Vitellogenin synthesis in the silkworm, Bombyx mori: Separate mRNAs encode two subunits of vitellogenin

Insect Biochem., Vol. 13, No. 1, pp. 81-85, 1983. Printed in Great Britain.

0020-1790/83/010081-05503.00/0 © 1983 Pergamon Press Ltd

VITELLOGENIN SYNTHESIS IN THE SILKWORM, B O M B Y X MORI: SEPARATE mRNAs ENCODE TWO SUBUNITS OF VITELLOGENIN SUSUMU IZUM! and SHIRO TOMINO Department of Biology, Tokyo Metropolitan University, Setagaya-ku, Tokyo, Japan (Received 7 June 1982)

Abstract--Vitellogenin synthesis was studied in the cultured fat body of Bombyx mori. Cultured fat body from female pupae immediately after the larval-pupal ecdysis synthesized and released into the medium both the heavy chain (VITL-H) and the light chain (VITL-L) vitellogenin. In the fat body cells a presumptive precursor of VITL-H was present in addition to two vitellogenin subunits. Kinetic studies revealed that the completion of VITL-L synthesis precedes at a faster rate than that of VITL-H synthesis in the fat body. Translation of mRNA isolated from the fat body of vitellogenic pupae under mild conditions directed the synthesis of VITL-H precursor and VITL-L in a reticulocyte lysate cell-free system. From these observations it can be concluded that two subunits of the B. mori vitellogenin are coded for by separate mRNAs. Key Word Index: Bombyx mori, fat body, mRNA, reticulocyte lysate system, vitellogenin

INTRODUCTION THE PRODUCTION of vitellogenin, the precursor of egg yolk protein, offers a suitable system for studying the developmental and hormonal regulation of protein synthesis in egg-laying vertebrates and insects (TATA, 1976; WYATX and PAN, 1978; ENGELMANN, 1979). In the silkworm, B o m b y x mori, vitellogenin is synthesized in the female fat body and selectively taken up into developing oocytes after being secreted into the haemolymph (IzuMI et al., 1980a,b). The B. mori vitellogenin and vitellin are almost indistinguishable from each other with respect to their molecular properties, Each molecule contains two different subunits termed the heavy chain (VITL-H, tool. wt 180,000) and the light chain (VITL-L, tool. wt 42,000), respectively and the native molecule is assumed to be a tetramer composed of two molecules of each of these subunits (IzuMI et al., 1980a). In a previous communication, we reported that the mRNAs for the major haemolymph proteins of B. mori are synthesized in the fat body in a time-dependent sequential fashion during the larval development, while the m R N A for VITL-L appears in the female pupae immediately after larval-pupal ecdysis (IzuMI et al., 1980b). However, since our previous experiment failed to detect the m R N A activity coding for VITL-H, it remained unclear whether VITL-H is synthesized de novo through the participation of m R N A or is derived from other molecules, such as storage proteins, in the fat body (ToJO e t al., 1980). The present experiments have been undertaken to study the kinetics of the syntheses of vitellogenin subunits in the fat body cells in culture. An attempt was also made to isolate RNA from the pupal fat body under mild conditions and to translate it in a cell-free system. In this communication we show that the two subunits of the B. mori vitellogenin are indeed coded 81

for by the separate mRNAs. Evidence is also presented that VITL-H is cleaved post-translationally in the fat body. MATERIALS AND METHODS Materials

Females of the silkworm, Bombyx mori, were used throughout the experiments. Larvae were reared on mulberry leaves at 25°C. Animals were sacrificed immediately after the larval-pupal ecdysis for the dissection of fat body. L-[4,5-3H]-Leucine (specific activity, 60 Ci/m-mole) and L-[asS]-methionine (specific activity, 900 Ci/m-mole) were obtained from New England Nuclear Co. Grace's insect medium depleted of leucine was prepared as described by GRACE (1962). Creatine kinase was isolated from rabbit skeletal muscle following the method of NODA et al. (1955). tRNA was purified from mouse liver as described (HANCHERet al., 1974). Other materials were obtained from commercial sources. Methods Isolation of vitellin and preparation of antibody. Vitellin was purified from the mature oocytes of B. mori and antiserum to vitellin was prepared as described previously (IzuMI et al., 1980a). Anti-vitellin lgG was separated from serum by ammonium sulphate precipitation at 40% saturation (OsAwA AND TOMINO, 1977; IZUMI et al., 1980a), In vitro culture o] fat body. Fat bodies were dissected from the female pupae, rinsed with Grace's insect medium (GRACE, 1962) and minced with scissors. Pieces of fat body (approx. 200/~g protein) were placed in Eppendorf centrifuge tubes each containing 50 #1 of Grace's insect medium supplemented with 50/ICi [3H]-leucine or [3sS]-methionine, five units of sodium penicillin G and 5 #g of streptomycin sulphate. Air space in the tubes were filled with a mixture of 5% CO2 and 95~0 02 and the tubes were incubated at 25°C for a defined time period. After incubation, fat body was separated from the medium and homogenized in 0.5 ml of 0.02 M Tris-HC1, 0.15 M NaCI, pH 7.5, 0.5%

SUSUMt5 IZUMI AND SHIRO TOM/NO

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Triton X-100 and 1% (w/v) sodium deoxycholate (Tris-buffeted saline, TBS). The culture medium was also diluted with 0.5 ml of the same buffer. (In the experiment shown in Fig. IB, fat body and medium were homogenized without separation). The mixture was centrifuged at 100,000O' for 30 min at 4 C and an aliquot (5 I~1} from the supernatant was assayed for acid-insoluble radioactivity as described (IzuMI et al., 1980b} and the rest was subjected to immunoprecipitation as described below. Isolation of the.tat body RNA. Fat bodies were placed in 10 vol (w/v) 7 M guanidine HCI containing 0.01 M EDTA and the mixture was gently stirred until a clear solution resulted. The solution was mixed with an equal vol of ethanol and kept at - 2 0 C for 90 rain. The precipitated RNA was collected by centrifugation at 10,000g for 10min at 4~C and further purified by repeating dissolution and precipitation as described previously (IzuMt et al., 1980b1. DNA and polysaccharide were removed from RNA preparation by washing with 3 M sodium acetate, pH6.0 (KIRr~Y, 19681. The final RNA preparation was dissolved in sterilized water and kept at - 8 0 C . In vitro translation of RNA. A rabbit reticulocyte lysate was prepared as described by SCHIMKFet al. (1974). Nuclease treatment of the lysate was performed according to PELHAM and JACKSON(1976), except that concentration of EGTA was raised to 6 raM. The incubation mixture for cell-free protein synthesis contained the following components in 50/~1:30 mM Hepes~ KOH, pH 7.6, 1 mM ATP, 0.2 mM GTP, 8 mM phosphocreatine, 5/~Ci [35S]-methionine, 0.04raM each of 19 unlabelled amino acids (see SCHUTZ el al.. 19721, 3 mM magnesmm acetate. 75 mM KC1. 1 mM dithiothreitol, 0.5 mg/ml creatine kinase, 2b~g/ml mouse liver deacylated tRNA, 201tl nuclease treated reticulocyte lysate and the fat body RNA. The mixture was incubated at 25 C for I hr and a 5,ul aliquot was assayed for total protein synthesis as described previously (OSAWAand TOM/NO, 1977: IZUMI et al., 1980bl. lmmunoprecipitation. After incubation for cell-free protein synthesis, the reaction mixture was diluted with 0.5 ml of TBS and centrifuged at 100,000 ,q for 30min at 4 C. The supernatant was mixed with 100 ~tg anti-vitellin antibody and incubated for 10min at 37C. Carrier vitellin (l()itg) was added and incubation continued for 30 min at 37C and overnight at 4:C. The extract of fat body or culture medium was treated with antibody and carrier vitellin as above. The immunoprecipitate was collected by centrifugation at 15000 for 10min at 4C, washed three times with 1.5 ml of cold TBS and dissolved in 1001d of SDS sample buffer by heating for 2 min in boiling water {LAI!MMI,I. 1970). Polyacrylamide

~,tel electrophoresis

aml jtuoroqraphy,

Polyacrylamide gel electrophoresis in SDS was performed as described by LAEMML/(1970) using 7.5 or 10~I~iacrylamide slab gel. Protein samples were treated with SDS sample buffer (LAEYJMLI,1970) as described above and electrophoresis was performed by applying a constant current of 25 mA until the bromophenol blue tracking dye reached the end of gel. Proteins were stained with Coomassie blue (WEBER et al., 1972), After electrophoresis and staining for proteins, the polyacrylamide gel was processed for fluorography according to the method of BONNERand LASKY(19741.

RESULTS AND DISCUSSION To ascertain whether the two subunits of the B, mori vitellogenin (VITL-H and VITL-L) are indeed synthesized de noco in the fat body cells, fat body was dissected from the female pupae immediately after larval-pupal ecdysis and cultured in vitro in the presence of radioactive methionine. After incubation for 20 hr, the fat body cells and culture medium were separated

and the extract from each fraction was treated with anti-vitellin antibody. As shown in Fig. IA, SDS gel electrophoresis revealed that the immunoprecipitate from the culture medium contained two radioactive peptides corresponding to VITL-H and VITL-L, respectively. When the extract of fat body was likewise analyzed, a pcptide with mol. wt approx. 200,000 was detected in the immunoprecipitate in addition to vitellogenin subunits (Fig. I B). Since this pcptide was reactive with anti-vitellin antibody and was detectable in the fat body cells but not in the secreted proteins, it is highly probable that the peptide in question is a precursor of VITL-H. The possibility that this peptide could be cleaved to give both VITL-H and VITL-L can be ruled out since its apparent tool. wt of 200,000 is insufficient to give peptides with tool. wts 180,000 (VITL-H) and 42,000 (VITL-L} (IzuMI et al., 1980al. The rates of syntheses of the vitellogenin subunits were compared as illustrated in Fig. 2, in which fat body of the vitellogenic female pupae was incubated with radioactive leucine for various time intervals and the radioactive peptides reactive with antibody were analyzed by SDS polyacrylamide gel electrophorcsis. It is evident from the figure thai radioactivity was detected in VITL-L after a 10rain incubation. whereas a 20 min incubation was required before the completion of the synthesis of the VITL-H or V1TL-H precursor. Thereafter, the amount of radioactivity in the labelled subunits increased with the incubation time. However. the densitometric tracing of the fluorogram revealed little increase in radioactivity in the VITL-H precursor during periods of 6%120min incubation, also indicating the precursor nature of this peptide. The result of amino acid analyses of the subunits of the B. mori vitellin (IzuMt et al., 1980a) indicated that the leucine contents of two subunits were almost identical, being 72/1000mole for VITL-H and 7(//1000 mole tbr VITL-L. Therefore, the present resuh implies that each subunit of the B. mori vitellogenin is encoded by the separate m R N A in the fat bod~ cells, assuming that the rates of peptide chain elongation of two subunits are similar. The occurrence of two m R N A species each liar VITL-H and VITL-L in the fat body was confirmed by means of in pitro translation of the fat body mRNA. Since our previous experiment using a heterologous translation system failed to detect mRNA activity for VITL-H (IzLMI el al., 1980b). special precautions were taken to circumvent either mechanical or enzymatical damage to the high molecular mRNA species. The fat body from the viteltogenic female pupae was dissected and gently stirred in a high concentration of guanidine HCI to extract RNA. The fat body RNA obtained through the procedure described in "Methods" was then translated in a mRNA-dependent reticulocyte lysate system (PELHAM and JACKS(IN, 1976) in the presence of radioactive methionine and the translation products wcre analyzed by SDS polyacrylamide gel electrophoresis and fluorography. As depicted in Fig. 3A, efficient translation of high molecular m R N A was noted in the reticulocyle lysate system as compared to the previously adopted wheal germ system (IZUMI et al., 1980b). In the immunoprecipitate of the translation prodncts, two radioactiv~ peptides were detected whose electrophoretic mobili-

Fig. 1. SDS polyacrylamide gel electrophoresis of immuno•eactive proteins synthesized in the cultured fat body. Fat ~odies (200 #g) dissected out from female pupae were incubated with Grace's insect medium containing [35S]-methonine (0.5 mCi/ml) at 25°C under an atmosphere of 95% D2 and 5 ~ CO2. After incubation for 4 hr fat bodies were ~eparated from medium and homogenized with TBS. The Fat body extract and medium were treated with anti-vitellin ~ntibody as described in "Methods". Immunoprecipitates from the medium (A) and from the extract of fat body (B) were electrophoresed using 10~o acrylamide gel and fluorographed.

VITL-L

Pre-VITL-H VITL-H

Fig. 2. SDS polyacrylamide gel electrophoresis of immuno-reactive proteins synthesized in the cultured fat body. Each 200 #g of the fat body was cultured in the Grace's insect medium containing [3H]-leucine for defined time as indicated. After incubation 1 ml of TBS was added to each culture and the mixture was homogenized. It was centrifuged and the supernatant was treated with anti-vitellin antibody as described in "Methods" and immunoprecipitate was electrophoresed using 7.5~o acrylamide gel and fluorographed. The following proteins were co-electrophoresed as mol. wt markers: dynein (tool. wt, 320,00(0, myosin (mol. wt, 200,000), phosphorylase (mol. wt, 94,000) bovine serum albumin (tool. wt, 67,000), egg albumin (mol. wt, 45,000), carbonic anhydrase (mol. wt, 30,000}, and soybean trypsin inhibitor (tool wt, 20,000).

VI TL-L

Pre-VITL-H VITL-H

l

A

---Pre-VITL-H

B

Fig. 3. SDS polyacrylamide gel electrophoresis of the translation products synthesized in ~,itro. Total fat body RNA (15 #g) isolated from the female pupae immediately after larval-pupal ecdysis was translaled in a mRNA dependent translation system from reticulocyte lysate (P~LnAM and JACKSON, 1976). After incubation for 60 min total translation products (A) and the immunoreactive translation products (BJ were subjected to SDS gel etectrophoresis on 10'~,, acrylamide gel and fluorographed as described in "'Methods". Several radioactive peptides seen in between the regions of pre-VITL-H and VITL-L could be the degradative products of pre-VITL-H (B). These peptides were frequently observed when the immunoprecipitate of the purified B. mori vitellin was analyzed by SDS polyacrylamide gel electrophoresis (Izu~l, unpublished result).

Vitellogenin synthesis in B. mori ties were indistinguishable from those of VITL-H precursor and VITL-L, respectively (Fig. 3B). The result together with that shown in Fig. 2 concludes that two m R N A species each coding for VITL-H precursor and VITL-L are present in the fat body of the vitellogenic female pupae. Vitellogenin of the B. mori silkworm is one of the best characterized egg yolk protein in insects (IzuMI et al., 1980a). F r o m what has been known on the structural properties, it was considered that the two protein subunits consisting of the B. mori vitellogenin might be encoded by the separate mRNAs. The results of present experiments not only substantiate the occurrence of m R N A for VITL-H in the fat body but demonstrate the participation of post-translational mechanisms in the synthesis and secretion of the B. mori vitellogenin. The post-translational cleavage of a single primary translation product to give a number of subunits has been demonstrated in the course of vitellogenin synthesis in Locusta mioratoria (CHEN et al., 1978). The result shown in Fig. 2 also suggests the possibility of the overproduction of VITL-L in the fat body in comparison with the cellular concentration of VITL-H. Since the molar ratio of VITL-H to VITL-L of the B. mori vitellogenin is 1 : 1, it would be possible to detect free form of VITL-L in the fat body or in the haemolymph. Cellular mechanisms involved in the post-translational processing and assembly of the B. mori vitellogenin are under study using a specific antibody prepared against VITL-H and VITL-L.

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