Purification and characterization of a lipid transfer particle in Rhodnius prolixus: phospholipid transfer

Insect Biochemistry and Molecular Biology 31 (2001) 563–571 www.elsevier.com/locate/ibmb

Purification and characterization of a lipid transfer particle in Rhodnius prolixus: phospholipid transfer Daniel M. Golodne, Miranda C. Van Heusden, Katia C. Gondim, Hatisaburo Masuda, Geo´rgia C. Atella * Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 21941-590 Received 19 June 2000; received in revised form 8 September 2000; accepted 12 September 2000

Abstract In this study we report the purification and characterization of a lipid transfer particle (LTP) from Rhodnius prolixus hemolymph, and its participation in phospholipid and diacylglycerol transfer processes. 3H-diacylglycerol labeled low density lipophorin from Manduca sexta (3H-LDLp) was incubated with R. prolixus lipophorin (Lp) in the presence of Rhodnius hemolymph. Following incubation and isolation, both lipoproteins showed equivalent amounts of 3H-labeled lipids. Hemolymph was subjected to KBr gradient ultracentrifugation. SDS–PAGE analysis of gradient fractions showed the enrichment of bands with molecular masses similar to the M. sexta LTP standard. LTP containing fractions were assayed and lipid transfer activity was observed. Purification of LTP was accomplished by (i) KBr density gradient ultracentrifugation, (ii) size exclusion, (iii) Cu++ affinity and (iv) ion exchange chromatographies. LTP molecular mass was estimated 苲770 kDa, comprising three apoproteins, apoLTP-I (315 kDa), apoLTP-II (85 kDa) and apoLTP-III (58 kDa). Phospolipid content of 32P-LTP was determined after two-dimensional TLC. 32P-phospholipidlabeled and unlabeled lipophorins, purified from R. prolixus were incubated in the presence of LTP resulting in the time-dependent transfer of phospholipids. LTP-mediated phospholipid transfer was not a selective process.  2001 Elsevier Science Ltd. All rights reserved. Keywords: Lipid transport; Lipophorin; Lipid transfer particle; Phospholipid transfer

1. Introduction In insects, lipids are transported throughout the hemolymph by means of a lipid-bearing protein analogous to mammalian lipoproteins, termed lipophorin (Chino and Gilbert, 1971; Chino et al., 1981; Chino, 1985; Shapiro et al., 1988). In certain insect species which rely on lipid resources for sustained flight activity, such as the tobacco hornworm Manduca sexta and the locust Locusta migratoria, lipophorin transports diacylglycerol from the fat body to the flight muscles where beta-oxidation of fatty acids supplies the great energy demand required by the active flight muscles (Beenakkers et al., 1981). Lipophorin acts as a reusable shuttle, i.e. lipids are trans-

* Corresponding author. Tel.: +55-21-590-4548 ext. 171; fax: +5521-270-8647. E-mail address: [email protected] (G.C. Atella).

ferred to the appropriate tissues and the lipophorin particle is recycled and reloaded with more lipids, e.g. at the fat body (Downer and Chino, 1985; Van Heusden et al. 1987, 1991; Soulages and Wells, 1994; Blacklock and Ryan, 1994). As a part of their lipid transport system, insects also possess a lipid transfer particle (LTP) circulating in hemolymph (Soulages and Wells, 1994; Blacklock and Ryan, 1994). Initially identified and purified from M. sexta, LTP catalyzes the in vitro transfer of several types of lipids in a reaction that can involve several different lipid donors and acceptors (Ryan et al., 1986a,b; Ryan et al., 1988a,b; Ryan et al., 1990a). Among the transferred lipids there are diacylglycerols (Ryan et al., 1988b), phospholipids (Ando et al., 1990; Singh et al., 1992), triacylglycerols (Ando et al., 1990), cholesteryl esters (Singh et al., 1992) and hydrocarbons (Takeuchi and Chino, 1993). Recently also the transfer of carotenoids mediated by LTP has been described (Tsuchida et al., 1998). Lipid

0965-1748/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 1 6 1 - 2

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donors and acceptors include lipophorins from different insect species (Takeuchi and Chino, 1993; Capurro and de Bianchi, 1990), human lipoproteins (Ryan et al., 1990a), microemulsions (Ando et al., 1990) and liposomes (Ryan et al., 1987). Recently, LTP from other insect species has also been identified (Capurro and de Bianchi, 1990) and purified (Hirayama and Chino, 1990; Takeuchi and Chino, 1993; Tsuchida et al., 1997). LTP also participates in the loading of diacylglycerol from the fat body to lipophorin (Van Heusden and Law, 1989) and in the lipid delivery to the eggs in M. sexta (Liu and Ryan, 1991). Insect LTP is a high molecular mass (⬎670 kDa) very high density lipoprotein composed of three apoproteins, apoLTP-I (310–320 kDa), apoLTP-II (85–94 kDa) and apoLTP-III (55–68 kDa), with non-covalently bound lipids (苲14% of total mass) and high-mannose carbohydrate (苲5% of protein mass) (Blacklock and Ryan, 1994). Lipids associated with LTP are directly involved in lipid transfer processes, and the composition is in dynamic equilibrium with the lipids found in lipophorin (Ryan et al., 1988b; Hirayama and Chino, 1990; Takeuchi and Chino, 1993; Tsuchida et al., 1997). The role of each LTP apoprotein has been studied using polyclonal and monoclonal antibodies. Blacklock and Ryan (1995) showed that anti-apoLTP-I, II, and III were all able to inhibit lipid transfer, but the inhibition pattern was quite different, with anti-apoLTP-II being the most effective. In contrast, Van Heusden et al. (1996) showed that a molar excess of antibodies for each of the three apoproteins inhibited lipid transfer activity equally and with a similar profile. A monoclonal anti-apoLTPIII was able to bind to the native particle but it did not inhibit the transfer, suggesting that some exposed epitopes are possibly not directly involved in the activity of the holoparticle. Due to its high molecular mass LTP can be directly visualized by electron microscopy, and it was shown to have a highly asymmetric morphology, with a roughly spherical head and a tail with a hinge (Ryan et al., 1990c; Takeuchi and Chino, 1993; Tsuchida et al., 1997). No structure–function relationship has yet been determined for LTP. In the blood-sucking insect, Rhodnius prolixus, lipophorin delivers phospholipids to the oocytes, and the particle can be reloaded with phospholipids at the fat body or midgut, according to the physiological state of the insect (Gondim et al., 1989b; Atella et al. 1992, 1995; Coelho et al., 1997). Since lipophorin can be reloaded with phospholipids, it was hypothesized that a LTP would also be present, associated with those tissues and/or free in the hemolymph. In this study we confirm the presence of a LTP in the hemolymph of R. prolixus, report its purification, and demonstrate its capacity for mediating the transfer of phospholipids between lipophorin particles.

2. Materials and methods 2.1. Insects Adult, mated Rhodnius prolixus females were reared at 28°C and 70–80% relative humidity, and fed on rabbit blood at 3-week intervals. 2.2. Hemolymph collection Five days after a blood meal, the first tarsi of the insects’ forelegs were cut and by applying a slight pressure on the abdomen, up to 10 µl of hemolymph per insect were collected in a 2-fold concentrated protease inhibitors mixture, containing phenylthiourea (3–13 mg/ml), 5 mM EDTA, 0.15 M NaCl, and final concentrations of 0.05 mg/ml soybean trypsin inhibitor, leupeptin, and antipain, and 1 mM benzamidine (final volume 苲800 µl). The collected hemolymph was centrifuged at room temperature for 5 min at 13,000g. The pellet containing the hemocytes was discarded and the supernatant stored under liquid nitrogen until use. 2.3.

32

Pi purification

Carrier-free 32Pi purchased from Comissa˜o Nacional de Energia Nuclear (CNEN, Sa˜o Paulo) was purified by means of a Dowex 1x-10 column (De Meis and Masuda, 1974). 2.4. Preparation of hemolymph metabolically labeled with 32Pi Adult females were fed on blood enriched with 32Pi (109 cpm/ml of blood) as described elsewhere (Gondim et al., 1989a) using a special feeder (Garcia et al., 1975). 2.5. Lipophorin purification Lipophorin (Lp) was purified from the final supernatant of the hemolymph collection (Gondim et al., 1989a). The supernatant was diluted to 5 ml using phosphate buffered saline (PBS, 10 mM sodium phosphate, 0.15 M NaCl, pH 7.4) with 5 mM EDTA and 1.25 g KBr was added. This material was ultracentrifuged at 159,000g in a fixed angle Beckman Type 50 rotor at 4°C for 20 h and lipophorin was collected from the top of the KBr gradient. The purified lipophorin was extensively dialyzed against PBS and stored under liquid nitrogen until use. Alternatively, lipophorin was obtained from the top fractions of the KBr gradient used in the first step of the purification of LTP (see below). 32P-lipophorin (32P-Lp) was purified starting from 32P-hemolymph and following the procedure described above. Protein concentration was estimated according to Lowry et al. (1951), using bovine serum albumin as standard.

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High density lipophorin (HDLp) from Manduca sexta was purified from adult male hemolymph by KBr gradient ultracentrifugation, essentially as described by Shapiro et al. (1984). Low density lipophorin (LDLp) also from M. sexta was purified from hemolymph of adult insects collected 90 min after the injection of synthetic M. sexta adipokinetic hormone (AKH, Peninsula Laboratories, Inc.) following the procedures described by Van Heusden and Law (1989). LDLp containing tritium-labeled neutral lipids (3H-LDLp) was obtained after the injection of [9,10⫺3H]oleic acid (Du Pont-New England Nuclear) followed by injection of AKH, hemolymph collection and KBr gradient ultracentrifugation, as above. 2.6. Treatment of lipophorin with phospholipase A2 Lipophorin completely depleted of phospholipids was obtained as described elsewhere (Gondim et al., 1992). Purified lipophorin was incubated for 3 h at 37°C in a reaction medium containing fatty-acid free albumin (Calbiochem Corporation, La Jolla, CA; 25 mg albumin/mg lipophorin), phospholipase A2 from Crotalus durissus terrificus (Boehringer Mannheim Biochemicals, Germany; approximately 3 U enzyme/mg lipophorin), 0.5 M NaCl, 0.02% sodium azide, and 3 mM CaCl2 in 50 mM Tris–HCl buffer, pH 7.4. In order to separate lipophorin particles from albumin, 4.3 ml of 0.1 M phosphate buffer, pH 7.0, 0.5 M NaCl and 5 mM EDTA containing 50% KBr (w/v) were added to the reaction mixture (0.7 ml). Five milliliters of 0.1 M phosphate buffer, pH 7.0, 0.15 M NaCl, and 5 mM EDTA containing 22.5% KBr (w/v), were gently overlaid on top of this mixture, and the material was ultracentrifuged at 4°C for 20 h at 183,000g in a Beckman 50 Ti rotor.

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exclusion volume, containing LTP, lipophorin and vitellogenin, was pooled. The material from two runs on the S-200 HR column was pooled, the NaCl concentration was adjusted to 0.5 M by adding solid NaCl, and applied to an IDA–Cu++ column (IMAC, immobilized metal ion affinity chromatography, 1.0 ml bed volume) prepared according to the manufacturer’s instructions (Sigma), in 20 mM sodium phosphate buffer, NaCl 0.5 M, pH 7.4 (buffer B). The column was eluted at room temperature with a stepwise imidazole gradient in buffer B, as follows: 5.0 mM imidazole, 5 ml; 7.5 mM imidazole, 10 ml; 10.0 mM imidazole, 3 ml; 30.0 mM imidazole, 3 ml; 50.0 mM imidazole, 3 ml. Fractions were collected and analyzed by SDS–PAGE. LTP was eluted from the column with a concentration of 7.5 mM imidazole, while other proteins eluted only with ⬎30 mM imidazole. The material obtained after Cu++ affinity chromatography was extensively dialyzed against 20 mM sodium phosphate buffer, pH 6.2, 0.01% sodium azide (buffer C) and it was applied to a DEAE–Toyopearl 650 M (Tosoh Corp.) column (1.5 ml bed volume) in buffer C. The column was washed with 10 ml of buffer C, and a stepwise NaCl gradient was used, as follows: 0.1 M NaCl, 5 ml; 0.3 M NaCl, 10 ml; 1.0 M NaCl, 5 ml. Fractions were analyzed by SDS–PAGE, and those containing purified and concentrated LTP (eluted with 0.3 M NaCl) were pooled and stored at 4°C. 32P-LTP was obtained using 32P-hemolymph as the starting material for the purification procedure. LTP from M. sexta was purified from the hemolymph of fifth instar larvae (Ryan et al., 1986a), after KBr density gradient ultracentrifugation, according to modifications described by Van Heusden and Law (1989). 2.8. Polyacrylamide gel electrophoresis

2.7. LTP purification As the first step, R. prolixus hemolymph was subjected to a KBr density gradient ultracentrifugation according to a modification of the method described by Van Heusden and Law (1989). To 5 ml of hemolymph, 4.4 g solid KBr were added, and the solution was brought to a final volume of 10 ml with PBS. This solution was gently overlaid with 10 mL of a solution containing 11% KBr (w/v) in PBS. The material was ultracentrifuged at 4°C for 17 h at 45,000 rpm in a Beckman 70 Ti rotor. Tube contents were fractionated from the top in 1 ml fractions and analyzed by SDS–PAGE. Fractions containing LTP were pooled and stored at 4°C. The material obtained after KBr gradient ultracentrifugation was dialyzed against PBS and applied to a Sephacryl S-200 HR column (1.5×90 cm) at room temperature, in PBS containing 0.01% sodium azide (buffer A), and eluted at 0.8 ml/min. Fractions were analyzed by SDS–PAGE, and the peak corresponding to the

Polyacrylamide slab gels were run both under denaturing conditions (with SDS; Laemmli, 1970) and under nondenaturing conditions (Davis, 1964), and stained with Coomassie Brilliant Blue R. For radioactive samples, the gels were stained, dried and autoradiographed. 2.9. Molecular mass determination Molecular mass of LTP was estimated in a Superose 6 HR 10/30 HPLC column equilibrated with 20 mM Tris–HCl, 1.0 M NaCl, pH 8.0, at 0.5 ml/min, and calibrated using the following protein standards: thyroglobulin, 669 kDa; apoferritin, 440 kDa; β-amylase, 200 kDa; bovine serum albumin, 66 kDa; soybean trypsin inhibitor, 20 kDa. Alternatively, determination was carried out on a pore limiting gradient PAGE (3–20%) as described by Nichols et al. (1986). The gels were subjected to 100 V for 24 h. Calibration standards used were: thyroglobu-

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lin, apoferritin, catalase (232 kDa) and bovine serum albumin. 2.10. Lipid transfer assay Initially, the lipid transfer assays employed 3H-LDLp (M. sexta, 500 µg) as lipid donor and HDLp (M. sexta, 500 µg) or Lp (R. prolixus, 500 µg) as lipid acceptor, incubated in the presence or absence of LTP (M. sexta, 50 µg) or hemolymph (R. prolixus, 10 mg of total protein) in PBS. Following incubation, lipophorins were isolated by KBr density gradient ultracentrifugation, as described by Shapiro et al. (1984). Lipid transfer assays involving the binding of lipophorins onto nitrocellulose membranes were performed essentially as described by Takeuchi and Chino (1993), with slight modifications. The incubations were conducted on 96-wells plates. The wells were blocked by incubation with 1% non-fat milk in PBS for 1 h; after each step the wells were rinsed 2–3 times with PBS. Nitrocellulose disks were placed in the wells (one disk per well), and incubated with 32P-Lp (R. prolixus, 40 µg) or 3HLDLp (M. sexta, 12 µg) for 3 h. Lipophorin that did not bind (20–25% of the amount added) was removed, and the disks were blocked by incubation with 0.2% non-fat milk in PBS for 1 h. The reactions were started by adding Lp or HDLp (80 µg) together with aliquots of the fractions from the first step of the LTP purification (KBr gradient) or purified LTP from R. prolixus, in a final volume of 120 µl/well. Following incubation, media were collected and subjected to scintillation counting for determination of the radioactivity transferred to the acceptor lipophorin (soluble). 2.11. Lipid analysis Lipid extraction was performed according to Bligh and Dyer (1959). Extracted lipids were analyzed by unidimensional thin-layer chromatography, for neutral lipids (Kawooya and Law, 1988) or by two-dimensional thin-layer chromatography, for phospholipids (Yavin and Zutra, 1977). The plates were stained with iodine vapor and autoradiographed. The spots were scraped, the silica collected and the radioactivity associated with each spot determined by scintillation counting.

3. Results 3

H-LDLp from M. sexta and non-radioactive Lp from R. prolixus were incubated and isolated by KBr gradient ultracentrifugation. 3H-lipids remained on the top fractions of the density gradient [Fig. 1(A)], while incubation in the presence of M. sexta LTP resulted in the presence of 3H-lipids in higher density fractions, where Lp from R. prolixus was located [Fig. 1(B)]. When 3H-

Fig. 1. Presence of a Lipid Transfer Particle (LTP) in R. prolixus hemolymph. 3H-LDLp from M. sexta was incubated with lipophorin from R. prolixus (Lp) in the presence or absence of LTP (M. sexta) and hemolymph from R. prolixus. One hour later, reaction media were subjected to KBr density gradient ultracentrifugation. Tubes were fractioned (1.0 ml fractions) from the top. Radioactivity present in the fractions was determined by liquid scintillation counting. (A) Incubation of 3H-LDLp and Lp. (B) 3H-LDLp and Lp incubated in the presence of LTP (M. sexta). (C) 3H-LDLp and Lp in the presence of hemolymph (R. prolixus).

LDLp and Lp were incubated in the presence of R. prolixus hemolymph, a great amount of labeled neutral lipids was transferred [Fig. 1(C)]. Lipid analysis of particles following incubation with hemolymph also showed the transfer of label (Table 1). These results confirmed the existence of a factor present in the hemolymph of R. prolixus that was capable of transferring lipids between lipophorins. In order to identify this factor, hemolymph was subjected to a KBr density gradient ultracentrifugation, and the fractions were assayed for lipid transfer activity (Fig. 2).

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Table 1 3 H-neutral lipids transferred from 3H-LDLp to Lp. 3H-LDLp from M. sexta and non-radioactive Lp from R. prolixus were incubated in the presence of R. prolixus hemolymph. After incubation, lipophorins were isolated, lipids were extracted and separated by thin-layer chromatography. As a control, lipids extracted from 3H-LDLp before incubation were used. The spots were scraped from the plate and subjected to scintillation counting. (DG) Diacylglycerol, (FA) fatty acids 3

Before After transfer

H-LDLp (M. sexta) DG (cpm)

FA (cpm)

Lp (R. prolixus) DG (cpm)

FA (cpm)

5240 2510

518 210

– 1920

– 160

hemolymph proteins were separated from the small amount of LTP (Fig. 3, lane 3). After size exclusion chromatography (Fig. 3, lane 4), there were still trace amounts of lipophorin and vitellogenin, two of the main constituents of the hemolymph; these contaminants were fully eliminated after the Cu++ affinity chromatography (Fig. 3, lane 5). After a concentration step using ion exchange chromatography, purified LTP was obtained (Fig. 3, lane 6). SDS–PAGE analysis of purified R. prolixus LTP (Fig. 3, lane 6) showed that it is composed of three apoproteins, apoLTP-I (苲315 kDa), apoLTP-II (85 kDa) and apoLTP-III (58 kDa). Apoprotein molecular mass values are in agreement with the data concerning LTP purified from the other insect species so far (Tsuchida et al., 1997). Densitometric analysis of LTP, together with the molecular mass of the individual apoproteins, indicated a mass ratio of 6.7:2.6:0.7 for apoLTP-I, II and III

Fig. 2. Localization of R. prolixus LTP in the KBr gradient fractions. Hemolymph was subjected to KBr density gradient ultracentrifugation. The tube was fractioned from the top and fractions were assayed for lipid transfer activity as described in the Materials and Methods section. (A) SDS–PAGE (5–15%) of the gradient fractions; fraction numbers are indicated, and the arrow shows the M. sexta LTP standard. The bar indicates the fractions showing the enrichment bands similar to the LTP standard. (B) Lipid transfer assay of the gradient fractions.

Analysis of the SDS–PAGE of the gradient fractions showed the enrichment of a high molecular mass band similar to apoLTP-I from M. sexta [Fig. 2(A)], which corresponded to the peak observed in the lipid transfer assay using the gradient fractions, further confirming the presence of a LTP in R. prolixus hemolymph [Fig. 2(B)]. In order to purify LTP, hemolymph was subjected to a KBr gradient ultracentrifugation, in which most of

Fig. 3. Summary of LTP purification. Samples from each purification step, containing LTP, were subjected to SDS–PAGE (5–15%) and the gels were stained with Comassie Brilliant Blue R. Lane 1: high molecular mass standards (myosin, β-galactosidase, phosphorylase b, phosphofructo kinase, bovine serum albumin, glutamic dehydrogenase, ovalbumin, glyceraldehyde 3-phosphate dehydrogenase); lane 2: hemolymph from R. prolixus females; lane 3: fraction from the KBr gradient step; lane 4: fraction from the gel filtration step (S-200 HR); lane 5: fraction from the Cu++ affinity step (IDA–Cu++); lane 6: purified LTP after the ion exchange step (DEAE). The three LTP apoproteins are also indicated.

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respectively, which corresponds to an apoprotein ratio of approximately 2:2:1. Native molecular mass determination of R. prolixus LTP by analytical gel filtration chromatography indicated a value of 苲745 kDa (Fig. 4) whereas by pore limiting gradient PAGE the value obtained was 苲800 kDa (Fig. 4, inset). Starting from metabolically labeled 32P-hemolymph, 32 P-LTP was purified and its 32P-phospholipid content was analyzed by thin-layer chromatography (Fig. 5). Lipids extracted from purified 32P-LTP contained ⬎99% of the total radioactivity associated with the particle (data not shown). The major phospholipids found were phosphatidylcholine (45% of total 32P-phospholipids) and phosphatidylethanolamine (39%), with traces of sphingomyelin, phosphatidylserine, cardiolipin and phosphatidic acid (1–3% each) (Fig. 5). To study the ability of R. prolixus LTP to promote phospholipid transfer between lipophorins, 32P-Lp was used as donor and Lp or a phospholipid depleted lipophorin (d-Lp), both non-radioactive, were used as acceptors in an assay involving binding of the donor lipophorin to nitrocellulose membranes and subsequent incubation with the acceptor, in the presence or absence of LTP. Using a fixed amount of LTP, a time-dependent phospholipid transfer was observed, from the donor lipophorin (bound to the membrane) to the acceptor lipophorin (free in the incubation media) (Fig. 6). Using Lp as

Fig. 4. Molecular mass determination. LTP and the following protein standards were separately applied on a Superose 6 HR 10/30 HPLC column: thyroglobulin, apoferritin, β-amylase, bovine serum albumin and soybean trypsin inhibitor. Absorbance at 280 nm was monitored and to each standard the peak elution volume was determined. The arrow indicates the position of LTP. Vo, exclusion volume; Ve, elution volume. Inset: pore limiting native PAGE (3–20%) of LTP; the arrow indicates the position of LTP. Lane 1: thyroglobulin; lane 2: apoferritin; lane 3: catalase; lane 4: bovine serum albumin; lane 5: purified LTP sample.

Fig. 5. Distribution of 32P-phospholipids found in 32P-LTP. Purified 32 P-LTP was subjected to lipid extraction, and the phospholipids were resolved using two-dimensional thin-layer chromatography followed by autoradiography. The spots corresponding to the different phospholipid fractions were scraped from the plate and subjected to scintillation counting. SM: Sphingomyelin; PS: phosphatidylserine; PC: phosphatidylcholine; PI: phosphatidylinositol; PE: phosphatidylethanolamine; CL: cardiolipin; PA: phosphatidic acid; nd: not determined.

acceptor, the lipid transfer increased until 10 h and approached equilibrium after 17 h [Fig. 6(A)]. The transfer observed when the acceptor was d-Lp proceeded with a similar profile, but, in contrast, a definite plateau was reached after 15 h, and it corresponded to approximately 60% of the transfer observed when Lp was used as the acceptor [Fig. 6(B)]. In both cases, the control values remained low even after 23 h. When more LTP or Lp were added after 15 h of incubation, there was no increase in the maximum transfer value (at 23 h, 苲30% of the total radioactivity initially present in the filter). In contrast, the addition of d-Lp (but not LTP) also at 15 h, to the incubation using d-Lp as acceptor, resulted in an increased transfer, up to the value observed for the Lp acceptor after 23 h (data not shown). Using 32P-Lp as donor and Lp as acceptor in the lipid transfer assay, the presence of increasing concentrations of purified LTP resulted in the concentration-dependent increase of transfer (Fig. 7). A plateau was reached with a LTP concentration of 苲8 µg/ml. To further characterize the LTP-mediated phospholipid transfer, the phospholipid content of the acceptor lipophorin was determined and compared to the content initially present in the donor particle. After incubation with the 32P-Lp donor in the presence of purified LTP, lipophorin and LTP in the incubation media were separated by means of a Cu++ affinity chromatography step identical to the one used in the purification of LTP. Quantification of the radioactivity associated with each phospholipid fraction confirmed the ability of LTP to transfer all phospholipid species, accordingly to the amounts associated with donor lipophorin (Table 2).

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Fig. 7. Effect of LTP concentration on phospholipid transfer between 32 P-Lp and Lp. 32P-Lp was fixed onto nitrocellulose membranes and incubated with lipophorin (Lp) in the presence of increasing concentrations of LTP. Twenty-two hours later the incubation media were subjected to scintillation counting. (쐌) 32P-Lp incubated in the presence of Lp and LTP; (왖) 32P-Lp incubated with LTP. Percentage of phospholipid transfer was determined from the ratio M/T of each individual point, where M stands for the radioactivity present in the reaction media and T to the total radioactivity present in the media plus the radioactivity bound to the nitrocellulose membranes. The bars represent the standard deviation (n=3). Table 2 P-phospholipids transferred from 32P-Lp to Lp. 32P-Lp was fixed onto nitrocellulose membranes and incubated with non-radioactive Lp in the presence of LTP. Nineteen hours later the incubation media (n=24) were pooled and applied on an IDA–Cu++ column in order to separate Lp from LTP. Lipids were extracted from the donor lipophorin (before incubation), and from the acceptor (repurified after incubation), and subjected to two-dimensional thin-layer chromatography. The spots corresponding to the different phospholipid fractions were scraped from the plate and subjected to scintillation counting

32 32

Fig. 6. Time-course of the LTP-mediated phospholipid transfer. Pphospholipid-labeled lipophorin from R. prolixus (Lp-32P) was fixed onto nitrocellulose membranes and incubated with lipophorin (Lp) (panel A) or a phospholipid depleted lipophorin (d-Lp) (panel B) in the presence of 30 µl of partially purified LTP from the KBr density gradient step. After the specified times incubation media were subjected to scintillation counting. (쐌) 32P-Lp in the presence of Lp and LTP; (䊏) 32P-Lp incubated with PBS only; (왖) 32P-Lp incubated with Lp; (왔) 32P-Lp incubated with LTP only; (䊊) 32P-Lp in the presence of d-Lp and LTP; (왕) 32P-Lp incubated with d-Lp. The bars represent the standard deviation (n=3).

Phospholipid

% 32P-phospholipids associated Lp after Lp-32P before incubation incubation

Sphingomyelin Phosphatidylserine Phosphatidylinositol Phosphatidylcholine Phosphatidylethanolamine Cardiolipin nd

0.26 0.53 0.47 41.91 55.68 0.88 0.27

4. Discussion Since the isolation and purification of LTP from M. sexta, similar proteins were purified from other insect species and their properties and modes of action compared. To verify the presence of a LTP in R. prolixus we initially used a ‘hybrid’ lipid transfer assay, consisting of lipophorins from different insect species (M. sexta and R. prolixus). Incubation of Rhodnius Lp in the presence of LTP from M. sexta resulted in the transfer of labeled neutral lipids [Fig. 1(B)], to a similar extent to that observed in a control assay using HDLp from M. sexta as acceptor (data not shown). Lipid transfer was also observed when hemolymph from R. prolixus was added, instead of Manduca LTP [Fig. 1(C) and Table 1]. The increased transfer observed after incubation of the lipo-

0.34 0.89 0.49 40.52 57.17 0.41 0.18

phorins with hemolymph was probably due to the higher amount of acceptor lipophorin present in this assay [Fig. 1(C)] since lipophorin is one of the major constituents of R. prolixus hemolymph. LTP purification was successfully accomplished in four steps (Fig. 3). The relatively low Cu++ affinity of LTP allowed the efficient separation from other contami-

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nant proteins, mainly lipophorin and vitellogenin, and suggested the presence of few exposed and/or available His residues in the molecule (Porath, 1990). After starting purification with 500 mg of hemolymph protein, the final step yielded 90 µg of LTP. The molecular mass of the native complex was determined by two different methods as 苲770±30 kDa. Using different methods, molecular mass estimates of LTP from M. sexta range from ⬎500 kDa by gel filtration (Ryan et al., 1986a), ⬎670 kDa by pore limiting PAGE (Ryan et al., 1988b), 苲900 kDa by analytical ultracentrifugation (Ryan et al., 1990b) and 1400 kDa by electron microscopy volume calculations (Ryan et al., 1990c). LTP from L. migratoria was estimated to have 苲600 kDa by gel filtration (Hirayama and Chino, 1990). These somewhat different results may possibly arise from the peculiar shape of the LTP holoparticle as seen by electron microscopy data (Ryan et al., 1990c; Takeuchi and Chino, 1993; Tsuchida et al., 1997). The major phospholipids associated with R. prolixus LTP were phosphatidylcholine and phosphatidylethanolamine. Although phospholipids account for ⬎50% of total lipids associated with LTPs from other insects (except for L. migratoria, 18%), the distribution of phospholipids in LTP from these species was not determined (Tsuchida et al., 1997), not allowing a direct comparison with the phospholipid composition determined in this study. To determine the LTP-mediated phospholipid transfer properties we have employed the method developed by Takeuchi and Chino (1993), which involves binding of the donor lipophorin to nitrocellulose membranes. This procedure does not require a posterior ultracentrifugation step to isolate the lipophorins used; this is particularly important in studies with insects that have no LDLp, such as P. americana and R. prolixus (Gondim et al., 1989a; Takeuchi and Chino, 1993). Time-dependent LTP-mediated phospholipid transfer was determined using 32P-Lp as donor and two distinct acceptors, Lp and d-Lp. The results suggest that, in the conditions employed, the maximum phospholipid transfer can only be attained with a certain amount of total lipid. When using d-Lp (and consequently less total lipid), this value is decreased, but it can be restored if more lipophorin is made available for the transfer to proceed. To date, the LTP-mediated phospholipid transfer has been the subject of few studies. Ando et al. (1990) demonstrated phospholipid transfer between a triolein/phospholipid microemulsion and human low density lipoprotein (LDL) in the presence of insect LTP. Singh et al. (1992) using HDLp as donor and human LDL as acceptor, incubated with LTP, showed an exchange of phospholipids between the lipoproteins. Tsuchida et al. (1997) employing lipophorins from adult M. sexta demonstrated phospholipid exchange, in con-

trast to the initial results from Ryan et al. (1986b), which indicated both lipid exchange and net transfer, but using lipophorins from different M. sexta life stages. These results are probably due to different physicochemical properties of the donor/acceptor pair. The present work further supports a phospholipid exchange process when using lipophorins from adult R. prolixus females. Furthermore, LTP is capable of transferring all phospholipid species present, while maintaining their relative amounts. In R. prolixus females, the transfer of phospholipids to the growing oocytes is of vital importance in their development. The present work has identified and purified LTP from R. prolixus hemolymph, confirming its ability to transfer phospholipids. The possibility that LTP is also involved in the transfer of lipids between lipophorin and tissues in R. prolixus is currently under study.

Acknowledgements We wish to express our gratitude to Rosane O.M.M. da Costa, Lilian Soares da Cunha Gomes and Heloisa S.L. Coelho for excellent technical assistance and Jose´ de S. Lima Junior for insect care. This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundac¸a˜o de Amparo a` Pesquisa do Rio de Janeiro (FAPERJ), Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Financiadora de Estudos e Projetos (Finep), Programa de Apoio ao Desenvolvimento Cientı´fico e Tecnolo´gico (PADCT) and Programa de Nu´cleos de Exceleˆncia (Pronex).

References Ando, S., Ryan, R.O., Yokoyama, S., 1990. Lipid transfer between human plasma low-density lipoprotein and a triolein/phospholipid microemulsion catalyzed by insect hemolymph lipid transfer particle. Biochim. Biophys. Acta 1043, 289–294. Atella, G.C., Gondim, K.C., Masuda, H., 1995. Loading of lipophorin particles with phospholipids at the midgut of Rhodnius prolixus. Arch. Insect Biochem. Physiol. 30, 337–350. Atella, G.C., Gondim, K.C., Masuda, H., 1992. Transfer of phospholipids from the fat body to lipophorin in Rhodnius prolixus. Arch. Insect Biochem. Physiol. 19, 133–144. Beenakkers, A.M.TH., Van der Horst, D.J., Van Marrewijk, W.J.A., 1981. Metabolism during locust flight. Comp. Biochem. Physiol. 69B, 315–321. Blacklock, B.J., Ryan, R.O., 1994. Hemolymph lipid transport. Insect Biochem. Molec. Biol. 24, 855–873. Blacklock, B.J., Ryan, R.O., 1995. Structural studies of Manduca sexta lipid transfer particle with apolipoprotein-specific antibodies. J. Lipid Res. 36, 108–116. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–913. Capurro, M.DE L., de Bianchi, A.G., 1990. A lipid transfer particle

D.M. Golodne et al. / Insect Biochemistry and Molecular Biology 31 (2001) 563–571

in Musca domestica haemolymph. Comp. Biochem. Physiol. 97B, 649–653. Chino, H., Gilbert, L.I., 1971. The uptake and transport of cholesterol by haemolymph lipoproteins. Insect Biochem. 1, 337–347. Chino, H., 1985. Lipid transport: biochemistry of hemolymph lipophorin. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 10, pp. 115–134. Chino, H., Downer, R.G.H., Wyatt, G.R., Gilbert, L.I., 1981. Lipophorins, a major class of lipoproteins of insect haemolymph. Insect Biochem. 11 (4), 491. Coelho, H.S.L., Atella, G.C., Moreira, M.F., Gondim, K.C., Masuda, H., 1997. Lipophorin density variation during oogenesis in Rhodnius prolixus. Arch. Insect Biochem. Physiol. 35, 301–313. Davis, B.J., 1964. Disc electrophoresis—II. Method and application to human serum proteins. Ann. NY Acad. Sci. 121, 407–427. De Meis, L., Masuda, H., 1974. Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate through two different reactions. Biochemistry 13, 2057–2062. Downer, R.G.H., Chino, H., 1985. Turnover of protein and diacylglycerol components of lipophorin in insect haemolymph. Insect Biochem. 15, 627–630. Garcia, E.S., Macarini, J.D., Garcia, M.L.M., Ubatuba, I.B., 1975. Alimentac¸a˜o do Rhodnius prolixus no laborato´rio. Ann. Acad. Bras. Cieˆnc. 47, 539–545. Gondim, K.C., Atella, G.C., Kawooya, J.K., Masuda, H., 1992. Role of phospholipids in the lipophorin particles of Rhodnius prolixus. Arch. Insect Biochem. Physiol. 20, 303–314. Gondim, K.C., Oliveira, P.L., Coelho, H.S.L., Masuda, H., 1989a. Lipophorin from Rhodnius prolixus: Purification and partial characterization. Insect Biochem. 19, 153–161. Gondim, K.C., Oliveira, P.L., Masuda, H., 1989b. Lipophorin and oogenesis in Rhodnius prolixus: transfer of phospholipids. J. Insect Physiol. 35 (1), 19–27. Hirayama, Y., Chino, H., 1990. Lipid transfer particle in locust hemolymph: purification and characterization. J. Lipid. Res. 31, 793– 799. Kawooya, J.K., Law, J.H., 1988. Role of lipophorin in lipid transport to the insect egg. J. Biol. Chem. 263 (18), 8748–8753. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685. Liu, H., Ryan, R.O., 1991. Role of lipid transfer particle in transformation of lipophorin in insect oocytes. Biochim. Biophys. Acta 1085, 112–118. Lowry, O.H., Rosebrough, N.J., Farr, A.R., Randall, R.J., 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265–270. Nichols, A.V., Krauss, R.M., Musliner, T.A., 1986. Nondenaturing polyacrylamide gradient gel electrophoresis. Meth. Enzymol. 128, 417–431. Porath, J., 1990. Amino acid side chain interaction with chelateliganded crosslinked dextran, agarose and TSK gel. J. Molec. Recog. 3, 123–127. Ryan, R.O., Wells, M.A., Law, J.H., 1986a. Lipid transfer protein from Manduca sexta hemolymph. Biochem. Biophys. Res. Commun. 136, 260–265. Ryan, R.O., Prasad, S.V., Henriksen, E.J., Wells, M.A., Law, J.H., 1986b. Lipoprotein interconversion of an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph. J. Biol. Chem. 261, 563–568. Ryan, R.O., Wells, M.A., Law, J.H., 1987. The dynamics of lipophorin

571

interconversions in Manduca sexta. In:. Law, J.H. (Ed.), Molecular Entomology, 49. Alan R. Liss Inc, USA, pp. 257–266. Ryan, R.O., Haunerland, N.H., Bowers, W.S., Law, J.H., 1988a. Insect lipid transfer particle catalyzes diacylglycerol exchange between high-density and very high-density lipoproteins. Biochim. Biophys. Acta 962, 143–148. Ryan, R.O., Senthilathipan, K.R., Wells, M.A., Law, J.H., 1988b. Facilitated diacylglycerol exchange between insect hemolymph lipophorins. Properties of Manduca sexta lipid transfer particle. J. Biol. Chem. 263, 14140–14145. Ryan, R.O., Wessler, A.N., Price, H.M., Ando, S., Yokoyama, S., 1990a. Insect lipid transfer particle catalyzes bidirectional vectorial transfer of diacylglycerol from lipophorin to human low density lipoprotein. J. Biol. Chem. 265, 10551–10555. Ryan, R.O., Hicks, L.D., Kay, C.M., 1990b. Biophysical studies on the lipid transfer particle from the hemolymph of the tobacco hornworm Manduca sexta. FEBS Lett. 267, 305–310. Ryan, R.O., Howe, A., Scraba, D.G., 1990c. Studies of the morphology and structure of the plasma lipid transfer particle from the tobacco hornworm Manduca sexta. J. Lipid. Res. 31, 871–879. Shapiro, J.P., Keim, P.S., Law, J.H., 1984. Structural studies on lipophorin, an insect lipoprotein. J. Biol. Chem. 259, 3680–3685. Shapiro, J.P., Law, J.H., Wells, M.A., 1988. Lipid transport in insects. Annu. Rev. Entomol. 33, 297–318. Singh, T.K.A., Scraba, D.G., Ryan, R.O., 1992. Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro. J. Biol. Chem. 267, 9275–9280. Soulages, J.L., Wells, M.A., 1994. Lipophorin: the structure of an insect lipoprotein and its role in lipid transport in insects. Adv. Protein Chem. 45, 371–415. Takeuchi, N., Chino, H., 1993. Lipid transfer particle in the hemolymph of the American cockroach: evidence for its capacity to transfer hydrocarbons between lipophorin particles. J. Lipid Res. 34, 543–551. Tsuchida, K., Soulages, J.L., Moribayashi, A., Suzuki, K., Maekawa, H., Wells, M.A., 1997. Purification and properties of a lipid transfer particle from Bombyx mori: comparison to the lipid transfer particle from Manduca sexta. Biochim. Biophys. Acta 1337, 57–65. Tsuchida, K., Arai, M., Tanaka, Y., Ishihara, R., Ryan, R.O., Maekawa, H., 1998. Lipid transfer particle catalyzes transfer of carotenoids between lipophorins of Bombyx mori. Insect. Biochem. Mol. Biol. 28, 927–934. Van Heusden, M.C., Law, J.H., 1989. An insect lipid transfer particle promotes lipid loading from fat body to lipoprotein. J. Biol. Chem. 264, 17287–17292. Van Heusden, M.C., Van der Horst, D.J., Voshol, J., Beenakkers, A.M.TH., 1987. The recycling of protein components of the flightspecific lipophorin in Locusta migratoria. Insect Biochem. 17, 771–776. Van Heusden, M.C., Van der Horst, D.J., Kawooya, J.K., Law, J.H., 1991. In vivo and in vitro loading of lipid by artificially lipiddepleted lipophorins: evidence for the role of lipophorin as a reusable shuttle. J. Lipid Res. 32, 1789–1794. Van Heusden, M.C., Yepiz-Plascencia, G.M., Walker, A.M., Law, J.H., 1996. Manduca sexta lipid transfer particle: synthesis by fat body and occurrence in hemolymph. Arch. Insect Biochem. Physiol. 31, 39–51. Yavin, E., Zutra, A., 1977. Separation and analysis of 32P-labeled phospholipids by a simple and rapid thin-layer chromatographic procedure and its application to cultured neuroblastoma cells. Anal. Biochem. 80, 430.