Marian Kochman

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 alpha-helix wrapped in a highly curved antiparallel beta-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 beta-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 F(1) 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.

Purification of the juvenile hormone esterase from the haemolymph of the wax moth Galleria mellonella (lepidoptera)

Juvenile hormone esterase (JHE) from the haemolymph of fully grown larvae of Galleria mellonella has been purified 2630-fold. The procedure involves a combination of anion exchange chromatography on DEAE-Sephacel, chromatography on SP-Sephadex C-50, P-cellulose chromatography and hydroxylapatite chromatography. The resulting JHE preparation which still has some detectable 1-naphthylacetate esterase activity was stable at pH range 6.5–8.5 for 14 hr at 5°C. The diluted 1 μg/ml JHE can be stored for two months at −20°C without any detectable loss of activity. This enzyme preparation has specific activity of 0.92 μ M JH-I hydrolyzed/min per mg protein, KmJH-I 2.1 × 10−8 M, optimum pH 6.5–9.5. The value for Km is the lowest yet observed for JHE from any source. Analytical electrofocusing of the 2630-fold purified JHE reveals two distinct JHE activity bands. One heavy JHE activity band (80% of total activity) does not contain any detectable 1-naphthylacetate activity and is comprised of three closely positioned protein bands of pH 4.75 ± 0.05. The second JHE activity band of pH 5.0 exhibits 1-naphthylacetate activity in addition to JHE activity.

Hydrolysis and binding of the juvenile hormone in the haemolymph of Galleria mellonella

Abstract Haemolymph of fully grown larvae of Galleria mellonella was chromatographed on Sephadex G-200 and the column eluate assayed for hydrolytic activity and juvenile hormone (JH) binding. Hydrolysis of JH-I and 1-naphthyl acetate (1-NA) by high molecular fractions ( 2 × 10 5 ) was sensitive to 2 × 10 −4 M diisopropylphosphofluoridate (DFP). JH-Hydrolysis by proteins ranging in molecular weight from 1.4 × 10 5 to 3.7 × 10 4 was unaffected by DFP and 2.1 × 10 −4 M p- chloromercuribenzoate (PCMB), whereas the overlapping 1-NA hydrolysis was inhibited by these reagents. Several of the JH-esterase peaks coincided with those which hydrolyzed 4-methylumbelliferyl esters of JH-II acid, JH-III acid and oleic acid. The enzymes hydrolyzing these substrates were also unaffected by DFP and PCMB. Highest non-enzymatic binding of JH occurred in the protein fraction with mol. wt of 2.5 × 10 4 . The binding protected JH against most of the blood esterases.

Age dependent changes in the binding and hydrolysis of juvenile hormone in the haemolymph of last instar larvae of Galleria mellonella

Abstract Binding and hydrolysis of juvenile hormone I (JH) and cleavage of four 1-naphthyl esters were determined in the haemolymph of Galleria mellonella throughout the last larval instar. JH binding per ml haemolymph parallels changes in the protein content with a maximum at the onset of spinning at 120 hr (152 mg protein/ml; binding of 20 n-mole JH/ml) and a drop at the pupal ecdysis. Activity of JH esterase(s) exhibits two maxima: first between cessation of feeding and the start of spinning at 96–120 hr (hydrolysis of 65 n-mole JH/ml/min) and second just before pupal ecdysis at 168 hr (52 n-mole JH/ml/min). Correlations between these changes and available data on the JH content in caterpillars indicate that JH binding protein and JH esterase(s) may play a role in clearing JH from its target tissues rather than in regulating its amounts. Fluctuations in the hydrolysis of 1-naphthyl esters of acetate (max. 4.5 μ-mole/ml/min), butyrate (max. 21 μ-mole/ml/min), palmitate (max. 0.5 μ-mole/ml/min), and laurate (max. 0.4 μ-mole/ml/min) reflect primarily the feeding activity of caterpillars and follow a very different course than changes in the potential to hydrolyze JH.

Purification of human liver fructose-1,6-bisphosphatase

Human liver fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) has been purified 1200-fold using a heat treatment step followed by absorption on phosphocellulose at pH 8 and specific elution with buffer containing the substrate (fructose 1,6-bisphosphate) and allosteric effector (AMP). The enzyme is homogeneous in electrophoresis in polyacrylamide gel, in the presence and absence of denaturing agent. It has a molecular weight of 144 000 and is composed of four identical or nearly identical subunits. Fluorescence spectra indicate that the enzyme does not contain tryptophan residues. The pH optimum is 7.5 and the Km is determined as 0.8 microM. The enzyme is inhibited by AMP in cooperative manner with a K0 x 5 of 6 microM.

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

Abstract 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 preprotein 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 fivefold 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 βbarrel motif flanked by αhelices and thus be evolutionary related to the same superfamily as calycins.

Biosynthesis and degradation of juvenile hormone in corpora allata and imaginal wing discs of Galleria mellonella (L.)

Abstract The rate of juvenile hormone (JH) biosynthesis by corpora allata-corpora cardiaca complex (CA/CC) during two last larval instars of Galleria mellonella was analysed. The rate of biosynthesis reaches maxima at the beginning of the VIth and VIIth instars. It is markedly reduced before the last larval ecdysis and after the first day of the last larval instar. After passing the second day of the last larval instar CA/CC exhibits again an increased ability for the biosynthesis of JH. The JH esterase activity in CA/CC is very low at the beginning of last larval instar and rapidly increases after the first day of this instar. Beginning on the second day of last larval instar the rate of JH hydrolysis is always higher than the rate of JH synthesis in CA/CC. It is concluded that the secretion of JH by CA/CC is possible until the second day of the last larval instar. After this, JH-acid can be supplied by CA/CC to peripheral tissues. The imaginal wing discs of mobile prepupa exhibit the ability to methylate JH-acid. It is concluded that some elevations of JH titre in G. mellonella haemolymph after the second day of VIIth instar are due in part to JH-acid methyltransferase activity in the imaginal discs.

Tissue specific juvenile hormone degradation in Galleria mellonella

Degradation of juvenile hormone III (JH III) in the fat body integument and silk glands of the last instar larvae of Galleria mellonella follows two major routes: the ester bond hydrolysis and the epoxide ring hydration. The juvenile hormone esterase activity is primarily localized in the cytosol as a 70–100 kDa protein fraction with an isoelectric point of pH 4.7. The epoxide hydrolase activity was found both in the 100,000 g supernatant and in the insoluble cell fraction. The soluble epoxide hydrolase occurs in the form of “an aggregated protein” of a molecular weight over 1000 kDa. On the first day of the last larval instar, the epoxide hydrolase activity accounts for approx. 50, 80 and 90% of JH III degradation in the fat body, integument and silk glands respectively. This activity is present both in the cytosol and in the insoluble cell fraction. The importance of the esterase activity for JH III degradation increases in the course of the instar. In the fat body it becomes dominant already on day 2, when the epoxide hydrolase activity completely disappears from the cytosol. The ester cleavage also becomes the major route of JH degradation in the integument from day 4, but in the silk glands it becomes equal to the rate of epoxide hydration only on day 6. In contrast to these tissues, JH III degradation in the haemolymph occurs only via esterolytic cleavage.

Juvenile hormone binding proteins from the epidermis of Galleria mellonella

Abstract Juvenile hormone binding proteins (JHBP) have been detected in cytosol of larval and pupal epidermis of Galleria mellonella. Juvenile hormone (JH) binding activity changes during insect development, reaching a maximum after each ecdysis. Using density gradient centrifugation, three distinct peaks of JHBP have been detected: 8.8–9.2 S, 4.2–4.6 S, and 3 S. Despite molecular mass heterogeneity of the JHBPs, only one class of JH binding sites has been detected. The equilibrium dissociation constants (Kd) of the larval JHBP for the natural (10 R, 11 S) JHs and their enantiomers are: Kd, (10 R, 11 S)-JH I = 24 ± 9 nM; Kd, (10 S, 11 R)-JH I = 51 ± 15 nM; Kd, (10 R, 11 S)-JH II = 9.7 ± 2.0 nM, Kd, (10 S, 11 R)-JH II = 18 ± 3 nM. From competitive binding assays using 3H-labeled (10 R, 11 S)-JH II, it was found that racemic JH III and the JH analog (10 R, 11 S)-12-iodo-JH I are bound with affinity close to the unnatural (10 S, 11 R) enantiomer of JH I. Juvenoid R 394 (a methoprene analog) is bound to JHBP with an affinity 100-fold lower than (10 R, 11 S)-JH II, whereas binding of the epoxygeranylphenyl ether juvenoid R 20458 is not detectable. Addition of 0.1% (w/v) Zwittergent 3–10 to the epidermal cytosol causes an increase of JH binding activity by 2–10-fold in penultimate and ultimate instars, respectively. This zwitterionic N-alkyl sulfobetaine detergent apparently converts a high molecular mass JHBP species into a 65-kDa component, but does not change the stereospecificity or the affinity of JHBP towards the JH homologs or analogs.