A lipid transfer particle in Musca domestica haemolymph

Comp. Biochem. Physiol. Vol. 97B, No. 4, pp. 649-653, 1990

0305-0491/90$3.00+ 0.00 © 1990PergamonPress plc

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A LIPID TRANSFER PARTICLE IN M U S C A D O M E S T I C A HAEMOLYMPH M. DE L. CAPURRO and A. G. DE BIANCHI Departamento de Bioquimica, Instituto de Quimica, Universidade de Silo Paulo, C.P. 20780, CEP 01498, $3.o Paulo, SP, Brazil (Received 17 May 1990) A b s t r a c t - - l . In Musca domestica haemolymph a lipid transfer particle (LTP) is present.

2. Musca domestica LTP is able to catalyze the transfer of lipids between different housefly lipophorin forms and also between lipophorins of Diptera and Lepidoptera. 3. The lipophorin of larval Dione juno (Lepidoptera) was purified and is composed of two apolipoproteins, apolipophorin I (Mr = 209,000) and apolipophorin II (Mr = 85,000) with a density of 1.124 g/ml. 4. The density of housefly lipophorin undergoes variations during the gonotrophic cycle. 5. The lipophorin density variation results suggest that when a high rate of lipid utilization occurs, the lipophorin has a higher density value.

INTRODUCTION

Lipophorin isolation

Larval haemolymph from Musca domestica and Dione juno were collected by puncturing the anterior end of the

In insect haemolymph, lipid transport is accomplished mainly by lipophorin (Chino, 1985; Shapiro et al., 1988). Lipophorin from Musca domestica is composed of two apoproteins, apolipophorin I and apolipophorin II, and several lipid types. During the insect life cycle M u s c a lipophorin shows density variations. However, during a specific and welldefined stage only one form is predominant in the haemolymph (Bianchi et al., 1987). These density variations of lipophorins are due to modifications in lipid content and compositions as shown to occur with larval Manduca sexta lipophorin (Prasad et al., 1986). The addition or removal of lipids from lipophorin is not very well understood. However, the Manduca sexta haemolymph contains a protein, denominated lipid transfer particle, that is able to catalyze the transfer of lipids between haemolymph lipoproteins (Ryan et al., 1986a,b, 1988a,b). This protein may also be involved in the transfer of lipids between lipophorin and cells (Van Heusden and Law, 1989). However to date, the lipid transfer particle has been shown only to occur in Manduca sexta. It would be interesting to identify and study similar proteins in other insect orders, since lipophorin occurs in all insect species examined. In this paper, we report on the presence of lipid transfer particle activity in the housefly haemolymph. This protein is able to catalyze the transfer of lipids between M u s c a lipophorins and also between M u s c a and Lepidoptera (Dione j u n o ) lipophorins.

animals. The haemolymph was pooled in centrifuge tubes containing 0.1 M NaH2PO4-NaOH, pH 7.0, 0.15 M NaC1, 5 mM EDTA, 1 mM benzamidine, 0.5 mM phenylthiocarbamide and 1 mM N-ct-p-tosyl-L-lysine chloromethylketone (buffer A). The diluted haemolymph (1:1) was centrifuged at 3000g for 10 rain at 4°C, and the supernatant was used for the experiments. The larval housefly lipophorin was isolated by a KBr density gradient ultracentrifugation followed by a glycerol gradient ultracentrifugation as previously described (Bianchi et al., 1987). The adult housefly and larval Dione juno lipophorins were isolated by KBr density gradient ultracentrifugation in the conditions described for adult housefly (Bianchi et al., 1987). Lipophorin radioiodination

Purified lipophorins were radioiodinated as previously described for Musca domestica storage protein (Marinotti and Bianchi, 1986). The radioiodinated larval housefly lipophorin was submitted to delipidation by the method of Folch et al. (1957) and the radioactivity associated with the lipidic and proteic fractions were determined with a gamma counter (Gamma 5500, Beckman Instruments). The radioiodinated lipophorins were also analyzed by SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) and autoradiography as anteriorly described for housefly storage protein (Marinotti and Bianchi, 1986). Preparation of crude lipid transfer particle (LTP) Manduca sexta haemolymph LTP is a lipoprotein with

MATERIALS AND METHODS

Animals Musca domestica was reared in the laboratory according to Targa and Peres (1979). Dione juno larvae were kindly supplied by Dr A. N. Cestari from Instituto de Biocirncias, USP, S~.o Paulo. 649

density 1.23 g/ml. Therefore, in the KBr density gradients used for lipophorin (density 1.08-1.18 g/ml) isolation, it is encountered at the bottom of ultracentrifugation tube (Ryan et aL, 1986a). Assuming that housefly LTP had a similar density to that one of Manduca sexta the material of the KBr density ultracentrifugation tubes, used for lipophorin isolation, with densities higher than 1.17 g/ml was dialyzed against 0.025 M Tris, 0.192 M glycine, pH 8.3, containing 5mM EDTA, overnight at 4°C. This material, that is a delipophorinated haemolymph, was used as crude LTP preparation. A similar type of preparation was utilized by Ryan et aL (1986a) to demonstrate the occurrence of Manduca sexta LTP.

650

M. DE L. CAPURROand A. G. DE BIANCHI

Lipid transfer assays For in vitro assays, purified adult Musca domestiea lipophorin was radioiodinated and incubated for 1 hr at 25°C, together with a large mass of purified larval lipophorin, (I) in the presence of LTP, (2) in the absence of LTP or (3) in the presence of LTP warmed to 100°C by 1rain. The incubation mixtures were submitted to KBr density ultracentrifugation as already described. After ultracentrifugation the tubes' contents were fractionated from the bottom, in fractions of 0.2 ml. The fractions were analyzed for the presence of radioactivity (Gamma counter 5500, Beckman Instruments); protein by absorbance at 280 nm; lipophorin by absorbance at 460 nm (since lipophorin contains carotenoids and is yellow) and density determined by refractive index (Refractometer PZO, Warszawa RLI, Poland). Similar experiments were performed using [13q]. labelled adult Musca domestiea lipophorin and larval Dione juno lipophorin. For in vivo assays, larval housefly lipophorin was radioiodinated and injected (1/~1) into adults. After 2 hr, the haemolymph was recovered and submitted to centrifugation at 11,500g for 10 min at 4°C. The supernatant was mixed with purified unlabelled larval lipophorin and then submitted to KBr density ultracentrifugation. The fractions were analyzed for radioactivity and density, as described above.

Fig. 1. Electrophoretic and autoradiographic analysis of [~3q]lipophorin from Musca domestica. The purified larval lipophorin from Musca dornestica was labelled with [1311] (see Materials and Methods) and submitted to SDS-PAGE (7%). After electrophoresis the gel plate was dried and exposed to an X-ray film. ApoLp I and ApoLp II are the lipophorin subunits. The arrow points to the front of the run where radioactivity of lipids and unbound 131I a r e detected.

RESULTS

Variations o f the lipophorin density Musca domestica lipophorin shows variations in its density during the insect life cycle, as reported by Bianchi et al. (1987). The larval lipophorin has a density of 1.152 _+ 0.006 g/ml and mature adults lipophorin has a density equal to 1.106 + 0.007 g/ml. In an attempt to understand these variations we measured the lipophorin density values during the first gonotrophic cycle of Musca. The gonotrophic cycle was divided in stages as described by Adams (1974). These results are shown in Table 1. The lipophorin of recently emerged flies has a density of 1.126_+ 0.002g/ml. Its density increases during the initial phases of vitellogenesis, but at the end of the gonotrophic cycle ($10), attains a minimum value. If recently emerged flies are maintained on a sugar water diet, the vitellogenesis is arrested at $4 and the lipophorin density shows a low value (1.109 4-_0.009 g/ml); but if the animals are then fed with protein the lipophorin density increases as the ooeytes develop along the gonotrophic cycle. Larval lipophorin injected into adults changes its density In order to study the lipophorin density modifications the lipophorin was labelled with [131I], as Table 1. Lipophorin density values during the first gonotrophic cycle of Musca domestica Rearing conditions Recently emerged Proteic food 24 hr Proteic food 48 hr Proteic food 72 hr Proteic food 120 hr Sugar food 72 hr Sugar food 72 hr + Proteic food 48 hr

Ovarian* stage

Density (g/ml)'~

S2 ---$10 S4

1.126 1.158 1.132 1.129 1.106 1.109

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1.138 ± 0.003

*Ovarian stages according to A d a m s (1974). tValues are mean + SD (n = 3).

_+ 0.002 + 0.007 + 0.003 + 0.001 ± 0.007 _+ 0.009

described in Materials and Methods. Under our conditions the radioactivity recovered with lipophorin was 71% protein bound and 29% lipid bound. Autoradiographic analysis of [131I]lipophorin preparation showed the two labelled subunits (Fig. 1). The density of larval lipophorin is not significantly modified by the iodination (Fig. 2A); larval labelled lipophorin (d = 1.145 g/ml) was injected into adult females and after 2 hr haemolymph from the flies was recovered and mixed with unlabelled purified larval lipophorin. The solution was analyzed by KBr gradient ultracentrifugation. The results show that the labelled lipophorin is recovered with a density smaller than the original larval lipophorin injected (Fig. 2B).

A factor that catalyzes lipophorin density modifications is present in the haernolyrnph Purified labelled adult lipophorin was mixed with a purified unlabelled larval lipophorin, incubated at 25°C for 1 hr before analysis by KBr density ultracentrifugation. In a second experiment purified labelled adult lipophorin was mixed with larval haemolymph and incubated at 25°C for 1 hr. The results (Figs 3A, B) show that haemolymph contains a factor capable of catalyzing the transfer of lipids between the two lipophorins. Similar experiments performed using delipophorinated haemolymph or purified lipophorins (not shown) indicated that the factor catalyzing the lipid transfer between the lipophorins has a density higher than 1.17 g/ml. It is not inactivated by dialysis but is inactivated by warming the delipophorinated haemolymph at 100°C by I min. Since these characteristics are very similar to LTP from Manuca sexta (Ryan et al., 1986a) we assumed that in Musca domestica haemolymph a LTP is also present. Musca domestica L T P catalyzes the transfer o f lipids between lipophorins o f two distinct insect orders In order to test the activity of Musca LTP in the lipid transfer between two distinct lipophorins

Musca domestica LTP

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Fig. 2. Lipophorin analysis by KBr density ultracentrifugation. (A) Purified larval lipophorin labelled with [13q] (0.2 mg) was mixed with unlabelled lipophorin (2 mg) and submitted to KBr density ultracentrifugation. The fractions were analyzed for total protein (A2s0), radioactivity and density by measuring the refraction index. (B) Larval lipophorin labelled with [~I] was injected into 10 adult females. After 2 hr the haemolymph of injected flies were recovered and mixed with urdabelled larval lipophorin. The solhtion was analyzed by KBr density ultracentrifugation.

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Fig. 3. Demonstration of LTP occurrence in the Musca domestica larval haemolymph. (A) Adult purified lipophorin labelled with [13tI](1.2 ttg) was mixed with unlabelled larval lipophorin (2mg), incubated for 1 hr at 25°C and then submitted to KBr density ultracentrifugation. The gradient fractions were analyzed for total protein (A280),radioactivity and density. (B) Adult purified lipophorin labelled with [13tI] (1.2#g) mixed with larval haemolymph (1 ml) was incubated for 1 hr at 25°C and submitted to KBr density ultracentrifugation. The gradient fractions were analyzed for lipophorin (A~; in this wavelength the lipophorin is detected due to its yellow colour); radioactivity and density.

we purified the Dione juno (Lepidoptera) larval to be occurring. Additionally, when flies are fed with lipophorin. The larval Dionejuno lipophorin contains sugar-water, and the vitellogenesis is arrested, the two apolipoproteins, ApoLp I (M, = 209,000) and lipophorin density value is small. These data support ApoLp II (Mr = 85,000) (Fig. 4) and has a density of the conclusion that when the rate of lipid transport 1.124 g/ml (Fig. 5A). is high the density of lipophorin is high. Purified Dione juno lipophorin was incubated with A simple model may explain our findings. If the adult Musca domestiea lipophorin labelled with [131I] utilization rate of lipids by organs undergoes variin the presence and absence of delipophorinated ations, but is never equal to zero and if the lipid Musca haemolymph. The results (Figs 5A, B) show loading rate of lipophorin is constant, the lipophorin that Musca LTP is able to catalyze the lipid transfer will show higher density at conditions of high lipid between the two lipophorins. It is interesting to note utilization by organs and lower density values at that the time required for complete equilibrium of the conditions of low lipid utilization. lipophorin forms was 4 hr. In the case of lipid transfer During the Manduca sexta life cycle lipophorin between adult and larval Musca lipophorin equi- also shows stage specific variations in density, (Ryan librium was attained after 1 hr of incubation. and Law, 1985; Prasad et al., 1986). However, during adult life the dynamics of lipid transport by lipophorin are different than for Musca domestica. In DISCUSSION adult Manduca, and also in adult Locusta migratoria The Musca domestica lipophorin shows variations (Beenakkers et al., 1988) the lipophorin may bind a in its density during the insect life cycle (Bianchi et al., third type of apolipoprotein, ApoLp III, that enables 1987) and also during the gonotrophic cycle (this the lipophorin to be loaded with very high levels of paper). During the stages of the gonotrophic cycle, in lipids (Ryan and Law, 1985; Kawooya et al., 1986). which great amounts of lipids are supposed to be In these insects the flight muscles utilize lipids as fuel transported, the lipophorin density is higher than in as opposed to Musca in which flight muscles utilize the stages in which lower lipid transport is supposed carbohydrates as fuel.

652

M. DE L. CAPURROand A. G. DE BIANCHI During the Musca domestica larval stage the lipids are transported from the midgut to the lipid utilizing organs and to the fat bodies where the lipids are stored. During the pupal life the lipids must be transported from the fat bodies to the other organs for utilization. The control mechanism that changes the function of fat bodies from lipid storage sites to lipid furnishing sites is still unknown. However, Van Heusden and Law (1989) reported that the presence of LTP is necessary for diacylglycerol transfer from fat bodies to lipophorin and that LTP is not necessary for diacylglycerol transfer from lipophorin to fat bodies of Manduca sexta. Thus, it may well be supposed that under some type of control (hormonal?) the larval fat bodies lipophorin receptors are modified, in order to be able to interact with LTP, and deliver lipid to lipophorin during the pupal stage. This is a working hypothesis that may be tested and efforts will be undertaken in order to confirm or disprove this idea.

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Fig. 4. Electrophoretic analysis of purified larval Dione juno lipophorin. The larval Dione juno haemolymph was submitted to a KBr density gradient ultracentrifugation (100,000 g for 16 hr at 4°C). The gradient tubes were fractionated from the bottom. The fraction with major contents of lipophorin was analyzed by SDS-PAGE (7% polyacrylamide). 1 and 2, molecular weight markers; 3, Dione juno lipophorin and 4, larval Musca domestica lipophorin obtained in identical conditions as described for Dione juno. The numbers to the left are the molecular weight of the markers.

Acknowledgements--This work was supported by grants from the Fundaq~o de Amparo fi Pesquisa do Estado de Sgo Paulo (FAPESP) and from Financiadora de Estudos e Projetos (FINEP), conv~nio No. 4.3.84.0725.00. We are grateful to Dr Ann Gunsalus Miguel for advice concerning the style of the manuscript; to Drs O. Marinotti and S. D. Pereira for helpful discussion. M. de L. Capurro is a staff member and A. G. de Bianchi is a staff member of the Biochemistry Department and a research fellow from CNPq.

The discovering and characterization of LTP which facilitates the exchange of lipids between different lipophorin forms was only recently reported for Manduca sexta (Ryan et al., 1986a,b; 1988a,b). Our data show the presence of LTP in Musca domestica. The M u s c a domestica LTP is able to catalyze the transfer of lipids between different forms of housefly lipophorin as well as between the housefly (Diptera) lipophorin and Dione juno (Lepidoptera) lipophorin. Manduca LTP is also able to catalyze the exchange of lipids between Manduca lipophorin and the very high density lipoprotein of Heliothis zea (Lepidoptera) (Ryan et al., 1988a).

REFERENCES

Adams T. S. (1974) The role of juvenile hormone in housefly ovarian follicle morphogenesis. J. Insect Physiol. 20, 263-279. Beenakkers A. M. Th., Chino H. and Law J. H. (1988) Lipophorin nomenclature. Insect Biochem. 18, 1~. Bianchi A. G. de, Capurro M. de L. and Marinotti O. (1987) Lipophorin in the larval and adult stages of Musca domestica. Arch. Insect Biochem. Physiol. 6, 39-48. Chino H. (1985) Lipid transport: biochemistry of hemolymph lipophorin. In Comprehensive Insect Physiology Biochemistry Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 10, pp. 115-135. Pergamon Press, Oxford.

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Fig. 5. Demonstration of Musca domestiea LTP activity in the lipid transfer between lipophorins of Musca and Dione. (A) Adult Musca lipophorin labelled with [1311] (1.2/~g) was mixed with larval Dione juno unlabelled lipophorin (1.0 ml) and submitted to KBr density ultracentrifugation. The gradient fractions were analyzed for lipophorin (A460; see Fig. 3 legend) radioactivity and density. (B) Adult Musca lipophorin labelled with [131I] (1.2#g) was mixed with larval Dione juno lipophorin (1.0ml) and delipophorinated Musca haemolymph (LTP; see Materials and Methods). The resultant solution was incubated for 4 hr at 25°C. After this the material was submitted to KBr density ultracentrifugation. The gradient fractions were analyzed for lipophorin (A460), radioactivity and density.

Musca domestica LTP

Folch J., Lees M. and Sloane-Stanley G. H. (1957) A simple method for the isolation and purification of total lipid from animal tissues. J. biol. Chem. 226, 497-509. Kawooya J. K., Meredith S. C., Wells M. A., Kezdy F. J. and Law J. H. (1986) Physical and surface properties of insect apolipophorin III. J. biol. Chem. 261, 13,588-13,591, Marinotti O. and Bianchi A. G. de (1986) Structural properties of Musca domestica storage protein. Insect Bioehem. 16, 709-716. Prasad S. V., Ryan R. O., Law J. H. and Wells M. A. (1986) Changes in lipoprotein composition during larval-pupal metamorphosis of an insect, Manduca sexta. J. biol. Chem. 261, 558-562. Ryan R. O. and Law J. H. (1985) Metamorphosis of a protein. Bio Essays 1, 250-252. Ryan R. O., Haunerland N. H., Bowers W. S. and Law J. H. (1988a) Insect lipid transfer particle catalyzes diacylglycerol exchange between high-density and very-highdensity lipoproteins. Biochim. Biophys. Acta 962, 143 148.

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