The Insect Hemolymph Protein HP19 Mediates the Nongenomic Effect of Ecdysteroids on Acid Phosphatase Activity*

The activity of acid phosphatase (ACP) in insect fat bodies is stimulated by the steroid hormone 20-hydoxyecdysone (20E) in vivo. However, in fat bodies kept in culture, a factor from the hemolymph is required to enhance the ACP activity. We identified the factor as a protein with a molecular mass of 19 kDa (HP19) from the hemolymph of a lepidopteran insect, the rice moth, Corcyra cephalonica. Western analysis of hemolymph proteins with denaturing and non-denaturing PAGE using antibodies raised against HP19 suggest that this protein exists as a monomer. It is synthesized by the hind gut-associated lobular fat body of the larvae and is released into the hemolymph. The stimulatory effect of HP19 on the ACP activity is developmentally regulated and exhibits its maximal effect shortly before the onset of metamorphosis. We cloned the HP19 cDNA by immunoscreening a hind gut-associated lobular fat body cDNA expression library. Analysis of the amino acid sequence shows that HP19 belongs to the family of glutathione S-transferase (GST) like proteins. However, affinity-purified GST from Corcyra failed to show any mediation effect on 20E-stimulated ACP activity, and HP19 lacks GST enzymatic activity. Notably, HP19 mediates the hormone-stimulated ACP activity in intact fat body tissue and homogenates even in the presence of inhibitors of transcription and translation, suggesting a nongenomic mode of action. In addition, we show that HP19 inhibits the 20E-induced phosphorylation of the hexamerin receptor protein.

Insect metamorphosis, the transition from the larval to the adult stage of insects, is controlled by ecdysteroid hormones (1)(2)(3). The ecdysteroids, like the steroids in vertebrates, regulate gene transcription by binding to the nuclear receptors, which are ligand-activated transcription factors that convert the hormonal stimulus into a transcription response (4 -7).
Metamorphosis involves the breakdown of larval structures and the formation of new tissues (1). As a part of cell remodeling during metamorphosis, acidic autophagic vacuoles accumulate in the cells of the fat body, and the activity of several lysosomal enzymes such as acid phosphatases increase and cause the lysis of larval tissues (8 -11). The fat body fills a large fraction of the insect, and its function has been considered equivalent to the role of the vertebrate liver in the intermediary metabolism (12). It has been demonstrated that the stimulation of the lysosomal activity is governed by ecdysteroids (11,(13)(14)(15)(16). There is an indication that in this case the hormone possibly acts on a nongenomic level (17). Although the molecular mechanism of the genomic mode of steroid action is well known, the mechanism of nongenomic steroid action remains unclear to date (18). Earlier studies show that 20E 1 stimulates ACP activity in fat bodies in vivo but not in vitro (19,20). This result suggests that 20E, the active form of ecdysone, requires an additional factor (or factors) to enhance ACP activity. Hence, we have focused on the process of acid phosphatase activation by 20E in the fat body cells of our model insect, the rice moth Corcyra cephalonica. We report the appearance of a stage and tissue-specificregulated protein, HP19, in the hemolymph of Corcyra responsible for the activation of the 20E-dependent stimulation of ACP activity. This hormone-triggered activation is independent of gene transcription.

EXPERIMENTAL PROCEDURES
Insects-Larvae of the rice moth, C. cephalonica (Stainton), were reared on coarsely crushed sorghum seeds at 26 Ϯ 1°C, 60 Ϯ 5% relative humidity, and a 14:10-h light:dark photo period. In the present study, the last (ϭVth) instar larvae, classified into early (ELI), mid (MLI), late-last instar (LLI) larvae and prepupae on the basis of body weight and head capsule size were used (21). The larvae were thoraxligated behind the first pair of prolegs by slipping a loop of silk thread around the head of the larvae as described earlier (20). The fat body and other insect tissue homogenates from ligated or unligated larvae were prepared as published earlier (22) and used after protein estimation in an aliquot of the homogenate (23).
ACP Assay-The enzyme assay was carried out according to the method of Henrickson and Clever (24). The reaction mixture contained 150 mM sodium acetate buffer (pH 5.0) and 20 g of tissue homogenate proteins. It was incubated at 37°C for 10 min to exclude glucose-6phosphatase activity (25). The reaction was initiated by the addition of 5 mol of substrate, p-nitrophenyl bisodium phosphate (Sigma) to the assay mixture followed by incubation for 1 h at 37°C. The reaction was terminated by the addition of 0.5 ml of 0.1 N NaOH, and the color was measured at 420 nm against a substrate blank. The p-nitrophenol was used for the preparation of a standard curve. The activity of the enzyme was expressed as nmol of p-nitrophenol released/h/g of fat body protein.
Studies on Fat Body Cultures-Ribbon-shaped visceral fat bodies from LLI larvae were dissected 24 h after ligation under sterile conditions in cold insect Ringer's solution and transferred to 100 l of TC-100 insect culture medium (JRH Biosciences Inc.) with traces of streptomycin sulfate. After rinsing, the tissue was transferred to 200 l of fresh culture medium, and 80 nM 20E was added while an equal volume of carrier solvent (ethanol) was added to the control cultures. The hormone 20E (Sigma) was dissolved in ethanol, the final concentration of which never exceeded 0.05% in any of the experiments. To study the hemolymph effect, the diluted (1:20) or fractionated hemolymph was added to the fat body culture in the presence or absence of 80 nM 20E. Studies with glutathione S-transferase (GST) were carried out by adding purified cytosolic GST from Corcyra to the fat body cultures in the presence of hormone. These cultures were then incubated for 4 h at 25°C with gentle shaking. At the end of incubation the tissue was removed, rinsed in ice-cold insect Ringer, homogenized, and used for ACP assay.
Fractionation and Purification of Hemolymph Proteins-Total hemolymph protein was loaded on a pre-equilibrated (10 mM Tris-HCl (pH 7.4)) Sephadex G-50 column and eluted with the equilibration buffer. The single fractions were checked for their ability to enhance the ACP activity in LLI fat bodies kept in culture. The apparent molecular mass of the active fraction was determined by gel electrophoresis. The purification of the active fraction was carried out by fractionating the hemolymph proteins followed by gel filtration chromatography. For fractionation we used molecular weight cut-off fractionators (Amicon Inc.). The hemolymph sample preparation (1 mg of protein/ml) was first transferred into 30-kDa cut-off fractionators (YM-30) and centrifuged at 4000 ϫ g for 20 min at 4°C to strip the protein fractions with mass above 100 kDa. The resultant filtrate as well as the retentate was collected separately. The filtrate with molecules Ͻ100 kDa was again subjected to fractionation using a 10-kDa cut-off fractionator (YM-10) for obtaining fraction of Ͻ30 kDa proteins. Once again, both filtrate and retentate were collected. Three fractions, having masses above 100 30 -100 and below 30 kDa thus obtained, were used to test their effect on 20E-dependent ACP activity as described above. For the purification of the active fraction, hemolymph from ϳ500 LLI larvae was fractionated using the 30-kDa cut-off fractionator. The filtrate was loaded on a Sephadex G-50 column pre-equilibrated with 10 mM Tris-HCl (pH 7.4). The protein fractions were eluted with the same buffer. An aliquot of each fraction was added to the fat body kept in culture in the presence of 20E (80 nM), and its enhancing effect on ACP activity was analyzed. Peak fractions that stimulated ACP activity were pooled, and the purity was checked by SDS-PAGE.
Production of Polyclonal Antibodies against HP19 -The HP19 protein band was electroeluted using a model 422 electroeluter (Bio-Rad) after slicing the protein band resolved by 12% SDS-PAGE and used as an antigen for raising polyclonal antibodies in 3-month-old male rabbits (New Zealand variety). The IgG fraction was purified by protein Aagarose chromatography (Bio-Rad).
Electrophoresis and Western Blotting-SDS-PAGE was carried out using 2.1% stacking and 12% resolving gel (26). Non-denaturing PAGE was carried out as described by Burmester et al. (27). The resolved proteins were visualized by silver staining (28). For Western blotting, the electrophoretically separated hemolymph proteins were transferred to a nitrocellulose membrane (29) and probed with polyclonal HP19 antibody to detect a polypeptide with the capability of mediating the ACP activity in the hemolymph as well as in different tissues. Visualization of the specific cross-reactivity was carried with anti-rabbit IgG coupled with alkaline phosphatase followed by nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate color reaction.
Preparation of a Hind Gut-associated Lobular Fat Body (HGLFB) cDNA Expression Library and Immunoscreening-The cDNA from the RNA of HGLFBs was generated and amplified using the SMART III TM kit (Clontech). Total RNA was isolated using the TriFast kit (peqLab). The library was cloned in a pTriplEx2 vector and screened with the IgG fraction of anti-HP19 antibody (30). By immunoscreening of 6 ϫ 10 9 recombinant phage plaques, 10 positives were obtained after 3 rounds, in vivo excised, converted into plasmids, and used for XL-1Blue cell transformation (Stratagene, La Jolla, USA). The restriction analysis revealed six of these clones to be of identical size.
Sequencing of HP19 Clone-The inserts were sequenced on a PerkinElmer 310 sequencer using forward and reverse primers provided with the kit. About 60% of the positives were found to be GST-like sequences (BLAST search). One such clone was analyzed in detail. A multiple sequence alignment of deduced amino acid sequence with best matching invertebrate GSTs was carried out using ClustalW and GeneDoc 2.6. Southern and Northern Blotting-The genomic DNA from total larval body was isolated as described in Birren et al. (31) and was digested with EcoRI and HinfI. The digested DNA was resolved on 0.8% agarose gel. The total RNA isolated from different insect tissue was resolved on 1.2% agarose/formaldehyde gel and transferred to a nylon membrane. The HP19 cDNA insert corresponding to whole open reading frame was excised from the pTriplEx2 vector by EcoRI and NotI digestion, gelpurified, and random prime-labeled (MBI Fermentas) using [␣-32 P]dATP (ϳ3000 Ci/mmol, Board of Radiation and Isotope Technology (BRIT), Hyderabad, India) to a specific activity of Ͼ10 9 dpm/g. The blots were worked up following standard procedures (30).
GST Assay-The GST activity was measured by the method of Habig et al. (32). A 1-ml reaction mixture contained 10 l of 100 mM 1-chloro-2,4dinitrobenzene, 10 l of 100 mM reduced glutathione (GSH), and 100 mM potassium phosphate buffer (pH 6.5). The reaction was initiated by the addition of the enzyme source and the product, i.e. the formation of thioether conjugate, was measured at 340 nm on a time scan of 0 -60 s.
Immunohistochemical Studies-The insect tissues were fixed in Carnoy's fixative (ethanol:chloroform:acetic acid, 6:3:1) for 4 h at room temperature and paraffin-embedded. Sections (5 m) were cut and mounted on glass slides. For immunohistochemical staining the sections were deparaffinized and treated with blocking solution (2% bovine serum albumin and 1% preimmune goat serum in 50 mM Tris-buffered saline (pH 7.4) containing 0.1% Triton X-100) for 1 h at 4°C. This was followed by anti-HP19 IgG treatment for 24 h at 4°C with gentle shaking. The slides were then treated with anti-rabbit IgG coupled with alkaline phosphatase for 1 h. Washing after each step was done with three changes of Tris-buffered saline. These slides were finally processed for staining using nitro blue tetrazolium chloride/5-bromo-4chloro-3-indolyl phosphate color reaction and mounted in glycerol gel (50% glycerol, 7.5% gelatin, and 0.1% azide in 0.1 M Tris-buffered saline) (33,34). The specificity of the antibodies was checked for all immunochemical experiments by treating parallel tissue sections using pre-immune rabbit serum.
[ 35 S]Methionine Incorporation Studies-In vitro incorporation of [ 35 S]methionine in the fat bodies kept in culture was carried out to compare the changes in the level of total protein synthesis and ACP activity stimulation in the presence of 20E and HP19. The fat bodies kept in culture were first preincubated for 2 h with 10 Ci of [ 35 S]methionine (ϳ1000 Ci/m mol, BRIT) at 25°C followed by incubations either with 20E (80 nM) alone, HP19 (40 ng) alone, or both along with actinomycin D (1 mM) or cycloheximide (1 mM) for 2 or 4 h. After incubation, the fat bodies were removed and homogenized as described above. Equal amounts of protein were used for radiolabel quantitation after trichloroacetic acid precipitation.
In Vitro Phosphorylation of Fat Body Proteins-Fat body homogenate from LLI larvae was incubated with 80 nM 20E and 40 ng of HP19 for 30 s, and phosphorylation was initiated by the addition of 4 Ci of [␥-32 P] ATP (ϳ3000 Ci/m mol, BRIT) as described earlier (22). The labeled proteins were separated by 10% SDS-PAGE. The gel was vacuum-dried and exposed to Kodak X-Omat AR film at Ϫ70°C for autoradiography.
Statistical Analysis-The mean and S.D. were calculated for the variables studied. The data were statistically analyzed by one way analysis of variance followed by comparisons of means by Tukey multiple comparison tests using Sigma Stat software (Jandel Corp.). The values were considered significantly different from each other when p Ͻ 0.05.

Identification of a Protein in the Hemolymph of Corcyra That
Mediates the 20E-stimulated ACP Activity in the Fat Bodies of Thorax-ligated, Hormone-deprived Larvae-When insect larvae are ligated behind the first pair of prolegs, i.e. behind the hormone producing glands, the posterior part of the animal is known to be relatively free of endogenous ecdysteroids (35)(36)(37). Fig. 1, a and b, shows the effect of thorax ligation and injection of exogenous 20E on the fat body ACP activity in LLI larvae. The ACP activity declined gradually from 6 to 72 h after ligation. Because the ACP activity in the fat body was significantly lower after 24 h of ligation, this time period was used for all hormone manipulation studies. Hormone injections of 80 nM 20E, i.e. the physiological concentration (22,36), to 24 h postligated LLI larvae caused a significant increase in the ACP activity in fat bodies after 24 h compared with the solventtreated larvae (Fig. 1b).
To study the effect of hormone on the ACP activity of fat bodies kept in culture, the tissue was dissected from 24-h post-ligated larvae and cultured for 4 h in the presence of 80 nM 20E. The results show that 20E did not elicit any stimulatory effect, and the activity was more or less the same as in the controls (Fig. 1c). However, the addition of hemolymph from LLI larvae together with 20E caused a significant increase in the ACP activity (Fig. 1c). This observation suggests that the hemolymph contains a factor (or factors) required by 20E to stimulate the ACP activity in fat body cultures. When the hemolymph was treated with alcohol, heat, acid, alkali, or protease, no stimulation of the ACP activity by 20E could be observed, suggesting the proteinaceous nature of the factor (Fig. 1d).
Purification and Characterization of the Hemolymph Factor as a Protein Mediating 20E-stimulated ACP Activity-After loading total hemolymph protein on a Sephadex G-50 column, we eluted several fractions (Fig. 2) and checked their ability to mediate the 20E-stimulated ACP activity. We found an active protein fraction with a molecular mass of ϳ22 kDa, calculated from the elution profile (Fig. 2, inset), or 19 kDa, calculated from the mobility on a SDS-PAGE (Fig 3b). On the basis of these results we purified the active hemolymph protein first by fractionating the total hemolymph protein followed by gel filtration chromatography. The fractionation was carried out using 30-and 10-kDa cut-off filters. The filtrate from the 10-kDa cut-off filter gave a protein fraction that mediated the 20Estimulated ACP activity of fat body cultures by an increase from 0.7 to 1.2 nmol of p-nitrophenol release/h/g of protein (Fig. 3a). However, the protein yield in the filtrate obtained from the 10-kDa cut-off filter was insufficient to proceed for further purification. Therefore, the filtrate from the 30-kDa cut-off filters, in which the HP19 was contained, was used for gel filtration. The protein fraction eluted from the Sephadex column that mediated the 20E-dependent ACP activity resulted in a contaminant-free pure polypeptide band of 19 kDa (Fig. 3b). Hence, the active hemolymph protein was named as HP19. Starting with 50 mg of total hemolymph protein, we obtained a 98.5-fold purification with 0.05% yield.
The Western blots presented in Fig. 3c show the specificity of the HP19 antibody both on denatured (lane 1) and non-denatured (lane 2) PAGE. A single protein band of 19 kDa suggested a monomeric structure of HP19. Southern analysis of genomic DNA (Fig. 3d) digested with EcoRI or HinfI probed with HP19-cDNA revealed HP19 as a single copy gene.
cDNA Cloning and Sequence Analysis of HP19 -To identify the cDNA encoding the HP19 protein, a cDNA expression library, prepared from the RNA of HGLFB of LLI larvae, was immunoscreened. We picked 10 positive cDNA clones for detailed examination. The restriction analysis revealed 6 of the 10 clones to be of identical size. Initial sequencing study dem- onstrated significant sequence similarity among these clones. Furthermore, they showed homology with invertebrate GSTs. One of our clones was sub-cloned and totally sequenced (Gen-Bank TM accession number AY369240). This HP19 cDNA was 634 nucleotides long, with an open reading frame of 585 bp, which encodes a protein of 195 amino acids. The calculated molecular mass of the translated unmodified protein was 22.95 kDa, which is close to the mass of HP19 detected in HGLFB, the tissue that synthesizes the protein (see Fig. 6e). The polypeptide comprises 12.3% basic (9 Arg, 1 His, and 14 Lys) and 13.3% acidic residues (10 Asp and 16 Glu) but no Cys residue. The estimated isoelectric point (pI) is 5.36. Comparison of the C. cephalonica HP19 (CcHP19) cDNA with the sequences in the GenBank TM showed 67% identity with Choristoneura fumiferana GST (CfGST) (38). Similarities of HP19 cDNA with other invertebrate GST were found to be less than 38%. The comparison of the amino acid sequences of CcHP19 with the four best matching invertebrate GSTs is shown in Fig.  4. Although the CcHP19 cDNA sequence revealed 67% identity with CfGST, affinity-purified GST from Corcyra had no enhancing effect on the 20E-dependent ACP activity when compared with purified HP19 or recombinant HP19 (Fig 5a). Furthermore, the hemolymph as well as the purified HP19 had negligible GST activity (Fig. 5b).
Tissue-specific Appearance of HP19 -Co-culturing of different larval tissues with fat body demonstrates that the HGLFB is the only HP19-synthesizing tissue. A stimulation of the ACP activity by 20E was only observed when it was co-cultured with HGLFB (Fig. 6a). The hemolymph used in all experiments was cell-free and, therefore, cannot be the site of HP19 synthesis. The tissue specificity of HP19 biosynthesis was further confirmed by immunohistochemical staining of different tissue sections using HP19 antibody. Again, HP19 was found to be localized only in HGLFB (Figs. 6, b and c). Western analysis of proteins from different tissues also revealed the presence of HP19 only in HGLFB (Fig. 6e, lane 2) and total larval body protein (Fig. 6e, lane 5). However, the apparent mass of HP19 in these was ϳ5 kDa higher than the HP19 present in hemolymph (Fig. 6e, lane 1). Northern hybridization with total RNA from different tissues and from total larval body displayed the tissue-specific expression of HP19 gene in HGLFB (Fig. 6f, lane  2). The faint band of HP19 in the Western blot (Fig. 6e, lane 5) and the apparent absence of HP19-mRNA in the total larval body, which comprises the HGLFB, is probably due to the limitation of the methods used.
Developmental Regulation of HP19 -The developmental profile studies of HP19 in Corcyra during the last (ϭVth) instar larval development suggest that only the hemolymph of LLI larvae could mediate the 20E-stimulated ACP activity (Fig.  7a). Western analysis of proteins from hemolymph (Fig. 7b) and HGLFB (Fig. 7c) of different developmental stages of Vth instar larvae show that HP19 is present at a maximal concentration in LLI (lane 3). HP19 is present in the hemolymph at all the developmental stages tested (Fig. 7b, lanes 1-4) but was not detectable in the HGLFBs of prepupae (Fig. 7c, lane 4). The intensity of the HP19 band in ELI and MLI was low compared with LLI (lanes 1-3). These results suggest that HP19 is synthesized throughout the complete last larval stage in HGLFB and is released into the hemolymph. The synthesis rate is low in ELI larvae, and the secretion into the hemolymph is rapid with exception of the LLI stage, when the HP19 synthesis is accelerated and is paralleled by the activation.
Nongenomic Regulation of 20E-stimulated ACP Activity by HP19 -When the fat bodies kept in culture were incubated with 20E and HP19 for 4 h, we observed that the stimulation of the ACP activity remained unaffected in the presence of transcriptional or translational inhibitors (Fig. 8a). The results in Fig. 8b further indicate the nongenomic regulation of 20Estimulated ACP activity by HP19 because the addition of protein directly to the fat body homogenate also mediated the steroid-stimulated action. This effect was rapid and could be observed within 30 s to 1 min (Fig. 8b). We further confirmed the nongenomic regulation of 20E-dependent ACP activity by incubating the fat bodies kept in culture first with [ 35 S]methionine for 2 h followed by incubation with hormone, HP19, and transcriptional or translational inhibitors. The results (Fig. 9) show that the total protein synthesis is induced under the influence of 20E, and this induction is inhibited by actinomycin D and cycloheximide (Fig. 9a). However, the inhibitors did not affect the ACP activity. The HP19 enhanced the 20E-stimulated ACP activity (Fig. 9b).
Phosphorylation of the Hexamerin Receptor by 20E-We have recently reported that 20E stimulates the phosphorylation of the Corcyra hexamerin receptor, which is partly mediated by a tyrosine kinase at a nongenomic level (22). Therefore, it was pertinent to check whether HP19 had any effect on hexamerin or tyrosine kinase phosphorylation. In vitro phosphorylation of fat body homogenate proteins from Corcyra resulted in the phosphorylation of mainly three proteins of masses 120, 60, and 48 kDa (Fig. 10). The addition of HP19 either in the absence (lane 3) or in the presence of 20E (lane 4) inhibited the basal as well as 20E-stimulated phosphorylation of the 120-kDa protein, which was identified earlier as the hexamerin receptor (22). DISCUSSION Insect development and reproduction are mainly controlled by a complex interaction between two groups of hormones, the ecdysteroids and the sesquiterpenoid juvenile hormones. The prominent functions of ecdysteroid hormones are their ability to trigger the temporal sequence of developmental process underlying molting and metamorphosis (1,2), one of the most dramatic event in the animal kingdom. The hemolymph, i.e. the insect "blood," is known as a source of several factors that do not only regulate ecdysteroid synthesis but also mediate ecdysteroid-dependent actions in larvae and pupae (39 -45). 20E has been shown to elicit effects on autophagic processes of the fat body by stimulating the lysosomal enzyme activity of ACP (10,11,(13)(14)(15)(16).
Thorax ligation that renders the posterior part in larvae relatively free of endogenous hormone showed a gradual decline in larval fat body ACP activity, pointing to the hormonal dependence of ACP activity (Fig. 1). The injection of a physiological dosage of exogenous 20E into ligated larvae caused a stimulation of ACP activity. However, when 20E alone was added to fat bodies kept in culture the hormone could not stimulate the enzyme activity. Only co-treatment of the cultured fat bodies with 20E together with hemolymph enhanced the ACP activity. This result confirmed our earlier findings of the presence of some hemolymph factor that mediated the 20E-dependent ACP activity in vivo (20). In this study we identified and characterized this hemolymph factor as a 19-kDa protein (HP19) and studied its synthesis as well as its developmental regulation. The novel function of the protein prompted us to devise a protocol for purification. Although a few nanograms of the protein were found to be sufficient for stimulation even in crude or partially purified fractions, the yield of the purified protein was very low. Other limitations were the requirement of a large quantity of hemolymph of a specific developmental stage (LLI) and removal of the major contaminating protein, hexamerin, which constitutes 75-80% of total hemolymph protein (46). Therefore, an antibody against HP19 was raised by electroeluting HP19, and its specificity against HP19 was  5. Effects of HP19 on the ACP activity in fat bodies compared with GST. a, ability of affinity-purified cytosolic Corcyra GST on 20E-dependent fat body ACP activity. Note that the presence of purified or cloned HP19 (CcHP19) mediated the 20E-stimulated enzyme activity, whereas the presence of GST did not show any effect. b, GST activity in different larval tissues and in purified HP19. Note that both hemolymph as well as purified HP19 have negligible GST activity. PNP, p-nitrophenol.

20E-stimulated ACP Activity Regulation by HP19
confirmed. When this antibody was added together with hemolymph to cultures of LLI fat body, the hemolymph failed to mediate the 20E-dependent action. Similarly, when the hemolymph was first immunoprecipitated using HP19 antibodies and the resulting complex and the supernatant were added to the fat body cultures, HP19 action was suppressed (data not shown).
Western analysis of denatured as well as non-denatured PAGE demonstrated that HP19 is a monomeric protein in Corcyra and is obviously the product of a single copy gene (Fig.  3d). Three independent methods, co-culturing, Western analysis, and immuno-histochemistry, revealed that HP19 is synthesized by the HGLFB, from where it is released into the hemolymph. This is further confirmed by the tissue-specific gene expression only in HGLFB. Western analysis provides evidence of a difference of ϳ5 kDa in the mass of HP19 in HGLFB (i.e. 24 kDa) and in hemolymph (19 kDa). The predicted mass of the unmodified translated protein from the HP19 cDNA was close to the HP19 synthesized in HGLFB. Furthermore, the 0.66kilobase HP19-mRNA (Fig. 6f) indicates a mass of about 24 kDa synthesized in HGLFB. The amino acid sequence deduced from HP19 cDNA did not show a typical signal peptide necessary for transmembrane transport (47), probably due to the lack of any hydrophobic sequence (38). Therefore, we conclude that HP19 might be cleaved before its release into the hemolymph. The presence of two putative N-glycosylation sites (Asn-51-Arg-52-Thr-53-Leu-54 and Asn-116 -Glu-117-Thr-118 -Ala-119) indicates that the protein can be secreted from the synthesizing cells.
The biosynthesis of HP19 takes place in the HGLFB during the total last larval instar. The protein is rapidly released into the hemolymph. The maximal HP19 concentration in the tissue as well as in the hemolymph could be observed in LLI. It is notable that only hemolymph from this developmental stage is capable of stimulating the 20E effect on ACP activity, i.e. the activity of HP19 is developmentally regulated. However, the molecular mechanism of activation of ACP by ecdysteroid hormone is unclear at this point.
To gain more insight into the nature and function of HP19, we produced and characterized CcHP19-cDNA clones and compared the sequence with those of related proteins, particularly with that of CfGST, which exhibits 67% identity. The CcHP19 does not contain specific amino acids that are responsible for glutathione binding or are involved in modulating the specific activity of mammalian , , and GSTs (38, 48 -50). The HP19 sequence also showed other putative post-translational modification sites like protein kinase C (189 -191), casein kinase II (81-90, 124 -127, 142-145, and 161-164), and tyrosine kinase (19 -25 and 33-41) phosphorylation sites as well as N-myristoylation (138 -143 and 150 -155) sites. However, in vitro phosphorylation studies with hemolymph did not reveal any phosphorylation of HP19 (data not presented).
GSTs with hormone-regulating actions are unknown, although few studies on vertebrate GSTs speculate about their steroid binding properties or their developmental and hormonal regulation (51)(52)(53)(54). In the present study we also checked the possibility of GST exhibiting HP19 function. For this purpose we tried to replace HP19 with affinity-purified GST and tested whether GST can exhibit the same effect as of HP19. The CfGST shows Ͼ60% sequence identity with HP19 (38), and the CfGST antibody revealed immunocross-reactivity with different tissue proteins in the mass range of HP19 (data not presented). The affinity-purified GST from Corcyra did not show any potentiation in 20E-stimulated ACP activity like that of HP19 purified from the hemolymph or recombinant HP19. In addition, the GST activity in the hemolymph was very low and negligible in the purified HP19. All these studies together show that HP19 has no GST activity. During the purification of cytosolic GST of Corycra by affinity chromatography, the flowthrough fractions that did not bind to the affinity matrix mediated the 20E effect on ACP activity, further indicating that HP19 is different from GSTs (data not presented).
A wealth of data on the molecular mechanism of ecdysteroid action shows that the transcriptional cascade leading to molting and metamorphosis is initiated when 20E binds to its nuclear receptor complex (2). For about four decades evidence has accumulated that some of the hormonally induced effects seemed to be too rapid for the classical model (18,55). This evidence casts doubt on the so-called genomic pathway as the sole mode of steroid action. Today, several modes for nongenomic steroid actions are examined. Most of them are thought to continuously modulate the long term program allowing cells or organs to adapt rapidly to environmental changes. Numerous experiments with many different species show that insect metamorphosis in general is under the genetic control of ecdysteroids. A few studies indicate that some events necessary for and accompanying metamorphosis are controlled by 20E at a nongenomic level. However, studies on these mechanisms are restricted to a small number of experimental systems, e.g. the activation of lysosomal enzymes and the hexamerin receptor (17,22,37).
To learn more about the molecular mechanisms of ecdysteroid in regulating the HP19-assisted ACP activity, the fat bodies in culture were incubated for several time periods with HP19 and 20E. A minimum incubation of 4 h was essential for the stimulation of the enzyme activity by 20E, time enough for a genomic hormone action. However, the measured stimulation was unaffected by transcriptional or translational inhibitors, indicating the independence of gene activation. Furthermore, the in vitro study with fat body homogenate showed a rapid stimulation (within seconds to 1 min) of the enzyme activity. That the homogenate preparation is essentially a cell or nucleus disintegrated fraction suggests cell/nuclear integrity is not an essential requirement for the effect of 20E on ACP activity. This possible nongenomic regulation was further strengthened by the results that in the presence of 20E fat body cultures showed a higher incorporation rate of [ 35 S]methionine, which was inhibited by transcriptional or translational inhibitors. This, however, had no effect on the ACP activity, and the presence of HP19 in the tissue culture rendered 20E capable of stimulating the enzyme activity even in the presence of inhibitors of transcription and translation.
The construction of adult tissues during metamorphosis requires large amounts of energy and building blocks, which are in all the insects investigated so far and are provided by "storage proteins." The major fraction consists of hexamerins, which are sequestered by the larval fat body by receptor-mediated endocytosis, stored in vacuoles, and utilized by the activity of lysosomal enzymes (46,56,57). The hexamerin receptor of Corcyra, a 120-kDa protein, is activated through phosphorylation by a tyrosine kinase in the fat body membrane (22). Therefore, it was tempting to extend our study and to check the effect of HP19 on the 20E-stimulated phosphorylation of the hexamerin receptor. As demonstrated, HP19 inhibits the 20E-induced phosphorylation of the hexamerin receptor. The physiological significance of this inhibition lies in the experimental evidence that uptake of hexamerin is a stage-specific event (46,56). As we have shown earlier, the receptor is present during the last larval instar in an inactive form and is not capable of sequestering hexamerins, i.e. binding its ligand (22,58). Furthermore, it is well documented that the ecdysteroid titer is very low during the last larval instar but increases rapidly (10-fold or more) before pupation (36). We could demonstrate that the hexamerin receptor becomes phosphorylated at this stage and is able to bind hexamerin. Moreover, we have shown that 20E is responsible for the phosphorylation (receptor activation) by enhancing the activity of a tyrosine kinase (22). From the present study it is evident that phosphorylation of the receptor is inhibited by HP19 in the larval stage, when hexamerin is not sequestered by the fat body. This suggests the existence of an inbuilt regulatory mechanism to prevent the ecdysteroid-dependent hexamerin uptake by the larval fat body cells, a stage when the fat body tissue actually synthesizes both the hexamerin and its receptor. Later, at the prepupal and pupal stage when hexamerin uptake occurs, HP19 gets inactivated, which is evident by its inability to mediate the 20E regulated ACP activity at this stage. However, like ACP, the dual requirement of both HP19 and 20E remains elusive, as HP19 alone could also inhibit the receptor phosphorylation. The receptor phosphorylation occurs in intact fat bodies in the membrane fraction from fat body as well as in the fat body homogenate, suggesting a nongenomic regulation of the phosphorylation of hexamerin receptor by 20E (22). The selective inhibition of the receptor phosphorylation in fat body homogenates suggests that HP19 most likely has a kinase inhibitory/phosphataseactivating effect on the phosphorylation of the hexamerin receptor and possibly acts by inhibiting the fat body tyrosine kinase activity. Further work needs to be done to broaden the understanding of nongenomic regulation by ecdysteroids and the regulatory role of HP19 on the 20E-stimulated acid phosphatase activity and herewith on the phosphorylation of hexamerin receptor in insects.