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 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.     

Juvenile hormone binding protein and transferrin from Galleria mellonella share a similar structural motif.

It has been previously suggested that juvenile hormone binding protein(s) (JHBP) belongs to a new class of proteins. In the search for other protein(s) that may contain structural motifs similar to those found in JHBP, hemolymph from Galleria mellonella (Lepidoptera) was chromatographed over a Sephadex G-200 column and resulting fractions were subjected to SDS-PAGE, transferred onto nitrocellulose membrane and scanned with a monoclonal antibody, mAb 104, against hemolymph JHBP. Two proteins yielded a positive reaction with mAb 104, one corresponding to JHBP and the second corresponding to a transferrin, as judged from N-terminal amino acid sequencing staining. Transferrin was purified to about 80% homogeneity using a two-step procedure including Sephadex G-200 gel filtration and HPLC MonoQ column chromatography. Panning of a random peptide display library and analysis with immobilized synthetic peptides were applied for finding a common epitope present in JHBP and the transferrin molecule. The postulated epitope motif recognized by mAb 104 in the JHBP sequence is RDTKAVN, and is localized at position 82-88.     

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.     

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.     

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.     

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.     

Photoaffinity labeling and biochemical characterization of binding proteins for pheromones,juvenile hormones,and peptides

Radiolabeled photoaffinity analogs can be used to purify and characterize proteins involved in pheromone perception, juvenile hormone (JH) action, and neuropeptide reception. Several photoaffinity analogs and purification strategies are described for each of these physiological targets. First, a diazoacetate photoaffinity label is used to selectively modify the pheromone binding protein of the gypsy moth, Lymantria dispar. Reverse-phase HPLC is then employed to fractionate the male antennal proteins. Second, a tandem procedure involving preparative isoelectric focusing (IEF) and ion-exchange (IEX) HPLC is employed for the purification of the Manduca sexta hemolymph juvenile hormone binding protein (JHBP), which has now been cloned and sequenced. A separate application of this strategy for the purification of the 29 kDa JH I/methoprene receptor proteins from epidermal nuclei of M. sexta larvae is outlined. A new photolabel, farnesyl diazoketone, has been employed for the characterization of crustacean hemolymph methyl farnesoate binding proteins. Third, the development of neuropeptide photoaffinity labels is described for two systems: (1) the red pigment concentrating hormone (RPCH) of shrimp and (2) the allatostatins isolated from the brain of the cockroach Diploptera punctata.     

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.     

Two disulphide bridges are present in juvenile hormone binding protein from Galleria mellonella

The hemolymph juvenile hormone binding protein (JHBP) from Galleria mellonella contains two disulphide bridges/molecule and no free Cys residues. An alignment of primary structures of other Lepidopteran JHBPs indicates that Cys residues, equivalent to Cys10,17,151,195 in G. mellonella JHBP, maybe involved in -S-S- bridge formation.     

Hybridoma antibodies as specific probes to Drosophila melanogaster yolk polypeptides

Hybridoma antibodies to Drosophila melanogaster soluble yolk proteins (YPs) were developed by both in vivo and in vitro immunizations followed by the fusion of SP2/0-Ag14 cells and splenocytes of BALB/c mice. Rabbit antiserum was made female specific by affinity column with male proteins as ligand. The binding sites of these hybridoma antibodies and rabbit antibodies towards different YP components were identified with a combination of gel electrophoresis, Western blotting and immunohistochemical staining. A double antibody sandwich enzyme-linked immunosorbent assay was developed with monoclonal antibodies from 2 cell lines and alkaline phosphatase labelled rabbit polyclonal antibodies as primary and secondary antibodies respectively. Yolk polypeptide levels in the haemolymph can be monitored in individual insect samples.     

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     

Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced by Bacillus cereus

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L₂ component, and antibody 1C2 was specific for the L₁ protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.     

Positions of disulfide bonds and N-glycosylation site in juvenile hormone binding protein

The juvenile hormone binding protein (JHBP) from Galleria mellonella hemolymph is a glycoprotein composed of 225 amino acid residues. It contains four Cys residues forming two disulfide bridges. In this study, the topography of the disulfide bonds as well as the site of glycan attachment in the JHBP molecule from G. mellonella was determined, using electrospray mass spectrometry. The MS analysis was performed on tryptic digests of JHBP. Our results show that the disulfide bridges link Cys10 and Cys17, and Cys151 and Cys195. Of the two potential N-glycosylation sites in JHBP, Asn4, and Asn94, only Asn94 is glycosylated. This site of glycosylation is also found in the fully biologically active recombinant JHBP expressed in the yeast Pichia pastoris.     

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.     

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).     

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 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-.     

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.     

Cuticle proteins of the boll weevil,Anthonomus grandis,abdomen: Structural similarities and glycosylation

Cuticle proteins are thought to be important in defining the structural and functional differences occurring in insect cuticle. In order to explain and better understand the structural similarities among the cuticle proteins of the cotton boll weevil, Anthonomus grandis Boheman, described in a previous study (Stiles and Leopold, 1990, Insect Biochem.20, 113–125) three series of monoclonal antibody producing hybridoma cell lines were produced. Larval, pupal or adult cuticle proteins were used as antigens. While some of the monoclonal antibodies were specific for one or two cuticle proteins from a single developmental stage, the majority showed multiple cuticle protein binding patterns on Western blots. To determine whether this cross-reaction was due to common oligosaccharide chains bound to the proteins, lectins were used to probe Western blots. Many of the cuticle proteins were found to be glycosylated. The majority of the Con A reactive carbohydrate could be removed from the protein by N-glycosidase F digestion (specific for N-asparagine linked carbohydrate). N-glycosidase F digestion did not reduce the multiple cross-reactions of the monoclonal antibodies, nor did periodate oxidation of the CP. The carbohydrate remaining after enzyme digestion is presumably O-linked to serine/threonine.