Prolyl carboxypeptidase purified from human placenta

Subtitle:its characterization and identification as an apelin-cleaving enzyme

Background The proteolytic regulation of peptides involved in feeding behavior is poorly understood. Prolyl carboxypeptidase (PRCP) is particularly known for its role in body weight control by converting the anorexigenic peptide, α-melanocyte-stimulating hormone 113 into the inactive form 112. The purpose of this study was to characterize purified human PRCP, to investigate its substrate specificity and to discover novel substrates linked to obesity. Pyroglutamated apelin-13, ghrelin, enterostatin and obestatin were investigated since these are feeding-regulating peptides with potential cleaving sites for PRCP. Methods PRCP was purified from human placenta and identified using western blotting and mass spectrometry. The kinetic parameters of purified and commercially available PRCP for known and potential peptide substrates were determined and compared using a RP-HPLC activity assay, isothermal titration calorimetry and mass spectrometry. Results PRCP was purified 575-fold from human placenta and succesfully identified as human lysosomal Pro-X carboxypeptidase. Purified and recombinant PRCP had similar substrate specificity with angiotensin III as the substrate of preference. Pyroglutamated apelin-13 was observed to be a novel substrate for human PRCP in vitro and PRCP-dependent cleavage was shown in a human umbilical vein endothelial cell culture experiment. Other potential substrates e.g. obestatin, ghrelin and enterostatin were not hydrolyzed by PRCP. Conclusion These results show that placenta is a good source of human PRCP and that PRCP removes the C-terminal phenylalanine from pyroglutamated apelin-13. For the first time, PRCP is identified as an apelin-cleaving enzyme. This finding adds evidence to the hypothesis that PRCP plays a role in energy homeostasis.

Publication year:2016

Keywords:A1 Journal article

BOF-keylabel:yes

BOF-publication weight:1

CSS-citation score:1

Authors from:Higher Education

Accessibility:Open