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IMMUNOLOGICAL STUDY OF JUVENILE HORMONE BINDING PROTEIN FROM HEMOLYMPH OF THE FALL WEBWORM (LEPIDOPTERA: ARCTIIDAE)
I. H. Lee1, Hak R. Kim1 and Richard T. Mayer2
1 2 Abstract
The juvenile hormone binding protein (JHBP) was purified from the hemolymph of Hyphantria cunea (Lepidoptera: Arctiidae) using anion exchange, gel filtration, and Mono P FPLC chromatofocusing chromatography. The protein is a single polypeptide (Mr 32,000) with an apparent dissociation constant of 0.43 µM for juvenile hormone III. An antibody developed against hemolymph JHBP (hJHBP) was prepared, and use of it showed that a protein immunologically identical with hJHBP occurs in fat body and ovary. The hJHBP of H. cunea was neither immunologically related to the lipophorin from H. cunea nor similar to the hJHBP of Bombyx mori and Periplaneta americana.
Key Words: Hemolymph, hemolymph proteins, insect, immunodetection, juvenile hormone III, lipoprotein, protein purification.
Resumen
La proteína de unión de la hormona juvenil (JHBP) fue purificada a partir de la hemolinfa de Hyphantria cunea. Para purificar la JHBP fueron usados el intercambio de aniones, la filtración en gel y cromatografías cromatofocales Mono P FPLC. La proteína es un polipéptido simple (Mr 32,000) con una constante de disociación aparente de 0.43 µM para la hormona juvenil III. Fue preparado un antibiótico desarrollado contra la hemolinfa JHBP (hJHBP) y su uso demostró que una proteína inmunológicamente idéntica a la hJHBP está presente en el cuerpo graso y el ovario. La hJHBP de H. cunea no estuvo inmunológicamente relacionada con la lipoforina de H. cunea ni fue similar a la hJHBP de Bombyx mori y Periplaneta americana.
Juvenile hormone is synthesized by the corpora allata, released into hemolymph, and transported to target tissues by the juvenile hormone binding protein (JHBP) (Whitmore & Gilbert 1972; Rudnica et al. 1979; Ozyhar et al. 1983). JHBP is synthesized by fat body and released into the hemolymph (Nowock et al. 1976; Ferkovich et al. 1977). Generally, high Mr (over 200,000) and low Mr (20,000-40,000) JHBPs are present in the hemolymph (de Kort & Granger 1981; de Kort et al. 1983). JHBP from hemolymph was first purified from Manduca sexta (Kramer et al. 1976) and then from Diatraea grandiosella (Dillwith et al. 1985), Diploptera punctata (King & Tobe 1988), and Platyprepia virginalis (Prestwich & Atkinson 1990). It has been reported that hemolymph juvenile hormone binding protein (hJHBP) is closely related to apoprotein-I of lipophorin (Rayne & Koeppe 1988; Koopmanschap & Dekort 1988).
Proteins with JH affinity were found to be present in ovary and fat body and their characteristics were also reported (van Mellaert et al. 1985; Koeppe et al. 1987; Shemshedini & Wilson 1993). However, information on the relationship between hJHBP and JH binding protein in these tissues is still limited, thus additional information is needed for a better understanding of the role of JHBP.
Here, we describe the presence of a JHBP in ovary and fat body which is immunologically identical with hJHBP by using an antibody against low Mr hJHBP from the fall webworm, Hyphantria cunea Drury (Lepidoptera: Arctiidae). In addition, the physicochemical characteristics of hJHBP are described.
Materials and Methods
Insects
Fall webworms, Hyphantria cunea, were reared on artificial diet (Dong Bang Oil Co., Ltd., Seoul, Korea) at 28 ± 1°C and 70 ± 5% relative humidity and with a photoperiod of 16:8 (L:D).
Collection of Hemolymph and Extraction of Ovary and Fat Body
Hemolymph was collected into a cold Eppendorf microcentrifuge tube from last instar larvae by cutting the forelegs. A few crystals of phenylthiourea were added to prevent melanization, and the hemolymph was centrifuged at 10,000 g for 10 min at 4°C to remove hemocytes and cell debris. The supernatant was stored at -70°C.
Ovaries were dissected from 5-day-old pupae in cold Ringer’s solution (128 mM NaCl, 1.8 mM CaCl2, 1.3 mM KCl; pH 7.4). They were used directly for electron microscopic observation or homogenized and centrifuged at 10,000 g for 10 min and the supernatant used as the sample.
Fat body was dissected from last instar larvae in Ringer’s solution. These tissues were stored at -70°C until used.
Dextran Coated Charcoal (DCC) Binding Assay
A DCC suspension (0.5% active charcoal and 0.05% dextran, Mr 80,000) in buffer (10 mM Tris, 5 mM MgCl2, 50 mM KCl, pH 7.4) was used to separate protein-bound JH from unbound hormone (Engelmann 1981). [3H]JH III (NEN Corp., Wilmington, DE; specific activity: 440 GBq/mmol; 50,000 cpm) was put into a microcentrifuge tube and the solvent evaporated with a gentle stream of nitrogen gas. Buffer (100 µl; 10 mM Tris, 5 mM MgCl2, 50 mM KCl, pH 7.4), and the sample used for the binding assay, were added to the hormone and incubated for 1 h at 4°C. DCC suspension (100 µl) was added to the tube, the contents incubated for 2 min and then centrifuged at 10,000 g to remove free hormone. An aliquot (10 µl) of supernatant was transferred to 10 ml of scintillation cocktail solution (toluene 2 liters, triton X-100 1 liter, 12 g Omnifluor, NEN) and the radioactivity was measured by liquid scintillation counter (Beckman LS 100°C, Palo Alto, CA).
Purification of JHBP
Anion exchange chromatography. Hemolymph (10 ml) was diluted 1:1 with TPN buffer [10 mM Tris, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.01% NaN3, pH 8.2] and dialyzed against the same buffer for 4 h. Dialyzed hemolymph was applied to a DEAE cellulose (DE 52, Whatman, Hillsboro, OR) column (1.8 × 20 cm) and eluted with 1 column volume of TPN buffer. This was followed by a NaCl concentration gradient (0-0.5 M) in buffer (10 mM Tris, 0.01% NaN3, 0.1 mM PMSF, pH 8.2, total 300 ml) at a flow rate of 18 ml per h and then monitored for the presence of JHBP by the DCC binding assay. Fractions containing JHBP were pooled, dialyzed against 10 mM Tris buffer (0.01% NaN 0.1 mM PMSF, pH 8.2) and applied to a Sepharose CL-6B (Pharmacia, Piscataway, NJ) column (0.9 × 15 cm). The column was washed with the same buffer using a gradient of 0.05-0.35 M NaCl. Each fraction (2.6 ml) was monitored for JHBP by the DCC binding assay. Fractions containing JHBP were lyophilized before the next purification step.
Gel Filtration. The lyophilized hemolymph sample was dissolved in 2 ml water and applied to a Sephadex G-100 column (1.8 × 55 cm) and eluted with buffer (25 mM bis-Tris, pH 7.2) at a flow rate of 12 ml per h. Each fraction (2.6 ml) was monitored for JHBP by the DCC binding assay. Fractions containing JHBP were pooled, concentrated by lyophilization, and applied to a Sephadex G-75 column (0.9 × 20 cm) and eluted with buffer (25 mM bis-Tris, pH 7.2) at a flow rate of 8 ml per h. Fractions of 1.5 ml were collected.
Mono P FPLC Chromatofocusing Chromatography. Fractions containing JHBP from the Sephadex G-75 column were subjected to FPLC chromatofocusing chromatography on a Mono P column (Mono P HR 5120, 5 × 200 mm, Pharmacia). Free proteins were eluted from the column with 25 mM bis-Tris buffer (pH 7.2, 10 ml), and then Polybuffer (pH 7.4, 52 ml) was used to elute resin-bound proteins. Each fraction was monitored for JHBP by the DCC binding assay.
Determination of Molecular Weight and Isoelectric Point
The molecular weights were determined on sodium dodecyl sulfate-polyacrylamide gel (10%) as described by Weber & Osborn (1969). Standard molecular weight markers were: bovine serum albumin, Mr 66,200; hen egg albumin, Mr 45,000; bovine carbonic anhydrase, Mr 31,000; soybean trypsin inhibitor, Mr 21,500; hen egg white lysozyme, Mr 14,400.
Isoelectric focusing was performed on 5% polyacrylamide gel according to Klages & Emmerich (1979) by using ampholytes in the pH range 3-10 (Sigma, St. Louis, MO).
Production of Antibody Against JHBP
Fractions (0.5 ml) containing JHBP after Mono P FPLC chromatofocusing were mixed thoroughly with Freund’s complete adjuvant (0.5 ml) and injected subcutaneously into a rabbit. Injections were repeated every other day for the first week. The fourth injection was conducted 1 week after the third injection, and a booster injection was given 2 weeks later. JHBP-containing fractions (0.5 ml) were mixed with incomplete adjuvant (0.5 ml) for the booster injection. Blood was taken 1 week after the booster injection and centrifuged at 10,000 g for 10 min. Antibody against JHBP was purified by immunoprecipitation of extraneous antibodies using JHBP-free fractions.
Immunodiffusion
Immunodiffusion was conducted using 1% agarose gel containing 0.1% (w/v) sodium azide in veronal buffer (pH 8.6) as described by Ouchterlony (1949). The plates were stained in 1% amido black 10B and destained in 2% acetic acid.
Electrophoresis and Immunodetection
Fractions from each purification step were electrophoresed on 10% SDS-polyacrylamide gel as described by Laemmli (1970) to determine purity. Gels were stained with silver nitrate according to Wray et al. (1981).
Western blotting was used to detect JHBP on SDS-polyacrylamide gels. Samples were electrophoresed as above and transferred to nitrocellulose sheets in Tris-glycine buffer (25 mM Tris, 92 mM glycine, 30% methanol, pH 8.3) at 100 V for 2 h (Towbin et al. 1979). After transfer, the nitrocellulose sheets were equilibrated in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) for 10 min and incubated in blocking solution (3% gelatin in TBS) for 30 min. The sheet was then washed with TTBS (0.05% tween 20 in TBS) twice, each time for 5 min, and incubated for 1 h in a solution containing a 300-fold dilution of the primary antibody against JHBP. This sheet was again washed with TTBS twice and incubated for 1 h in a solution containing a 3000-fold diluted secondary antibody (goat antirabbit-horseradish peroxidase conjugated IgG). After incubation, the sheet was again washed with TTBS twice and submerged in horseradish peroxidase color development reagent containing 4-chloro-1-naphthol in 20 ml ice-cold methanol + 0.015% H2O2 in 100 ml TBS for development of purple color.
Electron Microscopic Observation by Immunogold Labeling
Ovaries and fat body were dissected from pupae in Ringer’s solution and prefixed in 2.5% glutaraldehyde for 2 h at 4C. Tissues were washed with 0.1 M phosphate buffer (pH 7.2) 3 times at 15 min intervals and dehydrated in an ethanol series. Dehydrated tissues were then put into propylene oxide and embedded in an Epon-Araldite mixture. Embedded tissues were semithin-sectioned using an ultramicrotome (Sorvall MT-II, Wilmington, DE) and stained in 1% toluidine blue and attached to a grid. The thin section-attached grid was washed with TBS. This grid was reacted with the primary antibody solution 30-fold diluted with antibody buffer and again washed with TBS 3 times to remove nonspecifically attached antibody. This grid was then reacted with antirabbit IgG combined with protein-A gold particles (30 nm in diam) for 40 min, washed with TBS and distilled water 3 times each, stained in 2% uranyl acetate and observed under JEOL JEM 100 CX-II electron microscope at 80 kV.
Results
Purification of JHBP from Hemolymph
The DCC binding assay for each fraction showed that JHBP was eluted from the DE-52 column with a linear NaCl gradient (0.06-0.1 M) (Fig. 1A). Fractions containing JHBP were subjected to a second anion exchange chromatography treatment (Sepharose CL-6B). JHBP was found to be present in the backside of the first peak (0.1 M NaCl) (Fig. 1B). Fractions containing JHBP from the second ion exchange chromatography run were subjected to gel filtration (Sephadex G-100) and fractions containing radioactivity were located (Fig. 1C) and pooled. The pooled fractions were applied to Mono P FPLC chromatofocusing columns and 4 peaks isolated by using A280 (Fig. 1D). The fourth peak exhibited high binding activity for [3H]JH III (Fig. 1D, inset). Because the JHBP fractions were contaminated with other hemolymph proteins after Mono P chromatofocusing, gel filtration on Sephadex G-75 was necessary (data not shown). Fractions were applied to a 10% SDS gel and completely purified JHBP was confirmed (Fig. 2A).
Characterization of hJHBP
JHBP was electrophoresed with a low molecular weight standard marker to determine the Mr of the JHBP subunit. The Mr was estimated to be 32,000 (Fig. 2B). Also, the pI was determined to be 5.3 (data not shown). Saturation analysis of the purified hJHBP revealed a high affinity for JH III (KD of 0.43 µM) (Fig. 3).
Immunological Studies
Antibodies were made against fractions containing JHBPs (Fig. 4A, lane b) that had been separated by Mono P FPLC chromatofocusing. Extraneous antibodies, i.e., other than antibody against hJHBP, were precipitated with JHBP-free fractions (Fig. 4A, lane a). The resulting supernatant from this precipitation showed pure hJHBP antiserum (Fig. 4B).
A protein that is immunologically identical with hJHBP was found in ovaries by immunodiffusion and western blotting (Fig. 5). However, the hJHBP of H. cunea was neither immunologically related to the lipophorin from H. cunea nor was it similar to the hJHBP of Bombyx mori and Periplaneta americana (Fig. 5). The western blots shown in Figure 5 (left) show a crossreaction for the H. cunea hemolymph, ovarian extracts, and JHBP with the antibody against hJHBP. Likewise, the immunodiffusion test (Fig. 5, right) showed an immunopreciptation line extending from the JHBP well through the ovarian extract and ending at the H. cunea hemolymph well, thus corroborating the western blot data.
Electron microscopic observations showed that gold particles accumulated in ovarian protein bodies as well as the fat body (Fig. 6). These results clearly indicate protein that is immunologically identical with hJHBP is present in protein bodies of ovary.
Discussion
The DCC binding assay was employed to purify a low Mr JHBP. Fractions showing radioactivity according to the DCC binding assay were electrophoresed and silver stained and found to be a single polypeptide with an Mr 32,000. The protocols employed to purify hemolymph JHBP from H. cunea are similar to those used to purify JHBP of Diatraea grandiosella (Dillwith et al. 1985) and Manduca sexta (Kramer et al. 1976). The hJHBP of H. cunea has a pI of 5.3 (data not shown) and can be effectively separated from other hemolymph proteins with chromatofocusing. Low Mr JHBP has an Mr range of 20,000-40,000 in several insects (de Kort & Granger 1981; de Kort et al. 1983).
The dissociation constant of H. cunea hJHBP was estimated to be 0.43 µM. This value is similar to those of pyralid moth’s 0.08-0.28 µM (Lenz et al. 1986), D. grandiosella 0.31 µM, (Dillwith et al. 1985), and M. sexta, 0.44 µM (Kramer et al. 1976). The JHBP purified from hemolymph of H. cunea is of low Mr and high affinity.
Thus far, most JHBP antibody production employed large Mr proteins. Preparation of antibodies for the low Mr JHBP was difficult because of the small amount of material in hemolymph. In the present work with H. cunea, immunoprecipitation was very effective in purifying antibody against low Mr JHBP from other antibodies.
hJHBP was confirmed to be lipophorin in Leucophaea maderae (Koeppe et al. 1988) and Locusta migratoria (Koopmanschap & de Kort, 1988). Immunodiffusion studies conducted here indicate that the hJHBP was neither immunologically related to lipophorin in H. cunea nor the hJHBP of Bombyx mori and Periplaneta americana. The hJHBP of H. cunea appeared to be species-specific.
hJHBP is synthesized by fat body and released into hemolymph. Protein with JH affinity (JH binding protein) is also present in cytosol and nucleus of fat body (Shemshedini & Wilson 1993). A JH binding protein has been reported in the ovary (van Mellaert et al. 1985), but it is not known how it is related to hJHBP. We found a protein that is immunologically related to hJHBP that appears in fat body and the cytosol of ovarian protein body of H. cunea. This protein is assumed to be a JHBP. More information on the relationship between hJHBP and JHBP in the cytosol is required to better understand the transport of JH from secretory cells to target tissues and receptors.
Endnote
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Figure 1. Chromatography elution profiles for purification of H. cunea hemolymph JHBP. A, the first anion exchange chromatography of dialyzed hemolymph proteins on DE52. Hatched region (35-46) indicates fractions containing JHBP; B, the second anion exchange chromatography on Sepharose CL-6B. Fractions (47-55, hatched region) showing radioactivity in DCC binding assay were pooled and lyophilized for the next purification step; C, gel (Sephadex G-100) filtration of partially purified JHBP. JHBP containing fractions were confirmed (hatched region, 31-37); D, Mono P FPLC chromatofocusing column. Four major protein peaks were resolved and collected (fractions 26-29, 32, 35, 37-38). Each peak was also analyzed by DCC binding assay (inset). Arrow indicates peak containing JHBP.
Figure 2. A, electrophoretic profile of fractions from final purification step (gel filtration on Sephadex G 75). Anterior portions of the chromatographic profile still contain other proteins in addition to JHBP (A: a, b, c, d). Completely purified JHBP from the posterior portion of the chromatography was confirmed on lane e. The gel (10%) was stained with silver nitrate. B, SDS-polyacrylamide gel (15%) electrophoresis to determine the molecular weight of JHBP. The gel was stained with coomassie brilliant blue. Right lane contains the low molecular weight standard markers and the arrow in left lane indicates JHBP.
Figure 3. The saturation analysis (Scatchard plot) of the binding of JH III to the purified hJHBP. ¥¥¥, total binding; °°°, nonspecific binding; sss, specific binding. The specific binding was used for the Scatchard analysis (inset).
Figure 4. Western blot of purified JHBP. The JHBP band was detected by enzyme immunoassay using antibody against JHBP (B,b). Left panel (A) shows the electrophoretic patterns of fractions from the Mono P FPLC chromatofocusing chromatography. The JHBP-containing fractions (the fourth peak that the arrow indicates in Fig. 1D, lane b) were used for the production of antibody and fractions (anterior three peaks in Fig. 1D, lane a) that lack JHBP were used for the immunoprecipitation to remove other antibodies except antibody against JHBP.
Figure 5. Left panel: Western blots of proteins from the hemolymph (H), JHBP (J), ovarian extracts (O) of H. cunea, the hemolymph (B) of B. mori, the hemolymph (P) of P. americana. The JHBP bands were detected by enzyme immunoassay using antibody against the hemolymph JHBP. In three lanes (H,J,O), a single Mr 32,000 protein corresponding to the JHBP was detected by the antibody. Right panel: Immunodiffusion test to determine the relationship of several antigens (J, JHBP, 6 µg protein; Ov, ovarian extracts, 1.8 mg protein; H, hemolymph of H. cunea, 1 mg protein; B, hemolymph of B. mori 1.4 mg protein; L, lipophorin of H. cunea, 20 µg protein; P, hemolymph of P. americana, 1.2 mg protein; Ab, antibody against JHBP in hemolymph of H. cunea).
Figure 6. Immunoelectron micrographs of fat body (A) and ovary (B) from H. cunea. Immunogold particles were observed in large protein bodies (PB). Bar length = 0.2 µm (A), 0.5 µm (B).
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