Lipophorin: The Structure of an Insect Lipoprotein and Its Role in Lipid Transport in Insects

LIPOPHORIN: THE STRUCTURE OF AN INSECT LIPOPROTEIN AND ITS ROLE IN LIPID TRANSPORT IN INSECTS By JOSC L. SOULAGES and MICHAEL A. WELLS Department of Blochemlstry and Center for insect Sclence, Bloiogkal Sciences West, University of Arizona, Tucson, Arlzona 86721

1. 11.

111.

1V.

V.

VI. VII.

Introduction ....................................................... Lipid and Apolipoprotein Composition of Lipophorins ................. A. Lipid Composition of Lipophorins ............................... B. Apolipoproteins ................................................ Size, Molecular Weight, Heterogeneity, and Shape of Lipophorins ....... Organization of Lipids and Proteins in Lipophorins .................... A. Apolipoproteins ................................................ B. Location of Phospholipids ....................................... C. Location of Hydrocarbons ....................................... D. Location of Diacylglycerols ...................................... E. Lipophorin Models ............................................. Metabolism ........................................................ A. Biosynthesis ................................................... B. Physiological Roles of Lipophorin ................................ Metabolic Implications of Lipophorin Structure ........................ Concluding Remarks and Future Directions ........................... References .........................................................

371 372 373 375 384 388 389 390 390 390 391 393 394 397 405 408 409

I. INTRODUCTION Vertebrate, especially mammalian, lipoproteins have been extensively studied. In the invertebrate world, only insect lipoproteins have received serious attention. Whereas vertebrates rely on a battery of lipoproteins (chylomicrons, very low-density lipoproteins, low-density lipoproteins, and high-density lipoproteins) to effect lipid transport, insects use primarily a single type of lipoprotein, lipophorin, for lipid transport. Lipophorin is both more versatile than vertebrate lipoproteins in terms of the diverse lipids it transports and more efficient than vertebrate lipoproteins in that, for the most part, it delivers lipids to tissues without being internalized and destroyed. We believe that new insights can be obtained from an understanding of insect lipoproteins, and in this article we review the current state of knowledge about the structure and metabolism of lipophorins. Insects are the dominant terrestrial group of animals. Several hundred thousand species of insects have been described, and some investigators ADVANCES IN PROTEIN CHEMISTRY. Val. 45

37 1

Copyright Q1994 by Academic Press. Inc. All righu of reproduction in any form reserved.

372

JOSL L. SOULAGES AND MICHAEL A. WELLS

believe the total number of insect species may number in the millionsseveral times the total number of all other animal species. Insects first appeared about 350 million years ago and have evolved to utilize for food almost every available organic resource on the planet. Humans usually regard insects as adversaries, carriers of disease, and pillagers of food sources. However, life as we know it could not exist without insects: pollinating activities, scavenging and recycling refuse, and serving as a food source for other animals are only a few of the essential roles played by insects. Because of their extraordinary diversity, insects also have considerable potential as subjects for research; it is likely that new and novel solutions to biological problems can be found among insects. Generally, insect biochemistry and physiology resemble that of vertebrates: basic metabolic pathways and their control are similar and endocrine control is exerted by equivalent mechanisms, although the structures of the hormones differ. Insects have an open circulatory system in which blood (hemolymph) is enclosed by basement membranes that surround all tissues. Generally, unlike hemoglobin, hemolymph does not contain oxygen-carrying molecules; oxygen diffuses to the tissues through a network of trachea and tracheoles that are open to the atmosphere. A specialized tissue characteristic of insects is the fat body. Dispersed throughout the insect body, the fat body combines many of the functions of vertebrate liver and adipose tissue. T h e fat body is perhaps the most versatile metazoan tissue: it is the site of synthesis of most of the hemolymph proteins; it stores fat, glycogen, waste material, and specialized proteins; it produces most of the components that make u p the insect egg yolk and in some insects contains specialized endosymbionts. Detailed studies on insect lipoproteins have been carried out only in the last decade or so (for previous reviews, see Chino, 1985; Beenakkers et al., 1985; Shapiro et al., 1988; Law and Wells, 1989; Ryan, 1990; Van der Horst, 1990; Law et al., 1992). For the most part our understanding of insect lipoproteins and their metabolism have been derived from studies on only two species: Manduca sexta and Locusta migratoria. When other species have been studied, the picture developed using M. sexta and L. migratoria has been generally confirmed. However, more than 99.9% of all insect species have not been investigated and, considering the diversity in food sources and life histories, it would not be surprising if the analysis of more species revealed new and exciting variations on the theme developed in this review. 11. LIPIDAND APOLIPOPROTEIN COMPOSITION OF LIPOPHORINS

Insect lipoproteins are generally isolated by single-step ultracentrifugation in a density gradient. In all insects studied to date, the majority

LlPOPHORlN LIPID TRANSPORT IN INSECTS

373

of hemolymph lipids have been found associated with a single lipoprotein particle. Thus, even though there is a considerable variation in lipid content and composition among the insect lipoproteins, the common name lipophorin has been given to all insect lipoproteins (Chino et al., 1981a). Lipophorins are further named according to their buoyant density range: low-density lipophorin (LDLp), high-density lipophorin (HDLp), and very high-density lipophorin (VHDLp) (Beenakkers et al., 1988). Lipophorins can be further identified by indicating the life stage from which they were isolated: HDLp-L, high-density lipophorin from larvae; HDLp-A, high-density lipophorin from adults, etc. Two apolipoproteins have been observed in all lipophorins: apolipophorin I (apoLp-I) with a molecular mass of 230-250 kDa and apolipophorin I1 (apoLp-11)with a molecular mass of 70-85 kDa. In LDLp and some adult HDLp a third apolipoprotein is found, apolipophorin 111 (apoLp-111) with a molecular mass of 18-20 kDa. A. Lipid Composition of Lipophorins

The lipid composition of lipophorin and its concentration in hemolymph are functions of the age and developmental stage of the insects (Ziegler, 1984; Wheeler and Goldsworthy, 1983; Prasad et al., 1986a; de Bianchi et al., 1987; Telfer et al., 1991; Gonzalez et al., 1991). Significant changes in the lipid content and composition are also observed during starvation (Mwangi and Goldsworthy, 1977a; Tsuchida et al., 1987; Ziegler, 1991) and flight (Beenakkers, 1973; Justum and Goldsworthy, 1976; Van der Horst et al., 1978; Ziegler and Schulz, 1986). Table I contains a compilation of the lipid composition found in lipophorins. An obvious hallmark of lipophorins is their high content of diacylglycerol (DG), which in most cases is the main neutral lipid. sn-1,2Diacylglycerol accounts for most of the DG found in lipophorin. The small amounts of 1,3-DG occasionally reported probably represent an isomerization product of sn-1,2-DG (Tietz and Weintraub, 1980). Another unique characteristic of lipophorins is the presence of long chain ( C ~ O - ~normal, O), and methyl-branched aliphatic hydrocarbons, which in some insects or metabolic stages represents a significant proportion of the nonpolar lipid components (Katase and Chino, 1982; Blomquist et al., 1987; Katagiri et al., 1985; Katagiri and de Kort, 1991). Compared with vertebrate lipoproteins, lipophorins show a virtual absence of triacylglycerols and cholesterol esters, as well as a very low content of free sterols. The only common major lipid component of vertebrate and insect lipoproteins is phospholipid (PL). Lipophorin PLs have been characterized in only a few insect species (Table 11). The major PLs are phosphati-

374

JOSk L. SOULAGES AND MICHAEL A. WELLS

TABLE I L i e Compositwn and Density of Lipophminp Lipid

Insect Having lipophorins without apoLp-111 Acheta domesticus (A) A@ meUi@a (L) Diotrca grandiaulla (L) Drmophila melanogask-r(L) Lglinotarsa &cemlincata (A) Locurla migrolmia (A) Manduca smta Larvae Prepupal- 1 Prepupal-2 Pupae Larvae Larvae Musca domesfica (A) Periplaneta americana (A) P h i h m i a cynfia (L) Podisus maculivmtris (A) Tipula trivittafa (A) T d o m a infestam (A) Having lipophorins with apoLp-Ill Acheta domeslicus LDLp (A) Locurta migroforia LDLp (A) Manduca scxta HDLp (A) Manduca sexta LDLp (A)

DC

PL

HC

18.0 13.3 15.4 7.4 2.2 13.4

14.0 12.8 13.0 23.1 18.7 14.8

4.5 2.0

15.7 20.2 12.5 17.5 30.3 21.4 6.0 8.0 24.8 16.7 21.6 19.5

16.7 23.3 18.9 21.6 20.8 17.7 20.0 22.8 11.4 14.9 12.0 14.9

36.6 26.1 25.0 46.9

8.9 10.9 14.0 7.1

ST

(9%)

Density

Ref!

-

42.8 41.0 38.0 37.5 44.9 41.0

1.106 1.130 1.110 1.16 1.09 1.12

1 2 3 4 5 6

1.2 1.8 1.8 2.8 0.6

1.151 1.128 1.177 1.139 1.155 1.144 1.145 1.12

7

1.5 5.7

2.2 1.0 2.7 0.5 5.8 - ND 3.6 7.3 1.9 2.1

37.3 46.9 34.8 46.4 47.5 45.3 34.2 49.6 44 32.0 51.4 47.0

1.10

11 12 13 14

3.5 6.4 3.5 2.3

ND 0.5 2.5 1.7

ND 2.4 1.3 0.7

52.2 46.3 51.5 62.2

1.061 1.065 1.08 1.03

1 15 16 16

-

TG

3.9 3.2 5.4

0.9 20.4 8.7

0.7

2.8

1.1

0.6

1.1 1.0

0.5 0.4 2.3 3.1 6.0 15.0 0.6

-

-

1.0 0.7 2.1

-

6.0 6.4 0.7 1.2 3.2

1.0

-

1.16 1.117

7 7 7 8 9 10 11

a DG, Diacylglycerol; PL, phospholipid; HC, hydrocarbon, TG, triacylglycerol; ST, sterol; L, larval stage; A. adult stage; ND, not determined. Other lipophorins partially characterized: Bombyr mon' (Miura and Shimizu, 1989a,b),Rhodniusprolh (Gondim ct al., 1989a), C h i n a nwrsilans (Ochanda et al., 1991). Key to references: (1) Strobel et al., 1990; (2) Robbs et al., 1985; (3) Dillwith el al., 1986; (4) Fernando-Warnakulasuriya and Wells, 1988; (5) de Kort and Koopmanschap, 1987; (6)Chino and Kitazawa, 1981;(7) Prasad etal., 1986a;(8) Pattnaik etal., 1979; (9)Tsuchida etal., 1987; (10) Capurro. 1988; (11) Chinoetal., 1981b; (12) Haunerlandetal., 1992; (13) Neven et al., 1989; (14) Rimoldi et al.,1991; (15) Chino et al., 1986; (16) Ryan el al., 1986a.

dylethanolamine (PE) and phosphatidylcholine (PC). With the exception of L. mipatoria lipophorin, all insect lipophorins have a high content of PE. Lipophorin from the freeze-tolerant cranefly Tiplla trivzttutu contains significant amounts of phosphatidylinositol (PI) (Neven et al., 1989), which has not been detected in any other lipophorin. The presence of PI is essential for the ice nucleation activity of this lipophorin, an activity not shown by any other lipophorin. Low amounts of sphingomyelin, lyso-PC, lyso-PE, and acidic phospholipids have also been detected in lipophorin.

375

LIPOPHORIN LIPID TRANSPORT IN INSECTS

TABLE I1 Phospholipid Composition of Lipoplrorinp Insect

PC

PE

Diatraea grandiosello Leptinotarsa decemlineatu Locwta migratoria Mandwa sex& Periplaneta americana Philosomia cyntia Tipulo triuittatu Triatom infestans

40.0 54.4 95.0 34.3 68.0 48.0 16.0 35.9

51.0 45.6 5.0 54.4 32.0 32.0 62.0 64.1

SPH

PI

9.0

-

-

11.3 20.0 10.5

-

Ref. Dillworth ef al. (1986) Katagiri and de Kort (1991) Chino and Downer (1982) Pattnaik et al. (1979) Chino and Downer (1982) Chino and Downer (1982) Neven el al. (1989) Rimoldi et al. (1991)

PC, Phosphatidylcholine; PE, phosphatidylethanolamine; SPH, sphingomyelin; PI, phosphatidylinositol.

Although PLs are common components of vertebrate and insect lipoproteins, the high PE : PC ratio found in liphophorins distinguishes them from vertebrate lipoproteins, where PC is the predominant polar lipid (Chapman, 1986). Free fatty acids are also found in the hemolymph of most insects, presumably bound to lipophorin (Dillwith et al., 1986; Beenakkers, 1973; Van Marrewijk et al., 1984; Rimoldi et al., 1991; Pattnaik et al., 1979; Ryan et al., 1986a). Analysis of the fatty acid composition of lipophorin lipids has been performed in only a few insect species and myristate, palmitate, stearate, oleate, and linoleate were detected as the main fatty acids of PLs and DG (Beenakkers et al., 1985; Katagiri and de Kort, 1991; Miura and Shimizu, 1989a,b; Fichera and Brenner, 1982; Fernando- Warnakulasuriya and Wells, 1988; Kuthiala and Chippendale, 1989; Neven et al., 1989). B . Apolapopoteans 1 . Structural Proteins

There is one molecule each of apoLp-I and apoLp-I1 in each lipophorin molecule (Shapiro et al., 1984; Surholt et al., 1992). A similar amino acid composition, as well as the presence of oligosaccharide chains of the high-mannose type, has been observed for apoLp-I and apoLp-I1 from different insect species (Pattnaik et al., 1979; Ryan et al., 1984; Shapiro et al., 1984; Kashiwazaki and Ikai, 1985; Dillwith et al., 1986; Nagao and Chino, 1987; Rimoldi et al., 1991). Both apolipoproteins are water insoluble when separated from lipophorin, and in this regard resemble vertebrate apoB. No amino acid sequence data, either from direct or cDNA

376

JOSE L. SOULAGES AND MICHAEL A. WELLS

sequencing, has been published for either apoLp-I or apoLp-I1 from any insect. It has been shown that apoLp-I and apoLp-I1 are not immunologically related (Ryan et al., 1984;Schulz et al., 1987).Polyclonal antiserum against M. sexta apoLp-I1 showed cross-reactivity to the apoLps-I1 of seven insect orders, whereas antiserum against M. sexta apoLp-I did not cross-react with apoLp-I from any of the insect species tested (Ryan et al., 1984). On the other hand, monoclonal antibodies prepared against L. mzgratoria apolipophorin I, 11, or I11 did not cross-react with the corresponding apolipoproteins from three other species of insects (Schulz et al., 1987). In lipophorin, apoLp-I is much more susceptible to proteolytic cleavage than is apoLp-11, and iodination of native lipophorin results in iodination of apoLp-I but not apoLp-I1 (Pattnaik et al., 1979;Shapiro el al., 1984; Kashiwazaki and Ikai, 1985). Polyclonal antibodies raised against lipophorin, when analyzed by Western blotting, generally show stronger reactivity toward apoLp-I than toward apoLp-I I. Antibodies raised against purified apoLp-I will react with native lipophorin, whereas antibodies raised against purified apoLp-I1 generally do not react with native lipophorin (Shapiro et al., 1984).On the other hand, cross-linking experiments show that the two apolipoproteins are in close proximity in lipophorin (Kashiwazakiand Ikai, 1985),and monoclonal antibodies can be raised against apoLp-I1 when immunization is carried out with lipophorin (Schulz et al., 1987). These data suggest that apoLp-11, or a portion of apoLp-11, is somehow “sequestered” from the aqueous environment in native lipophorin. Of course, the environment and/or the folding of apoLp-I1 in lipophorin might render it protease resistant or unreactive with iodine without requiring that the protein be buried in the outer lipid layer of lipophorin. ApoLp-I and apoLp-I1 can be dissociated from lipophorin in 8 M guanidinium chloride (GdmCI) and isolated by gel-permeation chromatography (Kawooya et al., 1989).Under these conditions, apoLp-I1 is isolated as a lipid-free protein, which is, however, insoluble when GdmCl is removed, even in the presence of detergent. ApoLp-I isolated by this procedure has associated with it all the lipid present in lipophorin, but the lipid can be removed by extracting the GdmCl solution with ethanol : ether to yield a lipid-free protein solution. A soluble preparation of lipid-free apoLp-I can be obtained after removal of GdmCl if a detergent that forms large micelles is present, e.g., Triton X-100or lysophosphatidylcholine. In the presence of detergents that form small micelles, e.g., cholate or deoxycholate, apoLp-I does not remain in solution, in the absence of GdmCI. In this regard it differs from apoB. The detergent, preferably lysophosphatidylcholine, can be replaced by phosphati-

LlPOPHORlN LIPID TRANSPORT IN INSECTS

377

dylcholine to regenerate a particle about the same size as lipophorin (Kawooya et al., 1989).These data have been interpreted to indicate that apoLp-I plays the major structural role in stabilizing lipophorin. At present, there is no indication as to the role of apoLp-11. 2 . Exchangeable Apolipoproteins Only one water-soluble, exchangeable, apolipoprotein has been found associated with lipophorin, apolipophorin 111 (apoLp-HI), which has a molecular mass of 18-20 kDa. Generally, apoLp-111 is only found in insects that use lipid to fuel flight (Table HI), although some exceptions have been noted, e.g., the flightless grasshopper Balytetth psolus and the house cricket Acheta domesticus. ApoLp-111 has not been found in cockroaches (order Dictyoptera), bees (order Hymenoptera), or flies (order Diptera). In the family Acrididae, order Orthoptera (grasshoppers and locusts), apoLp-111 is glycosylated, but in all other cases reported to date, TABLE 111 Distribution of Apolipophorin III among Insects Species L M W migraftma ~ Bartlrtlu psolllr Mchnopw dflerc-nhal Casfnmargur a/ncanur Arhrla d m s f t r u r Crtllw inttgcr

T h a w ncubngulu Acanlhoccphala grnnulosa LIIhoccm m c d w A W u r herbmi Ranalra quadndentaro Podtsur macuhvmfru Rhodntllr prolcru Tnnloma tn/tsfaru Dtrobrorhu gminafur Coltnu lrxana Manduca scxb H y b hwab Achrronlia a h o p o s Hyalophora rccrofna Bombyx mon

Family

Order

Comments

Acrididae Acrididae Acrididae Acrididae Gryllidae Gryllidae Coreidae Coreidae Belastomatidae Belastomatidae Nepidae Peniatomidae Reduviidae Reduviidae Cerambycidae Scarabaeidae Sphingidae Sphingidae Sphingidae Saturnidae Bombycidae

Orthoptera Orthoptera Orthoptera Orthoptera Orthoptera Orthoptera Hemiptera Hemipiera Hemiptera Hemiptera Hemiptera Hemiptera Hemiptera Hemiptera Coleoptera Coleopiera Lepidopiera Lepidopiera Lepidoptera Lepidoptera Lepidoptera

Glycorylatedcomplete sequence, crystal structure Glycorylaied: N-terminal sequence Glycorylaied; N-terminal sequence Glycosylaied Complete sequence

1 2 2 3 4

N-terminal sequence

6 7

-

N-terminal sequence

-

-

Ref.”

5

8 7 7 9 10 II

Complete squence Complete sequence N-terminal sequence

-

-

7 7 12 7 13 14 15

a Key to references:( I ) Chino and Yazawa, 1986; Van der Horst et al., 199 1; Kanost et al., 1988; and Breiter et al., 1991; (2) Ryan etal., 199Od; (3) Haunerland etal., 1986; (4)Strobel et al., 1990; A. F. Smith and M. A. Wells, unpublished; (5) A. Hendrick, unpublished; (6) Wells et al., 1985; G . J. P. Fernando and M. A. Wells, unpublished; (7) A. F. Smith and M. A. Wells, unpublished;(8) M. K. Kanost and M. A. Wells, unpublished;(9) Haunerland et al., 1992; (10) Condim et al., 1989a; (11) J. L. Soulages, unpublished; (12) Kawooya et al., 1984; and Cole et al., 1987. (13) Surholt et al., 1992; (14) W. H. Telfer, unpublished; (15) Miura and Shimizu, 1989a,b.

378

JOSk L. SOULACES A N D MICHAEL A. WELLS

apoLp-I11 is not glycosylated. ApoLp-111 from L. mipatoria has been shown to contain N-linked oligosaccharide chains of the high-mannose type (Nagao and Chino, 1987). The function of apoLp-111is to facilitate transport of lipid from sites of storage in the fat body to sites of utilization in certain metabolic situations, e.g., flight. The triacylglycerolstores of the fat body are converted to DG, which leaves the fat body and becomes associated with preexisting HDLp in the hemolymph. In the process, HDLp is converted to LDLp and several molecules of apoLp-I11 become associated with LDLp. LDLp moves to the flight muscle, where the DG is hydrolyzed by a lipoprotein lipase. As the DG is removed, LDLp is converted back to HDLp and apoLp-111 dissociates. The HDLp and apoLp-I11 then cycle back to the fat body to carry more DG (see Section V for details). In contrast to the situation in vertebrates, in which most of the exchangeable apolipoproteins are bound to lipoproteins, most apoLp-I11 in insect hemolymph is free and represents a major component of lipophorin-free hemolymph (Wheeler and Goldsworthy, 1983; Kawooya et al., 1984; Van der Horst etal., 1984; Chino and Yazawa, 1986).Again in contrast to vertebrate apolipoproteins, apoLp-111 shows no tendency to self-associate in solution and this property has allowed extensive hydrodynamic characterization of the protein (Kawooya et al., 1986). ApoLp111binds to lipid surfaces with high affinity (Kawooya et al., 1986; Demel et al., 1992)and forms a stable complex with LDLp, which can be isolated by density gradient ultracentrifugation (Shapiro and Law, 1983) or by gel-permeation chromatography (Wheeler and Goldsworthy, 1983). However, apoLp-I11has a lower affinity for lipoprotein surfaces than do vertebrate apolipoproteins, because human apoA-I can displace apoLp111 from LDLp, whereas apoLp-I11 does not displace apolipoproteins from human HDL (Liu et al., 1991). The molecular structure of apoLp-111 from L. mipatoria has been determined at 2.5 8, (Breiter et al., 1991) (Fig. 1). The protein is composed of five long a helices connected by short loops and would be predicted to behave as a prolate ellipsoid in solution-a prediction in accord with hydrodynamic data suggesting that apoLp-I11 is a prolate ellipsoid with an axial ratio of about 3 (Kawooya et al., 1986).The helices are distinctly amphipathic with the hydrophilic side chains pointing toward the aqueous phase and the hydrophobic residues pointing into the interior of the protein. A similar structural motif, containing four a helices, was found in the N-terminal22-kDa receptor-binding domain of apoE (Wilson et al., 1991). Although the amphipathic a helix has been extensively discussed as a structural motif in exchangeable apolipoproteins (Segrest et al., 1992), these structural results are the first

LlPOPHORlN LIPID TRANSPORT IN INSECTS

379

FIG. 1. Molecular structure of apoLp-111. Ribbon drawing of the Locus& migratoria apoLp-I11 structure as determined by X-ray crystallography (Breiter el al., 1991). The helices are numbered beginning at the amino-terminal end of the protein. Reprinted with permission from Breiter el al. (1991).Copyright 1991 American Chemical Society.

demonstration that the amphipathic a helix is the predominant secondary structural element in such apolipoproteins. The complete amino acid sequences of apoLp-111 from M. sextu (Cole et ul., 1987), L. mzgrutoriu (Kanost et ul., 1988), Achetu domesticus (A. F. Smith and M. A. Wells, unpublished), and Derobruchus geminatus (A. F. Smith and M. A. Wells, unpublished) have been. determined from cDNA sequences and these sequences are aligned in Fig. 2;also shown in Fig. 2 are the location of the five a helices in the L. migrutoriu structure. The percent identities and similarities for pairwise comparisons between these sequences is shown in Table IV. A striking feature of these data is the low degree of sequence identity among these four apoLp-111 sequences, averaging only 23.3%. Note that the percentage identity between apoLps-111 from the fairly closely related species A. domesticus and L. mzgrutoriu is slightly higher than the percentage identity between apoLps111 from the very distantly related species L. migrutoriu and M. sextu. The overall low degree of sequence identity among the apoLps-111 is surprising considering the fact that all these apoLps-111can be found associated with LDLp, and it has been shown that M. sex& and L. migrutoriu apoLps111 are functionally equivalent in an in vitro system (Van der Horst et ul., 1988). Perhaps sequence identity is not as important for the function of apoLp-111 as is conservation of amino acids with similar physical properties, as might be suggested by the fact that percentage similarity between the four sequences is moderately high, averaging 43%. When the amino acid sequences in Fig. 2 are simplified by grouping the amino acids into

380

JOSt L. SOULACES AND MICHAEL A. WELLS

50 0

Map03 Dgapo3 Wapo3 hap03

0

DAGTTGADPN KVAEKS.QLQ DAPAGGNAFE DAA.GBVNIA 0

0

0

PANNLQAAAT LANSVQSVVD VLQQLSAFSS FKTKIAEVTT helix 2

0

0

QFNEKAAELS KIKTEIKNNQ SLQGAISDAN SLKQEAEKEQ

----------- I

I---------

0

0

LPSQ...EEV LPDS...KKV LVNSKNTQDF LGLPTPDEAC

0

0

0

SALAEV... KGLKDAVAQ AAVQTTVQE SALTNVGHQ helix 4

--------- I

RTQLQTHAQT VEVLNTNAQN NKALKDGSSS N.LLTEQANA 100

0

0

0

GDAQTAVRQA AQQLEQQVSN GEIDNvLlrQV SSKLSETAAE GKAKEALEQA RQNVEKTAEE GSVAEQLNAF ARNLNNSIHD I---- helix 3 ----I QEAEARRVQP VEKLTKAIEP SQKLAKEVAS WQDIATKTQA

0

I--------------

0

170 0

TVITNQVQQS VEVSNNVQQQ VKBAEEVQKK KEAAANLQNS helix 5

0

0

QAADKLKASIE KQAKEIKANLD KEANAFKDKLQ LNLQDQLNSLQ 0.

Adapo3 Dgapo3 Hsapo3 map03

0

---------- I

0

M a p 03 Dgapo3 MSap03 hp03

0

SLPEAAQRHF QNLTATIQNA ELAANAQQIV NNVTQTLQGN EMEKHAKEFQ KTFSEQF.NS EAVQQLNHTI VNAAHELEET helix 1

I--------

0

Map03 Dgapo3 Mmapo3 hp03

0

HADAVAESLK ETAKLKADLT NMEETNKKLA SAQEAWAPVQ

0

LRQQF.PDGA LQKQLGPEGQ LRK.AHPDVE A......ATS 150 0

0 0

TAARTAVEQA NAAKTFLDQI PKIKQAYDDF SALQEAAEKT

I------------

0

VQQAANAH.. VRATLDEKH. LBEAATKQ.. IQSAVQKPAN

------- I

FIG. 2. Alignment of apoLp-111 sequences. The amino acid sequences of apo1.p-Ill from Ache& domesticus (Ad, house cricket), Derobrachus geminatus (Dg, palo verde beetle), Locusta migratoria (Lm), and Mandwa sexla (Ms) were aligned by the method of Feng and Doolittle (1987). The positions of the five helices in the L. migratoria apoLp-I11 are indicated. ( 0 )Conserved residues; (0)hydrophobic residues.

five groups, i.e., neutral, acids and their amides, basic, hydrophobic, and proline, the results (Fig. 3) obtained show a much higher degree of “identity”between the sequences. These results suggest that it is indeed the properties of the side chain of the amino acid that are conserved TABLE 1V Painuise Identities and Similarities for Insect Apolipophorin 111

Percentage identity Percentage similarity Achetu domesticus Derobrahus geminatus Locustu migratoria Mandwa sexta

A. domesticus

45.0 48.6 41.0

D. geminatzcs

L. migratoria

M . sexla

25.0

33.8 15.7

21.1 19.1 25.5

36.5 42.0

45.4

38 1

LIPOPHORIN LIPID TRANSPORT IN INSECTS

** Adapo3 Dgapo3 Msapo3 hap03

**

**

DIGGGGIDID GIIDIIDHHI DDIGIGIDDI HIIDHG.DID DIIIDIDDII DDIGDGIDGD DIPIGGDIID DIDHHIHDID HGIGDD1D.G DII-GHIDII DIIDDIDHGI IDIIHDIHDG I-------- helix 1 IIDDIDIIIG DIDDHIIDIG IIDHIDGIID HIHGDIHDDD IIDDIGIIGG GIDGIIGDID IHGHIIDIGG GIHDDIDHHD helix 2 I

-----------

** Adapo3 Dgapo3 Msapo3 Lmapo3

DIIDHIHIGI HDIHDIHIDI HDIDIIHDHI IDIDDDIDGI (---------

**

I...PGDDDI I...PDGHHI IIDGHDGDDI IGIPGPDDII

DGIIID...I DDIDIHHIDP DHGIHDIIID IDHIGHIIDP DIIIDGGIDD GDHIIHDIIG DGIIGDIGHD IDDIIGHGDI helix 4 I 170

---------

BGDIDGHIDG IDIIDGDIDD DHIIBDGGDG D-IIGDDIDI

I_---------___-

100

*******

GDIDGIIHDI IDDIDDDIGD GDIDDIIHDI GGHIGDGIID GHIHDIIDDI HDDIDBGIDD GGIIDDIDII IHDIDDGIHD I---helix 3 ----I

** *

* * * *.

* * * * * * * *** Adapo3 Dgapo3 Msapo3 hap03

* * ***

**

50

***.**

**

---------- I

* * * * * * * **** Adapo3 Dgapo3 Msapo3 hap03

** ***

*.*

*

HIDIIIDGIB DGIHIHIDIG DIDDGDHHII GIDDIIIPID

IBDDI-PDGI IDHDIGPDGD 1HH.IHPDID I......IGG 150

***.***.

GIIHGIIDDI DIIHGIIDDI PHIHDIIDDI GIIDDIIDHG

I------------

GIIGDDIDDG IDDIIDIH.. IDIGDDIDDD IHIGIDDHH. IHHIDDIDHH IHDIIGHD.. HDIIIDIDDG IDGIIDHPID helix 5 ------- I

FIG. 3. Alignment of simplified apoLp-111 sequences. The sequences in Fig. 2 were simplified by grouping amino acids with similar side-chain properties: G = G , S, T; D = D, E, N, Q; H = H, K, R; I = A, L, I, M, V, F, Y,W; P = P. The asterisks indicate residues with similar side-chain properties found in all four sequences. Species acronyms as in legend to Fig. 2.

among these proteins, not the exact amino acid. Helical wheel analysis suggests that four or five amphipathic a helices are present in each protein, again emphasizing the importance of this secondary structural element in apolipoproteins. When spread as a monolayer at the air-water interface at low surface pressure, the molecular area of M. sexta apoLp-I11 (3800 A*) is nearly twice as large as would be predicted for a prolate ellipsoid of axial ratio 3 lying on its side (Kawooya et al., 1986). As the monolayer of apoLp-I11 is compressed, it undergoes a phase transition in which the area occupied by the protein is reduced to 480 A2.Under these conditions, the area occupied by the protein is about that predicted if apoLp-I11 were binding to the surface via one end of the ellipsoid. Demel et al. (1992) have criticized the monolayer results of Kawooya et al. (1986), claiming that their monolayer results with L. rnzgrutoria and M.sex& apoLps-111, which showed a molecular area for apoLp-I11 of 2300 A, do not support the

382

JOSI? L. SOULAGES AND MICHAEL A. WELLS

suggestion that apoLp-111 occupies an area larger than its cross-sectional area at the air-water interface. However, Demel et al. (1992) chose to measure the area occupied by apoLp-111 at the collapse pressure of the film, whereas Kawooya et al. (1986) measured the area at low surface pressure. Although there is obvious disagreement about the behavior of apoLp-111 at the air-water interface, it is the behavior of apoLp-111 on lipid surfaces that is relevant to its binding to lipoproteins. The area occupied by apoLp-111on PL- or DG-coated polystyrene beads was determined to be 4300 A2(Kawooya et al., 1986). Analysis of the area occupied by apoLp-111 on lipophorins showed two states: in one, the area, 630 A', was about that expected if the protein bound via one end; in the other state, the area, 3500 A2,was about twice as large as predicted from the largest cross-sectional area of the protein (Wells et al., 1987). Demel et al. (1992) reported the binding of apoLp-111 to a DG monolayer and suggested that each apoLp-111binds to 92 molecules of DG. Unfortunately, interpretation of these results is ambiguous. On one hand, the data can be interpreted to suggest that apoLp-111 covers the area occupied by 92 DG molecules, which, with a surface area of 58 A2 for a DG molecule, means that apoLp-111 occupies a surface area of 5300 A2,an area even larger than that found by Kawooya et al. (1986) and Wells et al. (1987). On the other hand, the data can be interpreted to mean that apoLp-111 binds to the free air-water interface generated as the DG film is compressed by adsorption of apoLp-111, i.e., the protein forces its way between DG molecules. The data of Demel et al. (1992) show that the increase in surface pressure caused by adsorption of apoLp-111 would reduce the area per DG molecule by 7.3 A*, and for 92 DG molecules this would correspond to an area of air-water interface occupied b apoLp-111 of 670 A'. If the minimal area per amino acid residue is 15 (Malcolm, 1973)) this would mean that only 45 residues, 28% of the protein, or about two helices, have actually penetrated to the air-water interface. This is a much lower value than that found for vertebrate apolipoproteins (Weinberg et al., 1992). These data suggest that, if apoLp-111 unfolds on the lipid surface, only a fraction of the protein actually penetrates between the polar head groups. Clearly, more data are needed to confirm this suggestion. Studies employing lipase treatment of apoLpIII-containing lipoproteins (Kawooya et al., 1991) and the characteristics of mixed apoLp-III-DG monolayers (Demel et al., 1992) suggest that apoLp-111 shows some specificity in binding to DG in the surface. These data are the basis for a model for the binding of apoLp-111 to lipophorin; the model suggests that apoLp-111binds to DG in the surface of lipophorin via one end and then spreads on the surface as depicted in Fig. 4 (Kawooya et al., 1986; Breiter et al., 1991). This model predicts an

i2

LlPOPHORlN LIPID TRANSPORT IN INSECTS

383

FIG.4. A model showing the proposed unfolding of apoLp-111on a lipoprotein surface, showing only the a-carbon backbone of the protein. It is proposed that the protein binds to the surface via one end and that helices 3 and 4, and helices 1,2, and 5 , then move relative to each other (filled arrows) around hinges in the loops connecting helices 4 and 5 , and 2 and 3 (unfilled arrows). The hydrophobic side chains, which are in the interior of the protein in the folded state, face the lipoprotein surface in the unfolded state.

initial binding via hydrophobic residues located in the loops between helices 1 and 2 and helices 3 and 4. These loops are relatively nonpolar and contain the only hydrophobic residues in the protein that are not buried-two leucines, which are conserved in all apoLp-111 sequences. According to the model, the protein spreads on the surface, without loss of its helical structure, via hinges located in the loops between helices 2 and 3 and helices 4 and 5. In essence, the hydrophobic interactions that hold the five helices together are replaced by energetically equivalent interactions that hold the apolipoprotein on the surface. T h e gene encoding M. sexta apoLp-I11 has been sequenced (Cole et al., 1990). It is composed of four exons; the first exon contains most of the signal sequence and the second exon contains the rest of the signal sequence, a Pro segment and some of the coding region for the mature protein; exons 3 and 4 contain the remainder of the coding region for the mature protein. This gene organization has some similarity to that of vertebrate apolipoprotein genes (Li el al., 1988), except that the coding region is divided among three exons in apoLp-I11 and only two exons in vertebrate apolipoproteins. What, if anything, can be said about the evolutionary relationship between apoLp-I11 and exchangeable vertebrate apolipoproteins? Both bind to lipoproteins and seem to have similar structural motifs. Whether apoLp-I11 is the ancestor of the vertebrate proteins can not be stated with

384

JOSE L. SOULAGES AND MICHAEL A. WELLS

any certainty. The evolutionary relation between insect apoLps-111, which are strictly orthologous proteins, is not easily demonstrated at the amino acid or nucleotide sequence level, therefore it is not surprising that insect apoLps-111 have little sequence homology to vertebrate apolipoproteins. If, as seems likely, amino acid sequence conservation is not strongly selected for in apolipoproteins, it may prove difficult to establish an evolutionary relationship between apoLp-111and the vertebrate apolipoproteins. It is also possible that apoLp-111 and the vertebrate apolipoproteins represent an example of convergent evolution and that there is not an ancestral relationship between apoLp-111 and the vertebrate apolipoproteins. In this regard, one must also consider the possibility that all insect apoLps-111 did not arise from a common ancestor, because some think that the ability to fly developed more than once among the insects. 111. SIZE,MOLECULAR WEIGHT,HETEROGENEITY, AND SHAPE OF LIPOPHORINS

Although lipoproteins are apparently easily purified by ultracentrifugation in a density gradient, it is actually impossible to obtain a chemically and physically homogeneous lipoprotein preparation. Even in a narrow density range a large polydispersity is expected and has been found when studied. The accurate determination of the molecular weight of a purified soluble protein can be achieved in several straightforward ways, e.g., gel electrophoresis, analytical ultracentrifugation, cDNA sequencing, and several other less commonly used methods. T h e shape of a pure protein in solution can be determined by spectroscopic techniques such as small-angle X-ray scattering (SAXRS) or can be inferred from its hydrodynamic properties. Some of the same techniques, e.g., sedimentation equilibrium, flotation rates, gel-filtration chromatography, electron microscopy, SAXRS, and gradient gel electrophoresis, can be used in determining the average molecular weight and shape of lipoproteins. Except for sedimentation equilibrium, most of the other experimental approaches are based on determination of an average radius from which, provided the density is known, the molecular weight can be estimated. However, as in any polydispersed system, the value of the average molecular weight determined depends on the method used. For example, electron microscopy gives a number-average radius from which one calculates a number-average molecular weight, whereas light scattering gives a Z-average radius from which one calculates a Z-average molecular weight. T h e ratio of the Z-average molecular weight to the number-average molecular weight can be significantly greater than one depending on the polydispersity of the system.

385

LlPOPHORlN LIPID TRANSPORT IN INSECTS

In order to compare the results of size and shape determinations for lipophorins and to discuss the differences observed using different methodologies, we have compiled most of the size-related data obtained for lipophorins in Table V. The data for some vertebrate lipoproteins are also included to permit a general discussion. Table V shows that there are large differences among the radii reported for lipoproteins and, at the same time, a systematic variation of the radius for any particular lipoTABLE V Comparison of Lipoprotein Sires Deduced Using Different Techniques" Volume ratios Lipoprotein

R,,

R,,,

Re,,

L. rnigratoria HDLpb P. amerzcana HDLp' P. cyntia H D L ~ ~ L. decemlineata HDLp' M . sexfa HDLP-LJ M. sexfa HDLp-A" M. sexta LDLph L. migratoria LDLp' HDL2 (human)' HDLs (human)' LDL, (human)h LDLy (porcine)h

59 59.5 59 67.3 85.2

90.3 90.3 77.4 98.1 -

78 80 65 82.5 80 57 85 67 128 147 50 44-49 38 39-41 110 107 -

-

50.5 40.8 86 81

-

60.7 54.1 137 129

R,,,

RdL sas:dc

58.3 61.7 59.2 59.3 60 65 87.1 69.9

-

3.7 3.1 2.3 4.5 -

-

-

1.7 2.3 4.0 4.0

em:dc em:gge 2.4 2.2 1.3 2.7 2.4 2.2 3.1 9.3

-

-

-

2.8 2.0

-

1 .o

0.8 2. I 2.3

" R,,, , Radius determined by analytical ultracentrifugation; Rsa,,radius determined from SAXRS; R,,, , radius determined by electron microscopy on negatively stained samples; R,, , radius determined by nondenaturing gradient electrophoresis; R&, radius obtained from the density and lipid content of the lipophorin, assuming one molecule of apoLp-1 and apoLp-I1 per particle. For those lipophorins containing apoLp-111, two, sixteen, and nine molecules of apoLp-I11 per particle were used, respectively, for M . sexta HDLp-A and LDLp, and L. migratoria LDLp. bRS,, (Katagiri el af., 1987); R,,,, (Nagao and Chino, 1991); R,, (Chino et al., 1981b). Re", = 85 was reported by Van Antwerpen et al. (1988). ' R,,, (Katagiri el a/., 1987);R,,, and Ra, (Chino ef al., 1981b). 'RSaJ(Katagiri etnl., 1987);R,,,, (Chino and Kitazawa, 1981). ' R,,, and R,,,, (Katagiri et af., 1991). /RaU (Pattnaik ef af., 1979);Re,,, (Ryan et al., 1990a; Kawooya et al., 1991); R,, (J. L. Soulages and M. A. Wells, unpublished). " R , , (Wells el af., 1987); Re,, (Ryan et a / . , 1990a; Kawooya ef al., 1991); R,,, 0. L. Soulages, and M. A. Wells, unpublished). R,, (Wells el af., 1987). ' R,,,, (Nagao and Chino, 1991);a similar value (140)was reported by Van Antwerpen et al. ( 1 988). Laggner and Muller (1978);Laggner (1982). R,,, (Jurgens el al., 1981);R,, (Jackson ef al., 1976).

'

386

JOSk L. SOULACES A N D MICHAEL A. WELLS

protein, depending on the technique used. With the exception of the human HDLs, it is clear that radii obtained by electron microscopy and SAXRS are the largest values, with SAXRS giving the largest dimensions. The large differences in the values for radii obtained with these two techniques, compared to those obtained by gradient gel electrophoresis and analytical ultracentrifugation, become even more pronounced if they are translated into volume or, what is equivalent, employed to estimate the molecular weight. It is noteworthy that in some cases up to a fourfold difference in the estimated molecular weight is observed when, for example, data from analytical ultracentrifugation are compared to those obtained with SAXRS or electron microscopy. Because such differences are sometimes overlooked and data obtained by different techniques are indiscriminately utilized to draw conclusions about lipoprotein structure, it seems appropriate to discuss the methodologies briefly. Although SAXRS has proved to be a powerful and accurate technique when applied to the structure of homogeneous, monodispersed particles in solution-or nonhomogeneous, but monodispersed, complexes of two components (Feigin and Sverdgun, 1987; Clatter and Kratky, 1982)-its application to the study of lipoprotein structure requires assumption of monodispersity. However, heterogeneity in terms of size has been found in most lipoprotein preparations analyzed by electron microscopy (Chapman, 1986). In addition, all lipoprotein fractions that have been analyzed by gradient gel electrophoresis show marked polydispersity characterized by populations that differ in size as well as in chemical composition (Nichols et al., 1983; Krauss and Burke, 1982; Cheung and Albers, 1984). Similar polydispersity has also been observed in lipohorin preparations from L. mzgratoria, using electron microscopy (Nagao and Chino, 1991), and M. sexta, using gradient gel electrophoresis (J. L. Soulages and M. A. Wells, unpublished). Thus, polydispersity is likely to be a common characteristic of insect lipoproteins. The extreme sensitivity of SAXRS to sample heterogeneity makes obtaining accurate data about the geometry, size, and internal organization of a lipoprotein a difficult, if not impossible, task. The natural heterogeneity of lipophorins and the presence of discreet aggregates, which are not uncommon, might account for the large radii estimated by SAXRS. In addition, the lack of a correction to infinite density contrast for the data on lipophorins might affect the calculated value of the radius of gyration (Laggner and Muller, 1978).Extensive treatments of the theoretical and experimental aspects of the study of lipoproteins and other particles by SAXRS have been published (Laggner, 1982; Feigin and Sverdgun, 1987). Electron microscopy is the other technique that is widely employed to

LIPOPHORIN LIPID TRANSPORT IN INSECTS

387

assess the size and geometry of lipoprotein particles. Among the methods employed, negative staining of the lipoprotein preparation has been most often utilized, but some studies involving cryofixation (freeze-fracture and etching) have been reported (Forte and Nordhausen, 1986). In principle, electron microscopy can give a number-average radii, and also provide a direct measure of sample heterogeneity. Unfortunately, the analysis of negatively stained samples by electron microscopy requires observation of samples in an environment in which the hydration shell is removed. This seems to be one of the sources of artifacts in samples containing lipids. The most important artifact, because it cannot be readily corrected, is a possible flattening of the lipoprotein particles, which increases as the lipid content of the lipoprotein increases (Forte and Nordhausen, 1986). It is probably for this reason that the sizes of lipoprotein particles are often overestimated by electron microscopy. The potentially more powerful freeze-fracture-etching technique has many technical problems and associated artifacts, e. g., sample heterogeneity makes conclusions about particle morphology essentially impossible (Forte and Nordhausen, 1986; Aggerbeck and Gulik-Krzywicki, 1986). Low-temperature electron microscopy is the most recently developed technique to observe frozen hydrated biological macromolecules (Adrian et al., 1984). This technique has the advantage that the sample solution is not fractured and the molecules can be observed in their hydrated states. It has been suggested that this technique might be useful in determining lipoprotein size (Aggerbeck and Gulik-Krzywicki, 1986), but no reports have yet appeared. In preliminary studies of adult M. sextu HDLp in a hydrated frozen sample, the average diameter obtained was very close to that obtained by analytical ultracentrifugation or gradient gel electrophoresis, and smaller than that obtained in negatively stained samples (R. Van Antwerpen, unpublished). The close agreement for the values of radii observed by analytical ultracentrifugation, gradient gel electrophoresis, and, in some cases, gel-filtration chromatography is consistent with a consensus basic structure for lipophorin consisting of one molecule each of apoLp-I and apoLp-11, and a variable lipid content. In some insect stages apoLp-111 binds to this basic structure in different amounts depending on the content of diacylglycerol. Assuming that this is the correct description of lipophorin structure, then from the molecular weights of the apolipoproteins and their relative amounts, and the lipid content and density of the lipoprotein, a compositional molecular weight can be calculated. As can be observed in Table V, the radii of the lipophorin particles calculated in this way show very close agreement to those determined by analytical ultracentrifugation and gradient gel electrophoresis. This con-

388

JOSB

L.

SOULACES AND MICHAEL A. WELLS

sistency gives further support to the previous comments about the necessity for analyzing carefully data obtained by electron microscopy and SAXRS. For example, in a recent report using electron microscopic observations of LDLp and HDLp of adult L. mzgratoria, it was concluded that the LDLp particles are the result of intermolecular fusion of HDLp particles (Nagao and Chino, 1991). This conclusion might be correct. However, because this conclusion was based on the comparison of sizes obtained by electron microscopy, which for HDLp gives a molecular weight twice the value obtained by analytical ultracentrifugation, it is reasonable to suggest that the large differences between the reported sizes of HDLp and LDLp are the effect of some artifact, such as flattening, which would markedly increase with the increase in lipid content. An independent measure of the size of LDLp will be required before 'the existence of fusion can be evaluated. It is generally agreed that lipoproteins have a spherical or sphericallike structure. However, there are not unambiguous methods to confirm this assumption. Two suitable techniques might be SAXRS and electron microscopy; however, for the reasons previously discussed, the accuracy and power of these techniques are obscured by the presence of artifacts. Electron microscopy of phosphotungstate negatively stained samples has been used to suggest that changes in the lipid content of lipophorin were accompanied by modifications in the morphology of the lipoprotein particles (Ryan et al., 1992). If this is the case, the assumption of spherical particles would not be appropriate for those lipophorins with a low lipid content. However, the features reported by Ryan et al. (1992) were not observed in uranyl acetate negatively stained samples of lipophorins (Ryan et al., 1990a; Kawooya et al., 1991). Technical artifacts, such as positive staining, might be present in either of these preparations, which would obscure interpretation of the data. Further work on this important point is needed. IV. ORGANIZATION OF LIPIDS AND PROTEINS I N LIPOPHORINS T h e elucidation of the structure of any lipoprotein, i.e., the organization of the protein and lipid components, is a challenging problem, because it relies on several techniques that give only partial and approximate information. Structural models should be consistent with experimental data that characterize the physiological role and the physicochemical properties of the lipoprotein and its components. Considerable effort has been expended to fit experimental data to structural models of mammalian lipoproteins (Zilversmit, 1965; Sata el al., 1972; Schneider et al., 1973; Havel, 1975; Verdery and Nichols, 1975; Shen et al., 1977;

LIPOPHORIN LIPID TRANSPORT IN INSECTS

389

Edelstein et al., 1979; Oeswein and Chun, 1981). The concept of a lipoprotein core of nonpolar lipids surrounded by an outer shell of phospholipids and proteins is a common theme in all these studies. T h e existence of the lipid core is supported by the properties of lipids, SAXRS studies, and is apparent for large lipoproteins with high lipid contents. On the basis of SAXRS studies this concept has also been applied to lipoproteins with low lipid contents, such as human HDL (Scanu, 1972; Laggner, 1982). In addition to the lipid core, experimental support for a surface localization of phospholipids and cholesterol has been obtained from NMR studies (Henderson et al., 1975; Yeagle et al., 1978; Lund-Katz and Phillips, 1986). Among the problems involved in constructing a general model for vertebrate lipoprotein structure, some of the more important are the heterogeneity in size and apolipoprotein composition and the small number of examples of lipoproteins containing similar apolipoprotein composition, which can be used to validate the model. Thus, in most cases, data as elementary as the stoichiometry of the apolipoproteins cannot be included in the models (Shen el al., 1977) or, if stoichiometry data are included, only a partial fit of the data to the model is observed (Edelstein et al., 1979). One of the most intriguing features of lipophorin composition is the large variation in lipid content and composition that can be accommodated without modifications in the apolipoprotein composition of the particles (Table I). This feature makes lipophorin a good system in which to analyze the structure of lipoproteins and the physicochemical factors that govern their structure and properties. In addition to the previously discussed data on the size and shape of lipophorins, several studies on other aspects of lipophorin structure have been performed and need to be discussed before describing models for lipophorin structure.

A . Apolipoproteim From the average circular dichroism (CD) spectra of locust and cockroach lipohorins a high content of P-sheet secondary structure was estimated for apoLp-I and apoLp-I1 (Kashiwasaki and Ikai, 1985). A similar result was obtained by Kawooya et al. (1989) with M.sexta lipophorin, where a secondary structure composed of 58% /3 structure, 30% a helix, and 12% random coil was estimated. As described earlier, most data are consistent with a surface localization of apoLp-I, whereas the localization of apoLp-I1 has been described as sequestered. However, apoLp-I1 can not be completely “buried” because water-soluble crosslinking reagents react with both apolipoproteins (Kashiwasaki and Ikai,

390

JOSk L. SOULACES AND MICHAEL A. WELLS

1985) and it is possible to generate monoclonal antibodies against apoLpI1 when immunization is carried out with intact lipophorin (Schulz et al., 1987). Although a portion of both apoLp-I and apoLp-I1 must be exposed to the aqueous media, the low solubility of both apolipoproteins in water (Pattnaik et al., 1979; Kawooya et al., 1989) and the stability of the lipid-apoLp-I interaction in GdmCl indicate a strong lipid-protein interaction in the native lipophorin particle. In this regard, the ordering effect of apoLp-I and apoLp-I1 on the lipid components of lipophorin, as measured by anisotropy of fluorescence (Soulages et ad., 1988a). which is unaffected after extensive proteolytic cleavage of apoLp-I (Rimoldi et al., 1991), supports the idea of a lipid-embedded localization, for at least a portion of the apolipoproteins.

B . Location of Phospholipids "P NMR studies on locust lipophorin indicate that a large proportion of the PLs reside on the surface of lipophorin (Katagiri et al., 1985). In addition to this study, the susceptibility of lipophorin phospholipids to hydrolysis by phospholipase A2 is consistent with a surface localization of PLs (Katagiri et al., 1985; Kawooya et al., 1991). Although the amphipathic nature of PLS makes this a logical conclusion, it has to be pointed out that the use of hydrolytic enzymes to demonstrate the location of any component would be valid only if a rearrangement of the lipid components in lipophorin does not occur on the time scale of the experiment. Because this condition is never met, a cautious interpretation of such data is necessary. C . Location of Hydrocarbons Experimental evidence for a core localization of hydrocarbon was obtained from 'C NMR, calorimetry, and SAXRS studies (Katagiri et al., 1985, 1987). Thus, from the similarity in thermotropic behavior of pure hydrocarbons isolated from locust lipophorin, and the same hydrocarbons in the native lipoprotein, it was concluded that hydrocarbons are partially segregated from the other lipophorin components forming a hydrocarbon-rich cluster. The presence of an internal region of low electronic density was observed by SAXRS in lipophorins containing hydrocarbons, suggesting that hydrocarbons form part of an inner lipid core, which is not exposed to the aqueous environment. D . Location of Diacylglycerols Another major component of lipophorin is DG. Because the hydroxyl group of DG gives some polarity to an otherwise highly nonpolar mole-

LIPOPHORIN LIPID TRANSPORT IN INSECTS

39 1

cule, and because of the active metabolism of DG, a partial exposure of lipophorin DG to the aqueous media has been proposed (Pattnaik et al., 1979; Soulages et al., 1988a; Shapiro et al., 1988; Van der Horst, 1990). However, it was also proposed that, due to the possible destabilizing effect of DG on the highly curved surface of lipophorin, a surface localization of DG is unlikely in those lipophorins without apoLp-111 (Soulages and Brenner, 1991). Kawooya et al., (1991) concluded that DG stabilizes the lipophorin particle: after removal of PLs from M . sexta LDLp, the integrity of the particle was maintained. However, because LDLp contains apoLP-111,it is also possible that apoLp-I11 stabilized the particle. When DG-labeled LDLp, produced in vivo, was treated with lipase, radioactive DG was hydrolyzed more rapidly than unlabeled DG (Kawooya et al., 1991). These data were interpreted as showing the existence of at least two, nonequilibrating, DG pools in lipophorin. However, the same result would be obtained if there were two or more species of lipophorin in the sample that had been labeled to different DG specific activities during in vivo labeling-once again lipophorin heterogeneity clouds interpretation of data. In order to study the effect of DG content on the structure and properties of lipophorin, we have recently developed a method that allows the specific modification of the DG content of lipoproteins employing sn-l,2dioctanoyl glycerol (diC8)(Soulagesand Wells, 1994a).We observed that the loading of lipophorin with diC8 promotes the binding of apoLp-111, indicating that the main requirement for apoLp-111binding to lipophorin is an increase in the content of surface DG. DiCs loading occurred in the presence or absence of apoLp-111, but, in the absence of apoLp-111, the DG-loaded particles are not stable and spontaneously aggregate. A sharp decrease in the degree of order of the lipid phase of the lipoproteins was observed by anisotropy of fluorescence of diphenyhexatriene as the content of diC8 was increased. A comparison of the lipid order of artifically loaded lipophorins, in the presence and absence of apoLp-111, natural lipophorins which differ in their DG content, and mammalian lipoproteins, clearly showed that DG has a extraordinary perturbing effect on the lipid order of lipoproteins. The result of this recent work provides further evidence for the destabilizing role of DG and we have suggested that the lipid disordering effect of DG plays a prominent role on the lipophorin surface in such a manner as to permit the binding of the weak lipid-binding apoLp-111 to the lipoprotein surface. E. Lipophorin Models There have been only a few attempts to fit composition and structure data for lipophorin to a model. Based on SAXRS studies of lipophorin

392

JOSr L. SOULACES AND MICHAEL A. WELLS

from L . mipatoria and Periplaneta americana (Katagiri et al., 1987), and Leptanotursa decemlineata (Katagiri et al., 1991), the distribution of PLs, apolipoproteins, hydrocarbons, and DG in each of the lipophorins was deduced. Employing electron density and composition data, the authors fit the distance distribution functions to a three-layer centrosymmetrical lipophorin model. This model proposes that lipophorins are composed of three radially symmetrical layers-an outer layer that contains apoLpI and PL, a middle layer that contains apoLp-I1 and DG, and a core that contains hydrocarbon. The exact composition of each layer depends on the lipophorin under consideration. However, in both studies, the authors used spherical radii that were large enough to accommodate from two to four times the number of electrons or molecules that constitute the lipophorin particles. Thus, the dimensions of the particles are clearly inconsistent with the molecular weight and radii of lipophorins (Table V), and, therefore, an accurate picture of the lipophorin particle cannot be inferred from these studies. Another approach has been to use lipophorin composition and size to develop a model. Two models were developed based on the paradigm of Shen et al. (1977) for mammalian lipoproteins. Pattnaik et al. (1979), on the basis of the compositional data for larval M. sexta lipophorin, concluded that the surface layer of lipophorin contains substantial quantities of DG in addition to PL. Shapiro et al. (1988), employing the same paradigm to study the organization of L. mipatoria and M . sexta HDLps and LDLps, also concluded that substantial amounts of DG would be in the surface layer, particularly in LDLp, where it might interact with apoLp-I1I. The last model for lipophorin was constructed on the basis of the density-composition data for 12 lipophorins that do not contain apoLp111 (Soulages and Brenner, 1991). The composition data were fit quantitatively to a lipophorin model wherein the particles were assumed to be spherical and to contain a hydrophobic lipid core composed of hydrocarbon, DG, and TG and a surface layer composed of apolipoproteins, PL, sterol, and small amounts of DG. This study showed good correlations between the proposed structure, the composition of lipophorins, and the space-filling requirements of the lipoprotein components. Thus, in spite of the apparently random variations in lipid content and composition of lipophorins, a model emerged that is consistent with the presumed properties of the lipids and apolipoproteins, and the physiological role of lipophorin. Among the findings of that study are the following conclusions: (1) as the size of the lipophorin increases, the content of PL increases, suggesting a fundamental role for PL in defining the size of the lipophorin surface layer (Fig. 5) and hence the volume of the particle; (2) in agreement with the known perturbing effect of DG on natural and

LIPOPHORIN LlPlD TRANSPORT IN INSECTS

75

393

125 150 175 200 moles PL/mole Lipophorin

100

FIG.5. Dependence of lipophorin surface area on the mole of phospholipid per particle. The data are from 15 lipophorins without apoLp-111, and calculations were done according to Soulages and Brenner (1991). The surface area of the lipophorin was related to the surface area occupied by apoprotein and lipid by the equation 4 P RZ - WCHOL ACHOL) = NPL(APL +

~

D

G

+) APROI..

where R2 is the radius of the particle, N is the number of molecules of lipid per particle, A is the molecular area of the component, and a is the number of DG molecules per molecule of PL on the surface. CHOL, Cholesterol; PL, phospholipid; DG, diacylglycerol; PROT, apolipoprotein. Assuming a molecular area of 20 A' for cholesterol, a Y intercept value, which is surface area occupied by the apolipoprotein, of 32,000 f 2000 A' is obtained. Thus, depending on the size of the lipophorin, apoLp-I and apoLp-I1 occupy from 62 to 82% of the surface area. From the slope of the plot (73.5 f 15.8 A'), and assuming that the molecular area of a PL may have a value between 75 and 96 A', a value for a near 0 is calculated, which suggests a virtual absence of DG on the lipophorin surface.

artificial membranes (Dawson et al., 1983, 1984; Das and Rand, 1984, 1986; Hamilton el al., 1991), only small amounts of DG seem to reside in the surface layer; (3) the apolipoproteins occupy a large proportion of the lipophorin surface, from 60 to 80%; (4) in conjunction with PLs, the apolipoproteins form an outer shell about 20 A thick; ( 5 ) the area occupied per apolipoprotein amino acid at the lipoprotein surface would be about 10 A2,which would be consistent with a high content o f p structure; and (6) a small proportion (5-10%) of the apolipoprotein could be embedded in the inner lipid core.

V. METABOLISM The key to understanding lipoprotein metabolism in insects was the discovery that lipophorin functions as a reusable shuttle (reviewed by Chino, 1985). Thus, lipophorin can be described as a basic apolipoprotein-phospholipid complex, which can carry a variety of lipids in

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its core and deliver these lipids to various tissues without internalization of the lipophorin particle and without destruction of the basic apolipoprotein-phospholipid complex. Electron microscopicevidence in support of this hypothesis has been presented for locust flight muscle (Van Antwerpen et al., 1988) and the midgut of the dragonfly, Aeshna cyanea, (Bauerfeind and Komnick, 1992a): lipophorin does not enter tissues to which it delivers lipid or from which it derives lipid. Although it was shown that lipophorin entered the fat body of A. cyanea, it was proposed that this intracellular lipophorin may be aged material in the process of degradation (Bauerfeind and Komnick, 1992b). Other hallmarks of lipophorin metabolism are its ability to deliver specific lipids to specific tissues (Chino and Kitazawa, 1981) and the fact that the same basic apolipoprotein-phospholipid complex changes its metabolic roles during the life of the insect. A. Biosynthesis

Lipophorin is biosynthesized only in the fat body, with no synthesis occurring in the midgut (Prasad et al., 1986b; Bauerfeind and Komnick, 1992b).In A. cyanea, immunocytochemical data suggest that the secretion of lipophorin follows a normal pathway for a secreted protein: lipophorin was found in the endoplasmic reticulum, Golgi bodies, and secretory vesicles (Bauerfeind and Komnick, 1992b). 1 . Larvae

In M. sexta larvae, a nascent lipophorin consisting of apoLp-I, apolp-

11, and phospholipid is assembled in the fat body and secreted into hemolymph as a very high-density lipophorin (Prasad et al., 1986b). A

similar VHDLp can be isolated from hemolymph of larvae fed a fat-free diet. When larvae raised on a fat-free diet are fed a bolus of triolein, and lipophorin is isolated 6 hr later, a lipophorin of normal density can be isolated in which diolein is the predominant DG (Prasad et al., 1986b). These data suggest that the nascent lipophorin secreted from the fat body is converted into mature lipophorin by picking up DG from the midgut. Bauerfeind and Komnick (1992a) arrived at a similar conclusion based on immunocytochemical studies on A. cyanea. FernandoWarnakulasuriya et al. (1988) showed in M. sexta larvae that lipophorin biosynthesis was independent of the amount of lipid in the diet: on a fat-free diet larvae produce a lipophorin with a very low lipid content; on a high-fat diet larvae produce a lipophorin with a higher than normal lipid content. However, in neither case was the lipophorin concentration in hemolymph different from that in normal larvae. It should be noted

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that insects raised on a fat-free diet deposit significant amounts of TG in their fat bodies, presumably derived from dietary carbohydrate (Fernando-Warnakulasuriya et al., 1988). Therefore, the lack of a DGrich lipophorin in the hemolymph of such insects is not due to lack of a DG precursor in the fat body, but must be due to lack of a DG precursor in the diet. These results show two distinct features of lipophorin biosynthesis during the larval stage. First, the nascent lipophorin produced in the fat body by de nouo synthesis is an apolipoprotein-phospholipid complex that derives its transported lipids from the midgut. Second, lipophorin biosynthesis is not coupled to fat intake, as is the case with vertebrates. These processes are illustrated in Fig. 6 and fit observations made on lipid storage in larvae. Thus, it has been shown that more than 70% of the fatty acids in the diet are stored as TG in the larval fat body (Tsuchida and Wells, 1988). Although the fat body can convert carbohydrates to fatty

FIG.6. Model for delivery of dietary lipid from the midgut to the fat body. Dietary lipid (triacylglycerol,TG) is hydrolyzed to fatty acid (FA) in the lumen of the midgut, absorbed into midgut epithelial cells, and used to synthesize diacylglycerol (DG). The DG is picked up by lipophorin via a mechanism that does not involve internalization of the lipophorin. Two cases are shown. In one case, newly synthesized (nascent) lipophorin (nLp), which is secreted from the fat body, picks up DG from the midgut and is converted to a DG-loaded HDLp. The DG-loaded HDLp then moves to the fat body, where it delivers the DG without internalization and is converted to a DG-unloaded HDLp. In the second case, the DGunloaded HDLp travels to the midgut, where it picks up DG and is converted to a DGloaded HDLp.

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acids (Horie and Nakasone, 1971), it would be inefficient for the fat body to produce and secrete a mature lipophorin, if most of the DG is going to be returned to the fat body. By synthesizing a nascent lipophorin particle, the larva produces the most efficient vehicle for transport of DG from midgut to fat body. The only other larval system that has been studied is the southwestern corn borer, Diatraea grandiosella (Venkatesh et al., 1987; Bergman and Chippendale, 1989). In both diapausing and feeding-stage larvae, it was shown that the fat body incubated in vitro released a mature, i.e., lipid-loaded, lipophorin. However, the data suggest a very low rate of lipophorin biosynthesis, and this may indicate that the fat body was not producing lipophorin at its in vivo rate, which could complicate interpretation of the results. High rates of protein synthesis and secretion are difficult to maintain in in vitro fat body incubations unless the system is kept well oxygenated (Noriega and Wells, 1992).

2. Pupae At the end of the larval stage, M . sexta enters the pupal stage, during which adult metamorphosis occurs. During the pupal stage there is no lipophorin biosynthesis (Prasad et al., 1987); however, lipophorin continues to play a central role in lipid transport, but now lipid is transported from fat body to developing adult tissues (Tsuchida and Wells, 1988). Therefore, lipophorin biosynthesized in the larval stage is important for lipid transport in both the larval and pupal stages: this may be the reason that lipophorin biosynthesis is uncoupled from fat intake. If the amount of lipophorin made during the larval period depended on the amount of fat in the diet, it is likely that pupae might not have sufficient lipophorin to support adult development. 3. Adults Lipophorin biosynthesis in adult insects has been studied in the house fly, Musca domestica (Capurro and d e Bianchi, 1990b), in L. migratoria (Weers et al., 1992), and in M . sexta (S. V. Prasad and M. A. Wells, unpublished). In each of these cases in vitro incubations of fat body resulted in the release of a lipophorin whose density and lipid composition closely resembled that of mature lipophorin. An important difference between larvae and adults is the rate of lipophorin biosynthesis. For example, in M . sexta larvae, which are rapidly growing, the amount of lipophorin per animal can increase u p to 10-fold in 3 days (Prasad et al., 1987), requiring a prodigious rate of lipophorin synthesis. In adults, lipophorin synthesis need only replace that which is lost from the hemolymph due to turnover: the half-life of lipophorin in adult L. migratm-ia is

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several days (Downer and Chino, 1985). In the adult, this low rate of lipophorin biosynthesis would not significantly deplete the fat body of lipid if a mature lipophorin were produced, whereas, in larvae, during the peak period of lipophorin synthesis, the fat body lipid stores would be signficantly depleted, if a mature lipophorin were produced. It is possible that lipophorin can be made either as a nascent particle o r as a mature particle, depending on the developmental stage and/or insect under investigation. A thorough understanding of the mechanism of lipophorin biosynthesis during insect development will require additional work. T o finish this duscussion on lipophorin biosynthesis we will mention studies on the origins of PLs, hydrocarbons, sterols, and carotenoids. It has been reported that in adult M.sexta and Rhodnius prolixzts PL can be transferred from fat body to lipophorin (Van Heusden et al., 1991; CorrCa et al., 1992). This transfer of PL is independent of de novo synthesis of lipophorin; however, the mechanism by which it occurs is unknown. Hydrocarbon transport by lipophorin has been studied only in P . americana. Katase and Chino (1982) have shown, in in vitro incubations, that a fat body rich in oenocytes, one type of cell in the hemolymph, which is the major site of hydrocarbon biosynthesis (Diehl, 1975), can release labeled hydrocarbon to lipophorin. It was also shown, using in vitro incubations, that the labeled hydrocarbon in lipophorin was delivered to the epidermis, the normal site of hydrocarbon deposition in insects. T h e sterols and carotenoids that are present in lipophorin must arise from the diet, because insects cannot biosynthesize either sterols or carotenoids de nova Chino and Gilbert (197 1) have shown that sterol can be transferred from the midgut to lipophorin, and the same is most likely true for carotenoids. The mechanism by which hydrocarbons, sterols, and carotenoids are transferred from either oenocytes or midgut epithelial cells to lipophorin is unknown.

B . Physiologacal Roles of Lipophorin In this section we describe what is known about lipophorin metabolism. First we discuss the lipid transfer particle, which may play an important role in transferring lipids to and from lipophorin, then we describe the different roles of lipophorin in lipid delivery. 1 . Lipid Transfer Particle

The lipid transfer particle (LTP) is a very high-density lipoprotein having a molecular mass greater than 650 kDa. LTP contains about 15% lipid and three apolipoproteins with M, = 320,000,85,000, and 55,000. First discovered in M. sexta (Ryan et al., 1986a), LTP has been purified

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A. WELLS

from M. sexta (Ryan et al., 1986b, 1988a), L. mipatoria (Hirayama and Chino, 1990), and M . domestica (Capurro and de Bianchi, 1990a) hemolymph. LTP catalyzes lipid exchange and net transfer between different lipophorins (Ryan et al., 1986a,b, 1988a,b; Hirayama and Chino, 1990) and between lipophorin and human HDL (Ryan et al., 1990b). A model of LTP has been developed based on electron microscopy (Ryan et al., 1990~).LTP differs from vertebrate lipid transfer proteins (Tall, 1986) in both its size and ability to catalyze net lipid transfer between lipoproteins. It has been shown that LTP can catalyze carrier-mediated transfer of DG between lipoproteins (Blacklock et al., 1992). It was speculated that LTP may function in vivo to catalyze lipid transfer between cells and lipophorin (Ryan et al., 1988a),but only one study has attempted to elucidate the physiological role of LTP (Van Heusden and Law, 1989). In this study, using the adult M. sextu fat body in an in vitro lipid transfer system, it was shown that antibodies against LTP inhibited transfer of DG from fat body to lipophorin, but had no effect on the transfer of DG from lipophorin to fat body. Thus, at present the only known physiological function of LTP is to facilitate transfer of DG from fat body to lipophorin and little is known about the mechanism by which LTP catalyzes either lipid exchange or transfer, except that the transferred lipid moves through the lipid pool of LTP (Ryan et al., 1988a). 2. Lipid Transport from Midgut to Fat Body

Fatty acids released from dietary lipids by midgut lipases (Bollade et al., 1970; Weintraub and Tietz, 1973, 1978; Hoffman and Downer, 1979a; Rimoldi et al.,1985; Tsuchida and Wells, 1988) are absorbed into midgut epithelial cells and transformed, by as yet uncharacterized reactions, to DG. Midgut of M . sexta contains fatty acid-binding proteins, which may play a role in fatty acid absorption by the epithelial cells (Smith et al., 1992). When labeled fatty acids, either as free fatty acids or TG, are fed to insects or placed in midgut sacs, which are then incubated in vitro in a lipophorin-containing medium, virtually all of the labeled fatty acid that leaves the midgut is found in lipophorin as DG (Chino et al., 1981b; Chino and Kitazawa, 1981; Rimoldi et al., 1985; Tsuchida and Wells, 1988; Bauerfeind and Komnick, 1992a). For example, using fatty acidlabeled triolein, Tsuchida and Wells (1988) showed in M . sextu larvae that during a 4-hr period nearly 90% of the fed fatty acid was absorbed, and, of that absorbed, more than 70% was found in the fat body as TG. In the hemolymph, more than 95% of the labeled fatty acids was present as DG, and all the hemolymph DG was present in lipophorin. When midgut sacs, containing labeled triolein, were incubated in vitro, it was shown that essentially no labeled lipid left the midgut unless lipophorin was present

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in the incubation medium, and then only labeled DG was found in the media. When lipophorin, containing labeled DG, was injected into M. sexta larvae, the DG disappeared from the hemolymph with a half-life of about 50 min, and after 4 hr about 60% of the injected label was found in fat body as T G (Tsuchida and Wells, 1988).When DG-labeled lipophorin was incubated in vitro with fat body, more than 95% of the label was taken up by fat body within 4 hr; the half-life of labeled DG in the incubation medium was about 60 min, a value comparable to the in vzvo measurement. During the incubation, after 95% of the DG had entered the fat body, there was no detectable loss of lipophorin apolipoproteins from the incubation medium. However, the density of the lipophorin in the incubation medium increased substantially, consistent with the loss of DG. These results are compatible with the hypothesis that lipophorin functions as a reusable, noninternalized shuttle. A few reports have appeared describing lipophorin receptors. Evidence for high-affinity lipophorin binding was demonstrated in adult L. migratoria flight muscle (Hayakawa, 1987;Van Antwerpen et al., 1990) and fat body (Van Antwerpen et al., 1989).A lipophorin receptor from larval M. sexta fat body has been purified and characterized (Tsuchida and Wells, 1990).The receptor has a molecular mass of 120 kDa, requires Ca2+ for activity, and is inhibited by suramin. In these properties the lipophorin receptor is similar to the human LDL receptor, although the insect receptor does not bind human LDL. T h e purified receptor has a single, high-affinity binding site for lipophorin. The fat body lipophorin receptor shows an 8-fold higher affinity for DG-rich lipophorin than for DG-poor lipophorin. In preliminary experiments, a lipophorin receptor from the midgut has been partially characterized (K. Tsuchida and M. A. Wells, unpublished). The midgut receptor differs from the fat body receptor in molecular mass (140kDa) and the fact that it does not require metal ions for activity. The midgut receptor has 12-fold higher affinity for DG-poor lipophorin than for DG-rich lipophorin. The mechanism by which these two receptors distinguish between a DG-rich and a DG-poor lipophorin is unknown. It should be remembered that both types of lipophorin have identical structural apolipoproteins (apoLp-I and apoLp-11), and differ only in lipid content. It may be that the two types of lipophorin have different surface areas, caused by different lipid core volumes, and that these different surface areas may result in different conformations of apoLp-I and apoLp-11, which are recognized by the two types of receptors. In this regard, it has been reported that apoLp-I and apoLp-I1 are more susceptible to proteolysis in DG-poor lipophorin than in DG-rich lipophorin (Ryan et al., 1992).It has also been reported

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JOSk L. SOULAGES AND MICHAEL A. WELLS

that monoclonal antibodies specific for apoLp-I1 inhibit DG uptake by the fat body in vitro (Hiraoka and Hayakawa, 1990) and that lipophorin binds fat body proteins (Schulz et al., 1991), which might suggest that some epitope on apoLp-I1 is recognized by the receptor. The different affinities of the midgut and fat body receptors for DGrich and DG-poor lipophorins suggest a model that explains why DG is transported from the midgut to the fat body (Fig. 7). This model proposes that a DG-poor lipophorin, e.g., the nascent lipophorin secreted from the fat body or a recently DG-depleted lipophorin, binds to the midgut receptor, which facilitates transfer of DG from the midgut to the DG-poor lipophorin, producing a DG-rich lipophorin. The DG-rich lipophorin dissociates from the midgut receptor and binds to the fat body receptor, which facilitates transfer of DG from the DG-rich lipophorin to fat body, producing a DG-poor lipophorin. The DG-poor lipophorin then dissociates from the fat body receptor and cycles back to the midgut receptor.

Receptor-I Fat Body

Receptor-It

1

\

J

Midgut DG '.I

FIG.7. Proposed role of midgut and fat b o d y receptors in lipid transport. The midgut receptor specifically recognizes diacylglycerol (DG)-poor lipophorins. When lipophorin is bound to the midgut receptor, DG is transferred from the midgut to lipophorin, producing a DG-rich lipophorin. The DG-rich lipophorin dissociates from the midgut receptor and binds to the fat body receptor, which specifically recognizes a DG-rich lipophorin. When lipophorin is bound to the fat body receptor, DG is transferred from lipophorin to the fat body, producing a DG-poor lipophorin. The DG-poor lipophorin dissociates from the fat body receptor and cycles back to the midgut. It is proposed that the differences in DG content between the two types of lipophorin translate into some structural differences between the apolipoproteins on the surfaces of the two types of lipophorins, which are recognized by the two receptors.

LIPOPHORIN LIPID TRANSPORT IN INSECTS

40 1

3. Lipid Transportfrom Fat Body to Flight Muscle This is the most intensively studied aspect of lipophorin metabolism. Lipid, mobilized from the fat body, is the primary substrate used by insects to fuel long-term flight (Beenakkers et al., 1984).The mobilization of lipid from the fat body is regulated by adipokinetic hormone (AKH), a peptide hormone released from the corpus cardiaca (Orchard, 1987). AKH can also activate glycogen phosphorylase in the fat body (Beenakkers et al., 1984): whether AKH activates lipolysis of glycogenolysis depends on the developmental stage of the insect. The details of the signal transduction pathways that are involved in AKH-dependent activation of lipolysis in the fat body are unknown, but the available information suggests that the effect of AKH on the fat body has many parallels with the effect of glucagon on vertebrate adipose tissue and liver: (1) AKH has been shown to elevate CAMPlevels in the fat body (Spencer and Candy, 1976; Gade and Holwerda, 1976; Gade and Beenakkers, 1977; Asher et al., 1984; Wang et al., 1990), (2) cyclic nucleotides have been shown to stimulate protein kinase (Pines and Applebaum, 1977) and lipase (Pines et al., 1981) activities in fat body homogenates, and (3) evidence has been presented to show that AKH treatment leads to phosphorylation and activation of a lipase in the fat body (E. L. Arrese and M. A. Wells, unpublished observations). It is also known that the activating effect of AKH is dependent on extracellular Ca2+ (Lum and Chino, 1990; Wang et al., 1990; Van Marrewijk et al., 1991) and that a Ca2+ionophore can mimic the effects of AKH on lipid mobilization both in vivo and in vitro (Lum and Chino, 1990; Wang et al., 1990). Developing these preliminary observations into a complete description of the regulatory pathways is clearly a fertile area for future research. The pathway for formation of DG from TG in the fat body is unknown. The lipolytic activity in fat body homogenates from L. migratoria (Tietz and Weintraub, 1978) and M . sextu (Arrese and Wells, 1992) converts T G primarily to free fatty acids (FFAs), whereas in the desert locust Schistocerca gregaria (Spencer and Candy, 1976) and the cockroach P. americana (Hoffman and Downer, 1979b)the end products were DG and FFA. Microsomes from the L. migratoria fat body can acylate 2-MG to produce DG (Tietz et al., 1975). It has been shown that the DG released from the fat body has the sn-1,2 configuration (Lok and Van der Horst, 1980; Tietz and Weintraub, 1980), therefore either a pathway involving de novo synthesis of DG via phosphatidic acid or the stereospecific hydrolysis of TG could be involved. Several studies have characterized the AKH-induced formation of LDLp, both in vivo and in vztro (Mwangi and Goldsworthy, 1977b, 1981;

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Van der Horst et al., 1979, 1981, 1984, 1987; Wheeler and Goldsworthy, 1983; Shapiro and Law, 1983; Shapiro et al., 1984; Kawooya et al., 1984; Van Heusden et al., 1984, 1987a; Goldsworthy et al., 1985; Chino et al., 1986, 1989; Wells et al., 1987; Surholt et al., 1988; Strobel et al., 1990; Nagao and Chino, 1991). Although there are some minor differences in details, all of these studies support the suggestion that the mechanism of AKH-induced formation of LDLp is the same in all insects. Indeed, it has been shown that mixtures of components, i.e., fat body, HDLp, and apoLp-111, from different insects will form normal LDLp (Van der Horst et al., 1988; Ziegler et al., 1988; Van Heusden and Law, 1989; Chino et al., 1992). Our current understanding of the formation and metabolism of LDLp during flight is summarized in Fig. 8. AKH induces DG formation from T G stores and the DG leaves the fat body, by an unknown mechanism, but with the assistance of LTP (Van Heusden and Law, 1989). and is delivered to HDLp. ApoLp-111 assists in the uptake of DG by HDLp by

FIG. 8. Role of lipophorin in DG delivery to flight muscle. Adipokinetic hormone (AKH) is released from the corpus cardiacum and binds to the fat body, where it cause production of CAMP and entry of Ca2+. These second messengers activate lipolysis of triacylglycerol(TG) and production of diacylglycerol (DG). The DG leaves the fat body with the assistance of a lipid transfer particle (LTP) and is taken up by HDLp. The capacity of HDLp to carry DG is increased by binding of apoLp-Ill to the surface. Ultimately, LDLp is formed and moves to the flight muscle, where a lipoprotein lipase hydrolyzes the DG to produce fatty acid (FA) and regenerate HDLp and apoLp-111. The FA enters the flight muscle, where it is oxidized to produce the ATP required to power flight. HDLp and apoLp-111 circulate back to the fat body to complete the cycle.

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binding to incipient hydrophobic patches on the lipoprotein surface caused by expansion of the lipoprotein as a result of the increased DG content of the core. Additional insight into LDLp formation has resulted from studies on insects that do not form LDLp in response to AKH, even though all of the hemolymph components necessary for formation of LDLp are present. Larval L. mipatoria produce only small amounts of LDLp when injected with AKH (Van der Horst et al., 1987). Furthermore, in vitro incubation of larval fat body in adult hemolymph did not lead to production of LDLp, although larval fat body has ample lipid to support formation of LDLp. On the other hand larval fat body does respond to AKH by elevating CAMP(Gade and Beenakkers, 1977) and glycogen phosphorylase is activated by AKH (Van Marrewijk et al., 1984). The flightless grasshopper Barytettix:psolus also does not form LDLp following injection of AKH (Ziegler et al., 1988). Yet, the hemolymph contains HDLp and apoLp-111, both of which showed normal function when tested in another grasshopper, Melanopus differentials, which does form LDLp. AKH stimulates glycogen phosphorylase in B. psolus fat body. In M . sexta larvae, AKH stimulates glycogen phosphorylase, but does not cause LDLp formation, although larval hemolymph contains HDLp and apoLp-111 (Ziegler et al., 1990). Because AKH causes activation of glycogen phosphorylase in all of these cases, it is unlikely that the AKH receptor is missing or that AKH does not cause elevation of cellular CAMP.The lack of a lipolytic response to AKH may involve the AKH-sensitive lipase: either the enzyme is missing or it cannot be activated. The cockroach P. americana does not form LDLp in response to AKH (Chino et al., 1992). In this case the hemolymph lacks apoLp-111, which would limit the amount of DG that could be carried by lipophorin. A particularly interesting result came from comparison of the solitary and gregarious phases of L. mipatoria (Chino et al., 1992). Solitary-phase locusts do not fly; gregarious-phase locusts are strong fliers and this is the phase that has been extensively studied. The transformation from solitary to gregarious phase occurs when the locusts are crowded, as occurs when food supplies become diminished. Solitary-phase locusts do not form LDLp when injected with AKH, although they have HDLp and apoLp-111 in their hemolymph. Solitary-phase locusts have less fat body (15%) and a lower TG content per milligram of tissue (5%) compared to gregarious-phase locusts. One possibility is that solitary-phase locusts cannot form LDLp because of insufficient TG stores. However, it is also known that juvenile hormone titers are higher in solitary-phase locusts than in gregarious-phase locusts and this may be an important factor in determining the metabolic state of the insect. It is also possible that the solitary locust fat body lacks AKH receptors.

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How does LDLp deliver fatty acids to the flight muscle? Flight muscle from L. migratoria (Wheeler et al., 1984,1986; Wheeler and Goldsworthy, 1985; Van Heusden et al., 1986, 1987a,b) and M. sexta (Van Heusden, 1993) has been shown to contain a membrane-bound lipoprotein lipase that hydrolyzes DG to FFA. T h e lipase has higher activity against LDLp than against HDLp, which would account for its selective hydrolysis of DG in LDLp. Once produced, the fatty acids are presumed to diffuse into the flight muscle cell, where they are oxidized. In this regard it should be noted that fatty acid-binding proteins have been identified in the flight muscle of L. mipatoria (Haunerland and Chisholm, 1990) and M. sexta (M. C. Pape and M. C. Van Heusden, unpublished). These fatty acidbinding proteins could play a role in uptake of fatty acids by flight muscle. Considering the high degree of analogy between lipid mobilization in insects and vertebrates, why d o insects use DG, instead of free fatty acids, to transport fatty acids from fat body to flight muscle? Insects are quite capable of metabolizing hemolymph FFA (Stanley-Samuelson et al., 1988): the half-life of hemolymph FFA in adult Triatoma infestam (Soulages et al., 1988b) and M. sexta (Soulages and Wells, 1994b) is only 2-3 min. In both insects FFAs are carried by lipophorin and no evidence could be found for an albumin-like molecule, which transports FFA in vertebrates. In adult M. sexta 75% of the FFA in hemolymph is reesterified into DG, TG, and PL in the fat body and the rest is oxidized. The fact that hemolymph FFAs are taken into the fat body so rapidly, coupled with the fact that insects have an open circulatory system, suggests that FFA would be a poor form in which to transport fatty acids to flight muscle, because most of the fatty acid released from fat body would be rapidly taken back into the fat body and reesterified, whereas with DG, the delivery of fatty acids to flight muscle is very efficient because flight muscle contains lipoprotein lipase. 4 . Lipid DeliveT to Developing Oocyte Eggs from Hyalophora cecropia (Telfer, 1960), Samina Cynthia (Chino et al., 1977), M . sexta (Kawooya et al., 1988), and R. p-olixus (Gondim et al., 1989b) have been shown to contain a very high-density lipophorin, VHDLp-E, which is derived from HDLp-A in the hemolymph by a receptor-mediated process (Kawooya et al., 1988; Telfer and Pan, 1988; Kulakosky and Telfer, 1990); this represents the only known exception to the generalization that lipophorin delivers its lipids to tissues without internalization. The conversion of HDLp-A to VHDLp-E involves removal of DG, which is catalyzed by a lipoprotein lipase found in the yolk body of the egg (Van Antwerpen and Law, 1992). However, in spite of the presence of lipophorin in the egg, 90% of the lipid in the egg is

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delivered by a noninternalizing mechanism, involving LDLp as the lipid carrier (Kawooya and Law, 1988; Telfer et al., 1991). At present it is not known whether a lipase, either the one described above or another, is involved in removal of DG from LDLp, a mechanism analogous to the situation in flight muscle, or whether some other mechanism exists to deliver DG to the ovary. 5. Changes in Lipophorin Metabolism during Development When M . sexta larvae reach the end of the larval period, they enter the prepupal stage, during which they stop eating, void their midgut, and, if in the wild, burrow in the ground or leaf litter to pupate. During the 3- to 4-day prepupal period, striking changes in lipophorin metabolism occur. Within 48 hr, the larval lipophorin (HDLp-L) is first converted to a higher density form, which is relatively depleted of DG (HDLp-Wz), and then to a lower density form (HDLp-W1), which is relatively enriched in DG (Prasad et al., 1986a). During this period apolipoprotein synthesis ceases (Prasad et al., 1987). The conversion of HDLp-L to HDLp-W2 seems to reflect continued delivery of DG to fat body, or other tissues, in the absence of feeding. The conversion of HDLp-W2 to HDLp-W1 may reflect a switch in metabolism in the fat body: it changes from a lipidstoring tissue to a lipid-mobilizingtissue. This hypothesis is supported by in vitro and in viuo experimental data demonstrating that fat bodies from prepupae did not take up labeled DG from lipophorin (Tsuchida and Wells, 1988). Little is known about lipophorin metabolism during the pupal period, except that there is a lipophorin whose density and lipid composition (HDLp-P)differ from those of lipophorins isolated from other life stages (Prasad et al., 1986a).However, it is known that HDLp-P is derived from HDLp-L by alterations in lipid composition,because no new lipophorin is produced by the fat body in the prepupal or pupal stages. It is likely that lipophorin metabolism is controlled by the changing hormonal milieu during the prepupal and pupal periods, but nothing is known of the details. VI. METABOLICIMPLICATIONS OF LIPOPHORIN STRUCTURE The lipid composition of a lipophorin depends on the steady-state relationship between thermodynamic and kinetic factors that dictate the movement of lipids between tissues and lipophorin. The thermodynamic factors are the metabolic state of the tissue and the stability of the lipophorin, and the lipoprotein will incorporate or release lipid depending on the magnitude and sign of the difference between these two thermo-

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dynamic contributions. However, the steady-state composition will also depend on the rate at which the lipid composition of lipophorin can be changed. For example, if a thermodynamically stable lipophorin takes up DG from the fat body, the DG content of the particle will be displaced from equilibrium. The particle can reestablish equilibrium by delivering the excess DG to some tissue, but the rate at which that occurs will depend on the rate of lipid transfer. It would not be possible to fit the widely varying lipid compositions of lipophorins without apoLp-111 to a single structural model, unless, in vivo, the lipid composition and content of lipophorins were determined primarily by the physiochemical requirements for particle stability. This suggests that lipophorins transport lipids in such a manner as to maintain a near-equilibrium composition, which could be accomplished by a rapid rate of lipid transfer or by transporting only a small amount of lipid in any one cycle, e.g., between the fat body and the tissue. In this regard, the data of Tsuchida and Wells (1988) on the half-life of DG in feeding M. sexta larvae, coupled with the content of lipophorin per animal (Prasad et al., 1987)and the composition of HDLpL (Prasad et al., 1986a), can be used to calculate that the rate of DG delivery from lipophorin is 20 nmol/min. This amounts to only 1% of the DG in HDLp-L delivered per minute, a rate that should not cause the lipid composition of HDLp-L to deviate very far from equilibrium. Assuming that the in vivo composition of lipophorin is not far from equilibrium, we can use the results of the composition-structure model to make some predictions about the role of PL and DG in regulating lipophorin metabolism. The PL content of lipophorin controls its core volume and hence its lipid-carrying capacity. In other words, the PL content determines an optimal value for the core volume that corresponds to the maximum stability of the particle. An increase in core volume above the optimal value would destabilize the particle, but stability can be reestablished by getting rid of DG, or some other core lipid; a decrease in core volume below the optimal value would also destabilize the particle, but stability can be reestablished by taking up DG, or some other core lipid. Although a lipophorin particle with a reduced core volume is less stable, there seem to be no physiological mechanisms to rapidly adjust the PL content in order to establish a new optimal core volume, because, in vivo, lipophorin PL turnover is slow. In fact, a slow adjustment of the PL content of lipophorin would be necessary if the optimal core volume is an important determinant in directing the movement of DG, or other core lipids, into or out of lipophorin. Even though the adjustment of the PL content of lipophorins is slow, there must be some mechanism to accomplish such an adjustment, as is most clearly demonstrated by the changes in PL content in lipophorin that accompany

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the changes in core lipid composition and content during the transition from the larval to pupal stage in M. sexta, a time when de nouo synthesis of lipophorin has stopped. Of particular importance to lipophorin metabolism are the properties of DG. The strongly perturbing effect of DG on phospholipid bilayers has been attributed to its relatively small polar head group in comparison to the volume occupied by the fatty acyl chains. This geometric disproportion, accentuated by the high radius of curvature of lipophorin particles, would promote the exposure of the hydrocarbon chains of DG and neighboring lipids to water, which would destabilize the particle due to the hydrophobic effect. In this context the high concentration of PE found in most of the lipophorins may also be important. It is well known that, although PC adopts bilayer structures, PE is a promoter of nonlamellar lipid structures, in a manner similar to that displayed by DG. Thus, it can be speculated that the presence of substantial amounts of PE on the surface of lipophorins would enhance the hypothesized destabilizing effects of DG. The combined destabilizing effects of DG and PE on the lipophorin surface would create a situation in which transfer of lipid from the particle would be energetically favorable, while at the same time, the disorder on the surface could lower the activation energy, and hence increase the rate, of processes involved in lipid transfer. For example, a small increase in the concentration of DG on the lipophorin surface, produced after DG loading in the midgut, would cause a strong hydration of the surface. This increased hydration could promote activation of lipolytic enzymes, or activation of lipid transfer proteins and/or spontaneous release of the DG for the surface. Any or all of these activities could play a role in reestablishing the stability of the lipophorin surface. Such considerations might account for the efficient lipid transport system of insects, which does not use a lipid transfer mechanism involving uptake and/or degradation of the whole lipoprotein particle. In LDLp, the relative excess of DG, compared to PL, must result in accumulation of DG on the surface of the particle. The surface disorder caused by the presence of DG and the increase in surface free energy caused by the hydrophobic effect could promote insertion of apoLp-111 into the lipoprotein surface. The increased hydration and disorder in the surface of LDLp might also be responsible for the fact that LDLp is a better substrate for muscle lipoprotein lipase than is HDLp, in which the surface content of DG is small. The accumulation of DG in LDLp suggests that the fat body is producing DG more rapidly than it can be consumed by the flight muscle. This probably occurs because HDLp is a poor substrate for lipoprotein lipase and does not deliver fatty acids to flight muscle very effectively. Therefore, when AKH stimulates produc-

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tion and release of DG from the fat body, the DG can not be delivered to flight muscles, but instead accumulates in HDLp, causing the production of LDLp. The net result is to produce, in the hemolymph and without requiring de nova synthesis, a lipophorin particle that carries much more DG and at the same time is an excellent substrate for the flight muscle lipoprotein lipase. A totally different rationale has to be applied to the transport of hydrocarbons. These extremely hydrophobic compounds seem to reside in the interior of the lipophorin particles. A mechanism involving uptake and degradation of the lipoprotein might be possible for the transport of hydrocarbons to the epidermal cells. This type of mechanism might be important in certain stages of insect development, when the lipoprotein could deliver amino acids and other lipid components necessary for the construction of the cuticle. A similar process may also exist for the delivery of carotenoids and sterols. A poorly understood aspect of lipophorin metabolism is tissue-specific delivery of lipids. At present mechanistic details are lacking, but some properties of the system are apparent. Lipid seems to be transferred from lipophorin to tissue only in those cases in which the tissue can carry out some additional reaction with the lipid, e.g., in the fat body DG is converted to TG, and in the epidermis hydrocarbon is secreted onto the outer surface of cuticle. In other cases there may be intracellular lipidbinding proteins that, by binding the lipid, drive lipid uptake into the tissue. These or other processes would drive the equilibrium of lipid transfer in favor of the tissue, irrespective of the mechanism by which lipid delivery occurs. VII. CONCLUDING REMARKS AND FUTURE DIRECTIONS Major progress has been achieved in understanding the structure and function of apoLp-111, including the amino acid sequence of the protein from four species and the molecular structure of one of them. These results are of significance not only for insect biochemistry but for an understanding of the function of apolipoproteins in general. T h e natural variability in the amino acid sequence of apoLps-111, coupled with the possibility for selective site-directed mutagenesis and the ability of the protein to exist in water-soluble or lipid-bound states, make this apolipoprotein an excellent model with which to analyze the details of lipidprotein interactions. Lipid metabolism in insects represents a system with many unique characteristics, which although complex, still appears simpler than that of vertebrates. Insects offer an especially attractive system in which to study

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the metabolism of DG, a lipid effector of importance in vertebrates. Because DG plays such a central role in lipid metabolism in insects, it is expected that the enzymes regulating DG metabolism might be more readily studied in insects. Although such studies are of importance for understanding lipid metabolism in insects, they may also provide information of relevance to the regulation of DG metabolism in vertebrates. With regard to lipoprotein metabolism in insects there remain several fundamental questions which need to be addressed. By what mechanism does DG move between lipophorin and tissues? What is the in uivo role of LTP? What are the structure and function of lipophorin receptors? How are apoLp-I and apoLp-I1 organized on the surface of lipophorin? What is the pathway for synthesis and secretion of lipophorin? What is the mechanism of LDLp formation? What are the details of how LDLp delivers DG to tissues? How does lipophorin achieve tissue-specific delivery of lipid? In addition, future studies should be directed toward refinement of the structural model of lipophorin. Such studies should take further advantage of the diversity of insects by obtaining more compositional data, and, perhaps more importantly, should involve collecting more, and better, physical data. As stated before, the fact that so many compositionally divergent lipophorins exist, which use the same basic apolipoprotein-phospholipid matrix, offers a unique opportunity to define the structure of a lipoprotein in considerable detail. ACKNO w LEDGMENTS We thank Drs. Estela Arrese, Carolina Barillas-Mury, Don Frohlich, John Law, Ann Peterson, Alan Smith, Rik Van Antwerpen, and Randi Van Heusden for helpful comments and criticisms during preparation of this review. Unpublished work from the authors’ laboratory was supported by NIH Grant HL 391 16.

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