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.     

Reduced activity of juvenile hormone esterase in microsporidia-infected Lymantria dispar larvae

Infection of the fat body of Lymantria dispar (Lep.: Lymantriinae) larvae with the microsporidium Vairimorpha disparis has severe effects on juvenile hormone (JH) metabolism of the host. Beginning 8 days postinfection, activity of the JH degrading enzyme JH-esterase was significantly lower in the hemolymph of infected than uninfected larvae. Activity remained low as microsporidiosis progressed. JH titers were slightly elevated in infected larvae; the difference was not significant in most cases. This disturbance of JH metabolism may be due to generally impaired fat body functions and high demand for resources by the developing pathogen.     

Purification and characterization of juvenile hormone esterase from hemolymph of the Colorado potato beetle

In the Colorado potato beetle (Leptinotarsa decemlineata), low juvenile hormone (JH) titers are necessary to initiate metamorphosis and diapause. Low JH titers coincide with high activities of JH esterase, which occur mainly in the hemolymph. The specific activity of JH esterase appeared to be highest in the last larval instar, at day 3 after the molt, and reached a value of 13.5 nmol/min/mg. JH esterase was purified from hemolymph collected at this stage by a sequence of separation systems, including preparative nondenaturing PAGE, isoelectric focusing, and SDS-PAGE. The enzyme had a molecular weight of 120,000 and was composed of two subunits with molecular weights of 57,000, which were not linked by disulphide bridges. Isoelectric focusing revealed two forms of the enzyme with isoelectric points of 5.5 and 5.6. The K_m and k_cat of the purified enzyme were determined. The major form with pI 5.6 had a K_m of 1.4 × 10^-6M and a k_cat of 0.9 s^-1 and the minor form with pI 5.5 had a K_m of 2.2 × 10^-6M and a k_cat of 1.9 s^-1. The quaternary structure of L. decemlineata JH esterase as a dimer, differs from JH esterases in other species, which are monomers. Arch. Insect Biochem. Physiol. 35:261-277, 1997.© 1997 Wiley-Liss, Inc.     

Purification and characterization of hemolymph juvenile hormone esterase from the cricket, Gryllus assimilis.

Juvenile hormone esterase (JHE) from the serum of the cricket, Gryllus assimilis, was purified to homogeneity in a four-step procedure involving polyethylene glycol precipitation, hydrophobic interaction FPLC, and ion exchange FPLC. This procedure could be completed in 4 days and resulted in a greater than 900-fold purification with greater than 30% recovery. The purified enzyme exhibited a single band on a silver-stained SDS PAGE gel and had an apparent subunit molecular mass of 52 kDa. The native subunit molecular mass, determined by gel permeation FPLC, was 98 kDa, indicating that JHE from Gryllus assimilis is a dimer of two identical or similar subunits. The turnover number of the purified enzyme (1.41 s(-1)), K(M(JH-III)) (84 +/- 12 nM) of nearly-purified enzyme, and k(cat)/K(M) (1.67 x 10(7) s(-1) M(-1)) were similar to values reported for other well-established lepidopteran and dipteran JHEs. JHE from Gryllus assimilis was strongly inhibited by the JHE transition-state analogue OTFP (octylthio-1,1,1-trifluoro-2-propanone; I(50) = 10(-7) M) and by DFP (diisopropyl fluorophosphate; I(50) = 10(-7) M). The shapes of the inhibition profiles suggest the existence of multiple binding sites for these inhibitors or multiple JHEs that differ in inhibition. Isoelectric focusing separated the purified protein into 4 isoforms with pIs ranging from 4.7-4.9. N-terminal amino acid sequences (11-20 amino acids) of the isoforms differed from each other in 1-4 positions, suggesting that the isoforms are products of the same or similar genes. Homogeneously purified JHE hydrolyzed alpha-napthyl esters, did not exhibit any detectable acetylcholinesterase, acid phosphatase, or aminopeptidase activity, and exhibited only very weak alkaline phosphatase activity. JHE exhibited a low (11 microM) K(M) for long-chain alpha-naphthyl esters, indicating that JHE may have physiological roles other than the hydrolysis of JH-III. Purification of JHE represents a key step in our attempts to identify the molecular causes of genetically-based variation in JHE activity in G. assimilis. This represents the first homogeneous purification of JHE from a hemimetabolous insect.     

Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae)

Glyptapanteles liparidis is a gregarious, polydnavirus (PDV)-carrying braconid wasp that parasitizes larval stages of Lymantria dispar. In previous studies we showed that parasitized hosts dramatically increase juvenile hormone (JH) titers, whereas JH degradation is significantly inhibited in the hemolymph. Here we (i) quantified the effects of parasitism on JH esterase (JHE) activity in hemolymph and fat body of penultimate and final instars of L. dispar hosts and (ii) assessed the relative contribution of individual and combined wasp factors (PDV/venom, teratocytes, and wasp larvae) to the inhibition of host JHE activity. The effects of PDV/venom was investigated through the use of gamma-irradiated wasps, which lay non-viable eggs (leading to pseudoparasitization), while the effects of teratocytes and wasp larvae were examined by injection or insertion of these two components in either control or pseudoparasitized L. dispar larvae. Parasitism strongly suppressed host JHE activity in both hemolymph and fat body irrespective of whether the host was parasitized early (premolt-third instar) or late (mid-fourth instar). Down-regulation of JHE activity is primarily due to the injection of PDV/venom at the time of oviposition, with only very small additive effects of teratocytes and wasp larvae under certain experimental conditions. We compare the results with those reported earlier for L. dispar larvae parasitized by G. liparidis and discuss the possible role of JH alterations in host development disruption.     

Juvenile hormone-binding protein and juvenile hormone esterase activity in hemolymph from last-instar nymphs of Leucophaea maderae

The concentration of the juvenile hormone-binding protein (JHB) in hemolymph was determined throughout the last nymphal instar. It was found to be 3.9 μM at the molt to the instar, rising to 13 μM by mid-instar, and dropping to 6.7μM the day before emergence. Endocrine control of its production during the last nymphal instar could not be established. The apparent juvenile hormone esterase (JHF) activity was low at the molt to the last instar, but rose about fivefold by mid-instar, and then modestly declined. On the day of emergence, JHF activity rose to the highest level observed. A four- to fivefold increase in absolute JHF activity was determined during the first half of the last nymphal instar. This increase is not regulated by JH. Removal of the JHB from hemolymph samples by precipitation with a polyclonal specific antibody increased the JHF activity up to 1,000-fold. Thus, changes in the concentrations of JHB can affect the apparent activity of JHE, which is unrelated to the production or degradation of the JHF.     

Immunochemical characterization of juvenile hormone esterase from different species of Lepidoptera

Cross-immunoreactivity of juvenile hormone esterase (JHE) from different species was tested using anti-JHE (Trichoplusia ni) (Noctuidae) polyclonal antibody. Partial cross-reactivity was observed between JHE from Hyphantria cunea, Isia isabella (Arctuidae) and Spodoptera exigua (Noctuidae) in immunoblot analysis. Soluble antigen-antibody complex formation was observed between anti-JHE (T. ni) and antigen(s) from Heliothis virescens (Noctuidae) during immunotitration of antigen(s). Using an ELISA method the highest cross-reactivity was observed for both species from Arctuidae and lower cross-reactivity for antigen(s) from H. virescens.     

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.     

In vitro expression and biochemical characterization of juvenile hormone esterase from Manduca sexta

Juvenile hormone esterase (JHE) is a selective enzyme that hydrolyzes the methyl ester of juvenile hormone. This enzyme plays an important role in the regulation of metamorphosis in caterpillars, and is implicated in additional roles in development and reproduction in this and other orders of insect. The full length coding region of the JHE cDNA from Manduca sexta was subcloned into the baculovirus AcMNPV genome under the control of the p10 promoter. The recombinant virus demonstrated the expression of high levels of JHE activity when infected into Hi5 cells from Trichoplusia ni. The recombinant protein was partially purified by anion exchange chromatography and its biochemical characterization showed similar features to the wild type protein. The recombinant JHE has an estimated MW of 66500 Da. Some heterogeneity with the enzyme was observed when analyzed by isoelectric focusing, although the peak of JHE activity was observed at pI=6.0. It is highly sensitive to trifluoroketone inhibitors and certain phosphoramidothiolates, while relatively insensitive to other common esterase inhibitors. Incubating the enzyme with various organic solvents and detergents showed that the enzyme is activated at lower concentrations of solvents/detergents and remains significantly active even at high concentrations. The high tolerance of organic solvents may make this JHE enzyme useful in future applications as a synthetic catalyst.     

Peptides that elicit midgut stem cell differentiation isolated from chymotryptic digests of hemolymph from Lymantria dispar pupae

Isolated stem cells of Heliothis virescens, cultured in vitro, were induced to differentiate by Midgut Differentiation Factors 3 and 4. These were peptides identified from a chymotrypsin digest of hemolymph taken from newly pupated Lymantria dispar. Partial purification was obtained by filtration through size exclusion filters. The most active preparation was subsequently subjected to a series of 3 Reverse Phase-HPLC procedures. Partial sequences of the peptides were identified via automated Edman degradation as the nanomers EEVVKNAIA-OH (MDF 3) and ITPTSSLAT-OH (MDF 4). These sequences were commercially synthesized. The synthetic compounds proved active in a dose-dependent manner. Stem cells responded to synthetic MDF 3 and MDF 4 as they did to previously identified peptides MDF 1 and 2, which have quite different amino acid sequences. All of the 4 MDFs administered singly induced statistically similar differentiation responses at 2 x 10(-8), 2 x 10(-9), and 2 x 10(-10) M. However, pairs of the 4 MDFs produced even more differentiation, the same response as one alone, no response, or were inhibitory, dependent on the MDF pair and its concentration. The data suggests complicated receptor interactions.     

舞毒蛾保幼激素结合蛋白家族基因克隆及对CO2胁迫的响应

保幼激素结合蛋白(JHBP)是存在于血淋巴和细胞内的一类载体蛋白,与保幼激素结合运送到靶标组织。本文研究舞毒蛾JHBP基因特性及其对高浓度CO_2胁迫的响应,为明确全球气候变化下舞毒蛾适应性机制提供理论依据。通过舞毒蛾转录组文库分析结合RT-PCR克隆鉴定出7个JHBP基因,并进行基因特性和发育阶段特异性分析,同时利用密闭式CO_2人工气候箱在不同CO_2浓度(397μL/L、550μL/L和750μL/L)下将舞毒蛾卵饲养至3龄幼虫,利用qRT-PCR技术测定3龄幼虫JHBP基因表达量变化。结果表明,舞毒蛾JHBP家族7个基因全长开放阅读框大小为714～756 bp,编码237～251个氨基酸,分子质量为28.22～28.54 kDa,理论等电点为5.33～8.47。7个基因在不同发育阶段的表达存在差异,幼虫期LdJHBP1、2、5、6基因表达量较高,而LdJHBP3、4和7在蛹期和成虫期高表达。进化树分析表明舞毒蛾LdJHBP1、LdJHBP2、LdJHBP6分别与棉铃虫Helicoverpa armigera、家蚕Bombyx mori、冬尺蛾Operophtera brumata的JHBP亲缘关系较近。高浓度CO_2下舞毒蛾3龄幼虫的LdJHBP表达量下降。JHBP基因表达可能影响JH结合与运输从而调节生长发育。     

Juvenile hormone biosynthesis by corpus cardiacum-corpus allatum complexes of larval Lymantria dispar

• 1.1. A radiochemical assay was used to examine juvenile hormone (JH) synthesis and secretion in vitro by incubating two pairs of larval corpus cardiacum-corpus allatum complexes (CC-CA) from, Lymantria dispar, in 50 μl of osmotically balanced Grace's medium containing 1 μC1 [³H-methyl]-methionine for 6 hr.
• 2.2. For CC-CA of fourth instar female larvae, maximal incorporation of ³H-methyl was 0.15 pmol/pr/hr between days 2 and 3. High pressure liquid chromatographic (HPLC) analysis suggested that the biosynthetic products are mainly JH III with a little JH II at times.
• 3.3. For CC-CA of last instar female larvae, incorporation of ³H-methyl was 0.48 pmol/pr/hr at the beginning of the stadium and decreased to negligible levels by day 10. HPLC analysis suggested that CC-CA of last instar larvae produced only JH III. Volume increases in CA during the last instar were associated with declining activities of JH secretion.
• 4.4. Comparisons of maximal rates of ³ H-methyl incorporation by each unit volume of CA revealed that in the last instar each unit volume (μm³) of glandular tissue secreted 50% more JH than in the fourth instar.

     

Prepupal regulation of juvenile hormone esterase through direct induction by juvenile hormone

The regulation of the prepupal peak of juvenile hormorne esterase activity was investigated and found to be directly induced by juvenile hormone. Allatectomy and reimplanation as well as juvenile hormone application experiments all indicated that the appearance of prepupal juvenile hormone esterase activity was in response to a prepupal burst of juvenile hormone. Implantation experiments indicated that the effect of juvenile hormone is not mediated through the isolated brain or subesophageal ganglion.     

Vitellogenin synthesis in female larvae of the gypsy moth,Lymantria dispar (L.): suppression by juvenile hormone

• 1.1. Fat body from feeding-phase, last instar gypsy moth females incorporates l-[³⁵S]methionine in vitro into two vitellogenins with the same molecular masses (165 and 180 kDa) as the apo-vitellogenins found in teh hemolymph and the apo-vitellins in teh eggs.
• 2.2. Both apo-vitellogenins are observed in the medium of fat body cultures, but only the 180 kDa apo-vitellogenin is observed in extracts of cultured tissue.
• 3.3. Synthesis and accumulation of the apo-vitellogenins are suppressed in a dose-dependent manner by topical treatment with the juvenile hormone analog, methoprene, prior to day 4.
• 4.4. This suppression suggests that a declining juvenile hormone titre is involved in the initiation of vitellogenin synthesis.

     

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.     

Isolation of juvenile hormone esterase and its partial cDNA clone from the beetle, Tenebrio molitor

Juvenile hormone esterase (JHE) plays an essential role in insect development. It is partially responsible for the clearance of juvenile hormone (JH) which regulates various aspects of insect development and reproduction. Because of its role in regulating JH titer, this enzyme has been targeted for development of biologically-based insecticides. JHE was partially purified from the beetle, Tenebrio molitor, using a transition state analog as the affinity ligand. Two forms of JHE were characterized by activity analysis, isoelectric focusing, two-dimensional SDS-PAGE and N-terminal sequence analysis. The esterase is associated with two proteins of sizes 71 and 150 kDa, both of which are active on JH III. A partial cDNA clone for the enzyme was isolated based on the sequence of N-terminal and internal peptides. Its sequence indicates that JHE from T. molitor and Heliothis virescens may have a common origin.     

Characterization of affinity-purified juvenile hormone esterase from Trichoplusia ni

Juvenile hormone (JH) esterase was purified greater than 1000-fold in one step from hemolymph and whole larval homogenates from the last larval instar of Trichoplusia ni to give a single diffuse band that migrates at Mr = 64,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification was based on an affinity chromatography procedure that employs trifluoromethyl ketone ligands. Isoelectric focusing of the purified preparations resulted in multiple bands that coincided to all significant hydrolysis of juvenile hormone detected in this manner. Kinetic experiments using optically pure enantiomers of JH II as substrates showed the two main electromorphs of JH esterase from the hemolymph to have apparently identical kinetic parameters as well as a similar capability to distinguish between substrates that differ in the orientation of the epoxide moiety of JH. However, the enzyme could hydrolyze esters lacking the JH structure. The proteins were shown to be monomers and to have asparagine-linked oligosaccharides, most likely of hybrid structure. Immunochemical and other evidence showed that the affinity-purified proteins were responsible for all significant JH esterase activity during periods of rapid esterolysis in vivo.     

Juvenile hormone esterases of Lepidoptera II. Isoelectric points and binding affinities of hemolymph juvenile hormone esterase and binding protein activities

Summary The juvenile hormone esterase (JHE) and juvenile hormone binding protein (JHBP) activities from the last larval instar of 14 species of Lepidoptera (Pieris rapae, Colias eurytheme, Danaus plexippus, Junonia coenia, Hemileuca nevadensis, Pectinophora gossypiella, Spodoptera exigua, Trichoplusia ni, Heliothis virescens, Orygia vetusta, Ephestia elutella, Galleria mellonella, Manduca sexta andEstigmene acrea) were analyzed by analytical isoelectric focusing (IEF). While the multiplicity and isoelectric point of these proteins varied, all of them were mildly acidic (pI 4.0–7.0), and a large number of the species possessed only a single JHE and/or JHBP activity. The Michaelis constants (K _m's) of the whole hemolymph JHE activities from selected species for JH III were in the range of 10^–7M. The equilibrium dissociation constantK _d of the JHBP was determined by Scatchard analysis for selected species as well, with the majority of species having aK _d near 10^–7M. This information is consistent with JHE acting as a scavenger for JH at various times during development and relying entirely on mass action to remove JH from its protective JHBP complexes. The JHBP should limit nonspecific binding and thus facilitate the rapid transport of the intact hormone through-out the hemocoel. These data indicate that the species currently used in the study of the developmental biology of the Lepidoptera are biochemically similar to a variety of other species in this order.Abbreviations JH juvenile hormone - JHE juvenile hormone esterase - JHBP juvenile hormone binding protein - IEF isoelectric focusing - EPPAT O-ethyl-S-phenyl phosphoramidothiolate - DFP O O-diisopropyl phosphofluoridate