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PMID:9774412

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Citation

Baset, HA, Ford-Hutchinson, AW and O'Neill, GP (1998) Molecular cloning and functional expression of a Caenorhabditis elegans aminopeptidase structurally related to mammalian leukotriene A4 hydrolases. J. Biol. Chem. 273:27978-87

Abstract

In a search of the Caenorhabditis elegans DNA data base, an expressed sequence tag of 327 base pairs (termed cm01c7) with strong homology to the human leukotriene A4 (LTA4) hydrolase was found. The use of cm01c7 as a probe, together with conventional hybridization screening and anchored polymerase chain reaction techniques resulted in the cloning of the full-length 2.1 kilobase pair C. elegans LTA4 hydrolase-like homologue, termed aminopeptidase-1 (AP-1). The AP-1 cDNA was expressed transiently as an epitope-tagged recombinant protein in COS-7 mammalian cells, purified using an anti-epitope antibody affinity resin, and tested for LTA4 hydrolase and aminopeptidase activities. Despite the strong homology between the human LTA4 hydrolase and C. elegans AP-1(63% similarity and 45% identity at the amino acid level), reverse-phase high pressure liquid chromatography and radioimmunoassay for LTB4 production revealed the inability of the C. elegans AP-1 to use LTA4 as a substrate. In contrast, the C. elegans AP-1 was an efficient aminopeptidase, as demonstrated by its ability to hydrolyze a variety of amino acid p-nitroanilide derivatives. The aminopeptidase activity of C. elegans AP-1 resembled that of the human LTA4 hydrolase/aminopeptidase enzyme with a preference for arginyl-p-nitroanilide as a substrate. Hydrolysis of the amide bond of arginyl-p-nitroanilide was inhibited by bestatin with an IC50 of 2.6 +/- 1.2 microM. The bifunctionality of the mammalian LTA4 hydrolase is still poorly understood, as the physiological substrate for its aminopeptidase activity is yet to be discovered. Our results support the idea that the enzyme originally functioned as an aminopeptidase in lower metazoa and then developed LTA4 hydrolase activity in more evolved organisms.

Links

PubMed

Keywords

Amino Acid Sequence; Aminopeptidases/antagonists & inhibitors; Aminopeptidases/biosynthesis; Aminopeptidases/genetics; Animals; Arginine/analogs & derivatives; Arginine/metabolism; Caenorhabditis elegans/enzymology; Caenorhabditis elegans/genetics; Cloning, Molecular; DNA, Complementary/genetics; Epoxide Hydrolases/genetics; Epoxide Hydrolases/metabolism; Evolution, Molecular; Expressed Sequence Tags; Gene Expression; Genes, Helminth; Helminth Proteins/biosynthesis; Helminth Proteins/genetics; Leucine/analogs & derivatives; Leucine/pharmacology; Leukotriene B4/biosynthesis; Molecular Sequence Data; Physical Chromosome Mapping; Protease Inhibitors/pharmacology; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity

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