Rapid purification and N-terminal amino acid sequence of a photoaffinity-labeled juvenile hormone binding protein from an arctiid moth larva,Platyprepia virginalis

A novel two-step procedure has been developed for the purification of juvenile hormone binding proteins (JHBP) from caterpillars. Crude hemolymph was photoaffinity labeled with [³H]EHDA, a JH II analog. After removal of excess ligand, 40 ml of buffer-diluted hemolymph containing over 200 mg protein was submitted to preparative isoelectric focusing (IEF) using a Rotofor device. After removal of ampholytes by dialysis, the ³H-labeled fractions were purified to > 95% homogeneity by anion-exchange HPLC. Over 1000-fold purification could be achieved in a few days on a scale which provides 100–1000 μg of purified JHBP. Proteins thus obtained can be used for proteolytic digestion or can be sequenced after electroblotting from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel onto a polyvinylidene fluoride (PVDF) membrane. This protocol is illustrated for the purification and N-terminal amino acid sequencing of a hemolymph JHBP from an arctiid wooly bear caterpillar, Platyprepia virginalis.     

Juvenile hormone binding components of locust fat body

Juvenile hormone (JH) binding components from the fat body of the African migratory locust were analyzed in a search for a potential nuclear JH receptor. Biosynthetically prepared 10R[³H]JH III gave a high proportion of specific binding to isolated nuclei and extracted proteins; data obtained with the JH analogs, [³H]methoprene and [³H]pyriproxyfen, on the other hand, were obscured by abundant non-specific binding. The vast majority of the high affinity JH III binding activity present in cytosolic and nuclear extracts was due to a high molecular weight JH binding protein (JHBP) which has previously been identified in locust hemolymph. This protein has several chromatographic forms which interfered in the search for a nuclear JH receptor. When specific antiserum was used to remove JHBP from nuclear extracts, a novel JH binding activity (NBP) was detected. NBP could be separated from JHBP by precipitation with ammonium sulfate. NBP displayed a high affinity for JH III (Kd = 0.25 nM) and JH I and JH II competed strongly for JH III binding, whereas methoprene and pyriproxyfen showed apparent competition when present in 1,000-fold excess. NBP was present in nuclear extracts at approximately 25,000 sites per cell; levels were similar in male and female locusts and were not greatly affected by the presence or absence of JH. The characteristics of NPB make it a strong candidate for a nuclear JH receptor. © 1995 Wiley-Liss, Inc.     

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.     

Photoaffinity labelling of juvenile hormone binding proteins in the cockroach Leucophaeamaderae

The synthesis and testing of several diazocarbonyl JH analogs (diazo JHA) which act as photoaffinity labels for insect juvenile hormone binding proteins are described. The best competitor, 10,11-epoxyfarnesyl diazoacetate, has been shown to irreversibly reduce [³H]-JH III binding to both ovarian and hemolymph JHBP from Leucophaeamaderae after irradiation at 254 nm for 20 seconds. No loss of activity was observed after incubation of JHBP and diazo JHA without irradiation. Protection from photoinactivation by diazo JHA II was achieved by the presence of an equimolar amount of JH III during the photolysis. Photoaffinity labeled proteins show loss of binding capacity without alteration of the binding affinity. This is the first example of the use of a photoaffinity label in the study of JH action on a molecular level, and may become a valuable tool in the elucidation of JH-receptor-chromatin interactions.     

JUVENILE HORMONE BINDING PROTEIN IN THE OVARIES OF HOUSEFLY MUSCA DOMESTICA VACINA

Abstract  By using charcocal binding assay, the juvenile hormone binding protein (JHBP) was determined in the ovaries of houseflies. This ovarian JHBP possesses high affinity with juvenile hormone III (JH III) and has a K_d of 2.1 III 10^--⁸ M. The binding of ³H-juvenile hormone III (³H-JH III) to this protein was inhibited by unlablled JH III, but not by juvenile hormone analog ZR 512 or ZR 515. The level of this ovarian JHBP reached the highest in houseflies 48 h after emergence, and was 6. 5-fold and 15. 5-fold higher than that in housefIies 60 h and 72 h after emergence, respectively. No binding activity was detected in the ovaries of houseflies 24 h or 36 h after emergence. The absence of JHBP in the ovaries of houseflies 36 h after emergence could be reversed by applying JH III to newly emerged houseflies. The data suggest that the fluctuation of the JHBP concentration might associate with the action of juvenile hormone (JH) on housefly vitellogenesis.     

A specific photoaffinity label for hemolymph and ovarian juvenile hormone-binding proteins in Leucophaea maderae

A tritium-labeled diazocarbonyl juvenile hormone (JH) analog, (10-[10,11-3H]epoxyfarnesyl diazoacetate, [3H]EFDA), covalently bound to proteins in both hemolymph and ovarian extracts when reaction mixtures were irradiated with UV light. The addition of various concentrations of unlabeled JH III selectively inhibited [3H]EFDA photoattachment to proteins. Using the Scatchard method of analysis, [3H]EFDA bound specifically and with relatively high affinity (KD = 1.5 X 10(-6) M) to a macromolecule in each extract, although nonspecific binding to other molecules was also present (20-50%). To determine if [3H]EFDA bound at the JH III-binding site on the binding proteins, radioactive [3H]JH III or [3H]EFDA was complexed with proteins in the presence of various concentrations of either unlabeled JH III or JH I under equilibrium conditions. The results demonstrated that the natural hormone, JH III, displaced both bound labeled ligands 4.1 +/- 0.5 times better than the homolog JH I. Thus, the photoaffinity label [3H]EFDA bound at the same site on the protein as [3H] JH III. Fluorescent autoradiography of [3H]EFDA-labeled proteins separated by sodium dodecyl sulfate electrophoresis revealed that several proteins in both hemolymph and ovarian extracts bound [3H]EFDA. To determine the specificity of binding, extracts were irradiated with UV light in the presence of unlabeled JH III and [3H]EFDA. The results demonstrated that JH III prevented photoattachment of [3H]EFDA to a major protein in each extract. The molecular weight of these proteins was estimated at approximately 200,000 for both the hemolymph protein and the ovarian protein.     

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.     

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.     

Synthesis and secretion of a precursor hemolymph juvenile hormone-binding protein in the adult female cockroach Leucophaea maderae

Hemolymph juvenile hormone-binding protein (JHBP) is synthesized and secreted from fat body in the adult female cockroach, Leucophaea maderae. The data in this paper suggest it is initially secreted from the fat body as a larger peptide whereas data in the accompanying paper demonstrate that JHBP is apolipophorin I. Using media from cultures of fat body maintained in vitro, a JH-binding component was found that is JH III saturable, has a K_D of 1.5 × 10⁻⁸ M, binds JH III > JH II > JH I, and has a sedimentation value of 6.5S on high salt sucrose gradients. Each of these properties is identical to those of the JHBP extracted from the hemolymph. To identify the protein that bound JH, media proteins were photoaffinity labeled with 10-[10,11-³H]epoxyfarnesyl diazoacetate ([³H]EFDA). The results revealed that two media proteins bound [³H]EFDA in the absence of JH III, but not in the presence of 100-fold excess JH III. The molecular weights of the two media peptides were estimated by SDS-PAGE to be 275,000 and 220,000.To determine if the JHBP found in media of fat body cultures was due to hemolymph contamination of fat body, incorporation of [³H]leucine into newly synthesized and secreted fat body proteins during a 48 h culture period was monitored. During the culture period, linear increases in the concentrations of radiolabeled 275 and 220 kD JHBP were observed. Monoclonal antibodies specific for the 220 kD hemolymph JHBP were found to recognize both the 275 and 220 kD JHBPs in the media.To investigate the possibility that the 275 kD protein is a precursor to the 220 kD protein and that components of the hemolymph process or modify the precursor, hemolymph was introduced into fat body cultures and relative concentrations of the 275 and 220 kD media JHBPs were determined. Addition of hemolymph to these organ cultures resulted in an increase in the concentration of radiolabeled 220 kD JHBP and a proportional decrease in the concentration of radiolabeled 275 kD JHBP, suggesting that the 275 kD protein is a precursor to the 220 kD hemolymph JHBP. The mechanism of processing or modification remains undetermined.     

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.     

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.     

Cloning and sequence analysis of Galleria mellonella juvenile hormone binding protein--a search for ancestors and relatives

Juvenile hormone binding proteins (JHBPs) serve as specific carriers of juvenile hormone (JH) in insect hemolymph. As shown in this report, Galleria mellonella JHBP is encoded by a cDNA of 1063 nucleotides. The pre-protein consists of 245 amino acids with a 20 amino acid leader sequence. The concentration of the JHBP mRNA reaches a maximum on the third day of the last larval instar, and decreases five-fold towards pupation. Comparison of amino acid sequences of JHBPs from Bombyx mori, Heliothis virescens, Manduca sexta and G. mellonella shows that 57 positions out of 226 are occupied by identical amino acids. A phylogeny tree was constructed from 32 proteins, which function could be associated to JH. It has three major branches: (i) ligand binding domains of nuclear receptors, (ii) JHBPs and JH esterases (JHEs), and (iii) hypothetical proteins found in Drosophila melanogaster genome. Despite the close positioning of JHEs and JHBPs on the tree, which probably arises from the presence of a common JH binding motif, these proteins are unlikely to belong to the same family. Detailed analysis of the secondary structure modeling shows that JHBPs may contain a beta-barrel motif flanked by alpha-helices and thus be evolutionary related to the same superfamily as calycins.     

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.     

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).     

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.     

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.     

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.     

Further characterization of the juvenile hormone binding protein from the cytosol of a Drosophila cell line: Use of a photoaffinity label

Further characterization of the juvenile hormone (JH) binding protein from the cytosol of Drosophila melanogaster Kc cells has been accomplished with the use of a photoaffinity analogue of JH. The analogue, 10,11-epoxy(2E,6E)farnesyl diazoacetate (EFDA), is tritiated in the 10-position. Following photolysis with short-wave ultraviolet light, it can be demonstrated that [³H]EFDA binds specifically to the cytosolic JH binding protein. This binding is inhibited if irradiation occurs in the presence of either unlabelled JH I or JH III. Both JH homologues protect the binding site equally against [³H]EFDA. No protection is observed with either methoprene or farnesyl acetate, a close structural analogue of EFDA that lacks the diazo photoactivatable group.The cytosolic JH binding protein, following covalent labelling with tritiated EFDA, was characterized by gel filtration column chromatography, velocity sedimentation through sucrose gradients, both native and denaturing gels, and binding to DNA cellulose. The binding protein has a molecular weight of approx. 49,200 and may consist of two subunits.     

Juvenile hormone binding protein traffic — Interaction with ATP synthase and lipid transfer proteins

Juvenile hormone (JH) controls insect development, metamorphosis and reproduction. In insect hemolymph a significant proportion of JH is bound to juvenile hormone binding protein (JHBP), which serves as a carrier supplying the hormone to the target tissues. To shed some light on JHBP passage within insect tissues, the interaction of this carrier with other proteins from Galleria mellonella (Lepidoptera) was investigated. Our studies revealed the presence of JHBP within the tracheal epithelium and fat body cells in both the membrane and cytoplasmic sections. We found that the interaction between JHBP and membrane proteins occurs with saturation kinetics and is specific and reversible. ATP synthase was indicated as a JHBP membrane binding protein based upon SPR-BIA and MS analysis. It was found that in G. mellonella fat body, this enzyme is present in mitochondrial fraction, plasma membranes and cytosol as well. In the model system containing bovine F₁ ATP synthase and JHBP, the interaction between these two components occurs with K_d = 0.86 nM. In hemolymph we detected JHBP binding to apolipophorin, arylphorin and hexamerin. These results provide the first demonstration of the physical interaction of JHBP with membrane and hemolymph proteins which can be involved in JHBP molecule traffic.