Characterization of affinity-purified juvenile hormone esterase from the plasma of the tobacco hornworm, Manduca sexta

Juvenile hormone (JH) esterase found primarily in the hemolymph and tissues of insects is a low abundance protein involved in the ester hydrolysis of insect juvenile hormones, JHs. The enzyme was purified from the larval plasma of wild-type Manduca sexta using an affinity column prepared by binding the ligand, 3-[(4'-mercapto)butylthio]-1,1,1-trifluoropropan-2-one (MBTFP), to epoxy-activated Sepharose. The purification was greater than 700-fold with a 72% recovery, and the purified enzyme appeared as a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoelectrophoresis, reverse phase high performance liquid chromatography, and amino acid sequence analysis. The molecular weight was 66,000. The plasma JH esterase in wild-type, black, and white strains of M. sexta was similar when analyzed by immunotitration, wide range (pH 3.5-9.0) isoelectric focusing, and inhibition with MBTFP and 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP). Inhibition studies revealed a sensitive and insensitive form (I50 = 10(-9) and 10(-6) M, respectively) in these three biotypes. Narrow range isoelectric focusing (pH 4.0-7.0) indicated the presence of two major isoelectric forms with pI values of 6.0 and 5.5, but their inhibition kinetics with OTFP and O,O-diisopropyl phosphorofluoridate were identical.     

Preparation of homogeneous juvenile hormone specific esterase from the haemolymph of the tobacco hornworm,Manduca sexta

Juvenile hormone specific esterase has been purified to homogeneity from the haemolymph of Manduca sexta by a combination of gel permeation, ion exchange and hydroxylapatite chromatography. The pure esterase has a molecular weight of 68,000 and consists of a single peptide chain. Juvenile hormone specific esterase hydrolyzes JH I at about twice the rate as JH III, has appreciable activity against methyl farnesenate, but does not hydrolyze 1-naphthyl acetate. The homogeneous enzyme is stable for several months when stored at low temperature.     

A novel protein that binds juvenile hormone esterase in fat body tissue and pericardial cells of the tobacco hornworm Manduca sexta L

Juvenile hormone esterase degrades juvenile hormone, which acts in conjunction with ecdysteroids to control gene expression in insects. Circulating juvenile hormone esterase is removed from insect blood by pericardial cells and degraded in lysosomes. In experiments designed to characterize proteins involved in the degradation of juvenile hormone esterase, a pericardial cell cDNA phage display library derived from the tobacco hornworm moth Manduca sexta L. was constructed and screened for proteins that bind juvenile hormone esterase. A 732-base pair cDNA encoding a novel 29-kDa protein (P29) was isolated. Western and Northern analyses indicated that P29 is present in both pericardial cell and fat body tissues and is expressed in each larval instar. In immunoprecipitation experiments, P29 bound injected recombinant juvenile hormone esterase taken up by pericardial cells and native M. sexta juvenile hormone esterase in fat body tissue, where the enzyme is synthesized. Binding assays showed that P29 bound juvenile hormone esterase more strongly than it did a mutant form of the enzyme with mutations that perturb lysosomal targeting. Based on these data, we propose that P29 functions in pericardial cells to facilitate lysosomal degradation of juvenile hormone esterase.     

Soluble tyrosinases from pharate pupal integument of the tobacco hornworm,Manduca sexta (L.)

Two soluble phenoloxidases were partially purified from pharate pupal integument of Manduca sexta by gel filtration or sucrose density gradient centrifugation. Thiourea was used to retard the formation of higher molecular weight aggregates. Subsequent analysis by gel filtration HPLC showed that the apparent molecular weights were about 300 kD and >700 kD. The smaller phenoloxidase was further purified by anion exchange HPLC. In comparative studies with mushroom tyrosinase and a fungal laccase, the Manduca phenoloxidases were identified as tyrosinases, since they exhibited monophenol mono-oxygenase activity (EC 1.14.18.1) and catechol oxidase activity (EC 1.10.3.1). Both enzyme preparations catalyzed ortho-hydroxylation of tyrosine at a relatively show rate, oxidized o-diphenols at a much faster rate than p-diphenols or monophenols, had broad pH optima from about pH 5.5–7.5, and were completely inhibited by 1 μM phenylthiourea, N-β-alanyldopamine (NBAD) and N-β-alanylnorepinephrine (NBANE), which are both abundant in pupal cuticle, were oxidized to o-quinones by the tyrosinases, with the former catecholamine oxidizing at a 10-fold higher rate than the latter. NBANE was synthesized from NBAD, apparently by spontaneous tautomerization of the o-quinone to a p-quinone methide, which then reacted with water to form the β-hydroxylated product. The possible role of tyrosinase in insect integument is discussed.     

Iron binding proteins and their roles in the tobacco hornworm,Manduca sexta (L.)

Summary Manduca sexta larvae accumulate large amounts of iron during their larval feeding period. When⁵⁹Fe was fed to 5th instar larvae, it was evenly distributed among the hemolymph, gut and carcass until the cessation of feeding. By pupation 95% of the labelled iron was found in the fat body. In the adult a significant portion of this iron was found in flight muscle.Studies of the hemolymph disclosed two ironcontaining proteins. The first was composed of a single polypeptide chain of 80 kD, containing one atom of iron. This protein bound ionic iron in vitro and was able to transfer this iron to ferritin when incubated with fat body in vitro. Therefore, it appeared to serve a transport function. The second protein had a molecular weight of 490 kD with subunits of 24 and 26 kD and contained 220 g of iron/mg protein. Its chemical and ultrastructural characteristics were those of ferritin. These studies demonstrate the presence of both a transport protein and a unique circulating ferritin inManduca sexta, the latter serving a storage function during development and possibly also a transport function.     

PBAN regulation of pheromone biosynthesis in female tobacco hornworm moths,Manduca sexta (L.)

Manduca sexta females that were decapitated produced no pheromone during the scotophase following decapitation, indicating that they were free of pheromone biosynthesis activating neuropeptide (PBAN). When deuterated hexadecanoic or (Z)-11-hexadecenoic acid was applied to the sex pheromone glands of decapitated or intact females of the same age, and allowed to incubate in vivo for 24 h, deuterium labeled Δ-11- and Δ-10, 12-unsaturated 16-carbon fatty acids were produced in both types of females. Injection of PBAN into intact or decapitated females 23 h after application of labeled acids had no effect on the production of unsaturated labeled fatty acids. However, deuterium labeled aldehydes were produced only in females that were injected with PBAN. Therefore, in this species, PBAN activates the process by which fatty acyl precursors in the pheromone gland are converted into the pheromonal aldehydes. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Molecular cloning of the prothoracicotropic hormone from the tobacco hornworm,Manduca sexta

A cDNA encoding a putative precursor of prothoracicotropic hormone (PTTH) from the tobacco hornworm, Manduca sexta, was isolated and sequenced. This clone contains an open reading frame encoding a 226-amino acid prepropeptide hormone. The deduced amino acid sequence is composed of a signal sequence, a precursor domain and a mature hormone and shows similarities to the other PTTHs that have been cloned from closely related lepidopteran species, Bombyx mori, Samia cynthia ricini, Antheraea peryni, and Hyalophora cecropia. Although these cDNAs showed slightly less similarities in predicted amino acid sequences, seven cysteine residues and the hydrophobic regions within those mature peptides were conserved. In situ hybridization using a cDNA probe encoding the Manduca PTTH showed that PTTH mRNA was in two pairs of neurosecretory cells in the Manduca brain. The recombinant putative Manduca PTTH produced in E. coli was biologically active, both causing a larval molt in neck-ligated Manduca 4th instar larvae (ED(50)=50 pM) and the adult molt of diapausing Manduca pupae (ED(50)=79 pM), but was unable to stimulate molting of debrained Bombyx pupae.     

Characterization of a diuretic hormone receptor from the tobacco hornworm,Manduca sexta

We have characterized a diuretic hormone receptor from the tobacco hornworm, Manduca sexta. A single high affinity binding site for the 41 amino acid M. sexta diuretic hormone was found in membranes prepared from Malpighian tubules of fifth stadium larvae. The site has a Kd = 79 pM and Bmax = 3.1 pmol/mg protein. The dissociation rate constant was determined to be 0.11 min^?1 with a corresponding half-life of 6.4 min. Receptor binding of the hormone is inhibited by Ca²⁺ and Mg2⁺, while Na⁺ and K⁺ inhibit binding to a lesser extent. Truncated diuretic hormone analogs in which up to 20 amino acids were removed from the N-terminus maintain high affinity for the receptor. A diuretic hormone from Locusta migratoria which has 43% sequence identity with the M. sexta diuretic hormone also possesses a high affinity for the receptor. Conformational analysis of the M. sexta diuretic hormone indicates the core region of the peptide assumes a helical conformation, which may have implications in the binding of the peptide to the receptor. © 1993 Wiley-Liss. Inc.     

Ligand blot analysis of juvenile hormone esterase binding proteins in Manduca sexta L

Biotinylated recombinant juvenile hormone esterase (JHE) was used for ligand blotting of proteins from fat body tissue and pericardial athrocytes of Manduca sexta. Proteins were separated by SDS-polyacrylamide gel electrophoresis or by two-dimensional electrophoresis. Eight putative JHE binding proteins were detected in fat body tissue and in pericardial athrocytes of both M. sexta and Heliothis virescens. The predominant bands were 29, 72, 75, 125 and 240kDa, with minor bands at 50, 80 and 205kDa. All putative JHE binding proteins were present from the second through to the fifth instar larvae of M. sexta. On wide-range isoelectric focusing, the 29kDa JHE binding protein separated into three species with isoelectric points of 6.5, 6.6 and 6.8. Biotinylated-JHE did not bind recombinant M. sexta-derived juvenile hormone binding protein. The mutant JHE with mutations K29R and K524R binds weakly to the JHE binding protein P29, relative to binding of wild-type JHE [Shanmugavelu et al., J. Biol. Chem., 275 (2000) 1802-1806]. A similar reduction in binding was not seen for the 29kDa binding protein identified here in pericardial athrocytes by ligand blot. This result is discussed.     

Prothoracicotropic hormone activity in the embryonic brain of the tobacco hornworm,Manduca sexta

Summary Head segments and brains were extirpated from embryos of the tobacco hornworm,Manduca sexta, extracted and the resulting extracts assayed for prothoracicotropic hormone (PTTH) activity on prothoracic glands from day 3 fifth instar larvae and day 0 pupae. Dose-response curves were generated and indicated the presence of PTTH activity in embryonic brains and head segments, suggesting a role(s) for this neurohormone during embryogenesis. Maximal PTTH activity was found in brains from embryos 117 h post-oviposition, just prior to hatching, but activity was also noted in head segments as early as 24 h postoviposition. These data on PTTH and those on ecdysteroids and juvenile hormones in embryos suggest that these 3 classes of hormones which control insect post-embryonic development, may also be involved in the regulation of developmental processes in the embryo.     

Life cycle expression of a bombyxin-like neuropeptide in the tobacco hornworm,Manduca sexta

Immunocytochemistry was used to investigate the developmental expression of the insulin-like neuropeptide bombyxin in the tobacco hornworm, Manduca sexta. A mouse monoclonal antibody raised against a synthetic peptide corresponding to bombyxin's A-chain N-terminus was used to localize a bombyxin-like peptide to a group of cerebral medial neurosecretory cells, the M-NSC IIa(2). Immunostaining was first detected on day 0 of the second larval instar, localized in the M-NSC IIa(2) somata and in the neurohemal organ, the corpora allata (CA). By day 0 of the fourth larval instar, the peptide was present throughout the M-NSC IIa(2) somata, axons, dendritic fields and CA. Between days 7 and 9 of the fifth instar, a dramatic reduction in the dendritic fields and CA staining occurred, suggesting the peptide is released. After day 2 of the pupal period, only M-NSC IIa(2) somata immunostained, a pattern that persisted through day 2 of the adult stage. The specificity of immunostaining was demonstrated by using a synthetic bombyxin peptide to block staining. These developmental data reveal times of potential Manduca bombyxin-like peptide release which should provide insight into the peptide's function.     

Control of juvenile hormone production: the relationship between precursor supply and hormone synthesis in the tobacco hornworm,Manduca sexta

Tissue culture conditions for the production of juvenile hormone by excised corpora allata from the tobacco hornworm, Manduca sexta, were optimized and hormone output was monitored by incorporation of [¹⁴C-methyl]-methionine. Production was moderate in early fifth instar but undetectable in late fifth instar larvae. It was low in pupae, unmeasurable in adult males and high in adult females. Hormone production by glands of adult females was unaffected by the presence in the medium of juvenile hormone III, the hormone analogue, Methoprene, dibutyrylcyclic AMP, dibutyryl cyclic GMP and 25-hydroxycholesterol, an inhibitor of cholesterol synthesis in mammals.The assay of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in homogenates of corpora allata from M. sexta was optimized and activity in homogenates from animals at various life stages was determined. The correlation between the activity of the enzyme and hormone production by intact glands was excellent in all cases examined except for adult males, which have very high HMG-CoA reductase levels, but produce no hormone.Unlike the HMG-CoA reductase of mammalian tissue, the activity of corpus allatum enzyme is unaffected by Mg²⁺ ATP or fluoride ion, an inhibitor of phosphoprotein phosphatase. The activity is also unaffected by the presence of cAMP, cGMP, IBMX or 25-hydroxycholesterol.