©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Sequencing of Arylphorin-binding Protein in Protein Granules of the Sarcophaga Fat Body
IMPLICATION OF A POST-TRANSLATIONAL PROCESSING MECHANISM (*)

(Received for publication, August 15, 1994; and in revised form, November 29, 1994)

Sung Ook Chung Takeo Kubo Shunji Natori (§)

From the Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Previously, we identified an arylphorin-binding protein of Sarcophagaperegrina (flesh fly) with a molecular mass of 120 kDa and suggested its participation in the selective uptake of arylphorin from the hemolymph into the pupal fat body at metamorphosis (Ueno, K., and Natori, S.(1984) J. Biol. Chem. 259, 12107-12111). This paper reports the isolation and sequencing of cDNA for the 120-kDa protein. This protein consists of 1146 amino acid residues. Immunoblotting and RNA blotting experiments revealed that this protein is present as two fragments of 76 kDa (695 residues) and 53 kDa (451 residues) in the larval fat body. When larvae pupate, the 120-kDa protein gene is further activated and the complete 120-kDa protein is synthesized without fragmentation. This suggests a novel mechanism for the production of the 120-kDa protein regulated by a proteinase depending upon the stage of development of Sarcophaga. All of these proteins were found to be localized in protein granules in the adipocytes.


INTRODUCTION

In dipteran and lepidopteran insects, storage proteins are major larval serum proteins that are synthesized in the fat body and secreted into the hemolymph during the final larval instar(1, 2, 3, 4) . Their contents amount to 70-80% of the total serum proteins at this stage. They are believed to have no function during larval life but to be used as sources of amino acids and energy to construct adult structures during metamorphosis(5) . Because of their high aromatic amino acid contents, storage proteins in general are called arylphorin(6) . When larvae pupate, arylphorin in the hemolymph is almost completely taken up again by the fat body within a relatively short period and is accumulated in protein granules in adipocytes(3, 7) .

We are interested in the process of uptake of arylphorin by the fat body because it provides a clue to the mechanism of selective uptake of serum proteins by cells. Using Sarcophagaperegrina (flesh fly) larvae, we previously demonstrated that this process is regulated by a molting hormone, 20-hydroxyecdysone (20E), (^1)and suggested the participation of a specific binding protein for arylphorin with a molecular mass of 120 kDa (putative arylphorin receptor) in this process ( (8) and (9) , and for review, see Refs. 10 and 11). This 120-kDa protein was detected in the membrane fraction of pupal fat body but not in that of larval fat body. Instead of the 120-kDa protein, the larval fat body was found to contain a protein with a molecular mass of 125 kDa. Thus, we speculated that the 125-kDa protein is a cryptic receptor and that it is converted to the active 120-kDa receptor in the presence of 20E(9) .

This paper reports cDNA cloning and sequencing of the 120-kDa protein of Sarcophaga. Results suggested a novel mechanism of the production of the 120-kDa arylphorin-binding protein regulated by a specific proteinase. The 120-kDa protein was found to be present as two fragments of 76 and 53 kDa in the larval fat body, but the pupal fat body was shown to synthesize the complete 120-kDa protein. These proteins were found to be localized in protein granules in the fat body.


MATERIALS AND METHODS

Animals and Collections of Hemolymph and Fat Body

The flesh fly, S.peregrina, was reared by the method of Ohtaki(12) . When third instar larvae were transferred from wet conditions to dry conditions, they started to pupate 16 h later. Third instar larvae and early pupae (0-24 h after puparium formation) were used for experiments. Hemolymph was collected by cutting off the anterior tip of the larvae with fine scissors and collecting the drop of hemolymph that exuded in an ice-cold test tube. Fat body was excised from larvae or pupae under a binocular microscope and rinsed well in insect saline (130 mM NaCl, 5 mM KCl, and 1 mM CaCl(2)). About 30 ml of packed tissues was obtained from 800 insects.

Preparation of Arylphorin and Its Radioiodination

Arylphorin (storage protein) was purified from the hemolymph of third instar larvae as described previously(9, 13) . Briefly, hemolymph was diluted 100-fold with 5 mM Pipes buffer, pH 6.5, containing 130 mM NaCl, 5 mM KCl, and 1 mM CaCl(2), and then subjected to 5-20% sucrose density gradient centrifugation in the same buffer. After centrifugation for 15 h at 75,000 times g, fractions containing arylphorin were collected. Radioiodination of arylphorin was performed with NaI essentially as described previously, according to the method of Miyachi et al.(14) . The specific activity of radioiodinated protein was usually 5 times 10^6 cpm/µg.

Preparation of a Fat Body Particulate Fraction

Fat body from about 10 larvae or pupae was suspended in 5 ml of buffer A (50 mM Hepes, pH 8.5, 0.1 mM CaCl(2), and 1 mg/ml of bovine serum albumin) and homogenized in a glass homogenizer with a Teflon pestle at 4 °C. The homogenate was centrifuged at 800 times g for 10 min to remove nuclei, and the resulting supernatant was centrifuged at 10,000 times g for 20 min at 4 °C. The pellet was washed once with buffer A with centrifugation and then suspended in 0.5 ml of buffer B (10 mM Hepes, pH 7.0, 2 mM CaCl(2), 1 mg/ml of bovine serum albumin, and 10% (w/w) sucrose) and stored at -80 °C. Previously we defined this fraction as fat body membranes(8, 9, 13) . Subsequently, we found that it contains many protein granules in addition to membranes.

Preparation of Arylphorin-binding Protein

This protein was prepared essentially as described before(9, 13) . Briefly, the fat body particulate fraction from pupae was solubilized in buffer C (125 mM Tris-HCl buffer, pH 6.8, containing 2% (w/v) SDS, 2% (v/v) 2-mercaptoethanol, and 20% (v/v) glycerol), and the solubilized protein was subjected to preparative SDS-polyacrylamide slab gel electrophoresis. Protein bands were located by dipping the gel in cold 0.25 M KCl solution, and the 120-kDa band of arylphorin-binding protein was cut out from the gel as a single strip. The gel strip was rinsed in 10 ml of 0.1 M Tris-HCl buffer, pH 6.8, for 10 min at 37 °C to remove KCl and then suspended in 4 ml of buffer C and sonicated in a Branson sonifier at a power setting of 50 watts for 10 min to solubilize the gel. The resulting viscous solution was again subjected to 4 M urea-SDS-polyacrylamide gel electrophoresis by the method of Bowen et al.(15) . The section of gel containing the arylphorin-binding protein was then cut out, and the protein was eluted from the gel electrophoretically in a solution of 2.5 mM Tris base containing 19.2 mM glycine and 0.01% SDS in an electroelution apparatus (Atto, AE-6580).

Cloning Procedure and Sequencing of cDNA

Purified arylphorin-binding protein (120-kDa protein) was digested with lysyl endopeptidase, and the resulting peptides were separated by high performance liquid chromatography on a column of Synchropak RP-P (C(18)). Fractions containing each peptide were lyophilized and subjected to automated sequence analysis in a Shimadzu PPSQ-10 protein sequencer. By this procedure, the sequences of seven peptides were determined. We also determined the amino-terminal amino acid sequences of the 120-kDa protein and related proteins. For this, proteins were blotted onto polyvinylidene difluoride filters (Millipore), and the filters were directly applied to the protein sequencer.

Oligodeoxyribonucleotides designed to correspond to NANTNIDI and QVNMPIQN, which were contained in the two proteolytic fragments of the 120-kDa protein, were synthesized in an Applied Biosystems DNA synthesizer (model 381A). Their sequences were 5`-CA(A/G)GTIAA(T/C)ATGCCIAT(T/C/A)CA(A/G)AA (probe 1) and 5`-AA(T/C)GCIAA(T/C)ACIAA(T/C)AT(T/C/A)GA(T/C)AT (probe 2), respectively. The probes were 5`-end labeled with P by the method of Sgaramella and Khorana(16) .

A cDNA library for the larval fat body of S. peregrina was constructed with 20 µg of poly(A) RNA and 2 µg of vector DNA by the method of Okayama and Berg(17) . About 100,000 colonies of Escherichia coli HB101 carrying recombinant plasmid were transferred to duplicate sets of nitrocellulose filters (BA-85, Schleicher & Schuell). After baking the filters at 80 °C for 2 h, prehybridization was done in 3 times SSC (1 times SSC = 0.15 M NaCl, 0.05 M sodium citrate), 10 times Denhardt's solution (1 times Denhardt's solution = 0.02% (w/v) each of Ficoll-400, bovine serum albumin, and polyvinylpyrrolidone-40) containing 50 µg/ml of denatured and sonicated salmon sperm DNA at 60 °C for 24 h. After prehybridization, the filters were hybridized with 5`-end labeled probe 1 and 2 in 4 times SSC, 10 times Denhardt's solution containing 25 µg/ml of denatured and sonicated salmon sperm DNA for 18 h at 51 °C. Then, the filters were washed with 0.1 times SSC containing 0.1% SDS for 15 min each time at room temperature and 51 °C. The blot was then exposed to Kodak XAR-5 film at -80 °C, and colonies that gave positive signals were cloned. Seventeen independent hybridization-positive clones were further analyzed by Southern blot hybridization (18) using probes 1 and 2.

For nucleotide sequencing of cDNA, the insert DNA was subcloned into pBluescript (Strategene), and various deletion derivatives of the DNA fragment were prepared using exonuclease III and mung bean nuclease. Each deletion derivative was sequenced by the dideoxy chain termination method of Sanger et al.(19) using a Taq dye primer cycle sequencing kit (Applied Biosystems). The nucleotide sequences of both strands were determined.

RNA Blot Hybridization

RNA blot hybridization was performed in 50% (v/v) formamide, 5 times NaCl/Cit (1 times NaCl/Cit = 0.15 M NaCl and 0.05 M trisodium citrate), 2 times Denhardt's solution, 50 mM phosphate buffer, pH 6.5, 0.1% SDS, sonicated salmon sperm DNA solution (100 µg/ml) for 18 h at 42 °C. Then the filters were washed with 2 times NaCl/Cit containing 0.1% SDS for 15 min at room temperature and with 0.1 times NaCl/Cit containing 0.1% SDS for 15 min at 47 °C and autoradiographed at -80 °C. Nick-translated cDNA for the 120-kDa protein was used as a probe(20) .

Antibody against the Arylphorin-binding Protein

Antibody against the arylphorin-binding protein was raised by injecting homogenized SDS-polyacrylamide gel containing about 400 µg of the purified protein into a male albino rabbit and giving two booster injections of about 300 µg of the protein 10 and 20 days later. The resulting antibody was affinity-purified essentially as described before(21) . For this, 100 µg of the purified protein was first electrophoresed in 12.5% polyacrylamide gel containing 0.2% SDS, and then proteins were blotted onto a nitrocellulose filter. The small region of the filter on which the protein had been concentrated was excised and treated with 5% skim milk solution. Then, the strip of filter paper was incubated in 15 ml of 2-fold diluted antiserum at 4 °C for 12 h with gentle shaking. The strip was then rinsed well and cut into pieces, and the antibody specifically bound to the 120-kDa protein was extracted with 1 ml of 0.2 M glycine-HCl buffer, pH 2.8. The resulting extract was neutralized with 1 M KOH, and a final concentration of 1% bovine serum albumin was added.

Determination of Partial Amino Acid Sequences of the 76- and 53-kDa Proteins

Solubilized protein of the fat body particulate fraction from pupae was subjected to SDS-polyacrylamide slab gel electrophoresis, and the bands of 76- and 53-kDa proteins, respectively, were cut out from the gel. Each gel strip was further subjected to 4 M urea-SDS-polyacrylamide gel electrophoresis, and then proteins were blotted onto a polyvinylidene difluoride filter. The bands for the 76- and 53-kDa proteins were identified by immunoblotting. These bands were cut out from the filters and subjected to automated sequence analysis to determine amino acid sequences from the amino termini of these two proteins. Partial amino acid sequence of the 76-kDa protein was determined by sequencing a peptide obtained by digesting the protein with lysyl endopeptidase.

Electrophoresis and Immunoblotting

Electrophoresis in SDS-polyacrylamide slab gel was carried out by the method of Laemmli (22) . Samples were denatured by heating them for 20 min at 75 °C in 1% SDS and 2% 2-mercaptoethanol. After electrophoresis, the gels were stained by the method of Fairbanks et al.(23) .

Proteins separated by electrophoresis were transferred electrophoretically from the gel to polyvinylidene difluoride filters. The filters were immersed in 5% skim milk solution for 1 h and then transferred to rinse solution (10 mM Tris-HCl buffer, pH 7.9, containing 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, and 0.25% skim milk) containing affinity purified antibody (5 - 10 µg/ml) and kept at 4 °C for 2 h. They were then washed well with rinse solution, transferred to 5 ml of rinse solution containing radioiodinated donkey anti-rabbit IgG (3.7 kBq/ml, Amersham Corp.) and kept for 2 h at room temperature. Finally, they were washed well with rinse solution, dried, and subjected to autoradiography with Kodak XAR film.

Binding of Radioiodinated Arylphorin to the Fat Body Particulate Fraction

The standard reaction mixture contained in 100 µl of 5 mM Mes buffer, pH 6.5, 4 mM CaCl(2), 1 mg/ml of bovine serum albumin, 1 mg/ml of gelatin, 0.1-0.5 µg of protein equivalent of the particulate fraction, and 10 ng (5 times 10^4 cpm) of radioiodinated arylphorin. The mixture was incubated for 1 h at 4 °C. Then, 0.5 ml of Mes buffer was added, and the tubes were centrifuged at 10,000 times g for 20 min. The pellet was washed once with 0.5 ml of Mes buffer with centrifugation, and then the radioactivity in the pellet was counted.

Other Methods

DNA manipulations including digestions with restriction enzymes, gel electrophoresis, DNA ligation, plasmid isolation, and E. coli transformation were carried out by standard methods. An immunofluorescence study was done with affinity-purified anti-120-kDa protein antibody and fluorescein isothiocyanate-conjugated anti-rabbit pig IgG according to a standard procedure. Protein was determined by the method of Lowry et al.(24) .


RESULTS

Molecular Cloning of cDNA for Arylphorin-binding Protein

Previously, we showed that a protein with a molecular mass of 120 kDa is present in the membrane fraction of pupal fat body of Sarcophaga and has specific affinity to arylphorin (storage protein)(9, 10, 11) . As this protein is present only in the pupal fat body, which has the ability to take up arylphorin from the medium, we speculated that it is a receptor for arylphorin and that arylphorin is taken up into the fat body via this receptor(9) . To gain more insight into the mechanism of incorporation of arylphorin into fat body, we tried to isolate cDNA for this protein.

As is evident from Fig. 1, the 120-kDa protein was detected almost exclusively in the particulate fraction of the pupal fat body and was not detected in that of larval fat body. This protein was purified to homogeneity by repeating SDS-polyacrylamide gel electrophoresis (lane3), and its proteolytic fragments were obtained by digesting it with lysyl endopeptidase. We determined the amino acid sequences of six peptides and 20 residues from the amino terminus of the 120-kDa protein. These were LNDNINTQGRR, QQICTISNDRHVQQMVGLYRLLVGA, QGILGRIAERRNANTNIDITSRVSG, FILDVLQQVNMPIQNRELLV, YLEQTEVDLSNLM, VVQLPGQGLLTDDIGLKAY, and GIITDRIRGGLDMVAGLGIG, respectively. We synthesized two DNA probes corresponding to partial amino acid sequences in the third and fourth peptides. Using these probes, we screened a cDNA library for larval fat body poly(A) RNA and finally obtained 17 clones. All of these clones contained approximately 3.7-kilobase inserts and gave identical restriction maps, indicating that they were the same. So, we sequenced one of them named pSBP1.


Figure 1: Electrophoretic profiles of the arylphorin-binding protein. Particulate fractions from larval fat body, pupal fat body (25 µg each), and purified 120-kDa protein (1 µg) were subjected to SDS-polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue. Lane1, larval fat body; lane2, pupal fat body; lane3, purified 120-kDa protein. The gel was calibrated with myosin (200 kDa), beta-galactosidase (116 kDa), phosphorylase b (97 kDa), and bovine serum albumin (66 kDa). The arrow indicates the 120-kDa protein.



The complete nucleotide sequence of the insert of pSBP1 and the putative amino acid sequence of the arylphorin-binding protein are shown in Fig. 2. There is an open reading frame of 1,163 residues encoding a protein with a molecular mass of 133 kDa. All of the peptide sequences described above were present in this sequence, indicating that pSBP1 is the cDNA for the arylphorin-binding protein. As Gly at residue 18 was assigned as the amino terminus, 17 residues from the first Met are thought to be a signal sequence. Thus, the 120-kDa protein consists of 1146 residues. As seen in the hydropathy profile shown in Fig. 3, about 20 hydrophobic motifs are present in the amino-terminal 330 and the carboxyl-terminal 420 residues besides the signal sequence, but the intervening 410 residues are hydrophilic. Possibly, some of these hydrophobic motifs are membrane-spanning sequences.





Figure 2: Nucleotide sequence of cDNA for the 120-kDa protein and its predicted amino acid sequence. The deduced amino acid sequence of the 120-kDa protein is shown by one-letter symbols below the nucleotide sequence, and amino acid residues are numbered beginning with the first methionine residue. Numbers of nucleotide beginning with the first letter of the first methionine codon are given on the right of each line. Amino acid sequences corresponding to six proteolytic fragments are underlined, and the 20 amino-terminal residues of the 120-kDa protein are boxed. Clusters of more than 3 Glu and Asp residues are shown with dottedunderlines. The poly(A) addition signal is heavilyunderlined.




Figure 3: Hydropathy analysis of the arylphorin-binding protein. The distribution of hydrophobic and hydrophilic domains was analyzed by the method of Kyte and Doolittle(28) . Numbers of amino acid residues are shown at the bottom. Data presented as hydrophobic and hydrophilic portions are plotted above and below the verticalline, respectively.



We searched for proteins that exhibit significant sequence similarity to the 120-kDa protein in the PIR, SWISS-PROT, and PRF protein sequence data bases and found about 25% sequence identity between this protein and the protein encoded by the P1 gene of Drosophilamelanogaster(25, 26, 27) , suggesting that these proteins are related (Fig. 4). No other protein was found to have significant sequence similarity to the 120-kDa protein.


Figure 4: Comparison of amino acid sequences of the arylphorin-binding protein and the protein encoded by the P1 gene of Drosophila melanogaster. Gaps were introduced to obtain maximal matching. Identical residues are boxed.



Expression of the P1 gene is known to be induced specifically by 20E in the fat body of late third instar larvae(25, 26, 27) , but nothing is known about the function of the P1 protein. Therefore, we examined the expression of the 120-kDa protein gene by RNA blot hybridization. When third instar larvae of Sarcophaga are transferred from wet to dry conditions, the titer of 20E in the hemolymph starts to increase after 8 h, and the larvae pupate after 16 h. We isolated total fat body RNA with time after transfer of larvae to dry conditions and subjected it to RNA blot hybridization using 120-kDa protein cDNA as a probe.

As shown in Fig. 5, fat body from third instar larvae contained mRNA for the 120-kDa protein, and its content increased significantly from the white pupal stage to the early pupal stage, indicating that the 120-kDa protein gene is activated by 20E like the P1 gene of Drosophila(25, 26, 27) , although its expression at a basal level was detected throughout the larval stage.


Figure 5: RNA blot hybridization analysis of mRNA for the 120-kDa protein. Fat body was isolated from larvae with time after transferring them from wet conditions to dry conditions. Total RNA was extracted from the fat body, and samples of 10 µg of RNA were subjected to RNA blot hybridization using nick-translated cDNA for the 120-kDa protein as a probe. Mouse ribosomal RNA was used as a reference marker. Numbers on the top indicate times in hours after transfer of larvae to dry conditions, and horizontalarrows show the stage of the insects.



Novel Regulation of Production of the 120-kDa Protein

Previously we demonstrated that pupal fat body contains a significant amount of the 120-kDa protein and that it incorporated arylphorin when incubated with radioiodinated arylphorin in vitro, whereas the larval fat body contained little, if any, of the 120-kDa protein, and it had almost no ability to incorporate arylphorin(8, 9) . RNA blot hybridization revealed that not only the pupal fat body but also the larval fat body contained mRNA for the 120-kDa protein. Therefore, we performed immunoblotting experiments using affinity-purified antibody against the 120-kDa protein to compare the contents of the 120-kDa protein in the larval and pupal fat body more precisely.

As is evident from Fig. 6, three proteins in the fat body lysates cross-reacted immunologically with this antibody. One was the 120-kDa protein itself, and the other two were 76- and 53-kDa proteins. The contents of the latter two proteins were almost the same in the two fat body lysates, but the content of the 120-kDa protein was much higher in the pupal fat body than in the larval fat body, consistent with our previous results.


Figure 6: Immunoblot analysis of the 120-kDa protein. Particulate fractions were prepared from the larval fat body and pupal fat body, and samples of 20 µg were subjected to SDS-polyacrylamide gel electrophoresis (A) followed by immunoblot analysis (B) using affinity-purified antibody against the 120-kDa protein. The gel was calibrated as described for Fig. 1. Positions of immunoreactive bands are indicated by arrows at the right of each panel. Lane1, larval fat body; lane2, pupal fat body.



To examine the relationship between the 120-kDa protein and the other two proteins, we determined the sequences of 19 and 20 amino acid residues from the amino termini of these two proteins and found that they coincided exactly with those of residues 18-36 and 713-732 of the 120-kDa protein. We also determined the amino acid sequence of a fragment of the 76-kDa protein obtained by its digestion with lysyl endopeptidase. The sequence coincided with residues 602-621 of the 120-kDa protein. Therefore, we concluded that the 76- and 53-kDa proteins are processed fragments of the 120-kDa protein; namely, the 76-kDa protein corresponds to residues 18-712, and the 53-kDa protein corresponds to residues 713-1163 of the 120-kDa protein. Possibly, there is a novel proteinase that cleaves most of the 120-kDa protein to 76- and 53-kDa proteins in the larval fat body, whereas in the pupal fat body, this proteinase is inactivated or repressed in some way, and so intact 120-kDa protein with ability to incorporate arylphorin accumulates.

Localization of the 120-kDa Protein and Related Proteins

If the 120-kDa protein participates in the uptake of arylphorin, it should be located on the surface of adipocytes. As affinity-purified antibody against the 120-kDa protein also reacted with the 76- and 53-kDa proteins, we used this antibody for immunofluorescence staining of fat body slices to locate these proteins. As shown in Fig. 7, the pupal fat body and larval fat body gave almost the same staining patterns, and immunofluorescence was detected exclusively in the protein granules that are distributed as irregular clumps in the cytoplasm of adipocytes. No significant fluorescence was detected on the surface of adipocytes (data not shown). Therefore, it is clear that the 120-kDa protein is not located on the surface of adipocytes but in the protein granules, suggesting that this protein does not participate directly in the uptake of arylphorin. Probably, it participates in entrapping arylphorin in the protein granules after its uptake. The results also suggest that the protein granules of the larval fat body contain mainly the 76- and 53-kDa proteins, whereas those of the pupal fat body contain the 120-kDa protein in addition to these two proteins.


Figure 7: Localization of the 120-kDa protein and its fragments. Frozen sections of fat body (10 µm) were treated successively with affinity-purified antibody against the 120-kDa protein and fluorescein isothiocyanate-conjugated second antibody. A, pupal fat body; B, larval fat body; C, pupal fat body treated with control IgG; D, diamidinophenylindole-stained section of pupal fat body to locate the nucleus. The bar indicates 15 µm.



We examined if affinity-purified antibody against the 120-kDa protein inhibited the binding of arylphorin to the particulate fraction from pupal fat body, which contains numerous protein granules. As shown in Fig. 8A, only the particulate fraction from pupal fat body bound radioiodinated arylphorin, as we observed before (8) , and this binding was specifically inhibited by the antibody (Fig. 8B), indicating that the 120-kDa in the protein granules of pupal fat body has affinity to arylphorin. Possibly, the 76- and 53-kDa proteins in the protein granules of the larval fat body have essentially no affinity to arylphorin.


Figure 8: Selective binding of radioiodinated arylphorin to the particulate fraction of pupal fat body and its inhibition by anti-120 kDa protein antibody. A, samples of 0.5 µg of protein of particulate fractions of the pupal and larval fat body were incubated with increasing amounts of radioiodinated arylphorin at 4 °C for 1 h, and the radioactivity bound to the particulate fraction was measured. bullet, pupal fat body; circle, larval fat body. B, binding experiments of the particulate fraction of the pupal fat body with radioiodinated arylphorin were performed in the presence of increasing amounts of affinity-purified antibody against the 120-kDa protein. The binding of arylphorin as a percentage of the control (without antibody) is plotted. bullet, with antibody; circle, with control IgG. Verticalbars indicate deviations of duplicate measurements.




DISCUSSION

In this study, we isolated cDNA for the 120-kDa protein of Sarcophaga pupal fat body, which has been shown to have affinity to arylphorin, and determined its complete amino acid sequence. This protein is apparently the Sarcophaga homologue of the Drosophila P1 gene product(25, 26, 27) , judging from its sequence similarity and specificity of gene activation. Previously, we suggested that the 120-kDa protein is an arylphorin receptor present in the fat body membrane, and proposed that it may be derived from the 125-kDa cryptic receptor by partial proteolysis(9) .

Immunoblotting and immunofluorescence studies revealed that most of the 120-kDa protein is present as a split form of 76- and 53-kDa proteins in the protein granules of the larval fat body but that the content of intact 120-kDa protein increased greatly in the protein granules of the pupal fat body. Therefore, the 125-kDa protein that we previously thought to be a cryptic receptor for arylphorin turned out to be an independent protein. Moreover, the fraction that we previously defined as fat body membranes (8, 9, 13) was found to contain many protein granules. Thus, the binding of the 120-kDa protein to arylphorin is probably necessary to keep the arylphorin in the protein granules, and the 120-kDa protein is not a membrane receptor necessary for taking up arylphorin from the hemolymph into the fat body.

As the contents of the 76- and 53-kDa proteins in the fat body did not differ appreciably before and after pupation, we assume that the 120-kDa protein in the pupal fat body is synthesized de novo from its mRNA whose content was found to increase manyfold on pupation. Moreover, it is difficult to conceive of a specific mechanism for joining the two smaller proteins to form the 120-kDa protein in the pupal fat body.

Our findings suggest the presence of a specific proteinase that cleaves the 120-kDa protein between Arg-712 and Ser-713 to form the 76- and 53-kDa proteins in the larval fat body. Possibly, the activity of this proteinase is repressed in the pupal fat body, and so the newly synthesized 120-kDa protein remains intact in the protein granules. We assume that 20E participates in inactivation of this proteinase because the larval fat body was shown to acquire ability to incorporate arylphorin when incubated with 20E(8) .

The main function of the 120-kDa protein may be to anchor arylphorin in the protein granules of pupal fat body, and it may be a different protein from the arylphorin receptor, if any, needed for incorporation of arylphorin into the fat body. However, it is possible that the pupal protein granules containing the 120-kDa protein participate directly in the uptake of arylphorin from the hemolymph into the fat body because this 120-kDa protein is the only protein so far shown to have affinity to arylphorin(9) . The biological role of its two fragments in the protein granules of the larval fat body is totally unknown. We found many Asp clusters in the 76-kDa protein and Glu clusters in the 53-kDa protein. These acidic stretches may be related to the function of these proteins.

Finally, we should point out that there still remains a possibility that the 120-kDa protein is in fact a membrane arylphorin receptor of pupal fat body. We cannot exclude a possibility that the binding of arylphorin to the particulate fraction from pupal fat body (Fig. 8A) is due to the binding between arylphorin and the 120-kDa protein in the membrane vesicles, and not in the protein granules. Moreover, although we could not detect immunofluorescence on the surface of adipocytes, this might be explained as a technical reason due to high background immunofluorescence of protein granules.


FOOTNOTES

*
This work was supported by a grant-in-aid for specifically promoted research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D29741[GenBank].

§
To whom correspondence should be addressed. Tel.: 03-5684-2931; Fax: 03-3813-5099.

(^1)
The abbreviations used are: 20E, 20-hydroxyecdysone; Pipes, piperazine-N,N`-bis(2-ethanesulfonic acid); Mes, 2-(N-morpholino)ethanesulfonic acid.


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