Available online at www.sciencedirect.com ABB Archives of Biochemistry and Biophysics 472 (2008) 139–149 www.elsevier.com/locate/yabbi Identification of two epoxide hydrolases in Caenorhabditis elegans that metabolize mammalian lipid signaling molecules Todd R. Harris a, Pavel A. Aronov a, Paul D. Jones b, Hiromasa Tanaka a, Michael Arand c, Bruce D. Hammock a,* a Department of Entomology and Cancer Research Center, University of California, One Shields Avenue, Davis, CA 95616, USA b International Flavors and Fragrances, 1515 State Highway 36, Union Beach, NJ 07735, USA c Institute of Pharmacology and Toxicology, University of Zu¨rich, Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland Received 11 September 2007, and in revised form 12 January 2008 Available online 31 January 2008 Abstract We have identified two genes in the genomic database for Caenorhabditis elegans that code for proteins with significant sequence similarity to the mammalian soluble epoxide hydrolase (sEH). The respective transcripts were cloned from a mixed stage cDNA library from C. elegans. The corresponding proteins obtained after recombinant expression in insect cells hydrolyzed standard epoxide hydrolase substrates, including epoxyeicosatrienoic acids (EETs) and leukotoxins (EpOMEs). The enzyme activity was inhibited by urea-based compounds originally designed to inhibit the mammalian sEH. In vivo inhibition of the enzymes using the most potent of these com- pounds resulted in elevated levels of the EpOMEs in the nematode. These results suggest that the hydrolases are involved in the metab- olism of possible lipid signaling molecules in C. elegans. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Soluble epoxide hydrolase; Lipid signaling; Epoxyeicosatrienoic acid; Leukotoxin; 9,10-Epoxy-12-octadecenoate; 12,13-Epoxy-9-octadecenoate Soluble epoxide hydrolase (sEH)1, an enzyme first char- In mammals, the epoxide hydrolase activity of sEH has acterized in mammals, hydrolyzes an epoxide moiety been implicated in the metabolism of several epoxide-con- through the addition of water [1,2]. Interestingly, the mam- taining lipid signaling molecules [7,8]. This creates a diol malian soluble epoxide hydrolase possesses two catalytic species, which is more water-soluble than the epoxide and activities localized to distinct regions of the enzyme [3]. more readily eliminated through excretion, thereby deplet- The epoxide hydrolase active site is located on the globular ing the supply of the epoxide signal. Also, in some cases the C-terminal region of sEH, while an active site on the N-ter- diols may be biologically active [9]. The epoxyeicosatrie- minal region has been found to display phosphatase activ- noic acids (EETs) are epoxide-containing compounds pro- ity [4]. These domains are expressed separately in plants, duced by the metabolism of arachidonic acid by giving rise to the hypothesis that invertebrates may also cytochrome p450 oxygenases [10]. The EETs have anti- express the N- or C- terminal domain separately [5,6]. inflammatory, as well as vasoconstrictive or vasodilatory properties, depending on the physiological context [11– 13]. sEH has also been shown to metabolize 9,10-epoxy- 12-octadecenoate (called leukotoxin, coronaric acid, or * Corresponding author. Fax: +1 530 752 1537. 9,10-EpOME) and 12,13-epoxy-9-octadecenoate (called E-mail address: [email protected] (B.D. Hammock). isoleukotoxin, vernolic acid or 12,13-EpOME), cyto- 1 Abbreviations used: sEH, soluble epoxide hydrolase; EETs, epoxyei- cosatrienoic acids; EpOMEs, epoxyoctadecenoic acids; PUFAs, polyun- chrome p450 metabolites of linoleic acid [14–16]. These saturated fatty acids; ARDS, acute respiratory distress syndrome; HLD, compounds were identified as having a role in the develop- haloalkane dehalogenase; HAD, haloacid dehalogenase. ment of acute respiratory distress syndrome (ARDS) [17]. 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.01.016 140 T.R. Harris et al. / Archives of Biochemistry and Biophysics 472 (2008) 139–149 Several recent studies have explored the signaling in AATTCGG-30 and 3CEEH22: 50-CAGTCAGCCAGGCGGAACAACT Caenorhabditis elegans. C18 and C20 polyunsaturated fatty GGTCC-30 for CEEH2. CEEH2 50RACE experiments were performed on acids (PUFAs) have been shown to effect directed sperm the total RNA sample with the SMART RACE cDNA Amplification Kit (Clontech, Palo Alto, CA) using the gene specific primer 5CEEH21: 50- movement towards the spermatheca and the formation of GCTCCCCAATCATGAGCAGCAAGTG-30. The 50UTR of CEEH1 synaptic vesicles in neuronal tissues [18,19]. FAT-3 was determined by nested PCRs using the primers 5CEEH1F1: 50-CCACT mutants lack the delta6 fatty acid desaturase and so cannot GTCACCTGGTTGGACG-30 and 5CEEH1R1: 50-CCTTCCAAAACGT convert linoleic acid into C20 PUFAs because this desatur- TTGGCTTCTCCCGCTGC-30, followed by the primers 5CEEH1F2: 50- 0 0 ase activity is a prerequisite to subsequent elongase activity TATAACGCGTTTGGAATCACT-3 and 5CEEH1R2: 5 -CGAACCG AACGCAAGGTCGTGACGGGAGAG-30 using cDNA from a Pro- [20]. These mutants show a number of defects which are Quest C. elegans mixed stage library (Invitrogen, Carlsbad, CA). rescued by dietary supplementation of C20 and C18 PUFAs, including smaller brood size, alterations in the def- Cloning ecation cycle, and defects in chemotaxis [20,21]. These find- ings are exciting, because the nematodes amenability to A ProQuest mixed stage cDNA library from C. elegans was purchased genetic manipulation and the wealth of knowledge con- (Invitrogen, Carlsbad, CA). Primers for CEEH1 were designed to add 0 cerning its physiology make it an excellent model of lipid BglII endonuclease sites on both ends, and a six histidine tag on the 3 end of the coding sequence. Primers for CEEH2 were designed to add EcoRI signaling. Thus the nematode may have implications for endonuclease sites on both ends, and a six histidine tag on the 30 end of the the study of such signaling pathways in mammalian disease coding sequence. The primer pair for CEEH1 was 50-AGATCTATGTTG models. TTTGAAAGTATATACATACAATGT-30 and 50-AGATCTTTAGTG There are many similarities between fatty acid synthesis ATGGTGATGGTGATGCTGATACTTATTCAAAAATTTT-30. The 0 and metabolism in mammals and nematodes. Several primer pair for CEEH2 was 5 -GGATCCATGGGATTCTTTGCCGAC TTGTTCA-30 and 50-GGATCCCTAGTGATGGTGATGGTGATGCA desaturases and elongases in the fatty acid synthetic AGTGGGACTTGAAAGTTTTG-30. pathway of C. elegans have mammalian counterparts with parallel activities [22–26]. The cytochrome p450 monooxy- Baculovirus expression genases responsible for production of the EETs and EpO- MEs are present in the genome, though few have been Recombinant baculoviruses harboring the CEEH1 or CEEH2 cDNA characterized [27–29]. Finally, C. elegans contain arachi- sequence were generated by co-transfection of Sf21 cells derived from Spodoptera frugiperda donic acid and linoleic acid, the precursors of these signal- with the recombinant transfer vector plasmid pACUW21 and Bsu36I-cleaved BacPAK6 viral DNA (Clontech, Palo ing molecules [30,31]. Alto, CA) as previously described [33]. The genome of C. elegans contains five enzymes which display significant sequence similarity to the mammalian Protein purification sEH. Of the five enzymes, two align with the C-terminal region of sEH. This raises the possibility that epoxide Infected high five cells (250 mL) were pelleted and resuspended in hydrolases are involved in the metabolism of epoxide-con- phosphate buffer with 10 mM imidazole. The cells were homogenized with an Ultra-Turrax T25 homogenizer (IKA Works, Wilmington, NC) taining lipids in C. elegans. We report the cloning and char- rotating at 17,500 rpm for three 30 s intervals, with 15 s rests on ice acterization of both hydrolases which display significant between each grinding. The homogenate was centrifuged at 100,000g for sequence similarity to the C-terminal region of soluble 1 h at 4 °C. Ni-NTA HisBind Resin (EMD Biosciences, Inc., Madison, epoxide hydrolase, and their functional disruption in vivo WI) was used to purify the enzymes according to manufacturer’s protocol. using a small molecule inhibitor. Details can be found in supplementary materials. Protein analysis Materials and methods Protein concentration measurements were made using the BCA assay Nematode culture (Pierce, Rockford, IL) with BSA fraction V protein (Sigma–Aldrich, St. Louis, MO) to derive a standard curve. Polyacrylamide gel electrophoresis The N2 (Bristol) strain of C. elegans was used. Plated nematodes were was performed using Novex gels (Invitrogen, Carlsbad, CA) for both cultured on agar plates at 20 °C and fed the OP50 strain of Escherichia coli SDS–PAGE analysis and isoelectric focusing. 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 Radiotracer based epoxide hydrolase activity assay standard technique [32]. For the AUDA–BE liquid culture experiments, the worms were fed OP50. Epoxide hydrolase activity was measured using racemic [3H]-trans-1,3- diphenylpropene oxide (t-DPPO) as substrate as previously described at a Rapid amplification of cDNA ends final concentration of 50 lM [34]. t-DPPO was previously synthesized and purified [35]. Details of the kinetics and IC50 determination can be found Details of worm extract preparation and RNA extraction can be found in supplementary materials. in supplementary materials. CEEH1 and CEEH2 30RACE experiments were performed