THE JOURNAL 0 1993 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc

Vol. 268, No. 6, Issue of February 25, pp. 4300-4303, 1993 Printed in U.S.A.

Biosynthesis of Locust Lipophorin APOLIPOPHORINS I AND I1 ORIGINATE FROM A COMMON PRECURSOR*

(Received for publication, September 3, 1992)

Paul M. M. Weer&, Wil J. A. Van Marrewijk, Ad M. Th. Beenakkers, and Dick J. Van der Horst From the Department of Experimental Zoology, Biochemical Physiology Research Group, University of Utrecht, 8 Paduulaan, 3508 TB Utrecht, The Netherlands

Biosynthesis of apolipophorins of high density lipo- phorin of the locust Locusta migratoria was studied in vitro. Analysis of immunoprecipitates from homoge- nates of in vitro labeled fat body revealed a common precursor for apolipophorin I (apoLp-I, M, 220,000) and apolipophorin I1 (apoLp-11, M, 72,000) with a molecular mass of approximately 280 kDa. Pulse- chase experiments showed that this high molecular mass precursor is cleaved into apoLp-I and apoLp-I1 which subsequently are secreted as high density lipo- phorin from the fat body. The time required for the complete synthesis and secretion was estimated to be approximately 35 min. Both apolipophorins are gly- coproteins as demonstrated by the incorporation of [3H] mannose. Treatment of [3H]mannose-labeled apolipo- phorin with endoglycosidase H resulted in the complete removal of the incorporated [3H]mannose. Endoglyco- sidase H treatment of [3H]leucine-labeled apolipophor- ins caused a reduction in molecular mass of approxi- mately 3 kDa for apoLp-I and 3.5 kDa for apoLp-11, suggesting the N-linked carbohydrate content to be 1- 2 and 5%, respectively. Incubation of fat body tissue in the presence of low concentrations of tunicamycin led to the synthesis and release of nonglycosylated apolipophorins.

High density lipophorin (HDLp)’ is the principal lipopro- tein in the hemolymph of resting insects (for reviews see Beenakkers et al. (1985), Chino (1985), Shapiro et al. (1988), Ryan (1990), and Van der Horst (1990)). It is generally composed of two distinct apoproteins: 1 molecule of a 240- kDa apolipophorin (apoLp-I) and 1 molecule of a 80-kDa apolipophorin (apoLp-11). In addition, 1 or 2 molecules of a third small apolipophorin (apoLp-111, M, 18,000-20,000) may be bound (Kawooya et al., 1984; Surholt et al., 1992). The major lipid classes present in lipophorin are diacylglycerol and phospholipids (Chino and Kitazawa, 1981). During insect flight, the lipophorin particle is capable to transport large amounts of diacylglycerol from the fat body to the flight muscles. Simultaneously with the diacylglycerol loading of

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$To whom correspondence should be addressed. Tel.: 31-30- 533586; Fax: 31-30-532837.

‘The abbreviations used are: HDLp, high density lipophorin; LDLp, low density lipophorin; apoLp-I, apoLp-11, and apoLp-111, apolipophorin I (M, 220,000), I1 (M, 72,000), and I11 (M, 18,000- 20,000); mAbaI and mAbaII, monoclonal antibodies specific for apoLp-I and apoLp-11, respectively; Endo H, endoglycosidase H; PAGE, polyacrylamide gel electrophoresis.

HDLp at the fat body, 9-14 molecules of apoLp-111, which is abundantly present as free protein in the hemolymph, asso- ciate with this particle. As a result, HDLp is converted into low density lipophorin (LDLp). Upon diacylglycerol hydroly- sis at the flight muscles, LDLp dissociates into HDLp and apoLp-111, thus these hemolymph proteins act as a reusable shuttle (Van Heusden et al., 1987). This flight-directed lipid- mobilizing process is under the control of adipokinetic hor- mone.

The biochemical and molecular aspects of the structure of apoLp-I11 have been extensively studied (Chino and Yazawa 1986; Ryan et al., 1990; Van der Horst et al., 1991), and the complete cDNA sequence is known for apoLp-I11 of the hawk- moth Munduca sexta (Cole et al., 1987) and for apoLp-I11 of the locust Locusta migratoria (Kanost et al., 1988). However, data concerning the structural aspects of both apoLp-I and apoLp-I1 are still fragmentary (Shapiro et al., 1984; Kashi- wazaki and Ikai, 1985). Both apolipophorins are glycosylated (Ryan et al., 1984; Nagao and Chino, 1987), and recently we showed for the locust that apoLp-I and apoLp-I1 are released combined with lipids as a mature HDLp particle (Weers et al, 1992). The apolipoprotein composition in HDLp shows struc- tural resemblance with the female specific yolk protein vitel- logenin of the mosquito Aedes aegypti and of M. sexta in that vitellogenin of both insects is equally composed of a large and a small subunit (Raikhel et al., 1986; Osir et al., 1986). In A . aegypti the presence of a common precursor for both subunits has been reported (Bose and Raikhel, 1988). This led us to suppose that the maintenance of this ratio of the two subunits in HDLp could be accomplished by the synthesis of one large precursor molecule that is cleaved into apoLp-I and apoLp- 11. In the present report a common precursor for locust apoLp- I and apoLp-I1 is identified. In fat body tissue, the site of lipophorin biosynthesis (Prasad et al., 1986; Venkatesh et al., 1987; Weers et al., 1992), this high molecular mass precursor is cleaved into apoLp-I and apoLp-11. Additionally, some characteristics of the synthesis and secretion of both HDLp apoproteins are described.

EXPERIMENTAL PROCEDURES

Chemicals-~-[4,5-~H]Leucine (specific activity 120 Ci/mmol) and ~-[2-~H]mannose (specific activity 10-20 Ci/mmol) were obtained from Amersham International. Endoglycosidase H (Endo H), tuni- camycin, aprotinin, leupeptin, and pepstatin were from Boehringer Mannheim. Grace’s insect medium and phenylmethanesulfonyl flu- oride were from Sigma. Diisopropylfluorophosphate was from Fluka AG, Buchs, Switzerland, Goat anti-mouse immunobeads were from Bio-Rad.

Animals-Locusts, L. migratoria, were reared under crowded con- ditions as described previously (Van der Horst et al., 1987). For experiments, 8-day-old adult male locusts were used.

saline (10 mM HEPES, 150 mM NaC1, 10 mM KCl, 4 mM CaClz, 2 Fat Body Incubations-Fat body tissue was dissected and rinsed in

mM MgC12, pH 7.0). Fat bodies were preincubated in 250 p1 of Grace’s

4300

Lipophorin Biosynthesis in Locust Fat Body 4301

insect medium for a 1-h period, and subsequently transferred to Grace's insect medium without leucine. Then radiolabel was added (10 pCi of ['H]leucine or 150 pCi of ['Hlmannose per fat body) and the incubation continued. For pulse-chase experiments, a pulse of 5 min with [3H]leucine was given, followed by an incubation in complete Grace's insect medium. Medium was collected and fat bodies were homogenized in 100 p1 of homogenization buffer (50 mM Tris-HCI, pH 7.6, a t 4 "C, 50 mM NaCI, 0.2% Triton X-100) supplemented with 0.2 mM phenylmethanesulfonyl fluoride, 1 mM diisopropylfluoro- phosphate, 1 mM EDTA, and 2 pg/ml aprotinin, leupeptin, and pepstatin.

Immunoprecipitation-Fat body homogenate and medium were subjected to immunoprecipitation with two monoclonal antibodies (mAbs) which are specific for apoLp-I (mAbnI) and apoLp-I1 (mAbnII), respectively. The production and characteristics of these mAbs have been described (Schulz et al., 1987). Samples were dis- solved in radioimmune precipitation buffer (20 mM Tris-HCI, pH 7.6, at 4 "C, 150 mM NaCI, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) supplemented with mAb, 1% bovine serum albumin, and protease inhibitors (see homogenization buffer). After a 16-h incubation a t 4 "C, goat anti-mouse immunobeads were added and the incubation was continued for another 5 h. Finally, the immuno- precipitate was isolated by centrifugation and washed three times in radioimmune precipitation buffer, two times in radioimmune precip- itation buffer buffer without detergents, and subsequently dissolved in 2% SDS and 5% 8-mercaptoethanol. This solution was incubated for 20 min at 65 "C. After centrifugation the pellet was removed and the supernatant, containing labeled apolipophorin, collected and stored a t -20 "C until use. Radioactivity was measured by liquid scintillation counting; samples of the apolipophorin solution were dissolved in Emulsifier Safe scintillator, and radioactivity was meas- ured with a liquid scintillation spectrometer (Packard model 4550).

SDS-Polyacrylamide Gel Electrophoresis-Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on gradient gels (Van der Horst et al., 1987). Radiolabeled proteins were visualized by fluorography using ENHANCE. Kodak X-Omat films were exposed to the gels for 1 week.

Treatment ulith Tunicamycin-Fat bodies were divided into con- tralateral halves, one half serving as a control for the other half treated with tunicamycin. For each experiment, tunicamycin was freshly prepared as a stock solution of 1 mg/ml in 25 mM NaOH. Tunicamycin was intluded in the incubation medium throughout the preincubation and radiolabeling periods. Immunoprecipitation of pro- teins from media was followed by liquid scintillation counting.

Digestion ulith Endo H-Apolipophorins immunoprecipitated from the incubation medium as well as from fat body homogenates were dissolved in 0.25% SDS instead of the usual 2%. The digestion was carried out in 100 mM citrate buffer (pH 5.3). A sample of dissolved immunoprecipitate was incubated for 24 h in the presence or absence of 1 unit of Endo H a t 4 "C. After the incubation, SDS and p- mercaptoethanol were added to a final concentration of 2% and 5%, respectively, and the sample was subjected to SDS-PAGE and fluo- rography.

RESULTS

Identification of a Common Precursor for ApoLp-I and ApoLp-II-Following labeling in uitro with [3H]leucine, the proteins synthesized by fat body and immunoprecipitated with mAbnI or mAbnII were analyzed by denaturing SDS- PAGE and fluorography. In fat body homogenates this re- sulted in the recovery of three labeled protein bands: one of 220 kDa and one of 72 kDa representing apoLp-I and apoLp- 11, respectively, and an additional protein of approximately 280 kDa, as shown in Fig. 1. This 280-kDa protein was precipitated with either of both mAbs, suggesting that it contains both apoLp-I and apoLp-11. In addition, apoLp-I and apoLp-I1 are also coprecipitated using either mAbaI or mAbaII, which is indicative of an association of both apoli- pophorins in fat body cells. In apolipophorins isolated from medium by immunoprecipitation a similar coprecipitation was found which appeared to be due to the secretion of the apolipophorins by the fat body as mature HDLp (Weers et al., 1992). This coprecipitation is not due to immunochemical cross-reactivity, since immunoblot analysis and enzyme-

1 2 3 4

.- apolp-l- - 212 kDa

- 170 koa

- 116 kDa

apolpll-

FIG. 1. Immunoprecipitation of fat body homogenate and incubation medium with mAbs specific for apoLp-I and apoLp-11. Apolipophorins were labeled during in vitro fat body incubation with ['Hlleucine and homogenized. Media and homoge- nates were subjected to immunoprecipitation, proteins were separated by SDS-PAGE (gradient 3-8%) and visualized by fluorography. Lanes I and 2, immunoprecipitate of fat body homogenate using mAbaI (lane 1 ) and mAbnII (lane 2). Lanes 3 and 4, immunoprecipitate of proteins secreted in medium using mAbaI (lane 3 ) and mAbaII (lane 4 ) . An asterisk indicates the position of the 280-kDa protein. The molecular mass standards on the right were myosin (212 kDa), (YP-

macroglobulin (170 kDa), and p-galactosidase (116 kDa).

5 20 30 60 5 20 30 60 (min) chase time

pro-apoLp-

apoLp-I-

apoLpll-

- 220 kDa

- 72 kDa

1 2 3 4 5 6 7 8

FIG. 2. Pulse-chase experiment on the ['Hlleucine labeling of the precursor for apoLp-I and apoLp-11. Fat bodies were incubated in uitro, pulse labeled for 5 min with ['Hlleucine and chased for the times indicated. Fat body tissue was homogenized and sub- jected to immunoprecipitation with mAbnI (lanes 1-4) and mAbnII (lanes 5-8) followed by SDS-PAGE (gradient 4-10%) and fluorogra- phy.

linked immunosorbent assay experiments have shown that mAbcvI or mAbaII recognizes exclusively its epitope on apoLp- I or apoLp-11, respectively (Schulz et al., 1987). In order to test whether the 280-kDa protein is the precursor for both apolipophorins, the fat bodies were pulse-labeled with [3H] leucine, chased a t various times, and homogenized, and the homogenates subjected to immunoprecipitation with mAbaI or mAbaII. SDS-PAGE and fluorogram analysis (Fig. 2) shows that after a chase of 5 min only the 280-kDa protein was visible. After a 20-min chase both apoLp-I and apoLp-I1 appeared, whereas the 280-kDa protein simultaneously dis- appeared. These data strongly support the view that the 280- kDa protein is the precursor for apoLp-I and apoLp-11.

Synthesis Time of Lipophorin-The time required for the complete synthesis and secretion of lipophorin into the me- dium was measured by pulse-chase experiments. Fat body tissue was pulse-labeled with [3H]leucine and chased for up to 3 h. As can be seen in Fig. 3, during the first 35 min no radiolabel was recovered in the precipitated medium, whereas approximately 35 min after the ['Hlleucine pulse, labeled apoLp-I and apoLp-I1 appeared in the medium. It thus seems that a period of approximately 35 min is required for the synthesis, processing, assembly, and secretion of apoLp-I and apoLp-I1 as HDLp into the medium.

Glycosylation of ApoLp-I and ApoLp-II-N-Linked glyco-

4302 Lipophorin Biosynthesis in Locust Fat Body

0 1 0 30 60 90 120 150 180 210

chase time (min)

FIG. 3. Pulse-chase experiment on the synthesis and release of apolipophorins by fat body tissue. Fat bodies were incubated in the presence of ['H]leucine for 5 min, transferred to medium without radiolabel, and incubated for up to 3.5 h. Samples of incu- bation media were taken at the times given. Apolipophorins present in the samples were subjected to immunoprecipitation with mAbaI and mAbnII, followed by liquid scintillation counting. The radiolabel recovered in apoLp-I and apoLp-I1 after 210 min was adjusted at 100%. In addition, samples were subjected to SDS-PAGE (gradient 4-10%) and fluorography (inset).

apolp-ll

- 220 kDa

.72 koa . 68.5 kDa

1 2 3 4

FIG. 4. Endo H treatment of apoLp-I and apoLp-11. Apoli- pophorins were labeled by fat body in vitro with [3H]mannose (lanes 1 and 2) or ['H]leucine (lanes 3 and 4 ) . Media were subjected to immunoprecipitation and incubated in the presence (lanes 1 and 3) or in the absence (lanes 2 and 4 ) of Endo H.

sylation of both HDLp apolipophorins was investigated by incubating fat bodies in the presence of [3H]mannose. Analy- sis of immunoprecipitates of medium by SDS-PAGE and fluorography showed incorporation of ['Hlmannose in both apoLp-I and apoLp-I1 (Fig. 4). The [3H]mannose incorporated in uitro could be removed completely by a treatment with Endo H.

In order to determine the degree of glycosylation of apoLp- I and apoLp-11, both apoproteins were labeled with ['HI leucine and subsequently digested with Endo H. This Endo H treatment of labeled apolipophorin resulted in a clear shift in electrophoretic mobility of apoLp-I1 when subjected to SDS-PAGE. The molecular mass of apoLp-I1 following the treatment with Endo H was estimated to be 68.5 kDa. Since the molecular mass of native apoLp-I1 is 72 kDa, this implies a glycosylation grade of 5%. Endo H treatment caused only a slight shift in the electrophoretic mobility of apoLp-I, al- though incorporation of ['Hlmannose was confirmed. The difference in molecular mass between glycosylated and non- glycosylated forms of apoLp-I ( M , 220,000) was about 3 kDa, suggesting a carbohydrate content of approximately 1-2%.

Effect of Tunicamycin on Synthesis and Release of Apolipo- phorins-Tunicamycin, which is known to inhibit the glyco- sylation of proteins, was used to investigate the requirement

of the carbohydrate part of apoLp-I and apoLp-I1 for their release from the fat body. For that purpose, a dose-response relationship was established for the effect of tunicamycin on the synthesis and release of apoLp-I and apoLp-11. It was found that the use of 0.1, 1, and 5 pg/ml tunicamycin caused a reduction of 9.5 & 1.0%, 32.1 f 6.9%, and 58.1 f 10.0%, respectively, in the amount of apolipophorin recovered in the medium. When higher concentrations of tunicamycin were used the inhibition was almost complete (10 pg/ml, 80.7 f 3.0% reduction; 20 pg/ml, 90.9 +. 3.2% reduction). Analysis by SDS-PAGE and fluorography of the labeled apolipophor- ins, which were synthesized at low tunicamycin concentra- tions (1 pg/ml) and isolated from fat body homogenate, shows a reduction of the molecular mass of both the 280-kDa pre- cursor, and apoLp-I and apoLp-I1 (Fig. 5A). Apparently the cleavage of the nonglycosylated 280-kDa precursor is not affected by tunicamycin. Analysis of media from tunicamycin- treated fat body (0.1-5 pg/ml) shows secretion of ['HH]leucine- labeled nonglycosylated apoLp-I and apoLp-11. This is appar- ent from Fig. 5B since only the 68.5-kDa labeled apoLp-I1 is present. SDS-PAGE on 4% slab gels (not shown) revealed that also for apoLp-I only the lower molecular weight form was recovered. Although glycosylation is prevented, synthesis and release of the nonglycosylated forms of both apolipo- phorins from the fat body does occur at the tunicamycin concentrations used.

DISCUSSION

In this report it is clearly shown that apoLp-I and apoLp- I1 originate from a common precursor protein. Analysis of immunoprecipitates from fat body homogenates yielded a 280- kDa protein which was recognized by both mAbaI and mAbaII. We have shown by pulse-chase experiments that this 280-kDa protein is cleaved into its two subunits: apoLp-I and apoLp-11. Therefore, the presence of 1 molecule of apoLp-I and 1 molecule of apoLp-I1 in HDLp is accomplished by the cleavage of the high molecular weight precursor into its sub- units. Consequently, it seems that apoLp-I and apoLp-I1 are products of the same gene.

There is a notable similarity between lipophorin and insect vitellogenin synthesis. In the mosquito A. aegypti the protein part of vitellogenin is composed of a large (MI 200,000) and a small subunit (Mr 66,000). These two subunits are synthesized

1 2 1 2 3 4 5 6 - +

A - + - + - + tuniwmycin

€

pro-apolp- awlpi

a w l p l -

apolpli

apolp-ll-

FIG. 5. SDS-PAGE of apolipophorins synthesized and se- creted by fat body tissue in the presence or absence of tuni- camycin. Fat bodies were incubated in the presence or absence of tunicamycin and homogenized. Fat body homogenates ( A ) and media ( R ) were subjected to immunoprecipitation. Proteins were separated by SDS-PAGE followed by fluorography. A, fat body homogenate in the absence (lane 1) and presence (lane 2) of 1 pg/ml tunicamycin (3-876 slab gel). R, incubation medium; lane 1 , O . l pg/ml tunicamycin; lane 2, control; lane 3, 1 pg/ml; lane 4, control; lane 5, 5 pglml; lane 6, control (4-10% slab gel).

Lipophorin Biosynthesis in Locust Fat Body 4303

as a high molecular mass precursor ( k f r 250,000) that is cleaved and processed into both vitellogenin subunits that are assembled and released as mature vitellogenin (Bose and Raikhel, 1988, Dhadialla and Raikhel, 1990). In M. sexta vitellogenin, which closely resembles the structure of A. ae- gypti vitellogenin (Osir et al., 1986), the presence of a large molecular mass precursor has not yet been reported. For locust vitellogenin, a more complex situation has been described. Two distinct high molecular mass precursors coded by two different genes deliver the six protein fragments of vitello- genin (reviewed by Wyatt (1988)). In the fat body cells of Leucophaea maderae vitellogenin is synthesized as a large precursor that is cleaved into five subunits (Della-Cioppa and Engelmann, 1987). It thus seems that the synthesis of a large precursor protein that is cleaved into smaller fragments which are assembled and secreted is a common event in insect lipoprotein biosynthesis.

The time required for the complete synthesis and release of apoLp-I and apoLp-I1 by isolated fat body, estimated to be approximately 35 min, is somewhat shorter than the synthesis time reported for Diatraea grandiosella apolipophorin, i.e. 45 min (Shelby and Chippendale, 1991).

Incubation of fat bodies in the presence of [3H]mannose resulted in the incorporation of radiolabeled mannose into both apoLp-I and apoLp-11. The carbohydrate content was determined from the electrophoretic mobility shift observed after Endo H treatment of immunoprecipitated apolipophor- ins. It was found that apoLp-I1 has a N-linked carbohydrate content of approximately 5%, while apoLp-I appears to be less glycosylated a carbohydrate content of approximately 1- 2% was demonstrated. These quantities are in fair agreement with the carbohydrate content of 3.5% reported for intact locust HDLp, without a discrimination between individual apolipophorins (Chino and Kitazawa, 1981). The Endo H treatment of apolipophorin led to the complete removal of [3H]mannose, which is indicative of N-linked oligosaccharides of the high mannose type. This is in accordance with the structures proposed for both locust apoLp-I and apoLp-11, which mainly constitute MansGlcNacs and GlclMansGlcNacz (Nagao and Chino, 1987; Nagao et al., 1987).

Tunicamycin treatment of fat body tissue resulted in the inhibition of the glycosylation of both apolipophorins. At high concentrations, tunicamycin strongly inhibits both the syn- thesis and release of apolipophorins (Weers et al., 1992). At

low tunicamycin concentrations, however, fat body tissue is able to synthesize and secrete nonglycosylated apolipophorins into the medium. It thus appears that the carbohydrate com- ponent is not essential for the release of apolipophorin, which is in agreement with the recent results reported for the syn- thesis and release of lipophorin by tunicamycin treated fat bodies of D. grandioseh (Shelby and Chippendale, 1990, 1991).

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