title: Functional expression and characterization of the cytoplasmic aminopeptidase P of Caenorhabditis elegans.
abstract: Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9) cleaves the N-terminal X-Pro bond of peptides and occurs in mammals as both cytosolic and plasma membrane forms, encoded by separate genes. In mammals, the plasma membrane AP-P can function as a kininase, but little is known about the physiological role of the cytosolic enzyme. The C. elegans genome contains a single gene encoding AP-P (W03G9.4), analysis of which predicts regions displaying high levels of amino-acid sequence homology between the predicted gene product and mammalian cytoplasmic AP-P, with the absolute conservation of key catalytic residues. The sequence of an EST (yk91g4), 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 intestine of larvae and adult hermaphrodites, indicating that the intestine is a major site of W03G9.4 expression. yk91g4 tagged with a hexahistidine and DLYDDDDK peptide epitope was expressed in Escherichia coli to yield, after affinity purification, a recombinant protein with a molecular mass of 71 kDa. The recombinant W03G9.4 removed the N-terminal amino acid from bradykinin (RPPGFSPFR), a Caenorhabditis elegans neuropeptide (KPSFVRFamide) and Lem Trp 1 (APSGFLGVRamide), but did not display activity towards angiotensin I (NRVYIHPFHL), 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 microM), a selective inhibitor of mammalian AP-P. A Km of 45 microM and an optimum pH of 7-8 was observed with bradykinin as the substrate. The activity of the nematode AP-P, like its mammalian counterparts, was strongly influenced by metal ions, with Co2+, Mn2+ 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 physiological role in the intracellular hydrolysis of proline-containing peptides absorbed from the lumen of the intestine.
authors.affiliation: Molecular and Cellular Biosciences, Faculty of Biological Sciences, Miall Building, University of Leeds, UK.
authors.forename: V
authors.initials: V
authors.lastname: Laurent
authors.forename: D R
authors.initials: DR
authors.lastname: Brooks
authors.forename: D
authors.initials: D
authors.lastname: Coates
authors.forename: R E
authors.initials: RE
authors.lastname: Isaac
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001920:Bradykinin / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D020869:Gene Expression Profiling; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: D029742:Caenorhabditis elegans Proteins; D010455:Peptides; D011994:Recombinant Proteins; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; C097277:apstatin; D001920:Bradykinin