Phosphoconjugation and dephosphorylation reactions of steroid hormone in insects.

In insects, the major products of phase II metabolism of ecdysteroids, which include the molting hormone, are phosphate esters. The phosphoconjugation pathway is a reversible process, comprising two enzyme systems: ecdysteroid 22-kinase (EcKinase) and ecdysteroid-phosphate phosphatase (EPPase). We report here that: (1) the biochemical characteristics of EcKinase and EPPase, (2) the physiological significance of the reciprocal conversion of ecdysteroids and ecdysteroid phosphates in the ovary-egg system in insects, (3) the biochemical mechanism by which ecdysteroid phosphates are synthesized in the ovary, transferred to eggs, and finally dephosphorylated in eggs, and (4) the possible catalytic steps of EcKinase and EPPase on the basis of the data obtained by an in silico study. From these studies, it is obvious that ecdysteroid phosphates as well as steroid sulfates, which are major products of phase II metabolism in mammals, function as precursors for the formation of biologically active hormones.

The role of ecdysteroid 22-kinase in the accumulation of ecdysteroids in ovary of silkworm Bombyx mori.

In the silkworm Bombyx mori, ecdysteroid 22-kinase (EcKinase) has been shown to catalyze the conversion of free ecdysteroids to physiologically inactive ecdysteroid 22-phosphates. First, a phylogenetic analysis of EcKinase showed that many proteins homologous to B. mori EcKinase are found mainly in Ecdysozoa, including insects and nematodes. Next, by RT-PCR analysis combined with laser microdissection and by immunohistochemistry, we demonstrated that free ecdysteroids in the B. mori ovary are synthesized in follicle cells and transferred into the oocyte where they are phosphorylated by EcKinase whose mRNA originates from nurse cells and the oocyte itself.

Synthesis and phosphorylation of ecdysteroids during ovarian development in the silkworm, Bombyx mori.

In the silkworm, Bombyx mori, it has been demonstrated that most free ecdysteroids in the ovary are converted to physiologically inactive ecdysteroid 22-phosphates, which are then transformed back to free ecdysteroids during early embryonic development. Two specific enzymes involved in the reciprocal conversion of ecdysteroids, namely, ecdysteroid 22-kinase (EcKinase) and ecdysteroid-phosphate phosphatase, have been isolated and characterized. In this study, we first attempted a phylogenetic analysis of EcKinase. The resulting phylogenetic tree showed that many proteins homologous to B. mori EcKinase are found not only in ecdysozoa, including insects and nematodes, but also in teleosts, fungi, and bacteria. We then investigated the sites where free ecdysteroids are synthesized and phosphorylated in the ovary. We found that (1) the mRNAs of two P450 enzymes involved in ecdysteroidogenesis, CYP306a1 (25-hydroxylase) and CYP314a1 (20-hydroxylase), are expressed mainly in follicle cells, (2) EcKinase mRNA localizes in the oocyte and nurse cells, and (3) EcKinase immunoreactivity localizes mainly in the external region of the oocyte, not in nurse cells or follicle cells. From these results, we suggest that ecdysteroids in the B. mori ovary are synthesized in follicle cells and transferred into the oocyte, where they are phosphorylated by EcKinase, whose mRNA originates from nurse cells and the oocyte itself.

Molecular cloning of ecdysone 20-hydroxylase and expression pattern of the enzyme during embryonic development of silkworm Bombyx mori.

In various insects, 20-hydroxyecdysone (20E) is indispensable for embryonic development. In eggs of the silkworm Bombyx mori, 20E has been demonstrated to be produced by two metabolic pathways: de novo synthesis from cholesterol and dephosphorylation of ovary-derived physiologically inactive ecdysteroid phosphates. In the former, ecdysone 20-hydroxylase (E20OHase) has been suggested to be a key enzyme. In the latter, it has been demonstrated that the dephosphorylation of ecdysteroid phosphates is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (EPPase). In this study, a cDNA encoding E20OHase was cloned from 3-day-old nondiapause eggs of B. mori and sequenced using PCR techniques. The protein exhibited the signature sequences characteristic of P450 enzymes, and mediated the conversion of ecdysone to 20E using the baculovirus expression system. Semi-quantitative analysis revealed that the E20OHase mRNA is expressed predominantly during gastrulation and organogenesis in nondiapause eggs, but is scarcely detected in diapause eggs whose development is arrested at the late gastrula stage. The developmental changes in the expression patterns of E20OHase and EPPase suggest that both enzyme activities are regulated at the transcription level, and both enzymes contribute cooperatively to 20E formation during embryonic development.

Purification, kinetic characterization, and molecular cloning of a novel enzyme, ecdysteroid 22-kinase.

This is the first report succeeding in the isolation and characterization of an enzyme and its gene involved in the phosphorylation of a steroid hormone. It has been demonstrated that ecdysteroid 22-phosphates in insect ovaries, which are physiologically inactive, serve as a "reservoir" that supplies active free ecdysteroids during early embryonic development and that their dephosphorylation is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (Yamada, R., and Sonobe, H. (2003), J. Biol. Chem. 278, 26365-26373). In this study, ecdysteroid 22-kinase (EcKinase) was purified from the cytosol of the silkworm Bombyx mori ovaries to about 1,800-fold homogeneity in six steps of column chromatography and biochemically characterized. Results obtained indicated that the reciprocal conversion of free ecdysteroids and ecdysteroid 22-phosphates by two enzymes, EcKinase and ecdysteroid-phosphate phosphatase, plays an important role in ecdysteroid economy of the ovary-egg system of B. mori. On the basis of the partial amino acid sequence obtained from purified EcKinase, the nucleotide sequence of the cDNA encoding EcKinase was determined. The full-length cDNA of EcKinase was composed of 1,850 bp with an open reading frame encoding a protein of 386 amino acid residues. The cloned cDNA was confirmed to encode the functional EcKinase using the transformant harboring the open reading frame of EcKinase. A data base search showed that EcKinase has an amino acid sequence characteristic of phosphotransferases, in that it harbors Brenner's motif and putative ATP binding sites, but there are no functional proteins that share high identity with the amino acid sequence of EcKinase.

Molt-inhibiting hormone-mediated regulation of ecdysteroid synthesis in Y-organs of the crayfish (Procambarus clarkii): involvement of cyclic GMP and cyclic nucleotide phosphodiesterase.

Crustacean molt-inhibiting hormone (MIH), a polypeptide secreted by the X-organ/sinus gland complex of the eyestalks, regulates molting by inhibiting the synthesis of ecdysteroids by Y-organs. Previous results indicate the biosynthetic activity of Y-organs is likely controlled not only by the level of hemolymphatic MIH, but also by the responsiveness of Y-organs to MIH. The present studies were conducted to (a) identify the second messenger that mediates MIH-induced suppression of ecdysteroidogenesis, and (b) assess the possible involvement of cyclic nucleotide phosphodiesterase (PDE) in determining the responsiveness of Y-organs to MIH. Adding 8-bromo cAMP or 8-bromo cGMP to incubation medium significantly suppressed ecdysteroid production by Y-organs of the crayfish (Procambarus clarkii). Incubating Y-organs with MIH produced a significant increase in glandular cGMP, but MIH had no effect on glandular cAMP. The composite data indicate that MIH-induced suppression of ecdysteroidogenesis in Y-organs of P. clarkii is mediated by cGMP. Subsequently, Y-organs from various stages of the molt cycle were incubated with MIH, 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of PDE), or both. Y-Organs from middle and late premolt stages were poorly responsive to MIH alone. Including IBMX in the incubation medium enhanced the responsiveness of the Y-organs to MIH at these stages. Moreover, glandular PDE activity in the Y-organs at these stages was significantly higher than other stages. The combined results suggest that molt cycle-associated changes in PDE activity affect the ability of MIH to stimulate cGMP accumulation and suppress ecdysteroidogenesis in Y-organs of P. clarkii.

A designed curved DNA segment that is a remarkable activator of eukaryotic transcription.

To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly, T20 was found to activate transcription in all the stable transformants, irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes, which is of importance in many fields, and may also be useful for the construction of nonviral vectors for gene therapy.

Release of ecdysteroid-phosphates from egg yolk granules and their dephosphorylation during early embryonic development in silkworm, Bombyx mori.

Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.

Cloning and characterization of a molt-inhibiting hormone-like peptide from the prawn Marsupenaeus japonicus.

Recently, it was demonstrated by PCR amplification that an additional molt-inhibiting hormone (MIH)-like peptide was present in the kuruma prawn Marsupenaeus japonicus. In this study, a cDNA encoding this peptide designated Pej-MIH-B was cloned. The Pej-MIH-B gene was expressed strongly in the nerve cord, and weakly in the eyestalk. It was possible to isolate Pej-MIH-B from the sinus glands in the eyestalks. The recombinant Pej-MIH-B expressed in Escherichia coli showed low molt-inhibiting activity, but did not exhibit hyperglycemic activity. These results suggest that Pej-MIH-B does not function as MIH or CHH intrinsically, but may have some unknown functions.

Ecdysteroids during early embryonic development in silkworm Bombyx mori: metabolism and functions.

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various molecular species of ecdysteroids in free and conjugated forms. In B. mori eggs, 20-hydroxyecdysone (20E) is a physiologically active molecule. In nondiapause eggs, 20E is produced by the conversion of maternal conjugated ecdysteroids (ecdysteroid-phosphates) and by de novo biosynthesis. In contrast, in diapause eggs, neither of these metabolic processes occurs. In de novo biosynthesis of 20E in B. mori eggs, hydroxylation at the C-20 position of ecdysone, which is catalyzed by ecdysone 20-hydroxylase, is a rate-limiting step. Furthermore, we found that a novel enzyme, called ecdysteroid-phosphate phosphatase (EPPase), specifically catalyzes the conversion of ecdysteroid-phosphates to free ecdysteroids. The developmental changes in the expression pattern of EPPase mRNA correspond closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs. EPPase is localized in the cytosol of yolk cells, and the bulk of maternal ecdysteroid-phosphates is bound to vitellin and stored in yolk granules. The vitellin-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase. Therefore, to examine how ecdysteroid-phosphates are hydrolyzed by EPPase during embryonic development further investigations were focused on yolk granules. Recent data indicate that acidification in yolk granules, induced by vacuolar H(+)-ATPase, triggers the dissociation of ecdysteroid-phosphates from the vitellin-ecdysteroid-phosphates complex and the dissociated ecdysteroid-phosphates are released from yolk granules to the cytosol. To explain the process of the increase in the level of 20E during embryonic development in B. mori eggs, a possible model is proposed.

Regulation of ecdysteroid secretion from the Y-organ by molt-inhibiting hormone in the American crayfish, Procambarus clarkii.

In crustaceans, molt-inhibiting hormone (MIH) has been proposed to regulate molting by inhibiting the secretion of ecdysteroids from the Y-organ. Thus, MIH titer in the hemolymph should be inversely related to ecdysteroid titers during the molt cycle. However, it has not been demonstrated whether the MIH titer in the hemolymph changes during the molt cycle. The purpose of this study was to determine the changes in the MIH titers in the hemolymph during the molt cycle of the American crayfish, Procambarus clarkii, and to discuss the role of MIH in regulation of molting. As predicted by the hypothesis, the hemolymph MIH titer was high at the intermolt stage when the hemolymph ecdysteroid titer was low, and the MIH titer decreased to a basal level at the early premolt stage when the hemolymph ecdysteroid titer began to increase slightly. At the middle premolt stage when the hemolymph ecdysteroid titer increased, the MIH titer was restored to a level as high as that during the intermolt stage. This is in contradiction to the hypothesis. However, the Y-organs at this stage scarcely responded to MIH both in vitro and in vivo. The present findings suggest that ecdysteroid secretion from the Y-organ may be regulated not only by changes in the hemolymph MIH titer, but also by changes in the responsiveness of the Y-organ to MIH.

Measurement of molt-inhibiting hormone titer in hemolymph of the American crayfish, Procambarus clarkii, by time-resolved fluoroimmunoassay.

In order to determine the titer of molt-inhibiting hormone (Prc-MIH) in the hemolymph of the American crayfish Procambarus clarkii, a time-resolved fluoroimmunoassay (TR-FIA) was established using specific antibodies against N-terminal and C-terminal segments of Prc-MIH. The lowest limit of detection of Prc-MIH in TR-FIA was 10 amol/assay. The Prc-MIH titers in the hemolymph were 6.53 fmol/ml at the intermolt stage and 1.25 fmol/ml at the early premolt stage. This result is consistent with the long-known hypothesis that the Y-organ is inhibited by MIH during the intermolt stage, whereas the Y-organ is activated by being freed from the inhibitory regulation of MIH.

Purification, kinetic characterization, and molecular cloning of a novel enzyme ecdysteroid-phosphate phosphatase.

From eggs of the silkworm Bombyx mori, we isolated a novel enzyme that is involved in the conversion of physiologically inactive conjugated ecdysteroids, such as ecdysone 22-phosphate and 20-hydroxyecdysone 22-phosphate, to active free ecdysteroids. This enzyme, called ecdysteroid-phosphate phosphatase (EPPase), was located in the cytosol fraction and differed from nonspecific lysosomal acid phosphatases in various enzymic properties. EPPase was purified about 3,000-fold to homogeneity by seven steps of column chromatography. The cDNA clone encoding EPPase was isolated by reverse transcription polymerase chain reaction using degenerate primers on the basis of the partial amino acid sequence obtained from purified EPPase and by subsequent 3'- and 5'-rapid amplification of cDNA ends. The full-length cDNA of EPPase was found to be composed of 1620 bp with an open reading frame encoding a protein of 331 amino acid residues. A data base search showed that there was no functional protein with the amino acid sequence identical to that of EPPase. Northern blot analysis revealed that EPPase mRNA was expressed predominantly during gastrulation and organogenesis in nondiapause eggs but was not detected in diapause eggs whose development was arrested at the late gastrula stage. In nondiapause eggs, the developmental changes in the expression pattern of EPPase mRNA corresponded closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs.

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori.

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.

Studies on embryonic diapause in thepnd mutant of the silkworm,Bombyx mori : V. Identification of apnd + gene-specific protein.

Two-dimensional gel electrophoresis has been used to analyse patterns of proteins synthesized in the eggs from theBombyx mutantpnd, whose homozygous embryo never enters diapause owing to a genetic defect. At the middle to late stage of gastrulation the diapause type of the heterozygous embryo, derived from a homozygouspnd female mated to a wild-type male, synthesizes eight proteins which are not detected in the homozygouspnd embryo. To examine the relationship between embryonic diapause and the appearance of the heterozygote-specific proteins, the pattern of proteins synthesized in the heterozygotes of the diapause type was compared with that in heterozygotes which were artificially altered so that they would continue development. Only one of the eight heterozygote-specific proteins was constitutively synthesized according to the embryonic genome, irrespective of their developmental state, whereas appearance of the remaining seven proteins was exclusively dependent on their developmental nature. This finding strongly suggests that the unique protein might result from the expression of thepnd + gene, and the other proteins might be synthesized along with diapause initiation in the heterozygotes. The possible role of the putativepnd + gene-specific protein at the onset of embryonic diapause is discussed in relation to the action of the diapause factor, which predetermines embryonic diapause by affecting the developing oocytes.

Studies on the embryonic diapause of thepnd mutant of the silkworm,Bombyx mori : III. Accumulation of alanine in the diapause eggs.

The changing pattern in free amino acids following the embryonic development in the non-diapause and diapause eggs of thepnd mutant of theBombyx silkworm was studied. In the diapause eggs, heterozygous for thepnd gene, the levels of most of the amino acids increased concomitantly with the substantial decrease in oxygen consumption. Among the amino acids, alanine was the only amino acid that showed a large accumulation. The accumulation could be induced experimentally in the non-diapause eggs, homozygous for thepnd gene, by reducing the oxygen supply. In contrast, it was prevented in the diapause eggs by increasing the oxygen supply. From these results, it is suggested that the alanine accumulation is the consequence of anaerobic metabolism in the eggs during diapause. The possible significance of the alanine accumulation is discussed in relation to the anaerobic carbohydrate metabolism associated with the embryonic diapause in thepnd mutant.

THE DIAPAUSE FACTOR FROM THE SILKWORM, BOMBYX MORI L.: PURIFICATION AND INACTIVATION EXPERIMENTS.

The diapause factor, which is responsible for the induction of èmbryonic dia pause in the silkworm (Bombyx mori L.), has been partially purified from the extract of adult heads by means of protein purification procedures, including the use of gel filtration of Sephadex, column chromatography on Dowex 1, isoelectric focusing and phenol extraction. Two species of the diapause factor could be recognized in respect to their molecular weight. They were separated by Sephadex G-25 and their molecular weights were estimated to be about 2,000 and 5,000 from the gel filtration results. The smaller species was purified about 90-fold in specific activity, and its isoelectric point was determined by isoelectric focusing to be at about pH 4.5. The biological activity of the partially purified principle could be abolished by incubation with several proteolytic enzymes (trypsin, α-chymotrypsin and pronase), or by treatment with amino acid-modifying reagents such as tyrosinase, N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide, but was not affected by incubation with neuraminidase, cyanogen bromide or photooxidation in the presence of methylene blue.