Regulation of Ecdysteroid Signaling: Cloning and Characterization of Ecdysone Oxidase, a Novel Steroid Oxidase from the Cotton Leafworm, Spodoptera

One route of inactivation of ecdysteroids in involves ecdysone oxidase-catalyzed conversion into 3-dehydroecdysteroid, followed by irreversible reduction by 3DE 3 α -reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (2.8 kb) encoding this 65 kDa protein. Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and database searching revealed that the enzyme is an FAD flavoprotein which belongs to the Glucose-Methanol-Choline (GMC) oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P) + -linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5 ′ -flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a single copy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.

2 SUMMARY One route of inactivation of ecdysteroids in insects involves ecdysone oxidasecatalyzed conversion into 3-dehydroecdysteroid, followed by irreversible reduction by 3DE 3α-reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reversetranscriptase PCR-based approach has been used to clone the cDNA (2.8 kb) encoding this 65 kDa protein. Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and database searching revealed that the enzyme is an FAD flavoprotein which belongs to the Glucose-Methanol-Choline (GMC) oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P) + -linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5′-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a single copy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system. INTRODUCTION 4 significance of such competitive reaction between ecdysone oxidase and 3DE 3βreductase is uncertain. Furthermore, particularly puzzling is the occurrence of enzymes for reversible interconversion of ecdysteroid and their 3-dehydro-derivatives in tissues of several species that are incapable of producing 3-epiecdysteroids (12,19).
As part of our studies aimed at elucidating the regulation of ecdysteroid titer, including the reactions involved in ecdysteroid inactivation in S. littoralis, we have cloned and characterized the cDNA encoding the 3DE 3β-reductase (hemolymph) (20) and 3DE 3α-reductase (Malpighian tubules) (16). Furthermore, we have purified ecdysone oxidase from S. littoralis midgut plus attached Malpighian tubules and evidence suggests that the native enzyme consists of a trimer with apparent molecular mass of approximately 190 kDa and subunit molecular mass of approximately 64 kDa (21). Amino acid sequences of the NH 2 -terminus as well as of interior tryptic peptides of the purified enzyme have been determined.
We now report the molecular cloning, characterization and heterologous expression of the cDNA encoding ecdysone oxidase of S. littoralis, together with analysis of the organization of the corresponding gene and its promoter. Conceptual translation and amino acid sequence analysis indicates that ecdysone oxidase belongs to the Glucose-Methanol-Choline (GMC) oxidoreductase superfamily. In fact, the ecdysone oxidase is novel in being, hitherto, the only eukaryotic animal steroid-metabolizing oxidase enzyme to be reported and characterized at the molecular level. By contrast, oxidation of steroids in vertebrates occurs via NAD(P) + -linked dehydrogenases.

EXPERIMENTAL PROCEDURES
Protein Sequence -Ecdysone oxidase from S. littoralis was purified as described previously (21), subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel, electrotransferred to ProBlott™ membrane, and visualized by Coomassie staining. A single band was observed, which was excised and sequenced by an automated pulsed liquid-phase sequencer (Applied Biosystems 471A) giving the NH 2terminal amino acid sequence as XYAVGGXAGAGPAATYVA (where X represents an unidentified amino acid). To obtain sequences of the interior region of the enzyme, the purified protein was excised from an SDS-PAGE gel and cleaved with trypsin. The resulting tryptic peptides were purified by high-performance liquid chromatography and sequenced. The best internal sequence was: ETPYXWXFTTIXXGVT.
cDNA Cloning and Sequencing -A PCR-based cloning strategy was used to isolate a cDNA fragment encoding the region between the two peptide sequences described above. Four degenerate primers were synthesized. Primer EO3-3 and EO-5 were designed on the basis of a part of the NH 2 -terminal amino acid sequence (EO3-3, 5'-GTN GGN GCI GGI CCI GC, where I represents inosine, N is A/T/C/G; EO-5, 5'-GCN GGI CCN GCI GCN ACN TAY GT, where Y represents T/C); primers EO-AS3-2 and EO-AS3-3 were designed based on the internal proteolytic peptide fragment (EO-AS3-2, 5'-ATI GTI GTR AAI NIC CAI NIR TAI GG, where R represents A/G; EO-AS3-3, 5'-GTI GTR AAI NIC CAI NIR TAI GGI GT).  72°C for 7 min using EO3-3 and EO-AS3-2 primers. This PCR product was used as   template for the nested PCR, which was carried out as follows: 1 cycle of 94°C for 3   min and 30 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 3 min and 1 cycle of   72°C for 7 min using EO-5 and EO-AS3-3 primers. The nested PCR yielded a product of approximately 300 bp.
The purified PCR product was cloned into pGEM ® -T Easy Vector (Promega, Ltd.).
Transformants were screened by colony PCR using M13 forward and reverse primers The sequences of three independent clones were compared to detect errors that could have occurred during the reverse-transcription and PCR amplification.

SDS-PAGE -2x10 6 High Five cells infected at the multiplicity of infection (MOI)
of 10 with wild type Autographa californica nucleopolyhedrovirus (AcMNPV), or with recombinant baculovirus, were homogenized at different times post-infection in 1 ml of 100 mM Tris buffer (pH 8.0), containing 0.05% NaN 3 . The homogenate was centrifuged at 17,000 x g for 10 min and the supernatant was recentrifuged at 170,000 x g for 1 h to obtain the cytosolic fraction. 5 µl of each fraction was added to an equal volume of SDS sample buffer and boiled for 5 min before loading onto an 8% polyacrylamide gel (22).
After electrophoresis, the gel was stained with Coomassie brilliant blue and destained to allow visualization of the protein.
Enzyme Assay -Assay of the recombinant ecdysone oxidase activity was performed in triplicate by modification of the method described in (21). binding motifs (28) at -35 and -65 (Fig. 4B). There is a repeat sequence (TTA) 6 at -285, which is likely to be a microsatellite sequence. However, the 11 bp motif (CT G / C A G / C AGTAN A / T ) is repeated 5 times at -548, -226, -171, -131 and -107, and since it resembles the binding motif for FTZ-F1, a transcription factor that belongs to the nuclear hormone receptor superfamily (29,30), it is very likely that the ecdysone oxidase in S. littoralis is regulated by a factor of this type. Northern blot analysis of mRNA isolated from midgut at different developmental stages of the last larval instar (Fig. 7B) revealed that the ecdysone oxidase mRNA is mainly expressed in the prepupal stage of the instar and in the early pupal stage. The mRNA started to be detectable at 20 h into the last larval instar, although its expression level during the feeding stage was very low. It started to increase in intensity from 66 h, and reached a peak at 96 h, and quickly decreased just before pupation, rising again after pupation. The developmental profile of ecdysone oxidase mRNA expression slightly preceded that of the enzyme activity. Attempt to normalize the northern blot have proven problematic, because of developmental changes in the transcript levels of the probes and for normalization.

Induction of the Ecdysone Oxidase Transcript by 20-Hydroxyecdysone and RH-5992
-To examine possible induction of ecdysone oxidase mRNA transcription, we injected larvae twice at 42 h and 48 h into the last larval instar with 20hydroxyecdysone or RH-5992 i.e. before the natural increase in enzymatic activity.
Total RNA was isolated from the midgut of the larvae 4 h or 18 h after the final injection and analyzed by northern blotting. The mRNA encoding ecdysone oxidase was strongly induced by RH-5992 after the final injection (Fig. 8). in the 5′-flanking region (Fig. 4B).

Expression of Ecdysone Oxidase in High Five cells -Monolayers of High
Database searching revealed that ecdysone oxidase belongs to the GMC oxidoreductase superfamily (31,32 (33). However, in the case of this substrate, the conformation of the sterol ring structure is quite different from that in ecdysteroids owing to the A/B cis ring junction in the latter. Furthermore, the significance of the observation that the primary sequence alignment shows that ecdysone oxidase has highest similarity to glucose dehydrogenase from Drosophila is unclear. Although ecdysone oxidase activity has been reported in Drosophila (34), no sequence has been annotated as a putative ecdysone oxidase. There is some significant similarity in gene structure between the ecdysone oxidase in S. littoralis and the two Drosophila glucose dehydrogenase genes.
All 3 have a large intron close to the transcription start site and upstream of the start codon. The last, relatively small, intron largely separates the first FAD binding domain from the remainder of the coding region, and consequently the last exon is large.
However the S. littoralis ecdysone oxidase gene has an additional intron compared to these glucose dehydrogenase genes. The P. serotina (R)-mandelonitrile lyase gene has a different structure.
Ecdysone oxidase activity has been demonstrated in midgut, fat body, hemolymph and integument of larvae in various insect species, although the predominant activity appears to be in midgut during the larval stage (11,35,36). Our northern blot analysis shows that the mRNA for ecdysone oxidase was highly expressed in the midgut, but undetectable in other tissues examined at the prepupal stage in S. littoralis under our conditions (Fig. 6). These data indicate that midgut is the main tissue which can produce ecdysone oxidase during the prepupal stage.
The developmental profile of the enzymatic activity (Fig. 7)  the active molting hormone titer is highest (6), suggests that the enzyme may play a role in deactivation of endogenous ecdysteroids. In Manduca sexta, ecdysone oxidase activity also increases at a similar developmental stage as observed in S. littoralis (36).
Also, the developmental profile showed that ecdysone oxidase is highly expressed in the early pupal stage. In many insects, high ecdysone oxidase activity has been detected in pupae (35), but the significance of this enzyme in the pupal stage is unclear.
In M. sexta, it has been shown that ecdysone oxidase activity can be induced by the ecdysteroid agonist, RH-5849, but not by 20-hydroxyecdysone (37). Similarly, in the current work, mRNA for ecdysone oxidase is induced by the ecdysteroid agonist, RH-5992, with no clear induction by 20-hydroxyecdysone (Fig. 8). Furthermore, in vivo expression of this enzyme seems to developmentally follow to some extent the ecdysteroid titer determined previously in hemolymph of a different batch of insects (Fig. 7). These data suggest that the gene encoding ecdysone oxidase is ecdysteroidresponsive. Although there is no obvious match to the ecdysone receptor binding motif (38,39) in the 5'-flanking region of this gene (Fig. 4B), there is an apparent Broad-Complex binding motif (27), which is an ecdysone-responsive element that is known to be required for induction of late genes. Interestingly, there are five copies of a motif in this promoter region, that is similar to the FTZ-F1 binding motif (30). FTZ-F1 is a member of the nuclear hormone receptor superfamily and is expressed in the prepupal stage (40,41). It is induced by ecdysteroids and functions as a regulatory factor for the late genes (30,42). Therefore, it is quite possible that ecdysone oxidase is regulated by FTZ-F1 or a similar factor.
Ecdysteroid 26-hydroxylase, another ecdysteroid inactivation enzyme, is also induced by ecdysteroids in S. littoralis (43). It is possible that both these inactivation systems may have similar regulatory mechanisms. Both enzymes catalyze the first steps in ecdysteroid inactivation pathways and might be expected to be subject to regulation.