo, Javier Garcia-Pardo, Sven Degroeve, Lennart Martens, Francesc Xavier Aviles, Kris Gevaert, and Petra Van Damme. 2013. “Proteome-derived Peptide Libraries to Study the Substrate Specificity Profiles of Carboxypeptidases.” Molecular & Cellular Proteomics 12 (8): 2096–2110.
APA
Tanco, S., Lorenzo, J., Garcia-Pardo, J., Degroeve, S., Martens, L., Aviles, F. X., Gevaert, K., et al. (2013). Proteome-derived peptide libraries to study the substrate specificity profiles of carboxypeptidases. MOLECULAR & CELLULAR PROTEOMICS, 12(8), 2096–2110.
Vancouver
1.
Tanco S, Lorenzo J, Garcia-Pardo J, Degroeve S, Martens L, Aviles FX, et al. Proteome-derived peptide libraries to study the substrate specificity profiles of carboxypeptidases. MOLECULAR & CELLULAR PROTEOMICS. 2013;12(8):2096–110.
MLA
Tanco, Sebastian, Julia Lorenzo, Javier Garcia-Pardo, et al. “Proteome-derived Peptide Libraries to Study the Substrate Specificity Profiles of Carboxypeptidases.” MOLECULAR & CELLULAR PROTEOMICS 12.8 (2013): 2096–2110. Print.
@article{4247607,
  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.},
  author       = {Tanco, Sebastian and Lorenzo, Julia and Garcia-Pardo, Javier and Degroeve, Sven and Martens, Lennart and Aviles, Francesc Xavier and Gevaert, Kris and Van Damme, Petra},
  issn         = {1535-9476},
  journal      = {MOLECULAR & CELLULAR PROTEOMICS},
  keywords     = {IDENTIFICATION,METALLOCARBOXYPEPTIDASE,GENE FAMILY,I-TASSER,PROTEASE,SEQUENCE-ANALYSIS,PROTEOLYTIC-ENZYMES,PROCARBOXYPEPTIDASE A2,DIAGONAL CHROMATOGRAPHY,MAST-CELL CARBOXYPEPTIDASE},
  language     = {eng},
  number       = {8},
  pages        = {2096--2110},
  title        = {Proteome-derived peptide libraries to study the substrate specificity profiles of carboxypeptidases},
  url          = {http://dx.doi.org/10.1074/mcp.M112.023234},
  volume       = {12},
  year         = {2013},
}

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