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.
Highlights
Carboxypeptidases (CPs)1 catalyze the release of C-terminal amino acids from proteins and peptides [1, 2], and are grouped according to the chemical nature of their catalytic site
Proteome-derived Peptide Library Generation and Carboxypeptidase Assay Workflow—To profile the substrate specificity of CPs, we opted for the use of proteome-derived peptide libraries, which should allow comprehensive analyses of protease preferences [18]
When human metallocarboxypeptidases A1 (hCPA1) was incubated with the Lys-N peptide library for a short time, the number of peptides double processed by the CP diminished to ϳ5%, whereas the resulting specificity profile shows again the increased preference of hydrophobic amino acids at P1 position
Summary
CPs are exploited in biotechnological and biomedical applications. Peptidomic studies have made use of natural peptides isolates from cells and tissues as natural substrate pools to test cleavages by CPs [8, 21, 22] In this list of degradomic approaches, we can consider the protein-centric positional proteomics approaches; C-terminal COFRADIC [23] and C-TAILS [24], capable of identifying in vivo CP proteolytic events, based on the identification of protein neo-C termini. Using Lys-N proteome-derived peptide libraries and making use of shorter protease incubation times, information on sequential cleavages of these enzymes could be obtained This assay was applied to PRCP, a pharmaceutically relevant SCP that differs from MCPs in its enzymatic characteristics, further demonstrating the more universal applicability of our method
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