Embryonic expression of juvenile hormone binding protein and its relationship to the toxic effects of juvenile hormone in Manduca sexta

The juvenile hormones (JHs) regulate a diverse array of insect developmental and reproductive processes. One molecular target of JH action is its transporter, hemolymph JH binding protein (hJHBP); in the larva of the tobacco hornworm, Manduca sexta, low doses of JH can immediately increase hJHBP gene expression. Less explored are the effects of JH on embryological development, where early hormonal treatment has been shown to affect embryonic development and pupation. This study examines the egg form of JHBP and its gene expression during embryogenesis of M. sexta, as well as the phenotypic effect JH treatment has on embryos and on JHBP gene expression. We here demonstrate that the preponderance of JHBP found in the egg is maternally derived and that the embryonic gene and protein appear identical to those found in the larva. Expression of the JHBP gene begins in both the embryo itself and extra-embryonic tissues 15 h after fertilization, long before emergence of a functional fat body and circulatory system. Topical application of low JH doses to early embryos resulted in larval abnormalities while high doses of the hormone induced embryonic mortality. These effects are not mediated through regulation of the JHBP gene, since embryonic expression appears invariant in response to JH challenge. The toxicity of JH is tightly correlated with the concentration of unbound hormone.     

Identification of specific interaction of juvenile hormone binding protein with isocitrate dehydrogenase

Juvenile hormone (JH) is essential for multiple physiological processes: it controls larval development, metamorphosis and adult reproduction. In insect hemolymph more than 99 % of JH is bound to juvenile hormone binding protein (JHBP), which protects JH from degradation by nonspecific hydrolases and serves as a carrier to supply the hormone to the target tissues. In Galleria mellonella hemolymph, JHBP is found in a complex with lipid-binding high molecular weight proteins (HMWP) and this interaction is enhanced in the presence of JH. In this report, we present studies on the interaction of JHBP with low molecular weight proteins (LMWP) in the hemolymph. Using ligand blotting we found that JHBP interacts with a protein of about 44 kDa. To identify the protein that preferentially binds JHBP, a LMWP fraction was applied to a Sepharose-bound JHBP and, after washing, the column was eluted with free JHBP acting as a specific competitor or with carbonic anhydrase as a negative control. The eluted proteins were separated by SDS/PAGE and analyzed by mass spectrometry. Isocitrate dehydrogenase was identified as a component of the supramolecular complex of JHBP with hemolymph proteins.     

Effects of stress on the hemolymph juvenile hormone binding protein titers of Manduca sexta

External stressors disrupt physiological homeostasis; in insects, the response to stress may result in delayed development as the animal attempts to restore homeostasis before proceeding with its complex life cycle. Previous studies have demonstrated that exposure to stress leads to increased levels of the juvenile hormone (JH), a hormone responsible for maintaining the insect larval state. In Manduca sexta, JH is transported to target tissue by a high-affinity binding protein, hemolymph JH binding protein (hJHBP). Since JH titers are elevated in stressed Manduca, we examined levels of hJHBP to better understand (1) the role of JH in regulating hJHBP levels and (2) the hJHBP-regulated bioavailability of hormone at the target site. Fourth stadium Manduca (48 h post-ecdysis) were exposed for 24h to various stressors including nutritional deprivation, microbial infection, cutaneous injury, episodic movement, and temperature elevation. Insects raised on diets lacking nutritional content exhibited mean hJHBP levels that were less than half (45%) those of control insects. Similarly, insects injected with Escherichia coli demonstrated a 47% reduction in hJHBP titers. Cutaneous injury, episodic movement, and temperature elevation lowered hJHBP levels by 47%, 43%, and 38%, respectively. Total hemolymph protein concentration was not affected. After a stress event (injury), a 50% reduction in abundance of fat body hJHBP mRNA was observed within 4h; hJHBP levels did not drop until 24h after injury. Stress in the fourth stadium was manifest in fifth instars, with 100% of the injured insects displaying an extended larval stadium or failing to pupate. Computational modeling of the JH-hJHBP interaction indicates that unbound JH doubles in stressed insects. These results indicate that in response to stress larval hJHBP titers are significantly reduced, increasing JH bioavailability at the target site and thereby impacting development and survival of the insect. Treatment of unstressed insects with physiological doses of JH I did not affect hJHBP levels, suggesting that elevated JH levels were not solely responsible for the observed down-regulation in stressed insects.     

N-linked glycosylation of G. mellonella juvenile hormone binding protein - comparison of recombinant mutants expressed in P. pastoris cells with native protein

Juvenile hormone (JH) regulates insect growth and development. JH present in the hemolymph is bound to juvenile hormone binding protein (hJHBP) which protects JH from degradation. In G. mellonella, this protein is glycosylated only at one (Asn(94)) of the two potential N-linked glycosylation sites (Asn(4) and Asn(94)). To investigate the function of glycosylation, each of the two potential glycosylation sites in the rJHBP molecule was examined by site-directed mutagenesis. MS analysis revealed that rJHBP overexpressed in the P. pastoris system may appear in a non-glycosylated as well as in a glycosylated form at both sites. We found that mutation at position Asn(94) reduces the level of protein secretion whereas mutation at the Asn(4) site has no effect on protein secretion. Purified rJHBP and its mutated forms (N4W and N94A) have the same JH binding activities similar to that of hJHBP. However, both mutants devoid of the carbohydrate chain are more susceptible to thermal inactivation. It is concluded that glycosylation of JHBP molecule is important for its thermal stability and secretion although it is not required for JH binding activity.     

Unfolding and refolding of juvenile hormone binding protein

Juvenile hormone (JH) regulates insect development. JH present in the hemolymph is bound to a specific glycoprotein, juvenile hormone binding protein (JHBP), which serves as a carrier to deploy the hormone to target tissues. In this report structural changes of JHBP from Galleria mellonella induced by guanidine hydrochloride have been investigated by a combination of size-exclusion chromatography, protein activity measurements, and spectroscopic methods. Molecules of JHBP change their conformation from a native state via two unstable intermediates to a denatured state. The first intermediate appears in a compact state, because it slightly changes its molecular size and preserves most of the JHBP secondary structure of the native state. Although the second intermediate also preserves a substantial part of the secondary structure, it undergoes a change into a noncompact state changing its Stokes radius from approximately 30 to 39 A. Refolding experiments showed that JHBP molecules recover their full protein structure, as judged from the CD spectrum, fluorescence experiments, and JH binding activity measurements. The free energy of unfolding in the absence of the denaturant, DeltaG(D-N), is calculated to be 4.1 kcal mol(-1).     

NMR assignments of juvenile hormone binding protein in complex with JH III

A hemolymph juvenile hormone binding protein (JHBP) shuttles hydrophobic JH, a key hormone in regulation of the insect life cycle, from the site of the JH biosynthesis to the cells of target organs. We report complete NMR chemical shift assignments of Bombyx mori JHBP in the JH III-bound state.     

Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris

Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.     

Synthesis and binding affinity of an iodinated juvenile hormone

The synthesis of the first iodinated juvenile hormone (JH) in enantiomerically enriched form is reported. This chiral compound, 12-iodo-JH I, has an iodine atom replacing a methyl group of the natural insect juvenile hormone, JH I, which is important in regulating morphogenesis and reproduction in the Lepidoptera. The unlabeled compound shows approximately 10% of the relative binding affinity for the larval hemolymph JH binding protein (JHBP) of Manduca sexta, which specifically binds natural 3H-10R,11S-JH I (labeled at 58 Ci/mmol) with a KD of 8 X 10(-8) M. It is also approximately one-tenth as biologically active as JH I in the black Manduca and epidermal commitment assays. The 12-hydroxy and 12-oxo compounds are poor competitors and are also biologically inactive. The radioiodinated [125I]12-iodo-JH I can be prepared in low yield at greater than 2500 Ci/mmol by nucleophilic displacement using no-carrier-added 125I-labeled sodium iodide in acetone; however, synthesis using sodium iodide carrier to give the approximately 50 Ci/mmol radioiodinated ligand proceeds in higher radiochemical yield with fewer by-products and provides a radioligand which is more readily handled in binding assays. The KD of [125I]12-iodo-JH I was determined for hemolymph JHBP of three insects: M. sexta, 795 nM; Galleria mellonella, 47 nM; Locusta migratoria, 77 nM. The selectivity of 12-iodo-JH I for the 32-kDa JHBP of M. sexta was demonstrated by direct autoradiography of a native polyacrylamide gel electrophoresis gel of larval hemolymph incubated with the radioiodinated ligand. Thus, the in vitro and in vivo activity of 12-iodo-JH I indicate that it can serve as an important new gamma-emitting probe in the search for JH receptor proteins in target tissues.     

The morphostatic actions of juvenile hormone

The maintenance of "status quo" in larvae by juvenile hormone (JH) involves both the programming of ecdysteroid-dependent synthesis during the molt and the suppression of morphogenetic growth during the intermolt. The latter morphostatic action does not require ecdysteroids, and has been studied in the formation of imaginal discs in Manduca sexta. Preultimate larval instars have both invaginated discs and imaginal primordia, both of which grow isomorphically with the larva. In the last instar, the young discs/primordia initiate the morphogenesis and patterning that results in a mature disc. JH suppresses both the initiation and progression of the signaling that transforms immature discs or primordia into a fully patterned imaginal disc. This transformation normally occurs in the context of the rapid growth of the last larval stage, and nutrient-dependent factors appear to be able to override the JH suppression. The morphostatic action of JH may have been important for the evolution of the larval stage. Studies on embryos of basal, hemimetabolous insects show that their premature exposure to JH can truncate patterning programs and cause precocious tissue maturation, factors essential for organizing a novel larval form. This suppression of embryonic patterning then results in embryonic fields that remain dormant as long as JH is present. These are the primordia that can transform into imaginal discs once JH disappears in preparation for metamorphosis.     

Juvenile hormone acts at embryonic molts and induces the nymphal cuticle in the direct-developing cricket

During embryogenesis of hemimetabolous insects, the sesquiterpenoid hormone, juvenile hormone (JH), appears late in embryogenesis coincident with formation of the first nymphal cuticle. We tested the role of embryonic JH by treating cricket embryos with JH III, or the JH-mimic (JHM) pyriproxifen, during early embryogenesis. We found two discrete windows of JH sensitivity. The first occurs during the formation of the first (E1) embryonic cuticle. Treatment with JHM prior to this molt produced small embryos that failed to complete the movements of katatrepsis. Embryos treated after the E1 molt but before the second embryonic (pronymphal) molt completed katatrepsis but then failed to complete dorsal closure and precociously formed nymphal, rather than pronymphal characters. This second sensitivity window was further assessed by treating embryos with low doses of JH III prior to the pronymphal molt. With low doses, mosaic cuticles were formed, bearing features of both the pronymphal and nymphal stages. The nymphal characters varied in their sensitivity to JH III, due at least in part to differences in the timing of their sensitivity windows. Unexpectedly, many of the JH III-treated embryos with mosaic and precocious nymphal cuticles made a second nymphal cuticle and successfully hatched. JH treatment also affected the growth of the embryos. By focusing on the developing limb, we found that the effect of JH upon growth was asymmetric, with distal segments more affected than proximal ones, but this was not reflected in misexpression of Distal-less or Bric-a-brac, which are involved in proximal-distal patterning of the limb.Edited by P. Simpson     

Insect juvenile hormone binding protein shows ancestral fold present in human lipid-binding proteins

Low molecular weight juvenile hormone binding proteins (JHBPs) are specific carriers of juvenile hormone (JH) in the hemolymph of butterflies and moths. As hormonal signal transmitters, these proteins exert a profound effect on insect development. The crystal structure of JHBP from Galleria mellonella shows an unusual fold consisting of a long α-helix wrapped in a highly curved antiparallel β-sheet. JHBP structurally resembles the folding pattern found in tandem repeats in some mammalian lipid-binding proteins, with similar organization of one cavity and a disulfide bond between the long helix and the β-sheet. JHBP reveals, therefore, an archetypal fold used by nature for hydrophobic ligand binding. The JHBP molecule possesses two hydrophobic cavities. Several lines of experimental evidence conclusively indicate that JHBP binds JH in only one cavity, close to the N- and C-termini, and that this binding induces a structural change. The second cavity, located at the opposite end of the molecule, could bind another ligand.     

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     

Purification of the Drosophila Kc cell juvenile hormone binding protein

The juvenile hormone binding protein (JHBP) from the cytosol of Drosophila melanogaster Kc cells has been purified with the use of a juvenile hormone photoaffinity analog, 10,11-epoxy (2E, 6E) farnesyl diazoacetate (EFDA). The purification procedure consists of five chromatographic steps and the end product of the purification procedure showed homogeneity by means of both native and SDS polyacrylamide gel electrophoresis. Furthermore, using a racemic mixture of the natural hormone, [³H]juvenile hormone III (JH III), as the radioligand in this purification procedure, we demonstrate that the purified protein is likely the authentic intracellular JHBP.     

Purification, characterisation and titre of the haemolymph juvenile hormone binding proteins from Schistocerca gregaria and Gryllus bimaculatus

Juvenile hormone binding proteins (JHBPs) were extracted from the haemolymph of adult desert locusts, Schistocerca gregaria, and Mediterranean field crickets, Gryllus bimaculatus. The JHBPs were purified by polyethyleneglycol precipitation, filtration through molecular weight cut off filters and chromatography on a HiTrap heparin column. The juvenile hormone (JH) binding activity of the extracts was measured using a hydroxyapatite assay and the purification progress was monitored by native gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The haemolymph JHBPs of both insects are hexamers composed of seemingly identical subunits. The JHBP of the locust has a native Mr of 480 kDa with subunits of 77 kDa, whereas the JHBP of the cricket has a Mr of 510 kDa with subunits of 81 kDa. The locust JHBP binds JH III with moderate affinity (KD = 19 nM). Competition for binding of JH II and JH I was about 2 and 5 times less, respectively. The cricket JHBP also has a moderate affinity for JH III (KD = 28 nM), but surprisingly, competition for binding of JH II was equal to that of JH III and JH I competed about 3 times higher. No sequence information was obtained for the locust JHBP, but the N-terminal sequence of the cricket JHBP shows ca. 56% sequence homology with a hexamerin from Calliphora vicina. Antisera raised against the purified JHBPs were used to measure age- and sex-dependent changes in haemolymph JHBP titres and to confirm that the JHBPs of both species are immunologically different.     

Identification,characterization, and developmental profile of a high molecular weight,juvenile hormone-binding protein in the hemolymph of the migratory grasshopper,Melanoplus sanguinipes

In the hemolymph of Melanoplus sanguinipes, a high molecular weight juvenile hormone binding protein (JHBP) was identified by photoaffinity labelling and found to have a M_r of 480,000. The JHBP, purified using native gel electrophoresis followed by electroelution, has an equilibrium dissociation constant for JH III of 2.1 nM and preferentially binds JH III over JH I. Antibody raised against JHBP recognized only the 480,000 band. Under denaturing conditions the native JHBP gave a single band with a M_r 78,000. The antibody against native JHBP recognized only the 78,000 protein in SDS-treated hemolymph samples, indicating that JHBP is a hexamer in this species. The concentration of JHBP fluctuates in both the sexes during nymphal and adult development in parallel with total protein content of hemolymph. © 1995 Wiley-Liss, Inc.     

Purification and characterization of a juvenile hormone binding protein from hemolymph of the silkworm,Bombyx mori

A juvenile hormone binding protein (JHBP) has been isolated from Bombyx mori hemolymph by gel filtration, ion-exchange chromatography, chromatofocusing and hydroxyapatite column chromatography. Gel electrophoresis indicates that the isolated protein is homogeneous in the presence or absence of a denaturing agent. The JHBP in question has a relative molecular mass of 32 kDa, determined by denaturing gel electrophoresis. Chromatofocusing analysis indicated that the JHBP is an acidic protein with pI 4.9. The protein exhibits a dissociation constant of 9.0 × 10⁻⁸ M for JH I, 1.14 × 10⁻⁷ M for JH II and 3.9 × 10⁻⁷ M for JH III, and thus its affinity for JH analogues is in the order of JHI >JHII >JHIII. Its amino acid composition indicates that the protein consists of 297 residues of 18 kinds of amino acids. The sequence of the N-terminus of the polypeptide chain was determined for 34 of the first 36 residues: Asp-Gln-Asp-Ala-Leu-Leu-Lys-Pro-?-Lys-Leu-Gly-Asp-Met-Gln-Ser-Leu-Ser-Ser-Ala-Thr-Gln-Gln-Phe-Leu-Glu- Lys-Thr-Ser-Lys-Gly-Ile-Pro-?-Tyr-His-.