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Relationship of hemolymph juvenile hormone-binding protein to lipophorin in Leucophaea maderae
Insect Biochem. Vol. 18, No. 7, pp. 667~73. 1988 Printed in Great Britain. All rights reserved
0020-1790/88 $3.00+0.00 Copyright © 1988 Pergamon Press plc
RELATIONSHIP OF HEMOLYMPH JUVENILE HORMONE-BINDING PROTEIN TO LIPOPHORIN IN L E U C O P H A E A M A D E R A E RICHARD C. RAYNE and JOHN K. KOEPPE* Department of Biology, University of North Carolina, Chapel Hill, NC 27514, U.S.A. (Received 1 September 1987; revised and accepted 28 April 1988)
Abstract--Monoclonal antibodies (MAb) specific for the hemolymph 220 kD juvenile hormone-binding protein (JHBP) of the cockroach, Leucophaea maderae, were used to further characterize the JHBP. Spleen cells, from mice immunized with semipurified hemolymph JHBP, were fused with myeloma cells to produce hybridomas secreting antibodies specific for the hemolymph 220 kD JHBP. Positive clones were recloned and rescreened by the same methods, resulting in the establishment of five cell lines producing IgG2~ anti-hemolymph JHBP antibodies. Using MAb against the 220 kD hemolymph JHBP, it was demonstrated by Western blot analysis that the 220 kD JHBPs present in both ovarian and egg case extracts have antigenic determinants similar to those of the hemolymph JHBP. In addition, both the 275 and 220 kD JHBPs, synthesized and secreted by fat body maintained in vitro, were detected by anti-hemolymph JHBP antibody, demonstrating that the two media proteins and the JHBP in the ovary share identical antigenic determinants. To determine if there is a relationship between hemolymph JHBP and lipophorin, hemolymph proteins were separated on potassium bromide gradients. Gradient analysis confirmed that the lipophorin complex bound JH and that the 220 kD JHBP is apolipophorin I (apolp I). The apolp I specific MAb did not cross-react with apoliphorin II (apolp II). Key Word Index: juvenile hormone binding proteins, monoclonal antibodies, lipophorin, hemolymph, ovary, Leucophaea maderae
INTRODUCTION High molecular mass, high affinity juvenile hormonebinding proteins (JHBPs) have been identified in hemolymph and ovarian extracts of a number of insect species (Koeppe et al., 1981; Engelmann, 1984; de Kort et al., 1984; Koeppe and Kovalick, 1986). In the cockroach, Leucophaea maderae, the JHBPs extracted from either the hemolymph or the ovary display many of the same properties: (1) they both prefer to bind JH III, the endogenous homologue, (KD of 2 x 10 -s M); (2) they both sediment on sucrose gradients with an S value of 6.5; and (3) they both have a molecular mass by S D S - P A G E of approx. 220,000. Currently, the site of synthesis of the ovarian JHBP remains undetermined, although we have now demonstrated that the 220 kD hemolymph JHBP is synthesized and secreted by fat body (Koeppe and Kovalick, 1986; Koeppe et al., 1988). Since the 220 kD hemolymph JHBP and the 220 kD ovarian JHBP share many of the same properties (Koeppe et al., 1981), we postulate that the two proteins are identical and that the 220 kD JHBP is incorporated into the developing oocyte during vitellogenesis. Similarities between hemolymph and ovarian JHBP also exist in the fleshfly, Sarcophaga bullata, and it is postulated that S. bullata ovaries incorporate hemolymph JHBP during ovarian maturation (Van *To whom correspondence should be addressed.
Mellaert et aL, 1985). However, in vitro incorporation experiments using [3H]leucine-labeled JHBP and developing L. maderae ovarioles have been unsuccessful (Shearin, Vogler and Koeppe, unpublished data). In two other insect species in which high molecular weight, high affinity JH III JHBP have been detected in the hemolymph, it has recently been shown that JH III is bound by the lipid shuttle protein, lipophorin (de Kort and Koopmanschap, 1986, 1987; de Kort et al., 1987). The lipophorins are a class of insect hemolymph lipoproteins which apparently shuttle lipids (primarily diacyloglycerol) between tissues (Gilbert and Chino, 1974; Chino et al., 1981; Chino, 1985; Shapiro et al., 1988). In virtually all insect systems analyzed to date, lipophorin is comprised of two apoproteins, apolipophorin I (apolp I) of ~ 2 4 0 k D and apolp II of ~ 8 0 k D (Ryan et al., 1984). Since apolp I has a molecular weight similar to the hemolymph JHBP in L. maderae and because lipophorin binds JH in two other insect species (see above), it was suspected that the 220 kD JHBP might be apolp I in L. maderae. This paper describes the production of five hybridoma cell lines that secrete monoclonal antibodies specific for the 220kD hemolymph JHBP in L. maderae. In addition, results are presented which demonstrate that lipophorin binds JH III in L. maderae and that the 220kD hemolymph JHBP synthesized and secreted by fat body is a lipophorin subunit, apolp I.
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RICHARD C. RAYNEand JOHN K. KOEPPE MATERIALS AND METHODS
Insects and JHBP extraction Colonies of L. maderae were maintained as described in a previous publication (Koeppe and Wellman, 1980). Females were mated l0 days after adult ecdysis, and ovulation of the terminal oocyte occurred approx. 18-20 days after mating. In all studies, females 5-10 days post-mating (Ms-M10) were used. Extraction of tissues for proteins, preparation of JH stock solutions, and quantification of JHBP in tissue extracts using a polyethylene glycol (PEG) assay were performed as previously described (Kovalick and Koeppe, 1983). Hemolymph JHBP was semipurified by sucrose gradient sedimentation (Koeppe and Kovalick, 1986). Fat body was maintained in vitro to obtain [3H]leucine-labeled proteins (Koeppe and Ofengand, 1976). Chemicals All chemicals were purchased from Sigma or Fisher, except as noted. [2,3,4,5 -3H]leucine (120 Ci/mmol) was purchased from ICN and a racemic mixture of [10-3H(N)]JH III (11 Ci/mmol) was obtained from New England Nuclear. All cultureware was purchased from either Falcon or Costar, and 0.22#m nitrocellulose membrane from MSI (Fisher/Cat. No. EO2HY00010). Hybridomas: immunization of mice, fusion and cloning The immunization and fusion protocols were modifications of procedures outlined elsewhere (Zola and Brooks, 1982). After fusion, cells were plated in 96-well culture plates (Falcon) at approx. 2 x 1 0 6 cells/m1 in HAT medium [Dulbecco's Modified Eagles Medium (DMEM) supplemented with 20% fetal bovine serum (FBS; Hyclone) and hypoxanthine-aminopterin-thymidine (HAT)]. Penicillin (200 IU/ml media) and streptomycin (200/zg/ml) were also added to the medium to prevent bacterial contamination. Hybridoma colonies were visible within 2 weeks when the cells were grown in 5% CO 2 at 37°C in a Queue CO2 incubator. Hybridoma colonies secreting enzyme linked immunosorbent assay (ELISA) and immunosorbent JHBP capture assay (IJCA)-positive antibodies (see below) were cloned by limiting dilution (Goding, 1983). Supernatants from culture wells containing single colonies were tested for activity by ELISA. Positive clones were expanded and then tested using secondary screening procedures for JHBP specificity. Clones secreting JHBPspecific immunoglobulins were subsequently recloned and rescreened by the same methods, resulting in the establishment of five anti-JHBP hybridoma clones. The antibody subclass of the clones was determined by ELISA with a kit from Boehringer-Mannheim. Cells from positive cloned cultures were preserved by freezing (106 cells/ml) in 90% HAT medium/10% DMSO in a liquid nitrogen freezing tank (Union Carbide). Primary screening: ELISA A noncompetitive ELISA was set up as described elsewhere (Engvall, 1980). Semipurified JHBP from a sucrose gradient was used as the antigen in testing hybridoma supematants. The monoclonal antibodies were then detected using a horseradish peroxidase (HRP) conjugated anti-mouse antibody (Sigma) and o-phenylenediamine. The color reaction was terminated by adding an equal volume of 1.0 M H2SO4 to the wells. A Dynatech MicroElisa auto reader (MR 580) was used to determine the optical density of the solution at 492 nm. Secondary screening (a) IJCA. Antibodies specific for JHBP were detected by a solid phase JHBP capture method, the IJCA. Wells of Costar PETG assay plates were incubated overnight at 4°C with 50/~1 of the ELISA-positive hybridoma culture super-
natants. The wells were then washed with phosphate buffered saline (PBS: 10mM NaPO4, pH 7.4, 150mM NaC1) and blocked with bovine serum albumin (BSA: I mg/ml PBS) as in the ELISA. After a PBS rinse to remove unbound BSA, 100/zl of [3H]leucine-labeled proteins from fat body culture were added to each well and allowed to incubate for 4 h at 4°C. At the end of the incubation the wells were again PBS washed and 100/zl of SDS-PAGE sample buffer was added to solubilize the bound proteins. Solubilized proteins were separated by 7.5% T, 2.7% C SDS-PAGE and detected by ftuorography (Koeppe et al., 1984). (b ) Sucrose gradient sedimentation. [3 H]Leucine-labeled proteins produced by fat body maintained in vitro or unlabeled proteins from hemolymph extracts were incubated overnight at 4°C with equal volumes of either hybridoma culture supernatants (+ antibody) or HAT media alone (-antibody). After incubation, the proteins were precipitated with PEG (8000; final concentration 15%) and resuspended in 1/10 vol of TMK buffer (10 mM Tris, pH 7.4, 5 mM MgC12, 300 mM KCI). Proteins in the resolubilized mixture were then separated by sucrose gradient sedimentation (Koeppe and Kovalick, 1986). Proteins from each of the gradient fractions were separated by SDS-PAGE and detected by either fluorography or silver-staining. (c) Western blotting. Western blots were used to confirm the specificity of the monoclonal antibodies. SDS-PAGE-separated proteins from media of cultured fat body, hemolymph, ovarian and egg case extracts were transferred to nitrocellulose membranes as described elsewhere (Towbin et al., 1979; Gershoni and Palade, 1983). The nitrocellulose membranes were then incubated with supernatants of the hybridoma cultures for 1 2 h. The antigenbound monoclonal antibodies were detected by enzyme immunoassay using HRP conjugated anti-mouse IgG antibodies (Cappel) and the chromogenic HRP substrate, 4-chloro-l-naphthol (modified from Hawkes et al., 1982).
RESULTS Hybridoma production Hybridoma clones described in this paper were derived from a fusion in which 65% of the plated wells (325 out of 500 wells) contained growing hybrids within 2 weeks. O f the 325 hybridoma cultures, 140 produced antibodies which reacted with the immunogen in an ELISA. Hybrids secreting JHBPspecific antibodies were identified using the IJCA. A culture was considered positive if the [3H]leucine-labeled 275 and 2 2 0 k D J H B P bands (obtained from fat body in culture) were detected in the fluorograms. O f the 140 ELISA-positive cultures tested, only eight were positive in the I J C A (data not illustrated). Hybridomas from two of the eight cultures were cloned by limiting dilution (Goding, 1983). In total, 42 of the 186 resulting clones were, according to visual inspection, derived from single colonies. Of those 42, 32 colonies scored positive in the E L I S A and 15 of the 32 were chosen for expansion and further screening. All of the clones secreted IgG2a. Antibody specificity Specificity of the IgG for hemolymph J H B P was determined by sucrose gradient migration and confirmed by Western blotting; 12 of the 15 cultures produced IgG capable of increasing the sedimentation rate of the [3H]leucine-labeled J H B P on sucrose gradients. Figure 1 shows that the addition of
IgG-JHBP
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Fraction Number Fig. 1. Sucrose gradient separation of proteins synthesized and secreted from fat body maintained in culture. Fluorogram of SDS-PAGE-separated [3H]leucine_labeled media proteins. Proteins were initially separated by sucrose gradient sedimentation and then identified by SDS-PAGE as described in the text. Lane 1 of each fluorogram corresponds to the bottom of the sucrose gradient, lane 18 to the top. (A) [3H]leucine-labeled proteins secreted by fat body maintained in vitro were incubated with equal volumes of hybridoma culture medium containing monoclonal antibodies against the JHBP and treated as described in the text. The location of the JHBP is marked by arrows on the diagram. (B) [3H]leucine-labeled proteins secreted by fat body were incubated with equal volumes of hybridoma culture medium containing no monoclonal antibody (control). The location of the JHBP is marked by arrows on the diagram. Hemolymph Extract
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Fig. 2. Western blots of proteins from hemolymph, ovarian and egg case extracts. Transfers from SDS-PAGE gels to the nitrocellulose were accomplished as described in the text. The JHBP bands were detected by enzyme immunoassay using monocolonal antibodies against the hemolymph JHBP. In each lane, a single 220 kD protein corresponding to the JHBP is detected by the antibody. The concentration of extracted proteins for each tissue sample analyzed by SDS-PAGE was about 1 mg/ml. Equivalent aliquots were electrophoresed. 669
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Fig. 3. Silver stained SDS-PAGE-separated hemolymph proteins from fractions of a potassium bromide (KBr) gradient, fractions 7 12 contain the almost purified high density lipoprotein complex, lipophorin. Analysis of each fraction by PEG assay and by Western blot analysis revealed that JH binding activity and the immunoreactive 220kD protein were also localized to fractions 7 12 (data not illustrated). Fraction 1 is the bottom of the gradient.
670
Relationship of hemolymph JHBP to lipophorin antibodies to medium containing [3H]leucine-labeled proteins secreted by fat body in vitro increased the migration of both the 220 and 275 kD JHBP relative to the control (no antibody). Analogous results were obtained when unlabeled proteins from hemolymph were used and analyzed by silver-staining the SDS-PAGE-separated protein from each sucrose gradient fraction. The results confirmed that the migration of the IgG-complexed 220 kD JHBP was increased relative to the uncomplexed 220 kD JHBP (data not illustrated). Antibodies from the 12 positive clones were then tested against hemolymph extracts in Western blots. The typical results of these blots--a single band corresponding to the 220kD JHBP--is shown in Fig. 2 (lane 1). Of the eight cultures positive in both of these assays, five were recloned and rescreened using the same procedures. From these, five hybridoma cultures secreting anti-JHBP antibodies were established. Antibody cross-reactivities Initial tests using ELISA showed that the antihemolymph JHBP antibodies cross-reacted with antigens in ovarian extracts. These results were confirmed by testing antigenicity of ovarian proteins in Western blots. The results of these blots (Fig. 2) showed that a single band corresponding to the ovarian JHBP (Koeppe et al., 1984) was detected by the monoclonal antibody probe, thus demonstrating that the JHBPs extracted from both the hemolymph and the ovaries have similar antigenic determinants. To obtain an ovarian extract free of hemolymph contamination, extracts from egg cases (separated from the hemolymph by a brood sac) were prepared. Western blots of the egg case proteins (2-3 days after egg case formation) were then probed with the antihemolymph JHBP antibodies. The results of these studies (Fig. 2) demonstrated the presence of an antigenic 220 kD peptide. The similar staining intensities of the ovarian and egg case bands suggest that the concentration of JHBP in M18 ovarioles is approximately the same as that in the encapsulated ovarioles 2-5 days post egg case development. This result corroborates previous results from Scatchard analysis of ovarian and egg case extracts which demonstrated approximately equal numbers of JH binding sites in the two tissues at these developmental stages. (Shearin, Vogler and Koeppe; unpublished data). Potassium bromide density gradient separation of hemolymph proteins Hemolymph proteins were fractionated on a potassium bromide gradient as described elsewhere (Shapiro and Law, 1983). After centrifugation in a swinging bucket rotor (TST 41.4) at 40,000 rpm for 22 h at 4°C, the gradients were collected and assayed for the presence of JHBP. Proteins in each fraction were separated by SDS-PAGE and detected by silver-staining. A typical protein profile and the location of JH binding activity is illustrated in Fig. 3. As illustrated by the results in Fig. 3, this procedure did not give us pure lipophorin complex, probably due to proteolytic digestion of lipophorin during the long centrifugation.
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DISCUSSION
Radiolabeled JH III (Koeppe et al., 1981) and a radiolabeled photoaffinity analogue of JH III, PH]EFDA (Koeppe et al., 1984), have been the only markers for studying proteins that bind JH in L. maderae. Production of antibodies against JHBPs permits new approaches for studying the structure and potential functions of these proteins. The major problem in raising antibodies specific for the hemolymph JHBP was the inability to obtain pure protein. However, because of hybridoma technology, the problem was circumvented and five cell lines producing IgG2a antibodies against the hemolymph JHBP in the cockroach, L. maderae, were generated. Epitope specificity and antibody affinity are currently unknown; however, it is known that these cell lines are not producing antibodies against the JH binding site since the antibody-JHBP complex retains its ability to bind JH. This was demonstrated via typical binding assays using protein concentrations that bind 50% of 1 x 10-SM [3H]JH III. Addition of the anti-JHBP antibodies and control antiserum did not alter JH binding activity. Experimental results concerning cross-reactivities of the antibodies with proteins from the ovary show that the ovarian JHBP has antigenic determinants similar to hemolymph JHBP. It is possible that some or all of the JHBP present in the ovarian extracts is a result of surface hemolymph contamination. Resuits from Western blot analysis of egg case extracts confirmed that the JHBP associated with ovarioles is not due to surface hemolymph contamination, but that it is in fact an ovarian protein. The exact role or function of this ovarian protein is currently unknown. In several insect species it has been demonstrated that lipophorins bind JH III (de Kort and Koopmanschap, 1986, 1987; de Kort et al., 1987). In these studies it was not determined if both or only one of the apoprotein subunits bound the hormone. Data presented in this communication demonstrates that native lipophorin in hemolymph from the adult female cockroach, L. maderae, also binds JH III (Fig. 3). In addition, SDS-PAGE analysis of lipophorin reveals the expected subunit composition, apolp I and apolp II (Fig. 3). Results from other studies demonstrate that EFDA and JH III bind specifically to only apolp I (Koeppe et al., 1984). These results may be explained by the finding that in other insect species apolp II appears to be in the core of the lipophorin complex and that apoip I is therefore the only molecule with which JH could interact in the native state (Shapiro et al., 1988; Ryan et al., 1984; Kashiwazaki and Ikai, 1985). The significance for JH III binding to apolp I remains unknown, although potential hypotheses are discussed later. The monoclonal antibodies against apolp I do not recognize apolp II in Western blots, indicating that apolp I and apolp II share no common antigenic determinants (Fig. 2). This result is in accord with the results of immunological studies of the Manduca sexta and Locusta migratoria lipophorins (Ryan et al., 1984; Schulz et al., 1987). Hemolymph JH carrier proteins have been identified in numerous insect species (Koeppe and Kovalick, 1986) and reports regarding the similarities
672
RICHARD C. RAYNE and JOHN K. KOEPPE
between the ovarian JHBP and the hemolymph JHBP have been communicated for L. maderae (Koeppe et al., 1981, 1984) and S. bullata (Van Mellaert et al., 1985). The discovery that the hemolymph JHBP is actually apolp I supports the contention that the hemolymph and ovarian JHBP are identical. It is well documented that lipophorin is also found in the ovary. However, the presence of lipophorin in the ovary has been hypothesized to be the result of non-specific uptake that occurs during lipid shuttling to the ovary (Chino, et al., 1977; Beenakkers et al., 1985). This latter hypothesis may prove to be correct; however, one might postulate that the continued presence of lipophorin within the embryonic egg may be of some functional significance and that the uptake is indeed specific. If lipophorin is specifically internalized by the oocyte, it is likely that a receptormediated mechanism is responsible. This mechanism of protein accumulation by oocytes has been well documented in numerous invertebrate and vertebrate systems for the yolk protein vitellogenin (Roth and Porter, 1964; Roth et al., 1976). We are currently using immunoaffinity purified [35S]methioninelabeled lipophorin to determine specificity of lipophorin binding to ovarian membranes. Since JH binding to apolp I is saturable, is specific for the natural homologue, and is of high affinity, apolp I is a natural JH-binding protein. However, the significance of JH binding to apolp I is unknown. It is possible that the binding of JH to apolp I is involved in mediating the actions of lipophorin. For instance, the presence of bound JH might affect the ability of the lipophorin molecule to deliver lipids to a given target such as the oocyte. Perhaps bound JH might enable oocyte receptors to recognize the lipophorin molecule, thus regulating lipid delivery and/or the entry of lipophorin into the cell. It is also possible that JH itself may be transported into the ovary and stored there for release during embryogenesis as a developmental hormone. In L. maderae we have monitored concentrations of JH III (Koeppe et al., 1980) and of JH III binding sites in the hemolymph during ovarian maturation (Koeppe et al., 1981). The results (JH III titer = 2 x 10 -7 M; binding sites = 1 x 10 -6 M) show that the number of binding sites exceeds the JH by almost 10-fold. The significance of an excess number of binding sites is unknown, although it is certainly not an uncommon phenomenon when one considers that hormones elicit responses without occupying all of their binding sites. To determine if hemolymph lipophorin is specifically internalized, oocyte membrane receptors which are specific for hemolymph lipophorin, and which bind lipophorin during discrete stages of maturation must be found. The use of monoclonal antibodies (immunoaffinity chromatography) to purify lipophorin in a biologically active form has been accomplished (Rayne and Koeppe, unpublished data) and we are currently investigating the specificity and binding affinities of this protein to oocyte membranes at different stages of development. Acknowledgements--We thank Jane H. Brice and Andy C. Kiser for their technical assistance. We also thank D. Jeffrey Lopes and Eric A. Whitsel for their editorial assistance. This work was supported by grants from the National Institutes
of Health (HD21467), the National Science Foundation (PCM-8302624), the Burroughs Welcome Foundation, Monsanto Inc. and the University Research Council at UNC-CH, Chapel Hill, N.C. REFERENCES
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Relationship of hemolymph JHBP to lipophorin de Kort C. A. D. and Koopmanschap A. B. (1987) Molecular characteristics of lipophorin, the juvenile hormonebinding protein in the hemolymph of the Colorado potato beetle. Archs Insect Biochem. Physiol. 5, 255-269. de Kort C. A. D., Koopmanschap A. B. and Ermens A. A. M. (1984) A new class of juvenile hormone binding proteins in insect hemolymph. Insect Biochem. 14, 619-623. de Kort C. A. D., Khan M. A. and Koopmanschap A. B. (1987) Juvenile hormone and the control of adult diapause in the Colorado potato beetle, Leptinotarsa decemlineata. Insect Biochem. 17, 985-988. Kovalick G. E. and Koeppe J. K. (1983) Assay and identification of juvenile hormone binding proteins in Leucophaea maderae. Molec. Cell Endocr. 31, 271-286. Roth T. F. and Porter K. R. (1964) Yolk protein uptake in the oocyte of the mosquito, Aedes aegypti, L. J. Cell Biol. 20, 313-332. Roth T. F., Cutting J. A. and Atlas S. B. (1976) Protein transport: a selective membrane mechanism. J. supramolec. Struct. 4, 527(487)-548(508). Ryan R. O., Schmidt J. O. and Law J. H. (1984) Chemical and immunological properties of lipophorins from seven insect orders. Archs Insect Biochem. Physiol. 1, 375-383.
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Schulz T. K. F., Van der Horst D. J., Amesz H., Voorma H. O. and Bcenakkers A. M. Th. (1987) Monoclonal antibodies specific for apoproteins of lipophorins from the migratory locust. Archs Insect Biochem. Physiol. 6, 97-107. Shapiro J. P. and Law J. H. (1983) Locust adipokinetic hormone stimulates lipid mobilization in Manduca sexta. BBRC 115, 924-931. Shapiro J. P., Law J. H. and Wells M. A. (1988) Lipid transport in insects. A. Rev. Ent. 33, 297-318. Towbin H., Staebelin T. and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natn. Acad. Sci. U.S.A. 76, 4350-4354. Van Mellaert H., Theunis S. and de Loof A. (1985) Juvenile hormone binding proteins in Sarcophaga bullata hemolymph and vitellogenic ovaries. Insect Biochem. 15, 665-661. Zola H. and Brooks D. (1982) Techniques for the production and characterization of monoclonal hybridoma antibodies. In Monoclonal Hybridoma Antibodies: Techniques and Applications (Edited by HurreU J. G.), pp, 1-57. CRC Press, Florida.
0020-1790/88 $3.00+0.00 Copyright © 1988 Pergamon Press plc
RELATIONSHIP OF HEMOLYMPH JUVENILE HORMONE-BINDING PROTEIN TO LIPOPHORIN IN L E U C O P H A E A M A D E R A E RICHARD C. RAYNE and JOHN K. KOEPPE* Department of Biology, University of North Carolina, Chapel Hill, NC 27514, U.S.A. (Received 1 September 1987; revised and accepted 28 April 1988)
Abstract--Monoclonal antibodies (MAb) specific for the hemolymph 220 kD juvenile hormone-binding protein (JHBP) of the cockroach, Leucophaea maderae, were used to further characterize the JHBP. Spleen cells, from mice immunized with semipurified hemolymph JHBP, were fused with myeloma cells to produce hybridomas secreting antibodies specific for the hemolymph 220 kD JHBP. Positive clones were recloned and rescreened by the same methods, resulting in the establishment of five cell lines producing IgG2~ anti-hemolymph JHBP antibodies. Using MAb against the 220 kD hemolymph JHBP, it was demonstrated by Western blot analysis that the 220 kD JHBPs present in both ovarian and egg case extracts have antigenic determinants similar to those of the hemolymph JHBP. In addition, both the 275 and 220 kD JHBPs, synthesized and secreted by fat body maintained in vitro, were detected by anti-hemolymph JHBP antibody, demonstrating that the two media proteins and the JHBP in the ovary share identical antigenic determinants. To determine if there is a relationship between hemolymph JHBP and lipophorin, hemolymph proteins were separated on potassium bromide gradients. Gradient analysis confirmed that the lipophorin complex bound JH and that the 220 kD JHBP is apolipophorin I (apolp I). The apolp I specific MAb did not cross-react with apoliphorin II (apolp II). Key Word Index: juvenile hormone binding proteins, monoclonal antibodies, lipophorin, hemolymph, ovary, Leucophaea maderae
INTRODUCTION High molecular mass, high affinity juvenile hormonebinding proteins (JHBPs) have been identified in hemolymph and ovarian extracts of a number of insect species (Koeppe et al., 1981; Engelmann, 1984; de Kort et al., 1984; Koeppe and Kovalick, 1986). In the cockroach, Leucophaea maderae, the JHBPs extracted from either the hemolymph or the ovary display many of the same properties: (1) they both prefer to bind JH III, the endogenous homologue, (KD of 2 x 10 -s M); (2) they both sediment on sucrose gradients with an S value of 6.5; and (3) they both have a molecular mass by S D S - P A G E of approx. 220,000. Currently, the site of synthesis of the ovarian JHBP remains undetermined, although we have now demonstrated that the 220 kD hemolymph JHBP is synthesized and secreted by fat body (Koeppe and Kovalick, 1986; Koeppe et al., 1988). Since the 220 kD hemolymph JHBP and the 220 kD ovarian JHBP share many of the same properties (Koeppe et al., 1981), we postulate that the two proteins are identical and that the 220 kD JHBP is incorporated into the developing oocyte during vitellogenesis. Similarities between hemolymph and ovarian JHBP also exist in the fleshfly, Sarcophaga bullata, and it is postulated that S. bullata ovaries incorporate hemolymph JHBP during ovarian maturation (Van *To whom correspondence should be addressed.
Mellaert et aL, 1985). However, in vitro incorporation experiments using [3H]leucine-labeled JHBP and developing L. maderae ovarioles have been unsuccessful (Shearin, Vogler and Koeppe, unpublished data). In two other insect species in which high molecular weight, high affinity JH III JHBP have been detected in the hemolymph, it has recently been shown that JH III is bound by the lipid shuttle protein, lipophorin (de Kort and Koopmanschap, 1986, 1987; de Kort et al., 1987). The lipophorins are a class of insect hemolymph lipoproteins which apparently shuttle lipids (primarily diacyloglycerol) between tissues (Gilbert and Chino, 1974; Chino et al., 1981; Chino, 1985; Shapiro et al., 1988). In virtually all insect systems analyzed to date, lipophorin is comprised of two apoproteins, apolipophorin I (apolp I) of ~ 2 4 0 k D and apolp II of ~ 8 0 k D (Ryan et al., 1984). Since apolp I has a molecular weight similar to the hemolymph JHBP in L. maderae and because lipophorin binds JH in two other insect species (see above), it was suspected that the 220 kD JHBP might be apolp I in L. maderae. This paper describes the production of five hybridoma cell lines that secrete monoclonal antibodies specific for the 220kD hemolymph JHBP in L. maderae. In addition, results are presented which demonstrate that lipophorin binds JH III in L. maderae and that the 220kD hemolymph JHBP synthesized and secreted by fat body is a lipophorin subunit, apolp I.
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RICHARD C. RAYNEand JOHN K. KOEPPE MATERIALS AND METHODS
Insects and JHBP extraction Colonies of L. maderae were maintained as described in a previous publication (Koeppe and Wellman, 1980). Females were mated l0 days after adult ecdysis, and ovulation of the terminal oocyte occurred approx. 18-20 days after mating. In all studies, females 5-10 days post-mating (Ms-M10) were used. Extraction of tissues for proteins, preparation of JH stock solutions, and quantification of JHBP in tissue extracts using a polyethylene glycol (PEG) assay were performed as previously described (Kovalick and Koeppe, 1983). Hemolymph JHBP was semipurified by sucrose gradient sedimentation (Koeppe and Kovalick, 1986). Fat body was maintained in vitro to obtain [3H]leucine-labeled proteins (Koeppe and Ofengand, 1976). Chemicals All chemicals were purchased from Sigma or Fisher, except as noted. [2,3,4,5 -3H]leucine (120 Ci/mmol) was purchased from ICN and a racemic mixture of [10-3H(N)]JH III (11 Ci/mmol) was obtained from New England Nuclear. All cultureware was purchased from either Falcon or Costar, and 0.22#m nitrocellulose membrane from MSI (Fisher/Cat. No. EO2HY00010). Hybridomas: immunization of mice, fusion and cloning The immunization and fusion protocols were modifications of procedures outlined elsewhere (Zola and Brooks, 1982). After fusion, cells were plated in 96-well culture plates (Falcon) at approx. 2 x 1 0 6 cells/m1 in HAT medium [Dulbecco's Modified Eagles Medium (DMEM) supplemented with 20% fetal bovine serum (FBS; Hyclone) and hypoxanthine-aminopterin-thymidine (HAT)]. Penicillin (200 IU/ml media) and streptomycin (200/zg/ml) were also added to the medium to prevent bacterial contamination. Hybridoma colonies were visible within 2 weeks when the cells were grown in 5% CO 2 at 37°C in a Queue CO2 incubator. Hybridoma colonies secreting enzyme linked immunosorbent assay (ELISA) and immunosorbent JHBP capture assay (IJCA)-positive antibodies (see below) were cloned by limiting dilution (Goding, 1983). Supernatants from culture wells containing single colonies were tested for activity by ELISA. Positive clones were expanded and then tested using secondary screening procedures for JHBP specificity. Clones secreting JHBPspecific immunoglobulins were subsequently recloned and rescreened by the same methods, resulting in the establishment of five anti-JHBP hybridoma clones. The antibody subclass of the clones was determined by ELISA with a kit from Boehringer-Mannheim. Cells from positive cloned cultures were preserved by freezing (106 cells/ml) in 90% HAT medium/10% DMSO in a liquid nitrogen freezing tank (Union Carbide). Primary screening: ELISA A noncompetitive ELISA was set up as described elsewhere (Engvall, 1980). Semipurified JHBP from a sucrose gradient was used as the antigen in testing hybridoma supematants. The monoclonal antibodies were then detected using a horseradish peroxidase (HRP) conjugated anti-mouse antibody (Sigma) and o-phenylenediamine. The color reaction was terminated by adding an equal volume of 1.0 M H2SO4 to the wells. A Dynatech MicroElisa auto reader (MR 580) was used to determine the optical density of the solution at 492 nm. Secondary screening (a) IJCA. Antibodies specific for JHBP were detected by a solid phase JHBP capture method, the IJCA. Wells of Costar PETG assay plates were incubated overnight at 4°C with 50/~1 of the ELISA-positive hybridoma culture super-
natants. The wells were then washed with phosphate buffered saline (PBS: 10mM NaPO4, pH 7.4, 150mM NaC1) and blocked with bovine serum albumin (BSA: I mg/ml PBS) as in the ELISA. After a PBS rinse to remove unbound BSA, 100/zl of [3H]leucine-labeled proteins from fat body culture were added to each well and allowed to incubate for 4 h at 4°C. At the end of the incubation the wells were again PBS washed and 100/zl of SDS-PAGE sample buffer was added to solubilize the bound proteins. Solubilized proteins were separated by 7.5% T, 2.7% C SDS-PAGE and detected by ftuorography (Koeppe et al., 1984). (b ) Sucrose gradient sedimentation. [3 H]Leucine-labeled proteins produced by fat body maintained in vitro or unlabeled proteins from hemolymph extracts were incubated overnight at 4°C with equal volumes of either hybridoma culture supernatants (+ antibody) or HAT media alone (-antibody). After incubation, the proteins were precipitated with PEG (8000; final concentration 15%) and resuspended in 1/10 vol of TMK buffer (10 mM Tris, pH 7.4, 5 mM MgC12, 300 mM KCI). Proteins in the resolubilized mixture were then separated by sucrose gradient sedimentation (Koeppe and Kovalick, 1986). Proteins from each of the gradient fractions were separated by SDS-PAGE and detected by either fluorography or silver-staining. (c) Western blotting. Western blots were used to confirm the specificity of the monoclonal antibodies. SDS-PAGE-separated proteins from media of cultured fat body, hemolymph, ovarian and egg case extracts were transferred to nitrocellulose membranes as described elsewhere (Towbin et al., 1979; Gershoni and Palade, 1983). The nitrocellulose membranes were then incubated with supernatants of the hybridoma cultures for 1 2 h. The antigenbound monoclonal antibodies were detected by enzyme immunoassay using HRP conjugated anti-mouse IgG antibodies (Cappel) and the chromogenic HRP substrate, 4-chloro-l-naphthol (modified from Hawkes et al., 1982).
RESULTS Hybridoma production Hybridoma clones described in this paper were derived from a fusion in which 65% of the plated wells (325 out of 500 wells) contained growing hybrids within 2 weeks. O f the 325 hybridoma cultures, 140 produced antibodies which reacted with the immunogen in an ELISA. Hybrids secreting JHBPspecific antibodies were identified using the IJCA. A culture was considered positive if the [3H]leucine-labeled 275 and 2 2 0 k D J H B P bands (obtained from fat body in culture) were detected in the fluorograms. O f the 140 ELISA-positive cultures tested, only eight were positive in the I J C A (data not illustrated). Hybridomas from two of the eight cultures were cloned by limiting dilution (Goding, 1983). In total, 42 of the 186 resulting clones were, according to visual inspection, derived from single colonies. Of those 42, 32 colonies scored positive in the E L I S A and 15 of the 32 were chosen for expansion and further screening. All of the clones secreted IgG2a. Antibody specificity Specificity of the IgG for hemolymph J H B P was determined by sucrose gradient migration and confirmed by Western blotting; 12 of the 15 cultures produced IgG capable of increasing the sedimentation rate of the [3H]leucine-labeled J H B P on sucrose gradients. Figure 1 shows that the addition of
IgG-JHBP
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Fraction Number Fig. 1. Sucrose gradient separation of proteins synthesized and secreted from fat body maintained in culture. Fluorogram of SDS-PAGE-separated [3H]leucine_labeled media proteins. Proteins were initially separated by sucrose gradient sedimentation and then identified by SDS-PAGE as described in the text. Lane 1 of each fluorogram corresponds to the bottom of the sucrose gradient, lane 18 to the top. (A) [3H]leucine-labeled proteins secreted by fat body maintained in vitro were incubated with equal volumes of hybridoma culture medium containing monoclonal antibodies against the JHBP and treated as described in the text. The location of the JHBP is marked by arrows on the diagram. (B) [3H]leucine-labeled proteins secreted by fat body were incubated with equal volumes of hybridoma culture medium containing no monoclonal antibody (control). The location of the JHBP is marked by arrows on the diagram. Hemolymph Extract
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Fig. 2. Western blots of proteins from hemolymph, ovarian and egg case extracts. Transfers from SDS-PAGE gels to the nitrocellulose were accomplished as described in the text. The JHBP bands were detected by enzyme immunoassay using monocolonal antibodies against the hemolymph JHBP. In each lane, a single 220 kD protein corresponding to the JHBP is detected by the antibody. The concentration of extracted proteins for each tissue sample analyzed by SDS-PAGE was about 1 mg/ml. Equivalent aliquots were electrophoresed. 669
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Fig. 3. Silver stained SDS-PAGE-separated hemolymph proteins from fractions of a potassium bromide (KBr) gradient, fractions 7 12 contain the almost purified high density lipoprotein complex, lipophorin. Analysis of each fraction by PEG assay and by Western blot analysis revealed that JH binding activity and the immunoreactive 220kD protein were also localized to fractions 7 12 (data not illustrated). Fraction 1 is the bottom of the gradient.
670
Relationship of hemolymph JHBP to lipophorin antibodies to medium containing [3H]leucine-labeled proteins secreted by fat body in vitro increased the migration of both the 220 and 275 kD JHBP relative to the control (no antibody). Analogous results were obtained when unlabeled proteins from hemolymph were used and analyzed by silver-staining the SDS-PAGE-separated protein from each sucrose gradient fraction. The results confirmed that the migration of the IgG-complexed 220 kD JHBP was increased relative to the uncomplexed 220 kD JHBP (data not illustrated). Antibodies from the 12 positive clones were then tested against hemolymph extracts in Western blots. The typical results of these blots--a single band corresponding to the 220kD JHBP--is shown in Fig. 2 (lane 1). Of the eight cultures positive in both of these assays, five were recloned and rescreened using the same procedures. From these, five hybridoma cultures secreting anti-JHBP antibodies were established. Antibody cross-reactivities Initial tests using ELISA showed that the antihemolymph JHBP antibodies cross-reacted with antigens in ovarian extracts. These results were confirmed by testing antigenicity of ovarian proteins in Western blots. The results of these blots (Fig. 2) showed that a single band corresponding to the ovarian JHBP (Koeppe et al., 1984) was detected by the monoclonal antibody probe, thus demonstrating that the JHBPs extracted from both the hemolymph and the ovaries have similar antigenic determinants. To obtain an ovarian extract free of hemolymph contamination, extracts from egg cases (separated from the hemolymph by a brood sac) were prepared. Western blots of the egg case proteins (2-3 days after egg case formation) were then probed with the antihemolymph JHBP antibodies. The results of these studies (Fig. 2) demonstrated the presence of an antigenic 220 kD peptide. The similar staining intensities of the ovarian and egg case bands suggest that the concentration of JHBP in M18 ovarioles is approximately the same as that in the encapsulated ovarioles 2-5 days post egg case development. This result corroborates previous results from Scatchard analysis of ovarian and egg case extracts which demonstrated approximately equal numbers of JH binding sites in the two tissues at these developmental stages. (Shearin, Vogler and Koeppe; unpublished data). Potassium bromide density gradient separation of hemolymph proteins Hemolymph proteins were fractionated on a potassium bromide gradient as described elsewhere (Shapiro and Law, 1983). After centrifugation in a swinging bucket rotor (TST 41.4) at 40,000 rpm for 22 h at 4°C, the gradients were collected and assayed for the presence of JHBP. Proteins in each fraction were separated by SDS-PAGE and detected by silver-staining. A typical protein profile and the location of JH binding activity is illustrated in Fig. 3. As illustrated by the results in Fig. 3, this procedure did not give us pure lipophorin complex, probably due to proteolytic digestion of lipophorin during the long centrifugation.
671
DISCUSSION
Radiolabeled JH III (Koeppe et al., 1981) and a radiolabeled photoaffinity analogue of JH III, PH]EFDA (Koeppe et al., 1984), have been the only markers for studying proteins that bind JH in L. maderae. Production of antibodies against JHBPs permits new approaches for studying the structure and potential functions of these proteins. The major problem in raising antibodies specific for the hemolymph JHBP was the inability to obtain pure protein. However, because of hybridoma technology, the problem was circumvented and five cell lines producing IgG2a antibodies against the hemolymph JHBP in the cockroach, L. maderae, were generated. Epitope specificity and antibody affinity are currently unknown; however, it is known that these cell lines are not producing antibodies against the JH binding site since the antibody-JHBP complex retains its ability to bind JH. This was demonstrated via typical binding assays using protein concentrations that bind 50% of 1 x 10-SM [3H]JH III. Addition of the anti-JHBP antibodies and control antiserum did not alter JH binding activity. Experimental results concerning cross-reactivities of the antibodies with proteins from the ovary show that the ovarian JHBP has antigenic determinants similar to hemolymph JHBP. It is possible that some or all of the JHBP present in the ovarian extracts is a result of surface hemolymph contamination. Resuits from Western blot analysis of egg case extracts confirmed that the JHBP associated with ovarioles is not due to surface hemolymph contamination, but that it is in fact an ovarian protein. The exact role or function of this ovarian protein is currently unknown. In several insect species it has been demonstrated that lipophorins bind JH III (de Kort and Koopmanschap, 1986, 1987; de Kort et al., 1987). In these studies it was not determined if both or only one of the apoprotein subunits bound the hormone. Data presented in this communication demonstrates that native lipophorin in hemolymph from the adult female cockroach, L. maderae, also binds JH III (Fig. 3). In addition, SDS-PAGE analysis of lipophorin reveals the expected subunit composition, apolp I and apolp II (Fig. 3). Results from other studies demonstrate that EFDA and JH III bind specifically to only apolp I (Koeppe et al., 1984). These results may be explained by the finding that in other insect species apolp II appears to be in the core of the lipophorin complex and that apoip I is therefore the only molecule with which JH could interact in the native state (Shapiro et al., 1988; Ryan et al., 1984; Kashiwazaki and Ikai, 1985). The significance for JH III binding to apolp I remains unknown, although potential hypotheses are discussed later. The monoclonal antibodies against apolp I do not recognize apolp II in Western blots, indicating that apolp I and apolp II share no common antigenic determinants (Fig. 2). This result is in accord with the results of immunological studies of the Manduca sexta and Locusta migratoria lipophorins (Ryan et al., 1984; Schulz et al., 1987). Hemolymph JH carrier proteins have been identified in numerous insect species (Koeppe and Kovalick, 1986) and reports regarding the similarities
672
RICHARD C. RAYNE and JOHN K. KOEPPE
between the ovarian JHBP and the hemolymph JHBP have been communicated for L. maderae (Koeppe et al., 1981, 1984) and S. bullata (Van Mellaert et al., 1985). The discovery that the hemolymph JHBP is actually apolp I supports the contention that the hemolymph and ovarian JHBP are identical. It is well documented that lipophorin is also found in the ovary. However, the presence of lipophorin in the ovary has been hypothesized to be the result of non-specific uptake that occurs during lipid shuttling to the ovary (Chino, et al., 1977; Beenakkers et al., 1985). This latter hypothesis may prove to be correct; however, one might postulate that the continued presence of lipophorin within the embryonic egg may be of some functional significance and that the uptake is indeed specific. If lipophorin is specifically internalized by the oocyte, it is likely that a receptormediated mechanism is responsible. This mechanism of protein accumulation by oocytes has been well documented in numerous invertebrate and vertebrate systems for the yolk protein vitellogenin (Roth and Porter, 1964; Roth et al., 1976). We are currently using immunoaffinity purified [35S]methioninelabeled lipophorin to determine specificity of lipophorin binding to ovarian membranes. Since JH binding to apolp I is saturable, is specific for the natural homologue, and is of high affinity, apolp I is a natural JH-binding protein. However, the significance of JH binding to apolp I is unknown. It is possible that the binding of JH to apolp I is involved in mediating the actions of lipophorin. For instance, the presence of bound JH might affect the ability of the lipophorin molecule to deliver lipids to a given target such as the oocyte. Perhaps bound JH might enable oocyte receptors to recognize the lipophorin molecule, thus regulating lipid delivery and/or the entry of lipophorin into the cell. It is also possible that JH itself may be transported into the ovary and stored there for release during embryogenesis as a developmental hormone. In L. maderae we have monitored concentrations of JH III (Koeppe et al., 1980) and of JH III binding sites in the hemolymph during ovarian maturation (Koeppe et al., 1981). The results (JH III titer = 2 x 10 -7 M; binding sites = 1 x 10 -6 M) show that the number of binding sites exceeds the JH by almost 10-fold. The significance of an excess number of binding sites is unknown, although it is certainly not an uncommon phenomenon when one considers that hormones elicit responses without occupying all of their binding sites. To determine if hemolymph lipophorin is specifically internalized, oocyte membrane receptors which are specific for hemolymph lipophorin, and which bind lipophorin during discrete stages of maturation must be found. The use of monoclonal antibodies (immunoaffinity chromatography) to purify lipophorin in a biologically active form has been accomplished (Rayne and Koeppe, unpublished data) and we are currently investigating the specificity and binding affinities of this protein to oocyte membranes at different stages of development. Acknowledgements--We thank Jane H. Brice and Andy C. Kiser for their technical assistance. We also thank D. Jeffrey Lopes and Eric A. Whitsel for their editorial assistance. This work was supported by grants from the National Institutes
of Health (HD21467), the National Science Foundation (PCM-8302624), the Burroughs Welcome Foundation, Monsanto Inc. and the University Research Council at UNC-CH, Chapel Hill, N.C. REFERENCES
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