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J Biol Chem, Vol. 273, Issue 43, 27978-27987, October 23, 1998

Molecular Cloning and Functional Expression of a Caenorhabditis elegans Aminopeptidase Structurally Related to Mammalian Leukotriene A₄ Hydrolases

Heba Abdel Baset^§, Anthony W. Ford-Hutchinson^§, and Gary P. O'Neill^§

From the  Department of Pharmacology and Experimental Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada and the ^§ Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec H9R 4P8, Canada

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 µM. 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.

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