Functional expression and characterization of the cytoplasmic aminopeptidase P of Caenorhabditis elegans

Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9) cleaves the N-te rminal X-Pro bond of peptides and occurs in mammals as both cytosolic and p lasma membrane forms, encoded by separate genes. In mammals, the plasma mem brane AP-P can function as a kininase, but little is known about the physio logical role of the cytosolic enzyme. The C. elegans genome contains a sing le gene encoding AP-P (W03G9.4), analysis of which predicts regions display ing high levels of amino-acid sequence homology between the predicted gene product and mammalian cytoplasmic AP-P, with the absolute conservation of k ey catalytic residues. The sequence of an EST (yk9lg4), comprising the open reading frame of W03G9.4, confirmed the predicted genomic structure of the gene and the prediction that W03G9.4 codes for a nonsecreted protein with a molecular mass of 68 kDa. Nematodes transformed with a promoter reporter construct, W03G9.4::GFP, showed high levels of fluorescence in the intestin e of larvae and adult hermaphrodites, indicating that the intestine is a ma jor site of W03G9.4 expression. yk9lg4 tagged with a hexahistidine and DLYD DDDK peptide epitope was expressed in Escherichia coli to yield, after affi nity purification, a recombinant protein with a molecular mass of 71 kDa. T he recombinant W03G9.4 removed the N-terminal amino acid from bradykinin (R PPGFSPFR), a Caenorhabditis elegans neuropeptide (KPSFVRFamide) and Lem Trp 1 (APSGFLGVRamide), but did not display activity towards angiotensin I (NR VYIHPFHL), des-Arg bradykinin and AF1 (KNEFIRFamide). The activity towards bradykinin was inhibited by EDTA and 1, 10 phenanthroline, as expected for a metalloenzyme, and also by apstatin (IC50, 1 muM), a selective inhibitor of mammalian AP-P. A K-m of 45 muM and an optimum pH of 7-8 was observed wi th bradykinin as the substrate. The activity of the nematode AP-P, like its mammalian counterparts, was strongly influenced by metal ions, with Co2+ M n2+ and Zn2+ all inhibiting the hydrolysis of bradykinin. We conclude that W03G9.4 codes for a cytoplasmic AP-P with very similar enzymatic properties to those of mammalian AP-P, and we suggest that the enzyme has a physiolog ical role in the intracellular hydrolysis of proline-containing peptides ab sorbed from the lumen of the intestine.