Identification of two epoxide hydrolases in Caenorhabditis elegans that metabolize mammalian lipid signaling molecules
Section snippets
Nematode culture
The N2 (Bristol) strain of C. elegans was used. Plated nematodes were cultured on agar plates at 20 °C and fed the OP50 strain of Escherichia coli according to standard technique [32]. Liquid cultures of nematodes were grown in S-basal media and fed the NA22 strain of E. coli according to standard technique [32]. For the AUDA–BE liquid culture experiments, the worms were fed OP50.
Rapid amplification of cDNA ends
Details of worm extract preparation and RNA extraction can be found in supplementary materials. CEEH1 and CEEH2
Cloning of the hydrolases
A tBLASTx search of the NCBI genomic database of C. elegans employing Gallus gallus (GenBank Accession No. Q120010) and Xenopus tropicalis (GenBank Accession No. BC078066) sEH nucleotide and translated amino acid sequences returned five soluble epoxide hydrolase hits. Two of the predicted enzymes, GenBank Accession Nos. NM_064867 and NM_073261, displayed significant sequence similarity to the C-terminus of mammalian sEH, while the other three, GenBank Accession Nos. NM_072133, NM_063993 and NM_072107.3
Discussion
The mammalian sEH is an approximately 60 kDa enzyme composed of two globular regions connected by a short proline-rich linker [2]. Mammalian soluble epoxide hydrolase is thought to be a product of the fusion of two ancestral bacterial genes [5]. The C-terminal region contains the epoxide hydrolase active site and is descended from haloalkane dehalogenase (HLD), while the N-terminal region contains a phosphatase active site and is descended from haloacid dehalogenase (HAD) [5], [38]. When
Acknowledgments
We thank Lesilee Rose and Adam Hayashi for supplying nematodes and bacterial strains and advice on the maintenance of C. elegans. We also thank Kara Schmelzer, Christophe Morisseau and George Kamita for advice at various stages of this project, Katrin Georgi for discussion of oxylipin quantification and Hsing-Ju ‘Cindy’ Tsai for generously sharing instrument time and analytical columns. T.R.H. and P.A.A were supported by NIEHS Advanced Training in Environmental Toxicology Grant T32 ES007059.
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