Jian-Hua Chen

Regulation of ecdysteroid signalling: molecular cloning, characterization and expression of 3-dehydroecdysone 3 alpha-reductase, a novel eukaryotic member of the short-chain dehydrogenases/reductases superfamily from the cotton leafworm, Spodoptera littoralis.

One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalysed conversion into 3-dehydroecdysone (3DE), followed by irreversible reduction by 3DE 3 alpha-reductase to 3-epiecdysone. The 3DE 3 alpha-reductase has been purified and subjected to limited amino acid sequencing. It occurs as two distinct forms, including a probable trimer of subunit molecular mass of approx. 26 kDa. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.2 kb) encoding the 26 kDa protein. Northern blotting showed that the mRNA transcript was expressed in Malpighian tubules during the early stage of the last larval instar. Conceptual translation of the 3DE 3 alpha-reductase cDNA and database searching revealed that the enzyme belongs to the short-chain dehydrogenases/reductases superfamily. Furthermore, the enzyme is a novel eukaryotic 3-dehydrosteroid 3 alpha-reductase member of that family, whereas vertebrate 3-dehydrosteroid 3 alpha-reductases belong to the aldo-keto reductase (AKR) superfamily. Enzymically active recombinant 3DE 3 alpha-reductase has been produced using a baculovirus expression system. Surprisingly, we observed no similarity between this 3DE 3 alpha-reductase and a previously reported 3DE 3 beta-reductase, which acts on the same substrate and belongs to the AKR family.

Molecular cloning and characterization of hemolymph 3-dehydroecdysone 3beta-reductase from the cotton leafworm, Spodoptera littoralis. A new member of the third superfamily of oxidoreductases.

The primary product of the prothoracic glands of last instar larvae of Spodoptera littoralis is 3-dehydroecdysone (3DE). After secretion, 3DE is reduced to ecdysone by 3DE 3β-reductase in the hemolymph. We have previously purified and characterized 3DE 3β-reductase from the hemolymph of S. littoralis. In this study, cDNA clones encoding the enzyme were obtained by reverse transcription-polymerase chain reaction, employing primers based on the amino acid sequences, in conjunction with 5′- and 3′-rapid amplification of cDNA ends. Multiple polyadenylation signals and AT-rich elements were found in the 3′-untranslated region, suggesting that this region may have a role in regulation of expression of the gene. Conceptual translation and amino acid sequence analysis suggest that 3DE 3β-reductase from S. littoralis is a new member of the third superfamily of oxidoreductases. Northern analysis shows that 3DE 3β-reductase mRNA transcripts are widely distributed, but are differentially expressed, in some tissues. The developmental profile of the mRNA revealed that the gene encoding 3DE 3β-reductase is only transcribed in the second half of the last larval instar and that this fluctuation in expression accounts for the change in the enzyme activity during the instar. Southern analysis indicates that the 3DE 3β-reductase is encoded by a single gene, which probably contains at least one intron.

Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans

Numerous invertebrate species belonging to several phyla cannot synthesize sterols de novo and rely on a dietary source of the compound. SCPx (sterol carrier protein 2/3-oxoacyl-CoA thiolase) is a protein involved in the trafficking of sterols and oxidation of branched-chain fatty acids. We have isolated SCPx protein from Spodoptera littoralis (cotton leafworm) and have subjected it to limited amino acid sequencing. A reverse-transcriptase PCR-based approach has been used to clone the cDNA (1.9 kb), which encodes a 57 kDa protein. Northern blotting detected two mRNA transcripts, one of 1.9 kb, encoding SCPx, and one of 0.95 kb, presumably encoding SCP2 (sterol carrier protein 2). The former mRNA was highly expressed in midgut and Malpighian tubules during the last larval instar. Furthermore, constitutive expression of the gene was detected in the prothoracic glands, which are the main tissue producing the insect moulting hormone. There was no significant change in the 1.9 kb mRNA in midgut throughout development, but slightly higher expression in the early stages. Conceptual translation of the cDNA and a database search revealed that the gene includes the SCP2 sequence and a putative peroxisomal targeting signal in the C-terminal region. Also a cysteine residue at the putative active site for the 3-oxoacyl-CoA thiolase is conserved. Southern blotting showed that SCPx is likely to be encoded by a single-copy gene. The mRNA expression pattern and the gene structure suggest that SCPx from S. littoralis (a lepidopteran) is evolutionarily closer to that of mammals than to that of dipterans.

Molecular cloning and induction of nuclear receptors from insect cell lines.

Fragments of EcR and USP were cloned from two insect cell lines, Sf21 and High Five cells (derived respectively from Spodoptera frugiperda and Trichoplusia ni), using a PCR-based approach employing degenerate primers designed on the basis of conserved regions of nuclear receptors, together with 5- and 3-RACE. An additional orphan nuclear receptor, HR4 fragment, was cloned from High Five cells. Comparison of these fragments with Manduca sexta counterparts showed that the cloned SfEcR [ecdysone receptor (EcR) from Sf21 cells] had high similarity to MsEcR-B1, whereas the cloned SfUSP [ultraspiracle (USP) from Sf21 cells] and TnUSP (USP from High Five cells) matched more closely to MsUSP-2 than to MsUSP-1. The TnHR4 showed most similarity to a recently cloned Bombyx mori GRF. While EcR and USP were constitutively expressed in both cell lines, HR4 was barely detectable by Northern blot analysis in High Five cells. Treatment with 20-hydroxyecdysone (20E) and agonist RH-5992 enhanced transcription of EcR in both cell lines, while the transcription of USP was suppressed in High Five cells. Such suppressed USP transcription was not observed in Sf21 cells. Transcription of TnEcR could also be enhanced by ecdysone and 3-dehydroecdysone, whereas transcription of SfEcR was unchanged with these two ecdysteroid compounds. Induction of HR4 transcription was also observed with 20E, RH-5992, ecdysone and 3-dehydroecdysone. The protein synthesis inhibitor, cycloheximide, superinduced expression of EcR and HR4 and restored the 20E/RH-5992-suppressed expression of TnUSP in the cells. Northern blot analysis also revealed that PCR, using degenerate USP primers, was able to amplify some other orphan nuclear receptors and their expression was inducible by 20E and RH-5992 and some of them were superinducible by cycloheximide.

Purification of ecdysone oxidase and 3-dehydroecdysone 3α-reductase from the cotton leafworm, Spodoptera littoralis

Abstract The 3-epimerization of ecdysteroids (insect moulting hormones) is an inactivation pathway of the hormones that has been reported to occur in midgut cytosol of Lepidoptera. The pathway involves ecdysone oxidase-catalysed conversion of ecdysone into 3-dehydroecdysone, which is then irreversibly reduced to 3-epiecdysone by 3DE 3α-reductase. In this study, ecdysone oxidase and 3DE 3α-reductase from the cotton leafworm, S. littoralis, have been purified by extensive chromatography together with electrophoresis on native gels. Gel filtration suggested that the native ecdysone oxidase might be a trimer with apparent molecular mass of approximately 190 kDa, since the apparent molecular mass of the oxidase subunit was determined to be 64 kDa by SDS-PAGE. Two forms of 3DE 3α-reductase were observed during the purification, the 26 kDa form reductase has been purified to homogeneity and the second form of the reductase identified as a 51 kDa protein. The former reductase may be a trimer with apparent molecular mass of 76 kDa, whilst the latter was suggested to be a monomer by gel filtration. Chromatographic behaviour suggested that the 26 kDa form of the reductase has a lower pI value and a higher degree of hydrophobicity than that of the 51 kDa reductase. Substrate specificity and the tissue distribution of these enzymes are discussed.