Takehiko Ogura

Molecular cloning, expression analysis and functional confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata

cDNA cloning of ecdysone receptor (EcR) and ultraspiracle (USP) of the coleopteran Colorado potato beetle Leptinotarsa decemlineata (LdEcR and LdUSP) was conducted. Amino‐acid sequences of the proteins deduced from cDNA sequences showed striking homology to those of other insects, especially the coleopteran yellow mealworm Tenebrio molitor. Northern hybridization analysis showed a 12.4‐kb message for the LdEcR A‐isoform, a 10.5‐kb message for the LdEcR B1‐isoform and a 5.7‐kb message for the LdUSP, in fat body, gut, integument, testis and ovaries. In developmental profile studies, expression of both the LdEcR and LdUSP transcript in integument changed dramatically. In gel mobility shift assays, in vitro translated LdEcR alone bound weakly to the pal1 ecdysone response element, although LdUSP alone did not, and this binding was dramatically enhanced by the addition of LdUSP. LdEcR/LdUSP complex also showed significant binding to an ecdysone agonist, ponasterone A (KD = 2.8 nm), while LdEcR alone showed only weak binding (KD = 73.4 nm), and LdUSP alone did not show any binding. The receptor‐binding affinity of various ecdysone agonists to LdEcR/LdUSP was not correlated to their larvicidal activity to L. decemlineata. From these results, it was suggested that multiple factors including the receptor binding affinity are related to the determination of the larvicidal activity of nonsteroidal ecdysone agonists in L. decemlineata.

Molecular cloning of the ecdysone receptor and the retinoid X receptor from the scorpion Liocheles australasiae

cDNAs of the ecdysone receptor and the retinoid X receptor were cloned from the Japanese scorpion Liocheles australasiae, and the amino acid sequences were deduced. The full‐length cDNA sequences of the L. australasiae ecdysone receptor and the L. australasiae retinoid X receptor were 2881 and 1977 bp in length, respectively, and the open reading frames encoded proteins of 560 and 414 amino acids. The amino acid sequence of the L. australasiae ecdysone receptor was similar to that of the ecdysone receptor‐A of the soft tick, Ornithodoros moubata (68%) and to that of the ecdysone receptor‐A1 of the lone star tick, Amblyomma americanum (66%), but showed lower similarity to the ecdysone receptors of Orthoptera and Coleoptera (53–57%). The primary sequence of the ligand‐binding region of the L. australasiae ecdysone receptor was highly homologous to that of ticks (85–86%). The amino acid sequence of the L. australasiae retinoid X receptor was also homologous to the amino acid sequence of ultraspiracles of ticks (63%) and insects belonging to the orders Orthoptera and Coleoptera (60–64%). The identity of both the L. australasiae ecdysone receptor and the L. australasiae retinoid X receptor to their lepidopteran and dipteran orthologs was less than 50%. The cDNAs of both the L. australasiae ecdysone receptor (L. australasiae ecdysone receptor‐A) and the L. australasiae retinoid X receptor were successfully translated in vitro using a rabbit reticulocyte lysate system. An ecdysone analog, ponasterone A, bound to L. australasiae ecdysone receptor‐A (KD = 4.2 nm), but not to L. australasiae retinoid X receptor. The L. australasiae retinoid X receptor did not enhance the binding of ponasterone A to L. australasiae ecdysone receptor‐A, although L. australasiae retinoid X receptor was necessary for the binding of L. australasiae ecdysone receptor‐A to ecdysone response elements.

Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone A hybrid compounds and evaluation of their molting hormone activity.

Two stereoisomers of a castasterone/ponasterone A hybrid compound, the (20R,22R) and (20R,22S)-isomers of 2alpha,3alpha,20,22-tetrahydroxy-5alpha-cholestan-6-one, were synthesized stereoselectively and their binding activity to the ecdysteroid receptor was determined. From the concentration-response curve for the inhibition of the incorporation of tritiated ponasterone A into ecdysteroid receptor containing insect cells, the concentration (IC50) required to inhibit 50% of the incorporation of radioactivity into cells was evaluated. The IC50 values of the (22R)- and (22S)-isomers were determined to be 0.30 and 38.9 microM against Kc cells, respectively, indicating that the (22R)-isomer is about 100 times more potent than the corresponding (22S)-isomer. IC50 values of these compounds against lepidopteran Sf-9 cells were determined to be 0.36 and 12.9 microM, respectively. The molting hormonal effect was examined in a Chilo suppressalis integument system and the 50% effective concentration for the stimulation of N-acetylglucosamine incorporation into the cultured integument was determined to be 2.7 microM for the (22R)-isomer, while the (22S)-isomer was inactive. On the other hand, both isomers did not show brassinolide-like activity in the rice lamina inclination assay.

Molecular characterization and functional analysis of novel carboxyl/cholinesterases with GQSAG motif in the silkworm Bombyx mori

We have previously cloned and characterized BmJHE, a juvenile hormone (JH)-selective esterase (JHE) that is important for JH titer regulation in the silkworm Bombyx mori. Here, we sought to determine whether multiple genes might function as JH-specific esterase in this species. We searched for putative carboxyl/cholinesterase (CCE) genes having GQSAG, a highly conserved motif in JHE, by the use of silkworm genomic database. Five novel CCE genes (Bmcce-1-5) were identified and their cDNA sequences and intron-exon structures were determined. We investigated the developmental expression patterns of these CCE genes by real-time quantitative PCR analysis and found that their expression patterns varied among developmental stages and organs. Of the proteins produced by the five genes, only BmCCE-5 had the ability to degrade JH; however, this protein might not function as a JH-specific esterase in vivo as it had a high K(m) value for JH. On the other hand, BmCCE-5 degraded general esterase substrates efficiently. Since Bmcce-5 was strongly expressed in Malpighian tubules and the gut, it might function in digestion or xenobiotic metabolism. Our results suggest that of the CCEs containing a GQSAG motif only BmJHE can function as a JH-specific degradation enzyme in the silkworm.

Characterization of Juvenile Hormone Epoxide Hydrolase and Related Genes in the Larval Development of the Silkworm Bombyx mori

Juvenile hormone epoxide hydrolases (JHEHs) are a family of enzymes that hydrolyze juvenile hormones (JHs). They are important in terms of organ-specific regulation and irreversible degradation. In contrast to three JHEH genes (jheh) in Drosophila melanogaster and five jheh in Tribolium castaneum, only one jheh gene has been reported to date in lepidopteran insects. By searching a genome database of the silkworm, KAIKOBLAST, five JHEH-related genes (jheh-r), in addition to Bmjheh, were found. Developmental changes in mRNA expression were brought about revealing several unique patterns for each of jheh-r as to developmental stages and organ-specificity. Recombinant proteins of JHEH-r were expressed using a baculovirus system to evaluate their enzymatic activities. Three of the five JHEH-r recombinant proteins had JH hydrolytic activities. This is the first report on lepidopteran jheh-related genes and also provides the comprehensive analysis of multiple jheh-related genes in an insect species with respect to their functions in enzyme activities.

Virtual screening for ligands of the insect molting hormone receptor.

Insect growth is regulated by the orchestrated event of ecdysteroids and their receptor proteins. Agonists/antagonists of ecdysteroid receptor are predicted to disrupt normal growth, providing good candidates of new insecticides. A database of over 2 million compounds was subjected to a shape-based virtual screening cascade to identify novel nonsteroidal hits similar to the known EcR ligand ponasterone A. Testing revealed micromolar hits against two strains of insect cells. Docking experiments against EcR were used to support the predicted binding mode of these ligands based on their overlay to ponasterone A.

Identification and Expression Analysis of Ras Gene in Silkworm, Bombyx mori

Ras proteins play important roles in development especially for cell proliferation and differentiation in various organisms. However, their functions in the most insect species are still not clear. We identified three ras cDNAs from the silk worm, Bombyx mori. These sequences corresponded to three Ras of Drosophila melanogaster, but not to three mammalian Ras (H-Ras, K-Ras, N-Ras). Subsequently, the expression profiles of ras were investigated by quantitative real-time PCR using whole body of individuals from the embryonic to adult stages, and various tissues of 4th and 5th instar larvae. Each of three Bombyx ras showed different expression patterns. We also showed membrane localization of their products. These results indicate that the three Bombyx Ras are functional and have different roles.

Properties of ecdysteroid receptors from diverse insect species in a heterologous cell culture system – a basis for screening novel insecticidal candidates

Insect development is driven by the action of ecdysteroids on morphogenetic processes. The classic ecdysteroid receptor is a protein heterodimer composed of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle (USP), the insect ortholog of retinoid X receptor. The functional properties of EcR and USP vary among insect species, and provide a basis for identifying novel and species‐specific insecticidal candidates that disrupt this receptor’s normal activity. A heterologous mammalian cell culture assay was used to assess the transcriptional activity of the heterodimeric ecdysteroid receptor from species representing two major insect orders: the fruit fly, Drosophila melanogaster (Diptera), and the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera). Several nonsteroidal agonists evoked a strong response with the L. decemlineata heterodimer that was consistent with biochemical and in vivo evidence, whereas the D. melanogaster receptor’s response was comparatively modest. Conversely, the phytoecdysteroid muristerone A was more potent with the D. melanogaster heterodimer. The additional presence of juvenile hormone III potentiated the inductive activity of muristerone A in the receptors from both species, but juvenile hormone III was unable to potentiate the inductive activity of the diacylhydrazine methoxyfenozide (RH2485) in the receptor of either species. The effects of USP on ecdysteroid‐regulated transcriptional activity also varied between the two species. When it was tested with D. melanogaster EcR isoforms, basal activity was lower and ligand‐dependent activity was higher with L. decemlineata USP than with D. melanogaster USP. Generally, the species‐based differences validate the use of the cell culture assay screen for novel agonists and potentiators as species‐targeted insecticidal candidates.

Purification and cDNA Cloning of LaIT2, a Novel Insecticidal Toxin from Venom of the Scorpion Liocheles australasiae

The novel insecticidal toxin, LaIT2, was isolated from venom of the scorpion Liocheles australasiae. The amino acid sequence of LaIT2 was determined by an Edman degradation analysis and subsequent cDNA cloning. LaIT2 is composed of 59 amino acids with three disulfide bridges, and shares sequence similarity to the scorpion β-KTx peptides.