Juvenile hormone esterase (JHE) from Tenebrio molitor: full-length cDNA sequence,in vitro expression,and characterization of the recombinant protein

Juvenile hormone regulates the development and reproduction in a variety of insects. Juvenile hormone esterase (JHE) is a selective enzyme, which hydrolyzes the methyl ester of JH and alters its activity. In Tenebrio molitor, JHE has been previously purified from pupae and a partial cDNA was amplified by RT-PCR using fat body mRNA. The previous report indicated that several forms of the JHE protein were present in pupal homogenate. In this study, we report the full-length cDNA, which was obtained by RACE methods. The deduced protein sequence corresponds to peptides from two proteins of different molecular weights in the previous study. The coding region of the full-length cDNA was subcloned into the AcMNPV genome and high levels of expression of the JHE enzyme from the viral p10 promoter were demonstrated in cell culture. The majority of JHE is secreted from the cells as a soluble enzyme. The recombinant JHE enzyme was biochemically characterized. The recombinant protein appears by PAGE analysis as a monomer of approximately the same MW (66000) and pI (4.9) as was expected from the deduced amino acid sequence of the cDNA.     

Characterization of the esterase isozymes of Ips typographus (coleoptera,scolytidae)

Four esterase isozymes hydrolyzing α-naphthyl acetate (α-NA) were detected screening whole body homogenates of larvae and adults of Ips typographus by electrophoresis. Two of the four isozymes (isozymes 3 and 4) were not detected by α-NA staining in the pupal stage, but topical application of juvenile hormone III (JH III) on the pupa induced these isozymes. The JH esterase (JHE) activity on the gel was associated with the proteins of isozyme 2. The compounds OTFP, PTFP, and DFP inhibited this catalytic activity of isozyme 2 on the gel at low concentrations, whereas the proteins of isozyme 3 and 4 were affected only at higher concentrations. A quantitative developmental study was performed to characterize which of the esterases hydrolyzed JH III, using a putative surrogate substrate for JH (HEXTAT) and α-NA. The I₅₀ of several esterase inhibitors and the JH metabolites were also defined. All findings supported the results that a protein associated with isozyme 2 is catabolizing JH and that isozymes 3 and 4 are the main contributors to the general esterase activity on α-NA. The JHE from Tenebrio molitor was purified by affinity chromatography. Although the recovery was low, an analytical isoelectric focusing gel showed that the JHE activity of the purified enzyme. T. molitor cochromatographed at the same pl as the JHE activity of I. typographus. Arch. Insect Biochem. Physiol. 34:203–221, 1997. © 1997 Wiley-Liss, Inc.     

Gene cloning and characterization of a cold-adapted esterase from Acinetobacter venetianus V28

Acinetobacter venetians V28 was isolated from the intestine of righteye flounder, Poecilopsetta plinthus caught in Vietnam seawater, and the esterase gene was cloned using a shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,017 bp) corresponded to a protein of 338 amino acid residues with a molecular weight of 37,186. The esterase had 87% and 72% identities with the lipases of A. junii SH205 and A. calcoaceticus RUH2202, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein from the strain V28 was produced in both a soluble and an insoluble form when the Escherichia coli cells harboring the gene were cultured at 18 degrees C. The maximal activity of the purified enzyme was observed at a temperature of 40 degrees C and pH 9.0 using p-NP-caprylate as substrate; however, relative activity still reached to 70% even at 5 degrees C with an activation energy of 3.36 kcal/mol, which indicated that it was a cold-adapted enzyme. The enzyme was a nonmetalloprotein and was active against p-nitrophenyl esters of C4, C8, and C14. Remarkably, this enzyme retained much of its activity in the presence of commercial detergents and organic solvents. This cold-adapted esterase will be applicable as catalysts for reaction in the presence of organic solvents and detergents.     

Structural studies of a potent insect maturation inhibitor bound to the juvenile hormone esterase of Manduca sexta

Juvenile hormone (JH) is an insect hormone containing an alpha,beta-unsaturated ester consisting of a small alcohol and long, hydrophobic acid. JH degradation is required for proper insect development. One pathway of this degradation is through juvenile hormone esterase (JHE), which cleaves the JH ester bond to produce methanol and JH acid. JHE is a member of the functionally divergent alpha/beta-hydrolase family of enzymes and is a highly efficient enzyme that cleaves JH at very low in vivo concentrations. We present here a 2.7 A crystal structure of JHE from the tobacco hornworm Manduca sexta (MsJHE) in complex with the transition state analogue inhibitor 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) covalently bound to the active site. This crystal structure, the first JHE structure reported, contains a long, hydrophobic binding pocket with the solvent-inaccessible catalytic triad located at the end. The structure explains many of the interactions observed between JHE and its substrates and inhibitors, such as the preference for small alcohol groups and long hydrophobic backbones. The most potent JHE inhibitors identified to date contain a trifluoromethyl ketone (TFK) moiety and have a sulfur atom beta to the ketone. In this study, sulfur-aromatic interactions were observed between the sulfur atom of OTFP and a conserved aromatic residue in the crystal structure. Mutational analysis supported the hypothesis that these interactions contribute to the potency of sulfur-containing TFK inhibitors. Together, these results clarify the binding mechanism of JHE inhibitors and provide useful observations for the development of additional enzyme inhibitors for a variety of enzymes.     

Characterization of juvenile hormone esterase from genetically-determined wing morphs of the cricket, Gryllus rubens

Long-winged (LW) vs short-winged (SW) genetic stocks of the cricket Gryllus rubens differ in plasma juvenile hormone esterase (JHE) activity during the last stadium. These activity differences may be important in morph determination. In the present study, plasma JHEs from the LW vs SW stocks were characterized with respect to a variety of kinetic and physical characteristics. Gel permeation chromatography of LW or SW plasma each resulted in a single JHE peak of high molecular weight (190 kDa). This molecular weight is about twice as high as that of JHEs from most other insects. The apparent Michaelis constant for JH III ranged from 47 to 81 nM. Like JHEs from other insects, the enzyme from G. rubens was inhibited strongly by trifluoropropanone transition-state analogs and weakly by the general esterase inhibitors, eserine and DFP. JHEs from LW and SW plasma exhibited no significant differences in K_M, inhibition by trifluoropropanone or general esterase inhibitors, thermal denaturation profiles or pH profiles. The absence of K_M differences between LW and SW JHEs indicate that the 2–4 fold higher enzyme activity in LW plasma, previous documented in assays employing saturating substrate concentration, will exist under physiological substrate concentrations. Two isoforms (pI = 5.1, 4.2−4.1) were identified in SW plasma but only the more acidic form was observed in LW plasma. This is the first documentation of genetically-determined differences in JHE isozymes in any insect species. However, the functional significance of these isoform differences, if any, remains to be established. These results provide no evidence that the plasma JHE activity differences between LW and SW stocks results from allozymes or isozymes with altered kinetic or stability characteristics.     

Evolutionary Endocrinology of Juvenile Hormone Esterase in Gryllus Assimilis: Direct and Correlated Responses to Selection

Hemolymph juvenile hormone esterase (JHE) activity on the third day of the last stadium in the cricket, Gryllus assimilis, exhibited a significant response to selection in each of six replicate lines. Mean realized heritability was 0.26 +/- 0.04. The response was due to changes in whole-organism enzyme activity as well as to changes in the proportion of enzyme allocated to the hemolymph compartment. In vivo juvenile hormone metabolism differed between some lines selected for high vs. low enzyme activity. Only minimal differences were observed between lines with respect to hemolymph protein concentration or whole-cricket activity of juvenile hormone epoxide hydrolase, the other major JH-degrading enzyme. Dramatic correlated responses to selection, equal in magnitude to the direct response, were observed for JHE activity on each of three other days of the last juvenile stadium. In contrast, no correlated responses in JHE activity were observed in adults. This indicates that JHE activities throughout the last stadium will evolve as a highly correlated unit independent of adult activities and the evolution of endocrine mechanisms regulating juvenile development can be decoupled from those controlling adult reproduction. This study represents the first quantitative-genetic analysis of naturally occurring endocrine variation in an insect species.     

Localization of juvenile hormone esterase during development in normal and in recombinant baculovirus-infected larvae of the moth Trichoplusia ni.

The pathogenesis and cellular localization of juvenile hormone esterase (JHE) was examined in larvae of the moth Trichoplusia ni, infected with a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus: AcNPV) engineered to produce high levels of JHE (JHE virus). The course of JHE localization in the recombinant virus infected larvae was compared with that of both wild type AcNPV infected, and uninfected larvae, using immunogold electron microscopy. In the JHE virus infected insects, high levels of JHE were observed in the endoplasmic reticulum of all cells showing evidence of viral structures in the nucleus, except for gut cells which showed only background JHE levels. Tracheole cells and haemocytes appeared to play a role in the dissemination of infection. In uninfected larvae, fat body and epidermis were the major tissues staining for JHE, which was only detectable at peak times of JHE activity during the fifth instar: lower levels at other times could not be distinguished from background. JHE was also present in lysosomes of granular haemocytes: these lysosomes increased in number in the fifth instar compared to the fourth instar. Similar lysosome-like granules in the pericardial cells did not become highly positive for JHE antigen until the fifth instar.     

Multiple forms of juvenile hormone esterase active sites in the hemolymph of larvae of Trichoplusia ni

Kinetic analysis was performed on the juvenile hormone (JH) esterase activity in the hemolymph of feeding, last instar larvae of Trichoplusia ni (Lepidoptera: Noctuidae). When the results were analyzed by several different graphical and regression procedures, all approaches yielded the same conclusion that at least two forms of JH esterase active sites exist in the hemolymph. The apparent Km for one site for JH I, II and III was 8.5 X 10(-8) M, and 6.6 X 10(-8) M, respectively. The Km for the other site for JH I, II and III was 6.6 X 10(-7) M, 7.6 X 10(-7) M, 40 X 10(-7) M, respectively. When hemolymph JHE activity was subjected to high resolution isoelectric focusing (IEF), two distinct large peaks of JHE activity were observed, with pIs of 5.3 and 5.5, as well as a small peak at pI 5.1. Separate kinetic analysis of the JHE activity in each peak showed that only the higher Km active site for each substrate was present (in the 10(-7) M range). These data necessitate a change in the current model for JHE in T. ni, and some other insects, which states that a single active site is responsible for most or all of the JH esterase activity in vivo. The data also explain the different estimates of the Km of JHE in T. ni obtained by different laboratories. Studies on the purification of, and the development of inhibitors for, JHE esterase must consider the role of both JHE forms and sites in regulation of T. ni metamorphosis.     

Characterization of hemolymph juvenile hormone esterase from Lymantria dispar

A major peak of juvenile hormone esterase (JHE) activity approaching 330 nmol JH III hydrolyzed/min/ml of hemolymph was observed during the last larval growth stage in Lymantria dispar. A smaller peak of JHE occurred 3–5 days after pupation. The gypsy moth JHE was purified from larval hemolymph using a classical approach. A specific activity of 766 units per mg of protein and a K_m of 3.6 × 10⁻⁷ M for racemic JH III and the (10R, 11S) enantiomer of JH II was determined for the purified enzyme. The 62 kDa esterase was insensitive to inhibition by O,O-diisopropyl phosphorofluoridate (DFP), or by phenylmethylsulfonyl fluoride (PMSF). Two forms of JHE isolated by RP-HPLC were indistinguishable by HPLC tryptic peptide mapping and share an identical N-terminal amino acid sequence. Polyclonal antisera raised against gypsy moth enzyme cross-reacted with JHE from Trichoplusia ni but not with JHE from Manduca sexta. A weak cross-reactivity was observed with JHE from Heliothis virescens. Forty amino acid residues of the N-terminus were placed in sequence. The N-terminal sequence of JHE from L. dispar showed little homology to the sequence of JHE from H. virescens. The immunological and structural data support the conclusion that markedly different esterases, which catalyze the hydrolysis of juvenile hormone, are present in the hemolymph of different Lepidoptera.     

Isolation and characterization of juvenile hormone esterase from hemolymph of Lymantria dispar by affinity- and by anion-exchange chromatography

Juvenile hormone esterase (JHE), which catalyzes the hydrolysis of juvenile hormone, was isolated from the hemolymph of 5(th) instars of Lymantria dispar by two different procedures. One procedure was based on affinity chromatography and the other on anion-exchange chromatography. The material from both purifications showed bands of approximately 50 kDa when analyzed by SDS-PAGE. Isoelectric focusing (IEF) gels in combination with enzyme activity assays indicated two isoelectric forms with the same pI values (pH 5.1. and 5.3) from affinity purification and from anion-exchange chromatography. Amino acid sequencing of several internal peptides from the 50 kDa band following affinity purification and alignment of these sequences with JHEs from previously purified lepidopteran species (Heliothis virescens, Manduca sexta) showed high homology of these enzymes.The isolated JHE, at least in the stage of insect used, was different from the enzyme reported earlier [Valaitis, A.P., 1991. Characterization of hemolymph juvenile hormone esterase from Lymantria dispar. Insect Biochemistry 21, 583-595] to hydrolyze JH in the hemolymph of gypsy moth, based on molecular weight and amino acid sequence.     

Identification and partial characterization of juvenile hormone esterase from cotton pest Dysdercus cingulatus

Juvenile hormone esterase (JHE), a selective enzyme that hydrolyzes the methyl ester of insect juvenile hormone plays an important role in regulating metamorphosis in nymphs as well as reproduction in adults. Studies on JH degradation provide insight into the possibilities of physiological disruption in the insects. In the present study, the JH degrading enzyme, JHE from the cotton pest Dysdercus cingulatus (Heteroptera) is characterized. Electrophoretic analysis of haemolymph during various developmental stages showed the JHE bands prominent only on the final day of 5th instar nymph, and the esterase substrate specificity confirmed the presence of JHE isoforms. In an attempt to clone cDNA of JHE gene from the final instar nymphs, mRNA isolated from fat bodies was coupled with JHE gene-specific primers and the cDNA was synthesized using RT-PCR. The PCR amplified cDNA showed the presence of JHE isoforms in D. cingulatus.     

Potential ligands of DmP29, a putative juvenile hormone esterase binding protein of Drosophila melanogaster

We previously reported the identification of a putative juvenile hormone esterase (JHE) binding protein DmP29 in Drosophila melanogaster and its primary localization to the mitochondria [Liu, Z., Ho, L., Bonning, B.C., 2007. Localization of a Drosophila melanogaster homolog of the putative juvenile hormone esterase binding protein of Manduca sexta. Insect Biochem. Mol. Biol. 37(2), 155-163]. To further characterize DmP29, we identified potential ligands of this protein. Recombinant DmP29 was shown by ligand blot and co-immunoprecipitation analyses to bind recombinant JHE as well as to larval serum proteins (LSP). The possible biological relevance of the in vitro DmP29-JHE interaction is provided by detection of JHE activity in D. melanogaster mitochondrial fractions; 0.48 nmol JH hydrolyzed/min/mg mitochondrial protein, 97% of which was inhibited by the JHE-specific inhibitor OTFP. However, the DmP29-LSP interactions may not be biologically relevant. Given the high abundance, and "sticky" nature of these proteins, interaction of DmP29 with LSP may result from non-specific associations. No DmP29 interactions with non-specific esterases were detected by co-immunoprecipitation analyses. The potential role of DmP29 as a chaperone of JHE is discussed.     

Correlations between juvenile hormone esterase activity,ecdysone titre and cellular reprogramming in Galleria mellonella

Juvenile hormone esterase (JHE) activity, ecdysone titre, and developmental competence of the epidermis were determined in last instar larvae and pupae of Galleria mellonella. Haemolymph JHE activity reaches a peak before increases are observed in ecdysone titre both during larval-pupal and pupal-adult metamorphosis. JHE activity is low during the penultimate larval instar although general esterase activity is relatively high. In last instar larvae two ecdysone peaks are noted after the increase in JHE activity. Furthermore, epidermal cell reprogramming occurs just after the increase in haemolymph JHE activity and possibly before the first increase in ecdysone titre. This was tested by injection of high doses of β-ecdysone into last instar larvae of different ages resulting in rapid cuticle deposition. Reprogramming occurred if the resulting cuticle was of the pupal type. These correlative observations may increase our understanding of the relative importance of an ecdysone surge in the absence of JH in reprogramming of the insect epidermis.     

Ligand blot analysis of juvenile hormone esterase binding proteins in Manduca sexta L

Biotinylated recombinant juvenile hormone esterase (JHE) was used for ligand blotting of proteins from fat body tissue and pericardial athrocytes of Manduca sexta. Proteins were separated by SDS-polyacrylamide gel electrophoresis or by two-dimensional electrophoresis. Eight putative JHE binding proteins were detected in fat body tissue and in pericardial athrocytes of both M. sexta and Heliothis virescens. The predominant bands were 29, 72, 75, 125 and 240kDa, with minor bands at 50, 80 and 205kDa. All putative JHE binding proteins were present from the second through to the fifth instar larvae of M. sexta. On wide-range isoelectric focusing, the 29kDa JHE binding protein separated into three species with isoelectric points of 6.5, 6.6 and 6.8. Biotinylated-JHE did not bind recombinant M. sexta-derived juvenile hormone binding protein. The mutant JHE with mutations K29R and K524R binds weakly to the JHE binding protein P29, relative to binding of wild-type JHE [Shanmugavelu et al., J. Biol. Chem., 275 (2000) 1802-1806]. A similar reduction in binding was not seen for the 29kDa binding protein identified here in pericardial athrocytes by ligand blot. This result is discussed.     

An in vitro system for studying juvenile hormone induction of juvenile hormone esterase from the fat body of Trichoplusia ni (Hübner)

An in vitro fat body culture was used to study juvenile hormone esterase (JHE) regulation. The present study shows juvenile hormone can directly induce JHE activity to appear in the culture medium in a dose-dependent manner at physiological concentrations of JH. This induced appearance of JHE can be blocked with actinomycin D. Biochemical characterization of the in vitro produced JHE demonstrated that it had the same isoelectric points as that of the in vivo JHE activity. The JHE inhibitor 1-1-1 trifluoro-tetradecan-2-one gave the same inhibition profile and I₅₀ toward both in vivo and in vitro produced JHE activities. Finally, the JHE activity induced in vitro was immunologically similar to that occurring in vivo. The system should be useful for high resolution studies on the regulation of JHE.     

The source and action of head factors regulating juvenile hormone esterase in larvae of the cabbage looper, Trichoplusia ni

Brain (median or lateral regions) or suboesophageal ganglion (SOG) homogenates of Day 1 fifth instar larvae of Trichoplusia ni induced the appearance of haemolymph juvenile hormone esterase (JHE) when injected into Day 1, Day 2 or early Day 4 fifth instar ligated hosts. Brain and SOG homogenates of late fourth instars also induced JHE when injected into Day 1 hosts, whole late fifth instar and pupal tissue did not. The pattern of JHE induction by early fourth through Day 3 fifth instar brain and SOG homogenates correlated with natural haemolymph JHE activity occurring at these times. Implantation of late fourth and Day 1 fifth instar brains and/or SOG into similar age hosts similarly induced JHE activity while prothoracic and abdominal ganglia did not. The relative levels of induction following implantation were SOG<brain<brain+SOG. JHE activity which appears in the haemolymph following injection of brain homogenates appears to be largely due to a single enzyme which has an isoelectric point indistinguishable from that of the natural haemolymph enzyme. Evidence is presented which suggests that inhibitory as well as stimulatory brain factors are involved in JHE regulation.     

In vivo and in vitro effects of epofenonane on juvenile hormone esterase activity in tissues of last stadium larvae of Trichoplusia ni

Topical application of the juvenoid, epofenonane, to last stadium postwandering larvae of Trichoplusia ni caused a precocious elevation of juvenile hormone esterase (JHE) activity that was tissue speific and time dependent. This increase in enzyme activity over controls was most dramatic in the hemolymph, whereas increases in the fat body were lower. Antibodies raised against JHE reacted on Western blots with a fat body and hemolymph protein present in epofenonane treated and untreated last stadium day 3 larvae. The abundance of this protein, which comigrated with JHE, closely coincided with the temporal increases in JHE catalytic activity that occurred in response to treatment in vivo with epofenonane.The presence of epofenonane (5–10,000 nM) in the medium at the start of fat body incubations failed to shift the temporal appearance of JHE activity or boost activity levels significantly over those of controls. If larvae were treated in vivo with epofenonane before fat body tissue was removed, only a small, but significant increase in JHE activity was found in vitro. The rate of enzyme secretion was insufficient to account for the rapid increases in enzyme activity that occur in the hemolymph in response to epofenonane, even though tissue held in vitro was deemed viable by monitoring lactate dehydrogenase activity in the medium, fat body intracellular ATP, and the incorporation of [³⁵S]methionine into fat body protein. Fat body tissue removed from various aged last stadium larvae released enzyme at different rates in vitro.