The Conserved Rieske Oxygenase DAF-36/Neverland Is a Novel Cholesterol-metabolizing Enzyme*

Steroid hormones play essential roles in a wide variety of biological processes in multicellular organisms. The principal steroid hormones in nematodes and arthropods are dafachronic acids and ecdysteroids, respectively, both of which are synthesized from cholesterol as an indispensable precursor. The first critical catalytic step in the biosynthesis of these ecdysozoan steroids is the conversion of cholesterol to 7-dehydrocholesterol. However, the enzymes responsible for cholesterol 7,8-dehydrogenation remain unclear at the molecular level. Here we report that the Rieske oxygenase DAF-36/Neverland (Nvd) is a cholesterol 7,8-dehydrogenase. The daf-36/nvd genes are evolutionarily conserved, not only in nematodes and insects but also in deuterostome species that do not produce dafachronic acids or ecdysteroids, including the sea urchin Hemicentrotus pulcherrimus, the sea squirt Ciona intestinalis, the fish Danio rerio, and the frog Xenopus laevis. An in vitro enzymatic assay system reveals that all DAF-36/Nvd proteins cloned so far have the ability to convert cholesterol to 7-dehydrocholesterol. Moreover, the lethality of loss of nvd function in the fruit fly Drosophila melanogaster is rescued by the expression of daf-36/nvd genes from the nematode Caenorhabditis elegans, the insect Bombyx mori, or the vertebrates D. rerio and X. laevis. These data suggest that daf-36/nvd genes are functionally orthologous across the bilaterian phylogeny. We propose that the daf-36/nvd family of proteins is a novel conserved player in cholesterol metabolism across the animal phyla.

Steroid hormones are crucial for development, growth, and homeostasis in multicellular organisms. Cholesterol and other sterol(s) serve as indispensable precursors for the biosynthesis of steroid hormones, and the conversion of cholesterol to the next specific intermediate is a crucial biochemical step across species (1)(2)(3)(4). In the biosynthesis of vertebrate steroid hormones, cholesterol is commonly converted to pregnenolone by side-chain cleavage catalyzed by the cytochrome P450 monooxygenase P450scc/CYP11A1 (2). The step catalyzed by CYP11A1 is the key rate-limiting step in the synthesis of all vertebrate steroids, and it thus is controlled by numerous physiological responses throughout the life cycle (2,5).
Cholesterol is also required for steroid hormone biosynthesis in protostomes, including nematodes and arthropods (1,6). However, the molecular mechanisms of cholesterol metabolism in these ecdysozoan animals have not been fully elucidated. The principal steroid hormones in nematodes and arthropods are dafachronic acids and ecdysteroids, respectively, both of which play pivotal roles in the regulation of developmental timing, reproduction, and longevity (1,7,8). In the biosynthesis of both dafachronic acids and ecdysteroids, which is different from the biosynthesis of vertebrate steroid hormones, cholesterol is converted to 7-dehydrocholesterol (7dC) 5 by dehydrogenation of the carbons at positions 7 and 8 ( Fig. 1A) (9). Nematodes and arthropods lost the ability to synthesize sterol de novo and must take in exogenous cholesterol or plant sterols from their diet (1,6). Therefore, the conversion of cholesterol to 7dC is the first crucial step of steroid hormone bio-synthesis in these ecdysozoans. However, no enzyme responsible for cholesterol 7,8-dehydrogenation has yet been identified at the molecular level.
The DAF-36/Neverland (Nvd) proteins are strong candidates for harboring cholesterol 7,8-dehydrogenase activity (10,11). Both of the nematode Caenorhabditis elegans daf-36 mutants and the fruit fly Drosophila melanogaster nvd loss of function animals exhibit defects in their steroid hormone production. The developmental abnormalities caused by loss of either daf-36 or nvd function are rescued by the topical application of 7dC, but not cholesterol, strongly indicating that daf-36/nvd is involved in the cholesterol 7,8-dehydrogenation in both nematodes and arthropods. The DAF-36/Nvd proteins possess a Rieske [2Fe-2S] domain and a non-heme iron binding domain (Fig. 1B), and they are therefore classified as Rieske oxygenases (12). Among the identified Rieske oxygenases, bacterial KshA, which shows the highest amino acid similarity with the eukaryotic DAF-36/Nvd proteins (10,11), acts as a 3-ketosteroid 9␣-hydroxylase in the steroid catabolism (13,14). It is therefore likely that DAF-36/Nvd itself has an enzymatic activity on sterol(s).
Here we report biochemical and genetic evidence that the DAF-36/Nvd protein family indeed has cholesterol 7,8-dehydrogenase activity. Intriguingly, the enzymatic activity of DAF-36/Nvd is evolutionarily conserved not only in nematodes and insects but also in deuterostomes. We propose that the daf-36/ neverland genes encode novel cholesterol-metabolizing enzymes across animal phyla.

EXPERIMENTAL PROCEDURES
Molecular Cloning and Vector Construction-Details of the molecular cloning of nvd-Xl, nvd-Dr, nvd-Ci-1, nvd-Ci-2, and nvd-Hp are described in the supplemental material. We deposited the DNA and amino acid sequences of the nvd orthologs in GenBank TM (accession numbers AB607950 -AB607954). To generate the constructs expressing HA-tagged daf-36/nvd genes under the control of a GAL4/UAS system (15), each ORF region was ligated into the pUAST vector with sequences coding three tandem 3ϫ HA tags at the C terminus (16). The generation of the nvd point mutant constructs is described in the supplemental material.
Cell Culture and the in Vitro Enzymatic Activity Assay System-Cholesterol and 7dC were purchased from Sigma. Culturing and transfection of S2 cells were performed as previously described (16). UAS vectors were transfected with the Actin5C-GAL4 construct (a gift from Yasushi Hiromi). Two days after the transfection of S2 cells with the vectors in a 60-mm culture dish, the medium was replaced with fresh medium (2 ml) containing 50 M cholesterol with 0.9% 2-hydroxypropyl ␤-cyclodextrin (Wako) (17). After a 24-h incubation, the medium and cells were collected and mixed with an equal volume of ethyl acetate. Then an aliquot of supernatant (1.2 ml) was collected, desiccated, and redissolved in 200 l of methanol. Reverse phase high pressure liquid chromatography (RP-HPLC) analysis was performed using a 2695 Separations Module apparatus (Waters) equipped with a 996 Photodiode Array Detector (Waters) or a 2996 Photodiode Array Detector (Waters). The RP column used in this study was the Senshu Pak PEGASIL ODS column (4.6 ϫ 250 mm). The conditions were as follows: solvent, 100% methanol; flow rate, 1 ml/min; detection, UV absorption at 281 nm.
Derivatization of Steroids and HPLC-Electrospray Ionization Liquid Chromatography-Tandem Mass Spectrometry (ESI-MS/MS)-Derivatization of steroids using Girard P reagent was performed essentially as described (18). The HPLC fraction corresponding to 7dC was collected, evaporated to dryness, and dissolved in a mixture of 10 l of isopropyl alcohol, 2 l of 1 mg/ml cholesterol oxidase (ToYoBo), and 200 l of 50 mM KH 2 PO 4 (pH 7.0). The mixture was incubated in the dark at room temperature for more than 2 h. The reaction was stopped with 400 l of methanol and then mixed with 30 mg of Girard P reagent (Tokyo Kasei Kogyo) and 30 l of glacial acetic acid. The mixture was left overnight in the dark at room temperature. LC-ESI-MS/MS analyses were performed on an Agilent HPLC system equipped with a Qstar system (Applied Biosystems) and a PEGASIL ODS column (2 ϫ 100 mm, 3 m; Senshu Scientific). The flow rate was 0.2 ml/min, and the injection volume was 500 l (ϳ80% of the total reaction volume). After sample injection in 70% ethanol, the flow from the HPLC was discarded for the first 10 min of the run to remove salts and excess derivatization reagents. The Qstar was then reconnected, and the following gradient was applied: 0 -10 min, methanol 70%; 10 -30 min, methanol 70 -95%; 30 -40 min,  JULY 22, 2011 • VOLUME 286 • NUMBER 29 methanol 95%. All MS spectra were acquired in TOF-MS mode with the ion spray voltage adjusted to 5500 V and the curtain gas 30 V in the positive ion mode. MS/MS spectra of m/z 516.35 were acquired in product ion scan mode with the following parameters: MS range, 50 -550; Q1 resolution, Unit; collision energy, 50 eV.
Fly Strains and Rescue Experiments-D. melanogaster flies were reared on standard agar-cornmeal medium. RNAi to knock down nvd-Dm with 2-286-GAL4 (a gift from Carl S. Thummel) and UAS-nvd-Dm-IR-1 transgenic flies were described previously (11). Transgenic flies carrying HA-tagged nvd-Bm, daf-36, nvd-Dr, and nvd-Xl genes were established using standard protocols. See also supplemental Table S2 and its legend for more details.

RESULTS
Insect and Nematode DAF-36/Nvd Convert Cholesterol to 7dC-To examine whether DAF-36/Nvd proteins convert cholesterol to 7dC, we adopted an in vitro enzymatic assay system using cultured D. melanogaster S2 cells that has been well utilized to characterize the catalytic roles of the ecdysteroidogenic P450s (20,21). S2 cells expressing either the silkworm Bombyx mori nvd (nvd-Bm) (11) or the C. elegans daf-36 (10) gene were incubated with cholesterol, followed by analysis of the cell extracts and culture medium by RP-HPLC. A single HPLC peak was observed at a retention time identical to that of standard 7dC (Fig. 2, A and B), whereas no peak or only a much smaller peak was observed in the GFP-transfected control S2 cells (Fig.  2C). Over a 24-h incubation of S2 cells expressing nvd-Bm or daf-36 with 50 M cholesterol, ϳ0.9 or 2.6% of the cholesterol was converted to 7dC, respectively (supplemental Table S1). For an unknown reason, we did not obtain any detectable protein expression of Nvd-Dm in the S2 cells under our experimental conditions. However, HPLC peaks corresponding to 7dC were observed by incubating cholesterol with S2 cells expressing nvd genes from other drosophilids: Drosophila mojavensis and Drosophila acanthoptera. 6 The identity of the DAF-36/Nvd metabolite was confirmed by HPLC-ESI-MS/MS following derivatization with Girard P reagent (supplemental Fig. S1A), which is widely used for analyses of sterols (18,22). Derivatized 7dC standard gave a molecular ion at m/z 516 that generates fragment ions of m/z 437, 409, 151, and 137 from two major transitions ( Fig. 2D and supplemental Fig. S1B). The derivatized Nvd-Bm cholesterol metabolite generated fragments with mass spectra identical to those of the derivatized 7dC standard (Fig. 2E). These results indicate that both nematode and insect DAF-36/Nvd have cholesterol 7,8-dehydrogenase activity, consistent with the genetic predictions of previous reports (10,11).
Microsomal Localization of DAF-36/Nvd-A previous study reported that the insect cholesterol 7,8-dehydrogenation reaction occurs in microsomes (23). Consistent with this observation, the subcellular distribution of wild type Nvd-Bm with epitope tags (Fig. 3A) was mostly in the peripheral region of microsomes in S2 cells (Fig. 3B). Specifically, signals of wild type Nvd-Bm were detected in the periphery of 92% of the punctate microsomes observed in 51 puncta of 10 S2 cells. In contrast, Nvd-Bm protein lacking the N-terminal region, which contains a computationally predicted transmembrane domain (⌬N-Nvd-Bm; Fig. 3A), was more uniformly distributed in S2 cells (Fig. 3B). We observed that only 31% of microsomes overlapped signals of ⌬N-Nvd-Bm in 54 microsomal puncta of 10 S2 cells. Moreover, we also found that the cholesterol catalytic activity of S2 cells expressing ⌬N-Nvd-Bm was almost completely abolished compared with that of S2 cells expressing the wild type form (Fig. 3D), suggesting that the microsomal localization of  Table S1.
The Conserved Motifs of DAF-36/Nvd Are Crucial for Its Enzymatic Activity-We further investigated the contribution of the conserved protein motifs of DAF-36/Nvd to its catalytic activity. In the bacterial Rieske oxygenases, the Rieske [2Fe-2S] motif and the non-heme iron binding motif are involved in electron transfer and are critical for their catalytic function (24). We introduced site-directed mutations of the conserved amino acids to alanine in either the Rieske [2Fe-2S] motif or the non-heme iron binding motif (Fig. 3C). The single residue replacements H190A and H282A each totally abolished the cholesterol 7,8-dehydrogenase activity of Nvd-Bm (Fig. 3E). Substitutions of the conserved residues of DAF-36, C122A and D234A, also resulted in the loss of catalytic activity (Fig. 3F). These results demonstrate that the canonical conserved motifs of the Rieske oxygenase family are essential for the cholesterol 7,8-dehydrogenase activity of DAF-36/Nvd.

Enzymatic Activity of DAF-36/Nvd Is Required for D. melanogaster Development-
To assess the importance of DAF-36/ Nvd enzymatic activity during development, we examined whether expression of the catalytically inactive form of Nvd could rescue the lethality of transgenic nvd-Dm RNAi D. melanogaster animals (11). When nvd was silenced specifically in the prothoracic gland, which is the endocrine organ that synthesizes ecdysteroids during insect larval development (21), the nvd-Dm RNAi animals died at the first larval instar stage (11) ( Table 1 and supplemental Table S2). Wild type Nvd-Bm rescued the lethality of the nvd-Dm RNAi animals, whereas Nvd-Bm-H190A and Nvd-Bm-H282A did not (Table 1 and supplemental Table S2). In a wild type background, expression of either wild type Nvd-Bm or the catalytically inactive Nvd-Bm in the prothoracic gland had no visible effect on D. melanogaster development (Table 1 and supplemental Table S2). These results support the hypothesis that the catalytic activity of DAF-36/Nvd is required for its in vivo roles.

daf-36/nvd Genes Are Structurally Conserved across Animal
Phyla-A BLAST search using available genome information showed that one or two predicted orthologs of daf-36/nvd are encoded in each genome of several deuterostome species as well as in nematodes and insects (supplemental Fig. S2). cDNAs of putative orthologs of daf-36/nvd were successfully amplified by PCR using reverse transcribed templates prepared from embryos of the sea urchin Hemicentrotus pulcherrimus (nvd-Hp), the sea squirt Ciona intestinalis (nvd-Ci-1 and nvd-Ci-2), the fish Danio rerio (nvd-Dr), and the frog Xenopus laevis (nvd-Xl) ( Fig. 1B; see supplemental material). By determining the cDNA sequences, we confirmed that all of the deduced proteins (Nvd-Hp, Nvd-Ci-1, Nvd-Ci2, Nvd-Dr, and Nvd-Xl) possess the characteristic domains of DAF-36/Nvd, including the Rieske [2Fe-2S] domain, the non-heme iron domain, and the putative transmembrane domain (Fig. 1B and supplemental  Fig. S3). These data suggest that daf-36/nvd genes are structurally conserved and are expressed in vivo in both protostomes and deuterostomes.
daf-36/nvd Genes Are Functionally Orthologous between Protostomes and Deuterostomes-To examine whether deuterostome DAF-36/Nvd proteins also convert cholesterol to 7dC, we transfected S2 cells with plasmids expressing each of the deuterostome genes. In every case, an HPLC peak at a retention time identical to the 7dC standard was observed (Fig. 4, A and B, and supplemental Fig. S4). The conversion efficiencies from cholesterol to 7dC by S2 cells expressing these deuterostome daf-36/nvd genes were ϳ0.2-2.0% from 50 M cholesterol over a 24-h period, which are comparable with the conversion efficiencies of Nvd-Bm and DAF-36 ( Fig. 4C and supplemental Table S1). The enzymatic activity of deuterostome DAF-36/ Nvd also depends on the Rieske [2Fe-2S] motif because a Nvd-Xl protein with an amino acid substitution in the motif lost its enzymatic activity (Fig. 3G).
We next investigated whether daf-36/nvd genes from species other than D. melanogaster can substitute for nvd-Dm function during development. Indeed, expression of either C. elegans daf-36, nvd-Dr, or nvd-Xl allowed nvd-Dm RNAi animals to complete their development and grow into adult stages (Table 1  and supplemental Table S2). Taken together, our results suggest that the daf-36/nvd genes are functionally orthologous between protostomes and deuterostomes.

DISCUSSION
In this study, we have shown that the Rieske oxygenase DAF-36/Nvd is the enzyme responsible for cholesterol 7,8-dehydro-genation, which is the first crucial step in the production of steroid hormones in nematodes and arthropods. We have also found that the protein structures and enzymatic activities of DAF-36/Nvd are conserved not only in protostomes but also in deuterostomes, for which the biosynthesis of dafachronic acids and ecdysteroids have not been confirmed. Importantly, daf-36/nvd genes from C. elegans and vertebrates can substitute for D. melanogaster nvd function in vivo. We therefore propose that the DAF-36/Nvd family of proteins is a novel evolutionarily conserved player in cholesterol metabolism.
It has long been assumed that cholesterol 7,8-dehydrogenation in insects is mediated by a P450 enzyme based on experiments using pharmacological inhibitors (1,23). Although our findings in conjunction with previous data (10, 11) strongly suggest that DAF-36/Nvd is the true identity of the cholesterol   Table S1 for more details. The average amount of 7dC formation during incubation with the DAF-36 was set as 1. The error bars indicate the S.E. calculated from at least three independent experiments. * and **, statistical significance (p Ͻ 0.01 and p Ͻ 0.001 (Student's t test), respectively) of differences of the enzymatic activities between each DAF-36/Nvd and the negative control GFP. D, possible diversification of the molecular mechanisms regulating the metabolism of cholesterol (C) and 7dC for steroid hormone biosynthesis. See "Discussion" for more details. *, DHCR7 is not found in the available genome sequences of the sea squirt C. intestinalis (33).
7,8-dehydrogenase, this does not exclude the possible existence of such a P450. The activation mechanism of Rieske oxygenases by electrons has been proposed to be analogous to that of the P450s (25). Therefore, it would be intriguing to examine whether the P450 inhibitors also hamper the enzymatic activity of DAF-36/Nvd. Compared with the conversion rate (ϳ20 -80%) of previously reported ecdysteroidogenic P450s in S2 cells (21), our study yielded lower conversion (maximally 2.6%) of the substrate. This may partly reflect the insolubility of cholesterol. In our assay, cholesterol was provided in a stable water-soluble vehicle using cyclodextrin (see "Experimental Procedures"), and the enzyme-substrate complex might thus be formed in an irregular way. An alternative possibility is that DAF-36/Nvd requires an unknown cofactor to exhibit its maximal enzymatic activity. In bacteria, the Rieske oxygenases work with one or two partner proteins that transport electrons (12). For example, 3-ketosteroid 9␣-hydroxylation is catalyzed by a complex of the Rieske oxygenase KshA and the Rieske oxygenase reductase KshB (13,14). It is therefore likely that there is an unidentified innate reducing partner of Nvd that is not present or is little present in S2 cells.
Our study has demonstrated that several deuterostome DAF-36/Nvd exhibit the cholesterol 7,8-dehydrogenase activity. It is uncertain, however, whether the reaction of cholesterol to 7dC occurs physiologically in these animals (26). In vertebrates, the reduction of 7dC to cholesterol, which is the reverse of the reaction mediated by DAF-36/Nvd, is instead known to be the critical step in the cholesterol biosynthesis pathway (27). It has been demonstrated that the 7dC reductase (DHCR7) is essential for vertebrate development (28,29) and that deficiency in DHCR7 in humans leads to a metabolic and developmental disorder known as Smith-Lemli-Opitz syndrome (27). We speculate that daf-36/nvd is involved in cholesterol metabolism and homeostasis in deuterostomes by acting in concert with DHCR7 to maintain a balance between the amounts of cholesterol and 7dC (Fig. 4D). To test this hypothesis, detailed expression analyses and knockdown experiments of deuterostome daf-36/nvd are now under way. In addition, the biochemical properties of DAF-36/Nvd, especially its substrate specificity, should also be elucidated.
It should be noted that no orthologs of daf-36/nvd have been found in mammalian genomes (Fig. 4D). In parallel, no structural or functional orthologs of DHCR7 have been identified in the genome information available for insects (30), and a reducing reaction from 7dC to cholesterol is undetectable in C. elegans (31) (Fig. 4D). Curiously, the genome of the more primitive animal, sea anemone Nematostella vectensis (32), encodes both proteins highly similar to DAF-36/Nvd (Protein ID 183599; supplemental Fig. S2) and DHCR7 (Protein ID 229921). Thus, an interesting hypothesis is that during evolution, ecdysozoans lost the catalytic reaction from 7dC to cholesterol and mammals lost that from cholesterol to 7dC. Alternatively, ecdysozoans and mammals might be able to catalyze the interconversion of 7dC and cholesterol by other unique unidentified enzymes that have been evolved independently from the acquisition of daf-36/nvd and DHCR7. Our findings and further studies will shed light on the evolution of cholesterol metabolism and steroid hormone biosynthesis.