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J. Biol. Chem., Vol. 278, Issue 44, 42795-42801, October 31, 2003

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The Caenorhabditis elegans Orthologue of Mammalian Puromycin-sensitive Aminopeptidase Has Roles in Embryogenesis and Reproduction^*

Darren R. Brooks, Nigel M. Hooper, and R. Elwyn Isaac

From the Molecular and Cellular Biosciences, Faculty of Biological Sciences, University of Leeds, Miall Bldg., Leeds, West Yorkshire LS2 9JT, United Kingdom

Mammals possess membrane-associated and cytosolic forms of the puromycin-sensitive aminopeptidase (PSA; EC 3.4.11.14). Increasing evidence suggests the membrane PSA is involved in neuromodulation within the central nervous system and in reproductive biology. The functional roles of the cytosolic PSA are less clear. The genome of the nematode Caenorhabditis elegans encodes an aminopeptidase, F49E8.3 (PAM-1), that is orthologous to PSA, and sequence analysis predicts it to be cytosolic. We have determined the spatio/temporal gene expression pattern of pam-1 by using the promoter region of F49E8.3 to control expression in the nematode of a second exon translational fusion of the aminopeptidase to green fluorescent protein. Cytosolic fluorescence was observed throughout development in the intestine and nerve cells of the head. Neuronal expression was also observed in the tail of adult males. Recombinant PAM-1, expressed and purified from Escherichia coli, hydrolyzed the N-terminal amino acid from peptide substrates. Favored substrates had positively charged or small neutral amino acids in the N-terminal position. Peptide hydrolysis was inhibited by the metal-chelating agent 1,10-phenanthroline and by the aminopeptidase inhibitors actinonin, amastatin, and leuhistin. However, the enzyme was 100-fold less sensitive toward puromycin (IC₅₀, 135 µM) than other PSA homologues. Following inactivation of the enzyme, aminopeptidase activity was recovered with Zn²⁺, Co²⁺, and Ni²⁺. Silencing expression of pam-1 by RNA interference resulted in 30% embryonic lethality. Surviving adult hermaphrodites deposited large numbers of oocytes throughout the self-fertile period. The overall brood size was, however, unaffected. We conclude that pam-1 encodes an aminopeptidase that clusters phylogenetically with the PSAs, despite attenuated sensitivity toward puromycin, and that it functions in embryo development and reproduction of the nematode.

Received for publication, June 12, 2003 , and in revised form, August 19, 2003.

^* This work was supported by a Biotechnology and Biological Sciences and Research Council project grant (24/S12813) and forms part of the Medical Research Council Co-operative on Zinc Metalloproteinases in Health and Disease at the University of Leeds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-113-343-2891; Fax: 44-113-343-2835; E-mail: bgydrb{at}leeds.ac.uk.

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