Abstract
CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment.
Highlights
Based on the present results, CPA4 is clearly a CPA-like enzyme with specificity for C-terminal aliphatic and aromatic residues, consistent with the substrate binding pocket predicted from analysis of the crystal structure of CPA4 (14 –16)
The three types of substrates used in the present study provide complementary information
The small chromogenic substrates are ideal for kinetic analysis of kcat and Km values, these are not natural substrates, and the chromogenic group can potentially affect the cleavage specificity
Summary
Protein Production and Purification—Human pro-CPA4 (PCPA4) was produced using the vector pPIC9 and the methylotrophic yeast Pichia pastoris as an expression host and purified as described elsewhere [15]. The kinetic parameters, kcat and Km, were obtained using six experimental points by direct fit to a Michaelis-Menten curve using a nonlinear least-squares regression analysis For those substrates for which the solubility of the substrates limited the measurements to concentrations well below Km, the kcat/Km values were estimated from the slope of the linear portion of saturation curve using the simplified equation, v ϭ (kcat/Km)1⁄7[E0]1⁄7[S]. Peptides were extracted by sonication of each brain in 1.0 ml of ice-cold water followed by incubation at 70 °C for 20 min, cooling in an ice bath, acidification with 120 l of 0.1 M HCl to a final concentration of 10 mM HCl, and centrifugation at 13,000 ϫ g for 30 min at 4 °C. The peak intensities of the samples incubated with enzyme were compared with the peak intensity of the sample incubated without enzyme; previous studies examining four aliquots of brain extract with the four different isotopic tags showed average ratios of 1.00 [19]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
