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The structures of insect lipoproteins
The structures of insect lipoproteins Robert O Ryan U n i v e r s i t y of Alberta, Edmonton, Canada The insect hemolymph lipoprotein, lipophorin, exists as one of several distinct subspecies, whose lipid contents are correlated with physiological lipid transport needs. It has been shown that lipophorin subspecies are interconvertable, through the acquisition or loss of lipid from pre-existing subspecies. A hemolymph lipid transfer particle has been isolated and shown to function in facilitated lipid redistribution, giving rise to different subspecies. In the adult life stage, flight activity induces a dramatic enrichment of lipophorin with neutral glycerolipid as well as the exchangeable apolipoprotein, apolipophorin III. Apolipophorin III exists as an elongated bundle of five helices that is proposed to undergo a major conformational change upon lipoprotein binding whereby it exposes its hydrophobic interior which interacts with the lipid surface. Current Opinion in Structural Biology 1994, 4:499-506
Introduction
The structure and properties of lipophorin
Insects serve as excellent models for the study of many biochemical processes [1]. For studies of plasma lipid transport, insects provide a relatively simple, yet physiologically active, system which provides abundant biological material that can be easily manipulated. Insects require efficient lipid transport in their aqueous hemolymph (blood) for a wide variety of specialized purposes. Holometabolous species, which undergo complete metamorphosis, must acquire sufficient essential lipophilic nutrients from dietary sources prior to moulting. It is known that ingested lipids are transported from the midgut tissue to the fat body where dietary glycerolipid is deposited for storage. In the adult female, developing oocytes must acquire sufficient lipid reserves to support embryonic development following fertilization. Similarly, energy-demanding flight activity in certain species involves utilization of long chain fatty acids as fuel. Neutral glycerolipid, mobilized from fat body triacylglycerol stores and transported through hemolymph as diacylglycerol (DAG), serves as the source of this fuel [2]. The transport of hydrocarbons is also important. These long chain, unbranched and methyl-branched, aliphatic molecules are functionally essential as a protective cuticular wax (to prevent desiccation) and, in some species, as pheromones [3]. These materials are thought to be transported in the hemolymph from their site of origin to the cuticle [4].
The wide variety of lipophilic biomolecules transported in the hemolymph are predominantly complexed with a single major multifunctional lipoprotein particle, termed lipophorin. Lipophorin is synthesized by fat-body tissue and is secreted into the hemolymph either as a neutral lipid-deficient particle [5], or alternatively, with a full complement of lipid [6,7]. All lipophorin particles possess two integral, nonexchangeable apolipoproteins, which are invariably present in a 1:1 molar ratio. Apohpophorin I has an approximate molecular weight of 240 000Da, while that of apolipophorin II is -80 000 Da. Recent studies of early events in lipophorin biosynthesis, using pulse-chase experiments and specific immunoprecipitation, have shown that these apolipopro-teins arise from a common precursor protein that is posttranslationally cleaved into the observed apolipoprotein components [8°]. It is not yet clear whether this event is a prerequisite for lipid accumulation or is a requirement for proper folding and orientation of these polypeptides, during nascent particle assembly. Many studies have been made of the physical and structural properties of llpophorin particles and these are generally consistent with the stuctural paradigm of' an outer coat of amphiphilic phospholipid and pro-tein surrrounding, and thereby effectively solubilizing, a core of hydrophobic lipid (mostly DAG and hydro-
Abbreviations AKH--adipokinetic hormone; apoLp-III--apolipophorin III, CD--circular dichroism; DAG~iacylglycerol; DMPC~imyristoylphosphatidylcholine; HDL--high density lipoprotein; LDL--Iow density lipoprotein; LTP--lipid transfer particle.
© Current Biology Ltd ISSN 0959-440X
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500
Lipids carbons) [9,10]. Given the wide variety of insect species in nature, as well as their frequently unique physiological lipid transport requirements, large variations in the lipid compositions of lipophorins exist [11]. Indeed, careful examination of tipophorins present in the larval hemolymph of Manduca sexta during its various stages of development reveals the presence of distinct subspecies differing in hpid content and composition, and possessing apolipophorin I and II as their sole apolipoprotein components [12]. Compositional and morphological studies, fluorescence quenching and proteolysis susceptibility have shown that these putative subspecies are physically distinct [13]. Together with these and earlier studies, showing that the various subspecies of lipophorin share a precursor-product relationship, the concept that lipophorin subspecies are interconvertablg through the acquisition or loss of lipid has evolved. The corollary of this is the existence of a basic apolipoprotein matrix, to which hpids can be added, and from which they can be removed, giving rise to the stable, distinct subspecies observed in hemolymph during the specific stages of development. Importantly, measurements of lipid and apotipoprotein half-lives in hpophorin [14,15] have shown that the lipid component turns over at a much faster rate than the protein component. Taken together, these observations have led to the hypothesis that a factor(s) may be present in hemolymph whose function is to facilitate the redistribution of lipid between lipoproteins and tissues in response to physiological demands. In 1986, such a factor, now termed lipid transfer particle (LTP), was identified and purified [16,17]. The present review will focus on research progress over the past three years toward understanding the structure and function of the major lipid binding proteins in insect hemolymph.
Lipid transfer particle Early experiments had demonstrated that incubation of two distinct lipophorin subspecies in the presence of catalytic amounts of LTP in vitro resulted in a single density population [17]. Characterization of this reaction revealed that neither apolipoprotein transfer nor hpoprotein fusion was responsible for the observed changes. Rather, facilitated net lipid transfer from the lipid-rich lipophorin population to relatively lipid-poor lipophorin particles occurred resulting in the equilibration of the lipid and creation of a uniform distribution of particles with intermediate density. Subsequent experiments have shown that LTP is a high molecular weight particle comprising three distinct apolipoproteins and 14 % hpid [18]. Electron microscopic studies have shown that LTP has a highly asymmetric morphology comprising a spheroidal head region linked to an elongated cylindrical tail, which possesses a central hinge [19,20]. The ability of LTP to facilitate lipid redistribution among hpophorins in vitro is entirely consistent with its proposed role in modulating their lipid content and composition in vivo in
response to specific physiological lipid transport needs. Convincing evidence in support of this has been obtained using neutralizing antibodies directed against LTP [18] in studies ofhpid transfer from isolated fat-body tissue to hpophorin in response to the peptidergic adipokinetic hormone (AKH). Van Heusden and Law [21] have demonstrated that DAG transfer from fat body to lipophorin is inhibited by pre-incubation with LTP antibody. This inhibition is overcome, however, upon addition of exogenous LTP suggesting an integral role in flight-related hpoprotein conversions. In a similar manner Liu and Ryan [22] used LTP antibodies to demonstrate that LTP facilitates conversion of high density hpophorin into egg very high density lipophorin upon endocytosis by oocytes. In studies to determine the catalytic activity of LTP compared with other well characterized plasma hpid transfer proteins [23], human hpoproteins have been evaluated as potential substrates for LTP. Interestingly, incubation of catalytic amounts of LTP with human high density lipoprotein (HDL) alone induces a remarkable transformation reaction [24]. Some HDL particles are converted into large apolipoprotein A-I deficient particles. This presumably results from net vectorial hpid transfer as well as exchangeable apohpoprotein transfer. Net loss of lipid from the donor HDL particles ultimately results in their disintegration, accompanied by lipid assimilation by the acceptor particles. The lower surface area to core volume ratio of the larger product lipoproteins requires less surface components, which explains why considerable amounts of apolipoprotein A-I are recovered in the infranatant following.density gradient ultracentrifugation. Interestingly, similar results have been reported with HDL following incubation with dimyristoylphosphatidylcholine (DMPC) vesicles [25] or after heating HDL to 70°C [26]. The observed LTP-induced transformation of HDL also provides a unique method for the isolation of apolipoprotein A-I without the use of denaturants or organic solvents [27]. A different result is obtained when human lipoproteins, such as low density lipoprotein (LDL) which lacks exchangeable apolipoproteins, are used in transfer assays. No changes occur in the density distribution of LDL in the absence of an appropriate donor/acceptor lipid particle. In the presence of hpophorin, however, catalytic amounts of LTP facilitate a net vectorial transfer oflipophorin-associated DAG into LDL, where it accumulates [28]. Since LDL retains it original complement of hpid, the net effect of this reaction is a loss of lipid from lipophorin together with a corresponding increase in LDL-associated lipid. Since the product particles in this reaction are easily separated by density gradient ultracentrifugation, reaction progress can be readily monitored using a donor lipophorin which contains radiolabelled DAG. Indeed, this strategy has been adopted to assess the mechanism of LTP-mediated hpid transfer and distinguish between a carrier-mediated process and one requiring formation of a ternary collision complex between donor, acceptor and LTP. Using solid phase
The structures of insect lipoproteins
lipoprotein substrates packed into separate columns connected in series, the effect of circulating LTP or buffer was determined by monitoring the transfer of labelled DAG from donor lipophorin particles to acceptor LDL particles [29]. The results clearly demonstrate that LTP can facilitate net vectorial transfer of DAG via a carriermediated mechanism providing further support for the concept that the lipid component of LTP plays a key role in facilitated lipid transfer. In further characterization of this reaction, the effect of increasing the amount of DAG available for transfer into LDL has been examined. It was found that, beyond a threshold ratio of lipophorin:LDL, the sample becomes turbid in the presence of LTP [30]. Characterization of this reaction reveals that turbidity development is due to the aggregation of substrate LDL particles which have accepted lipophorin-derived DAG beyond their capacity to retain a stable particle structure. The concept of LTP-mediated DAG enrichment of the acceptor lipoprotein in this reaction is reminiscent oflipophorin conversions in vivo which occur in response to flight activity. In this case AKH induces mobilization of stored triacylglycerol, which is converted to DAG for transport via
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hemolymph lipophorin to the flight muscle. Pre-existing adult high density lipophorin accepts large amounts of DAG in an LTP-mediated process to become a DAGloaded low density lipophorin particle. Low density lipophorin transports DAG to flight muscle where hydrolysis generates the fuel molecules (free fatty acid) utilized by the oxidative machinery of the tissue (Fig. 1). Importantly, however, the conversion of high density lipophorin to low density lipophorin, which increases the DAG content from 300 molecules per particle to more than 1000 molecules per particle, requires the presence of an additional exchangeable apolipoprotein, apolipophorin III (apoLp-III, Mr=18000 ). In fact, AKH-induced conversion of high density lipophorin to low density lipophorin in vivo results in the association of up to 16 molecules of apoLp-III [31]. ApoLp-III is normally present in a state not associated with lipid, and binds to the lipophorin surface as its DAG content increases. By analogy, therefore, LDL particles enriched in DAG as a result of LTP-mediated vectorial lipid transfer from lipophorin in vitro may require additional exchangeable apolipoproteins to stabilize the increased lipid/water interface created by DAG
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Fig. 1. A model of the effect of adipokinetic hormone on fat mobilization and transport by lipophorin in insects that utilize lipid as a fuel for sustained flight. Abbreviations: apoLp-l--apolipophorin-I; apoLp-II--apolipophorin-ll; FABP--fatty acid binding protein; FFA~free fatty acid; HDLp-A--high density lipophorin-adult; LDLp~low density lipophorin; Lp-I--lipophorin intermediate; TAG--triacylglycerol. Note: depiction of apoLp-I and apoLp-II is not meant to imply their interaction with apoLp-III molecules. Adapted from [11].
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Lipids enrichment. Indeed, when exogenous apoLp-III is included in hpid transfer assays with LDL and lipophorin, turbidity development is prevented and stable DAG-enriched LDL particles of increased size and lower density are created [30]. The ability of LTP to induce turbidity in incubations of LDL and lipophorin has been established as a novel, convenient spectrophotometric assay of LTP activity [32]. Future work on LTP will involve characterization of its apohpoprotein components with a view to determining the structural and functional role of each of the three apohpoproteins in substrate interaction and/or catalytic activity.
Apolipophorin III Considerable attention has been focused on apoLp-III, as it is a member of the class of exchangeable apolipoproteins, as well as apparently being a water soluble hemolymph protein and a lipid-binding apolipoprotein. First characterized in 1983 as a hemolymph component that associates with lipophorin in response to AKH [33-35], apoLp-III has since been isolated and characterized from many species and, in general, these apoLp-IIIs share a similar size and function. Full length amino acid sequences (derived from the cDNAs) are available for four apoLp-IIIs [36,37]. These proteins possess 40-50 % sequence homology throughout, while secondary structure predictions, as well as circular dichroism (CD) spectroscopy, suggest that the proteins have high degrees of amphipathic 0t-helical content [36,38]. A major advance in our understanding of apoLp-III structure came with the determination, by X-ray crystallography, of the three dimensional structure of Locusta migratoria apoLp-III in the lipid-flee state by Holden and coworkers [39]. The protein is globular with five long ~-helices connected by short loops arranged as a helix bundle. The amphipathic helices are oriented such that their hydrophobic faces point towards toward the center of the bundle while their hydrophilic faces interact with the solvent. This explains the observed water solubility of apoLp-III as well as its existence as a monomer over a wide concentration range. Oligosaccharide moieties, containing highly unusual 2-aminoethylphosphohate groups, are covalently bound to Asn16 and Asn83 [40"]. Although not 'visible' in the X-ray crystal structure, these oligosaccharides appear to have a key role in the stabihzation of the solution conformation of apoLpIII. Indeed, enzymatic deglycosylation of the carbohydrate structures results in a time-dependent loss of 0thelix and subsequent denaturation of the protein [41"]. It will be important to determine how these moieties stabilize the helix bundle structure, and to assess whether they affect the lipid-bound conformation of the protein. Lipid-free apoLp-III is a labile protein that is sensitive to environmental perturbation [42"]. In denaturation studies of M. sexta apoLp-III, monitored by
CD and fluorescence spectroscopy, the free energy of stabilization, AGDH2 o , of 1.29kcalmol-1 is obtained. Comparison with other apolipoproteins and representative globular proteins [43], reveals that only human apolipoprotein A-II (1.0 kcal mo1-1) and apolipoprotein A-IV (0.02kcalmol-1) have values lower than that for apoLp-III. An examination of the effect of solvent pH on the stability of lipid-free M. sexta apoLp-III shows that below p H 4 and above pH 10, there is a marked loss of secondary structure [42°]. Examination of the amino acid sequence reveals an arrangement of 22 pairs of oppositely charged amino acid side chains, in an i,i+4 sequence configuration. On the basis of studies with 0thelical peptides it is known that salt bridge formation between charged residues can stabilize 0t-helical structures [44]. As such salt bridges between the 23 lysine and 27 aspartate and glutamate residues would be disrupted at pH values above 10 and below 4, it is reasonable to speculate that these salt bridges exert a stabilizing effect on lipid-free M. sexta apoLp-III. Interestingly, such an effect has not been observed in the case ofL. migratoria apoLpIII, which contains far fewer charged residues. Consistent with this is the fact that no specific salt bridges have been noted in the X-ray crystal structure of the L. migratoria apoLp-III [39]. Studies of the lipid-binding interaction ofapoLp-III have been performed. In a classical study of the properties of apoLp-III at the air/water interface using a monolayer balance, Kawooya et ah [38] found evidence that the molecular area occupied by M. sexta apoLp-III could not be accounted for by the globular structure of the protein in solution. These authors therefore proposed that a conformational change occurs upon the binding of lipid, in which the protein spreads to occupy a greater area than the solution conformation. A further suggestion from the crystal structure of L. migratoria apoLp-III is that the protein opens about two hinge regions in the loops between helices two and three, and between helices four and five. This putative conformational change would result in exposure of the hydrophobic faces of the amphipathic 0t-helices, thereby permitting direct interaction with lipid surfaces. Key unresolved questions pertain to the structural features which initiate lipid binding and the precise conformational state of the protein in the lipid bound state. With regard to the former, it has been proposed that apoLp-III initially binds lipid surfaces via an exposed hydrophobic region at one of its ends, whereupon the protein opens to expose its hydrophobic interior [31,39]. In contrast, studies by Zhang et ah [45"] of the interaction of apoLp-III with various phospholipids have provided evidence that M. sexta apoLp-III interacts strongly with negatively charged lipids, perhaps through ionic interactions with positive charges on the side chains of the 23 lysine, two arginine and four histidine residues present. Furthermore, on the basis of the distribution of positively charged residues at the interface between the hydrophobic and hydrophilic faces of the amphipathic 0t-helices, it is postulated that ionic interactions can bring the protein into the proximity of the
The structures of insect lipoproteins Ryan
bilayer, inducing a conformational change that results in bilayer disruption and protein-lipid interaction. In further studies of apoLp-IlI-phospholipid interactions, it has been shown that the protein spontaneously disrupts sonicated bitayers of DMPC resulting in formation of a uniform population of disk-like particles [46"]. Characterization of these particles reveals a DMPC:apoLp-III molar ratio of 102:1 with each disk containing six molecules of apoLp-III. Calculations based on disk composition, dimensions and molecular weight, together with the dimensions of lipid-free apoLp-III obtained from X-ray crystallography, have led to a model in which apoLp-III undergoes a conformational change exposing its hydrophobic interior, which interacts with the exposed fatty acyl chains of the disklike DMPC bilayer. Consideration of possible orientations of the protein resulted in the concept that the protein orients with its Ix-helices perpendicular to the DMPC fatty acyl chains (Fig. 2). Recent spectroscopic studies of apoLp-III have provided further support for a major conformational change upon interaction with lipid surfaces. In the case of M. sexta apoLp-III, which lacks tryptophan and contains a single tyrosine residue at position 145, fluorescence spectroscopy has been employed. In the lipid-free helix bundle conformation, tyrosine fluorescence is highly quenched with an observed quantum yield of 0.015. Upon interaction with DMPC, however, there is a dramatic increase in tyrosine fluorescence quantum yield to 0.110 [46"]. Since CD spectroscopy data reveal that the protein retains its ix-helix content in both states, it is unlikely that the fluorescence enhancement observed upon lipid binding is the result of changes in helix content. Alternatively, it is conceivable that tyrosine fluorescence is quenched in the lipid-free helix bundle conformation of M. sexta apoLp-III as a result of the proximity of an amino acid side chain carboxyl group on a neighboring helix, which can serve as an acceptor of the hydroxyl proton of the excited tryosine, thereby quenching fluorescence [47]. When the protein alters its conformation upon interaction with a lipid surface, however, the orientation of neighboring helices is altered, causing the repositioning of the putative quenching carboxyt side chain, which, as
a result, is too distant to serve as an acceptor of the excited tyrosine side chain hydroxyl proton. In studies of L. migratoria apoLp-III, near-UV CD spectroscopy provides evidence that the two tryptophans and two phenylalanines adopt a new conformation in a more hydrophobic environment upon binding to DMPC [41"]. This is indicated by the dramatic complete reversal in sign and red shift ofnear-UV CD extrema attributed to these residues in the lipid-bound as opposed to the lipid-free state (Fig. 3). Importantly, far-UV spectra reveal a similar amount of Ix-helix content in both states [41"]. The unusually well defined near UV spectra obtained with L. migratoria apoLp-III compared with other proteins and apolipoproteins [48] is likely to be due to the absence of tyrosine and cysteine in this protein. A key question unanswered by studies of model complexes pertains to the conformation and orientation adopted by apoLp-III when it binds to the surface of lipophorin. On the basis of the observed close correlation between apoLp-III binding and DAG accumulation, it has been suggested that these components may interact in some manner. Thus it can be envisioned that as the DAG content of lipophorin increases, some portion of this lipid may partition to the surface monolayer. This partitioning, which is consistent with the composition of the particles, may have a destabilizing effect on particle structure (similar to that of DAG on phospholipid bilayer membranes [49]), due to the lack of a significant polar head group on DAG compared with phospholipid. Thus it is envisioned that-apoLp-III may associate with the lipoprotein surface in order to occupy defects in the surface monolayer created by the presence of DAG. In studies employing 31p-NMR to examine the diffusion coefficient and intrinsic viscosity of the phospholipid components of different lipophorins, evidence has been obtained that, in those subspecies that lack apoLp-III, there is greater phosphollpid mobility [50]. On the basis of this, and other data, it has been hypothesized that interactions between charged groups of apoLp-III and those present in the polar head group of lipophorin phospholipids may have a role in stabilizing apoLp-III interactions with the lipophorin surface. A model of such an interaction between an Ix-helical
Fig. 2. Hypothetical representaion of an apoLp-III-DMPC complex in cross-section. Six apoLp-III molecules surround the DMPC bilayer via binding of the amphipathic (x-helices to the phospholipids, in a manner perpendicular to the fatty acyl chains. Reproduced with permission from [46"].
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As illustrated in this review, lipid transport processes in insect hemolymph offer a system whereby structural information on lipid binding protein can be obtained. Data presented herein support the concept that major parallels exist between processes in insect hemolymph and those in vertebrate species. Future studies designed to address key questions related to the mechanism and specificity of lipid-protein interactions in this model system should increase our understanding oflipoprotein metabolism in general.
Acknowledgements
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Fig. 3. Far-UV and aromatic (inset) circular dichroism specta of L. migratoria apoLp-III. Lipid-free apoLp-III is shown as solid line and apoLp-III-DMPC complexes as dashed line. Spectra were recorded in 5 0 m M sodium phosphate (pH 7.0) and l O0mM KCl. Reproduced with permission from [41•].
seganent of apoLp-Ill and a DAG-containg lipoprotein surface monolayer has been presented [50]. In an effort to gain further evidence for the concept that the presence of DAG in the lipoprotein surface provides a binding site for apoLp-IIl which, in turn, stabilizes the particle structure, phospholipase C has been used to convert lipoprotein phospholipid into DAG. On the basis of previous studies demonstrating that treatment of human LDL particles with phospholipase C causes aggregation [51], we have demonstrated that aggregation of LDL in response to phospholipase C is prevented when apoLpIII, or human apolipoprotein A-I, is included in the incubation [52]. Furthermore, phospholipase C treatment induces a stable association of apolipoprotein with the lipoprotein surface, providing evidence that the presence of surface DAG provides a site for interaction. Recently, using lipophorins enriched with exogenous short chain DAG, a correlation between DAG enrichment and apoLp-III association has been observed [53]. Since other apolipoproteins, including human apolipoprotein A-I, function in a similar manner, these observations provide support for the concept that exchangeable apolipoproteins from vertebrate and invertebrate sources are functionally homologous. This concept is also con-
Work from the author's laboratory was supported by grants from the Medical Research Council of Canada and the Alberta Heart and Stroke Foundation. The author is a recipient o f a Scientist Award from the Medical Research Council and a Scholarship Award from the Alberta Heritage Foundation for Medical R e search. I thank P D ' O b r e n a n and L Hicks for assistance with the figures and my colleagues within the Lipid and Lipoprotein Research Group and the Department o f Biochemistry for continued collaborative support.
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chemistry and Physiology of Insect Cuticular Lipids. Arch Insect Biochem Physiol 1987, 6:227-265. 4.
Chino H: Lipid Transport: Biochemistry of Hemolymph Lipophorin. In Comprehensive Insect Physiology Biochemistry and Pharmacology. Edited by Kerkut GA, Gilbert LI. Oxford: Pergamon Press; 1985, 10:115-136.
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Prasad SV, Fernando-Warnaknlasuriya GJP, Sumida M, Law JH, Wells MA: Lipoprotein Biosynthesis in the Larvae of the Tobacco Hornworn, Manduca sexta. J Biol Chem 1986, 261:17174-17176.
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Weers PMM, Van der Horst DJ, Van Marrewijk WJA, Van den Eijnden M, Van Doom J, Beenakkers AMTh: Biosynthesis and Secretion of Insect Lipoprotein. J Lipid Res 1992, 33:485-491.
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Shelby KS, Chippendale GM: Assembly and Secretion of Lipo-
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Weers PMM, Van Marrewijk WJA, Beenakkers AMTh, Van der Horst DJ: Biosynthesis of Locust Lipophorin: Apolipophorins I and II Originate from a Common Precursor. J Biol Chem 1993, 268:4300-4303. Pulse chase experiments reveal that the two integral apolipoproteins of lipophorin arise from a common precursor protein through post-translational cleavage of a proapolipophorin. These results explain the constant 1:1 stoichiometry of these apolipophorins in all subspecies.
sity Lipoprotein Without Denaturation. Biochemistry 1992, 31:4509-4514. 28.
Ryan RO, Wessler AN, Ando S, Price HM, Yokoyama S: Insect Lipid Transfer Particle Catalyzes Bidirectional Vectorial Transfer of Diacylglycerol from Lipophorin to Human Low Density Lipoproteln. J Biol Chem 1990, 265:10551-10555.
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18.
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19.
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Ryan RO, Howe A, Scraba DG: Studies of the Morphology and Structure of the Plasma Lipid Transfer Particle from the Tobacco Hornworm, Manduca sexta. J Lipid Res 1990, 31:871-879. Takeuchi N, Chino H: Lipid Transfer Particle in the Hemolymph of the American Cockroach: Evidence for its Capacity to Transfer Hydrocarbons Between Lipophorin Particles. J Lipid Res 1993, 34:543-551.
21.
Van Heusden MC, Law JH: An Insect Lipid Transfer Particle Promotes Lipid Loading from Fat Body to Lipoprotein. J Biol Chem 1989, 264:17287-17292.
22.
Liu H, Ryan RO: Role of Lipid Transfer Particle in Transformation of Lipophorin in Manduca sexta Oocytes. Biochim Biophys Acta 1991, 1085:112-118.
23.
Tall AR: Plasma Cholesteryl Ester Transfer Protein. J Lipid Res 1993, 34:1255-1274.
24.
Silver ET, Scraba DG, Ryan RO: Lipid Transfer Particle-Induced Transformation of Human High Density Lipoprotein into Apolipoprotein A-I-Deficient Low Density Particles. J Biol Chem 1990, 265:22487-22492.
25.
Tall AR, Small DM: Solublization of Phospholipid Membranes by Human Plasma High Density Lipoproteins. Nature 1977, 265:] 63-164.
26.
Tall AR, Deckelbaum RJ, Small DM, Shipley GG: Thermal Behaviour of Human Plasma High Density Lipoprotein. Biochim Biophys Acta 1977, 487:145-153.
27.
Ryan RO, Yokoyama S, Liu H, Czarnecka H, Oikawa K, Kay CM: Human Apolipoprotein A-I Liberated From High Den-
40. •
H~rd K, Van Doom JM, Thomas-Oates JE, Kamerling JP, Van der Horst DJ: Structure of the Asn-Linked Oligosaccharides of Apolipophorin III From the Insect Locusta ml. gratorla. Carbohydrate-Linked 2-aminoethylphosphonate as a Constituent of a Glycoprolein. Biochemistry 1993, 32:766-775. The structure of the carbohydrate moieties of L. migratoria apoLp-III are reported. A remarkable feature is the presence of 2-aminoethylphosphonate bound to mannose and/or N-acetylglucosamine moieties. This is the first example of a glycoprotein containing 2-aminoethylphosphonate, which explains the previously observed lack of interaction of this protein with various lectins. 41. •
Weers PMM, Kay CM, Oikawa K, Wientzek M, Van der Horst DJ, Ryan RO: Factors Affecting lhe Stability and Conforma.. lion of Locusta migratorla Apollpophorin III. Biochemistry 1994 33:3617-3624. It is demonstrated that enzymatic deglycosylation of L. migratoria apoLpIII induces a time-dependent loss of helical structure with complete denaturation occurring over several days. The data suggest that the carbohydrate moiety of the protein functions in stabilization of the helix bundle. Also, spectroscopic data are presented which support a major reorientation of the protein upon interaction with a lipid surface. 42. •
Ryan RO, Oikawa K, Kay C: Conformational, Thermodynamic and Stability Properties of Manduca sexta Apolipophorin III. J Biol Chem 1993, 268:1525-1530. The structural stability of lipid-free M. sexta apoLp-III is determined. It is reported that apoLp-IH adopts a loosely folded structure that is sensitive to
505
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Lipids environmental perturbation. It is postulated that salt bridge formation between oppositely charged amino acid side chains serves as a stabilizing force for the protein in the lipid-free helix bundle conformation. 43.
Wetterau JR, Aggerbeck LP, Rail, SC Jr, Weisgraber KH: Human apolipoprotein E3 in Aqueous Solution I. Evidence for Two Structural Domains. J Bio/ Chem 1988, 263:6240-6248.
44.
Marqusee S, Baldwin RL: Helix Stabilization of Glu-...Lys÷ Salt Bridges in Short Peptides of de novo Design. Proc Natl Acad
45. •
47.
48.
49.
Sci U S A 1987, 84:8898-8902.
50.
ZhangY, Lewis RNAH, McEIhaney RN, Ryan RO: Calorimetric and Spectroscopic Studies of the Interaction of Manduca sexta Apolipophorin III with Zwitlerionic, Anionic and Nonionic Lipids. Biochemistry 1993, 32:3942-3952.
51.
Differential scanning calorimetry and NMR spectroscopy are employed to evaluate the interaction of M. sexta apoLp-III with a series of lipids. The data indicate a strong interaction of apoLp-III with anionic lipids consistent with the concept that the binding of apoLp-III to lipid bilayers is mediated through ionic interactions at the lipid surface. Initial interaction of the protein is proposed to be followed by a conformational change to expose its hydrophobic interior which results in bilayer disruption. Wientzek M, Kay CM, Oikawa K, Ryan RO: Binding of Insect Apolipophorin III to Dimyristoylphosphalidylcholine Vesicles: Evidence for a Conformational Change. J Biol Chem 1994, 269:4605-4612. The interaction of M. sexta apoLp-III with DMPC multilamellar vesicles revealed the formation of a uniform population of disk-like particles whose composition and properties are consistent with apoLp-III interaction with DMPC fatty acyl chains around the perimeter of the bilayer disk. Calculations based on the particle dimensions, apoLp-III size and lipid content suggest apoLp-III may open about a hinge region as predicted by Holden and coworkers (see ref [39]). Fluorescence data are reported which also suggest a conformational change in the protein upon lipid interaction.
52.
53.
46. •
54.
Stryer /: Excited-State Proton-Transfer Reactions. A Deuterium Isotope Effect on Fluorescence. J Am Chem Soc 1966, 88:5708-5712. Lux SE, Hirz R, Shrager I, Gotto AM: The Influence of Lipid on the Conformation of Human Plasma High Density Apolipoproteins. J giol Chem 1972, 247:2598-2606. Das, S, Rand, RP: Modification by Diacylglycerol of the Structure and Interaction of Various Phospholipid Bilayer Systems. Biochemistry 1986, 25:2282-2289. Wang J, Liu H, Sykes BD, Ryan RO: 31p-NMR Study of the Phospholipid Moiety of Lipophorin Subspecies. Biochemistry 1992, 31:8706-8712. Suits AG, Chait A, Aviram M, Heinecke JW: Phagocytosis of Aggregated Lipoproteins by Macrophages: kow Density Lipoprotein Receptor-Dependent Foam Cell Formation. Proc Nat/ Acad Sci USA 1989, 86:2713-2717. Liu H, Scraba DG, Ryan RO: Prevention of Phospholipase-C Induced Aggregation of Low Density Lipoprotein by Amphipathic Apolipoproteins. FEBS Lett 1993, 316:27-33. Soulages JL, Wells MA: Effect of Diacylglycerol Content on Some Physicochemical Propedies of the Insect Lipoproteln, Lipophorin. Correlation with the Binding of Apolipophorin III. Biochemistry 1994, 33:2356-2362. Wilson C, Wardell MR, Weisgraber KH, Mahley RW, Agard DA: Three Dimensional Structure of the LDL ReceptorBinding Domain of Human Apolipoprotein E. Science 1991, 252:1817-1822.
RO l,Lyan, Lipid and Lipoprotein Research Group, 328 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
Introduction
The structure and properties of lipophorin
Insects serve as excellent models for the study of many biochemical processes [1]. For studies of plasma lipid transport, insects provide a relatively simple, yet physiologically active, system which provides abundant biological material that can be easily manipulated. Insects require efficient lipid transport in their aqueous hemolymph (blood) for a wide variety of specialized purposes. Holometabolous species, which undergo complete metamorphosis, must acquire sufficient essential lipophilic nutrients from dietary sources prior to moulting. It is known that ingested lipids are transported from the midgut tissue to the fat body where dietary glycerolipid is deposited for storage. In the adult female, developing oocytes must acquire sufficient lipid reserves to support embryonic development following fertilization. Similarly, energy-demanding flight activity in certain species involves utilization of long chain fatty acids as fuel. Neutral glycerolipid, mobilized from fat body triacylglycerol stores and transported through hemolymph as diacylglycerol (DAG), serves as the source of this fuel [2]. The transport of hydrocarbons is also important. These long chain, unbranched and methyl-branched, aliphatic molecules are functionally essential as a protective cuticular wax (to prevent desiccation) and, in some species, as pheromones [3]. These materials are thought to be transported in the hemolymph from their site of origin to the cuticle [4].
The wide variety of lipophilic biomolecules transported in the hemolymph are predominantly complexed with a single major multifunctional lipoprotein particle, termed lipophorin. Lipophorin is synthesized by fat-body tissue and is secreted into the hemolymph either as a neutral lipid-deficient particle [5], or alternatively, with a full complement of lipid [6,7]. All lipophorin particles possess two integral, nonexchangeable apolipoproteins, which are invariably present in a 1:1 molar ratio. Apohpophorin I has an approximate molecular weight of 240 000Da, while that of apolipophorin II is -80 000 Da. Recent studies of early events in lipophorin biosynthesis, using pulse-chase experiments and specific immunoprecipitation, have shown that these apolipopro-teins arise from a common precursor protein that is posttranslationally cleaved into the observed apolipoprotein components [8°]. It is not yet clear whether this event is a prerequisite for lipid accumulation or is a requirement for proper folding and orientation of these polypeptides, during nascent particle assembly. Many studies have been made of the physical and structural properties of llpophorin particles and these are generally consistent with the stuctural paradigm of' an outer coat of amphiphilic phospholipid and pro-tein surrrounding, and thereby effectively solubilizing, a core of hydrophobic lipid (mostly DAG and hydro-
Abbreviations AKH--adipokinetic hormone; apoLp-III--apolipophorin III, CD--circular dichroism; DAG~iacylglycerol; DMPC~imyristoylphosphatidylcholine; HDL--high density lipoprotein; LDL--Iow density lipoprotein; LTP--lipid transfer particle.
© Current Biology Ltd ISSN 0959-440X
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Lipids carbons) [9,10]. Given the wide variety of insect species in nature, as well as their frequently unique physiological lipid transport requirements, large variations in the lipid compositions of lipophorins exist [11]. Indeed, careful examination of tipophorins present in the larval hemolymph of Manduca sexta during its various stages of development reveals the presence of distinct subspecies differing in hpid content and composition, and possessing apolipophorin I and II as their sole apolipoprotein components [12]. Compositional and morphological studies, fluorescence quenching and proteolysis susceptibility have shown that these putative subspecies are physically distinct [13]. Together with these and earlier studies, showing that the various subspecies of lipophorin share a precursor-product relationship, the concept that lipophorin subspecies are interconvertablg through the acquisition or loss of lipid has evolved. The corollary of this is the existence of a basic apolipoprotein matrix, to which hpids can be added, and from which they can be removed, giving rise to the stable, distinct subspecies observed in hemolymph during the specific stages of development. Importantly, measurements of lipid and apotipoprotein half-lives in hpophorin [14,15] have shown that the lipid component turns over at a much faster rate than the protein component. Taken together, these observations have led to the hypothesis that a factor(s) may be present in hemolymph whose function is to facilitate the redistribution of lipid between lipoproteins and tissues in response to physiological demands. In 1986, such a factor, now termed lipid transfer particle (LTP), was identified and purified [16,17]. The present review will focus on research progress over the past three years toward understanding the structure and function of the major lipid binding proteins in insect hemolymph.
Lipid transfer particle Early experiments had demonstrated that incubation of two distinct lipophorin subspecies in the presence of catalytic amounts of LTP in vitro resulted in a single density population [17]. Characterization of this reaction revealed that neither apolipoprotein transfer nor hpoprotein fusion was responsible for the observed changes. Rather, facilitated net lipid transfer from the lipid-rich lipophorin population to relatively lipid-poor lipophorin particles occurred resulting in the equilibration of the lipid and creation of a uniform distribution of particles with intermediate density. Subsequent experiments have shown that LTP is a high molecular weight particle comprising three distinct apolipoproteins and 14 % hpid [18]. Electron microscopic studies have shown that LTP has a highly asymmetric morphology comprising a spheroidal head region linked to an elongated cylindrical tail, which possesses a central hinge [19,20]. The ability of LTP to facilitate lipid redistribution among hpophorins in vitro is entirely consistent with its proposed role in modulating their lipid content and composition in vivo in
response to specific physiological lipid transport needs. Convincing evidence in support of this has been obtained using neutralizing antibodies directed against LTP [18] in studies ofhpid transfer from isolated fat-body tissue to hpophorin in response to the peptidergic adipokinetic hormone (AKH). Van Heusden and Law [21] have demonstrated that DAG transfer from fat body to lipophorin is inhibited by pre-incubation with LTP antibody. This inhibition is overcome, however, upon addition of exogenous LTP suggesting an integral role in flight-related hpoprotein conversions. In a similar manner Liu and Ryan [22] used LTP antibodies to demonstrate that LTP facilitates conversion of high density hpophorin into egg very high density lipophorin upon endocytosis by oocytes. In studies to determine the catalytic activity of LTP compared with other well characterized plasma hpid transfer proteins [23], human hpoproteins have been evaluated as potential substrates for LTP. Interestingly, incubation of catalytic amounts of LTP with human high density lipoprotein (HDL) alone induces a remarkable transformation reaction [24]. Some HDL particles are converted into large apolipoprotein A-I deficient particles. This presumably results from net vectorial hpid transfer as well as exchangeable apohpoprotein transfer. Net loss of lipid from the donor HDL particles ultimately results in their disintegration, accompanied by lipid assimilation by the acceptor particles. The lower surface area to core volume ratio of the larger product lipoproteins requires less surface components, which explains why considerable amounts of apolipoprotein A-I are recovered in the infranatant following.density gradient ultracentrifugation. Interestingly, similar results have been reported with HDL following incubation with dimyristoylphosphatidylcholine (DMPC) vesicles [25] or after heating HDL to 70°C [26]. The observed LTP-induced transformation of HDL also provides a unique method for the isolation of apolipoprotein A-I without the use of denaturants or organic solvents [27]. A different result is obtained when human lipoproteins, such as low density lipoprotein (LDL) which lacks exchangeable apolipoproteins, are used in transfer assays. No changes occur in the density distribution of LDL in the absence of an appropriate donor/acceptor lipid particle. In the presence of hpophorin, however, catalytic amounts of LTP facilitate a net vectorial transfer oflipophorin-associated DAG into LDL, where it accumulates [28]. Since LDL retains it original complement of hpid, the net effect of this reaction is a loss of lipid from lipophorin together with a corresponding increase in LDL-associated lipid. Since the product particles in this reaction are easily separated by density gradient ultracentrifugation, reaction progress can be readily monitored using a donor lipophorin which contains radiolabelled DAG. Indeed, this strategy has been adopted to assess the mechanism of LTP-mediated hpid transfer and distinguish between a carrier-mediated process and one requiring formation of a ternary collision complex between donor, acceptor and LTP. Using solid phase
The structures of insect lipoproteins
lipoprotein substrates packed into separate columns connected in series, the effect of circulating LTP or buffer was determined by monitoring the transfer of labelled DAG from donor lipophorin particles to acceptor LDL particles [29]. The results clearly demonstrate that LTP can facilitate net vectorial transfer of DAG via a carriermediated mechanism providing further support for the concept that the lipid component of LTP plays a key role in facilitated lipid transfer. In further characterization of this reaction, the effect of increasing the amount of DAG available for transfer into LDL has been examined. It was found that, beyond a threshold ratio of lipophorin:LDL, the sample becomes turbid in the presence of LTP [30]. Characterization of this reaction reveals that turbidity development is due to the aggregation of substrate LDL particles which have accepted lipophorin-derived DAG beyond their capacity to retain a stable particle structure. The concept of LTP-mediated DAG enrichment of the acceptor lipoprotein in this reaction is reminiscent oflipophorin conversions in vivo which occur in response to flight activity. In this case AKH induces mobilization of stored triacylglycerol, which is converted to DAG for transport via
AKH ATP"~
Ryan
hemolymph lipophorin to the flight muscle. Pre-existing adult high density lipophorin accepts large amounts of DAG in an LTP-mediated process to become a DAGloaded low density lipophorin particle. Low density lipophorin transports DAG to flight muscle where hydrolysis generates the fuel molecules (free fatty acid) utilized by the oxidative machinery of the tissue (Fig. 1). Importantly, however, the conversion of high density lipophorin to low density lipophorin, which increases the DAG content from 300 molecules per particle to more than 1000 molecules per particle, requires the presence of an additional exchangeable apolipoprotein, apolipophorin III (apoLp-III, Mr=18000 ). In fact, AKH-induced conversion of high density lipophorin to low density lipophorin in vivo results in the association of up to 16 molecules of apoLp-III [31]. ApoLp-III is normally present in a state not associated with lipid, and binds to the lipophorin surface as its DAG content increases. By analogy, therefore, LDL particles enriched in DAG as a result of LTP-mediated vectorial lipid transfer from lipophorin in vitro may require additional exchangeable apolipoproteins to stabilize the increased lipid/water interface created by DAG
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Fig. 1. A model of the effect of adipokinetic hormone on fat mobilization and transport by lipophorin in insects that utilize lipid as a fuel for sustained flight. Abbreviations: apoLp-l--apolipophorin-I; apoLp-II--apolipophorin-ll; FABP--fatty acid binding protein; FFA~free fatty acid; HDLp-A--high density lipophorin-adult; LDLp~low density lipophorin; Lp-I--lipophorin intermediate; TAG--triacylglycerol. Note: depiction of apoLp-I and apoLp-II is not meant to imply their interaction with apoLp-III molecules. Adapted from [11].
501
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Lipids enrichment. Indeed, when exogenous apoLp-III is included in hpid transfer assays with LDL and lipophorin, turbidity development is prevented and stable DAG-enriched LDL particles of increased size and lower density are created [30]. The ability of LTP to induce turbidity in incubations of LDL and lipophorin has been established as a novel, convenient spectrophotometric assay of LTP activity [32]. Future work on LTP will involve characterization of its apohpoprotein components with a view to determining the structural and functional role of each of the three apohpoproteins in substrate interaction and/or catalytic activity.
Apolipophorin III Considerable attention has been focused on apoLp-III, as it is a member of the class of exchangeable apolipoproteins, as well as apparently being a water soluble hemolymph protein and a lipid-binding apolipoprotein. First characterized in 1983 as a hemolymph component that associates with lipophorin in response to AKH [33-35], apoLp-III has since been isolated and characterized from many species and, in general, these apoLp-IIIs share a similar size and function. Full length amino acid sequences (derived from the cDNAs) are available for four apoLp-IIIs [36,37]. These proteins possess 40-50 % sequence homology throughout, while secondary structure predictions, as well as circular dichroism (CD) spectroscopy, suggest that the proteins have high degrees of amphipathic 0t-helical content [36,38]. A major advance in our understanding of apoLp-III structure came with the determination, by X-ray crystallography, of the three dimensional structure of Locusta migratoria apoLp-III in the lipid-flee state by Holden and coworkers [39]. The protein is globular with five long ~-helices connected by short loops arranged as a helix bundle. The amphipathic helices are oriented such that their hydrophobic faces point towards toward the center of the bundle while their hydrophilic faces interact with the solvent. This explains the observed water solubility of apoLp-III as well as its existence as a monomer over a wide concentration range. Oligosaccharide moieties, containing highly unusual 2-aminoethylphosphohate groups, are covalently bound to Asn16 and Asn83 [40"]. Although not 'visible' in the X-ray crystal structure, these oligosaccharides appear to have a key role in the stabihzation of the solution conformation of apoLpIII. Indeed, enzymatic deglycosylation of the carbohydrate structures results in a time-dependent loss of 0thelix and subsequent denaturation of the protein [41"]. It will be important to determine how these moieties stabilize the helix bundle structure, and to assess whether they affect the lipid-bound conformation of the protein. Lipid-free apoLp-III is a labile protein that is sensitive to environmental perturbation [42"]. In denaturation studies of M. sexta apoLp-III, monitored by
CD and fluorescence spectroscopy, the free energy of stabilization, AGDH2 o , of 1.29kcalmol-1 is obtained. Comparison with other apolipoproteins and representative globular proteins [43], reveals that only human apolipoprotein A-II (1.0 kcal mo1-1) and apolipoprotein A-IV (0.02kcalmol-1) have values lower than that for apoLp-III. An examination of the effect of solvent pH on the stability of lipid-free M. sexta apoLp-III shows that below p H 4 and above pH 10, there is a marked loss of secondary structure [42°]. Examination of the amino acid sequence reveals an arrangement of 22 pairs of oppositely charged amino acid side chains, in an i,i+4 sequence configuration. On the basis of studies with 0thelical peptides it is known that salt bridge formation between charged residues can stabilize 0t-helical structures [44]. As such salt bridges between the 23 lysine and 27 aspartate and glutamate residues would be disrupted at pH values above 10 and below 4, it is reasonable to speculate that these salt bridges exert a stabilizing effect on lipid-free M. sexta apoLp-III. Interestingly, such an effect has not been observed in the case ofL. migratoria apoLpIII, which contains far fewer charged residues. Consistent with this is the fact that no specific salt bridges have been noted in the X-ray crystal structure of the L. migratoria apoLp-III [39]. Studies of the lipid-binding interaction ofapoLp-III have been performed. In a classical study of the properties of apoLp-III at the air/water interface using a monolayer balance, Kawooya et ah [38] found evidence that the molecular area occupied by M. sexta apoLp-III could not be accounted for by the globular structure of the protein in solution. These authors therefore proposed that a conformational change occurs upon the binding of lipid, in which the protein spreads to occupy a greater area than the solution conformation. A further suggestion from the crystal structure of L. migratoria apoLp-III is that the protein opens about two hinge regions in the loops between helices two and three, and between helices four and five. This putative conformational change would result in exposure of the hydrophobic faces of the amphipathic 0t-helices, thereby permitting direct interaction with lipid surfaces. Key unresolved questions pertain to the structural features which initiate lipid binding and the precise conformational state of the protein in the lipid bound state. With regard to the former, it has been proposed that apoLp-III initially binds lipid surfaces via an exposed hydrophobic region at one of its ends, whereupon the protein opens to expose its hydrophobic interior [31,39]. In contrast, studies by Zhang et ah [45"] of the interaction of apoLp-III with various phospholipids have provided evidence that M. sexta apoLp-III interacts strongly with negatively charged lipids, perhaps through ionic interactions with positive charges on the side chains of the 23 lysine, two arginine and four histidine residues present. Furthermore, on the basis of the distribution of positively charged residues at the interface between the hydrophobic and hydrophilic faces of the amphipathic 0t-helices, it is postulated that ionic interactions can bring the protein into the proximity of the
The structures of insect lipoproteins Ryan
bilayer, inducing a conformational change that results in bilayer disruption and protein-lipid interaction. In further studies of apoLp-IlI-phospholipid interactions, it has been shown that the protein spontaneously disrupts sonicated bitayers of DMPC resulting in formation of a uniform population of disk-like particles [46"]. Characterization of these particles reveals a DMPC:apoLp-III molar ratio of 102:1 with each disk containing six molecules of apoLp-III. Calculations based on disk composition, dimensions and molecular weight, together with the dimensions of lipid-free apoLp-III obtained from X-ray crystallography, have led to a model in which apoLp-III undergoes a conformational change exposing its hydrophobic interior, which interacts with the exposed fatty acyl chains of the disklike DMPC bilayer. Consideration of possible orientations of the protein resulted in the concept that the protein orients with its Ix-helices perpendicular to the DMPC fatty acyl chains (Fig. 2). Recent spectroscopic studies of apoLp-III have provided further support for a major conformational change upon interaction with lipid surfaces. In the case of M. sexta apoLp-III, which lacks tryptophan and contains a single tyrosine residue at position 145, fluorescence spectroscopy has been employed. In the lipid-free helix bundle conformation, tyrosine fluorescence is highly quenched with an observed quantum yield of 0.015. Upon interaction with DMPC, however, there is a dramatic increase in tyrosine fluorescence quantum yield to 0.110 [46"]. Since CD spectroscopy data reveal that the protein retains its ix-helix content in both states, it is unlikely that the fluorescence enhancement observed upon lipid binding is the result of changes in helix content. Alternatively, it is conceivable that tyrosine fluorescence is quenched in the lipid-free helix bundle conformation of M. sexta apoLp-III as a result of the proximity of an amino acid side chain carboxyl group on a neighboring helix, which can serve as an acceptor of the hydroxyl proton of the excited tryosine, thereby quenching fluorescence [47]. When the protein alters its conformation upon interaction with a lipid surface, however, the orientation of neighboring helices is altered, causing the repositioning of the putative quenching carboxyt side chain, which, as
a result, is too distant to serve as an acceptor of the excited tyrosine side chain hydroxyl proton. In studies of L. migratoria apoLp-III, near-UV CD spectroscopy provides evidence that the two tryptophans and two phenylalanines adopt a new conformation in a more hydrophobic environment upon binding to DMPC [41"]. This is indicated by the dramatic complete reversal in sign and red shift ofnear-UV CD extrema attributed to these residues in the lipid-bound as opposed to the lipid-free state (Fig. 3). Importantly, far-UV spectra reveal a similar amount of Ix-helix content in both states [41"]. The unusually well defined near UV spectra obtained with L. migratoria apoLp-III compared with other proteins and apolipoproteins [48] is likely to be due to the absence of tyrosine and cysteine in this protein. A key question unanswered by studies of model complexes pertains to the conformation and orientation adopted by apoLp-III when it binds to the surface of lipophorin. On the basis of the observed close correlation between apoLp-III binding and DAG accumulation, it has been suggested that these components may interact in some manner. Thus it can be envisioned that as the DAG content of lipophorin increases, some portion of this lipid may partition to the surface monolayer. This partitioning, which is consistent with the composition of the particles, may have a destabilizing effect on particle structure (similar to that of DAG on phospholipid bilayer membranes [49]), due to the lack of a significant polar head group on DAG compared with phospholipid. Thus it is envisioned that-apoLp-III may associate with the lipoprotein surface in order to occupy defects in the surface monolayer created by the presence of DAG. In studies employing 31p-NMR to examine the diffusion coefficient and intrinsic viscosity of the phospholipid components of different lipophorins, evidence has been obtained that, in those subspecies that lack apoLp-III, there is greater phosphollpid mobility [50]. On the basis of this, and other data, it has been hypothesized that interactions between charged groups of apoLp-III and those present in the polar head group of lipophorin phospholipids may have a role in stabilizing apoLp-III interactions with the lipophorin surface. A model of such an interaction between an Ix-helical
Fig. 2. Hypothetical representaion of an apoLp-III-DMPC complex in cross-section. Six apoLp-III molecules surround the DMPC bilayer via binding of the amphipathic (x-helices to the phospholipids, in a manner perpendicular to the fatty acyl chains. Reproduced with permission from [46"].
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As illustrated in this review, lipid transport processes in insect hemolymph offer a system whereby structural information on lipid binding protein can be obtained. Data presented herein support the concept that major parallels exist between processes in insect hemolymph and those in vertebrate species. Future studies designed to address key questions related to the mechanism and specificity of lipid-protein interactions in this model system should increase our understanding oflipoprotein metabolism in general.
Acknowledgements
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Fig. 3. Far-UV and aromatic (inset) circular dichroism specta of L. migratoria apoLp-III. Lipid-free apoLp-III is shown as solid line and apoLp-III-DMPC complexes as dashed line. Spectra were recorded in 5 0 m M sodium phosphate (pH 7.0) and l O0mM KCl. Reproduced with permission from [41•].
seganent of apoLp-Ill and a DAG-containg lipoprotein surface monolayer has been presented [50]. In an effort to gain further evidence for the concept that the presence of DAG in the lipoprotein surface provides a binding site for apoLp-IIl which, in turn, stabilizes the particle structure, phospholipase C has been used to convert lipoprotein phospholipid into DAG. On the basis of previous studies demonstrating that treatment of human LDL particles with phospholipase C causes aggregation [51], we have demonstrated that aggregation of LDL in response to phospholipase C is prevented when apoLpIII, or human apolipoprotein A-I, is included in the incubation [52]. Furthermore, phospholipase C treatment induces a stable association of apolipoprotein with the lipoprotein surface, providing evidence that the presence of surface DAG provides a site for interaction. Recently, using lipophorins enriched with exogenous short chain DAG, a correlation between DAG enrichment and apoLp-III association has been observed [53]. Since other apolipoproteins, including human apolipoprotein A-I, function in a similar manner, these observations provide support for the concept that exchangeable apolipoproteins from vertebrate and invertebrate sources are functionally homologous. This concept is also con-
Work from the author's laboratory was supported by grants from the Medical Research Council of Canada and the Alberta Heart and Stroke Foundation. The author is a recipient o f a Scientist Award from the Medical Research Council and a Scholarship Award from the Alberta Heritage Foundation for Medical R e search. I thank P D ' O b r e n a n and L Hicks for assistance with the figures and my colleagues within the Lipid and Lipoprotein Research Group and the Department o f Biochemistry for continued collaborative support.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •, of outstanding interest 1.
Law IH, Ribeiro JMC, Wells MA: Biochemical Insights Derived from Insect Diversity. Annu Rev Biochem 1992, 61:87-111.
2.
Van der Horst DJ: Lipid Transport Function of Lipoproteins in Flying Insects. Biochim Biophys Acta 1990, 1047:195-211.
3.
Blomquist GJ, Nelson DR, de Renobales M: Chemistry, Bio-
chemistry and Physiology of Insect Cuticular Lipids. Arch Insect Biochem Physiol 1987, 6:227-265. 4.
Chino H: Lipid Transport: Biochemistry of Hemolymph Lipophorin. In Comprehensive Insect Physiology Biochemistry and Pharmacology. Edited by Kerkut GA, Gilbert LI. Oxford: Pergamon Press; 1985, 10:115-136.
5.
Prasad SV, Fernando-Warnaknlasuriya GJP, Sumida M, Law JH, Wells MA: Lipoprotein Biosynthesis in the Larvae of the Tobacco Hornworn, Manduca sexta. J Biol Chem 1986, 261:17174-17176.
6.
Weers PMM, Van der Horst DJ, Van Marrewijk WJA, Van den Eijnden M, Van Doom J, Beenakkers AMTh: Biosynthesis and Secretion of Insect Lipoprotein. J Lipid Res 1992, 33:485-491.
7.
Shelby KS, Chippendale GM: Assembly and Secretion of Lipo-
phorin by the Larval Fat Body of the Southwestern Corn Borer, Diatraea grandiosella: an in vitro Study. Arch Insect Biochem Physiol 1991, 18:203-217.
The structures of insect l i p o p r o t e i n s Ryan 8. •
Weers PMM, Van Marrewijk WJA, Beenakkers AMTh, Van der Horst DJ: Biosynthesis of Locust Lipophorin: Apolipophorins I and II Originate from a Common Precursor. J Biol Chem 1993, 268:4300-4303. Pulse chase experiments reveal that the two integral apolipoproteins of lipophorin arise from a common precursor protein through post-translational cleavage of a proapolipophorin. These results explain the constant 1:1 stoichiometry of these apolipophorins in all subspecies.
sity Lipoprotein Without Denaturation. Biochemistry 1992, 31:4509-4514. 28.
Ryan RO, Wessler AN, Ando S, Price HM, Yokoyama S: Insect Lipid Transfer Particle Catalyzes Bidirectional Vectorial Transfer of Diacylglycerol from Lipophorin to Human Low Density Lipoproteln. J Biol Chem 1990, 265:10551-10555.
29.
Blacklock BJ, Smillie M, Ryan RO: Manduca sexta Lipid Transfer Particle Can Facilitate Lipid Transfer via a Carrler-Mediated Mechanism. J Biol Chem 1992, 267:14033-14037.
9.
Soulages JL, Brenner RR: Study of the Composition-Structure Relationship of Lipophorios. J Lipid Res 1991, 32:407-415.
10.
Katagiri C, Sato M, Tanaka N: Small-Angle X-Ray Scattering Study of Insect Lipophorin. J Biol Chem 1987, 262:15857-15861.
30.
Singh TKA, Scraba DG, Ryan RO: Conversion of Human Low Density Lipoprotein Into a Very Low Density Lipoproteln-Like Particle in vitro. J Biol Chem 1992, 267:9275-9280.
11.
Ryan RO: Dynamics of Insect Lipophorin Metabolism. J Lipid Res 1990, 31:1725-I 739.
31.
Wells MA, Ryan RO, Kawooya JK, Law JH: The Role of Apolipophorin III in in vivo Lipoproteln Interconversions in Adult Manduca sexta. J Biol Chem 1987, 262:4172-4176.
12.
PrasadSV, Ryan RO, Law JH, Wells MA: Changes in Lipoprotein Composition During Larval-Pupal Metamorphosis of an Insect, Manduca sexta. J Biol Chem 1986, 261:558-562.
32.
Singh TKA, Blacklock BJ, Wientzek M, Ryan RO: A Turbidimetric Assay of Facilitated Lipid Transfer. Annal Biochem 1992, 206:137-141.
13.
Ryan RO, Kay CM, Oikawa K, Liu H, Bradley R, Scraba DG: Effect of Particle Lipid Content on the Structure of Insect Lipophorins. J Lipid Res 1992, 33:55-63.
33.
14.
Surholt B, Goldberg J, Schulz TKF, Beenakkers AMTh, Van der Horst DJ: Lipoproteins Act as a Reusable Shuttle for Lipid Transport in the Flying Death's-Head Hawkmoth, Acherontia atropos. Biochim Biophys Acta 1991, 1086:15-21.
Wheeler CH, Goldsworthy GJ: Protein-Lipoprotein Interactions in the Haemolymph of Locusta During the Action of Adlpokinetic Hormone: the Role of Ct-Proteins. J Insect Physiol 1983, 29:349-354.
34.
Shapiro JP, Law JH: Locust Adipokinetlc Hormone Stimulates Lipid Mobilization in Manduca sexta. Biochem Biophys Acta 1983, 15:924-931.
Downer RGH, Chino H: Turnover of Protein and Diacylglycerol Components of Lipophorin in Locust Hemolymph. Insect
35.
Van der Horst DJ, Van Doom JM, Beenakkers AMTh: Hormone Induced Rearrangement of Locust Haemolymph Lipoproteins: the Involvement of Glycoprolein C2. Insect Biochem 1984, 14:495-504:
36.
Cole KD, Fernando-Warnakulasuriya GJP, Boguski MS, Freeman M, Gordon JI, Clark WA, Law JH, Wells MA: Primary Structure and Comparative Sequence Analysis of an Insect Apolipoprotein. Apolipophorin III from Manduca sexta. ] Biol Chem 1987, 262:11794-11800.
37.
Soulages JL, Wells MA: Lipophorin: the Structure of an Insect Lipoprotein and its Role in Lipid Transport in Insects. Adv Protein Chem 1994, 45:371-414.
38.
Kawooya JK, Meredith SC, Wells MA, K~zdy FJ, Law JH: Physical and Surface Properties of Insect Apollpophorin III. J Biol Chem 1986, 261:13588-13591.
39.
Breiter DR, Kanost MR, Benning MM, Wesenberg G, Law JH, Wells MA, Rayment I, Holden HM: Molecular Structure of an Apolipoprotein Determined at 2.5-,~ Resolution. Biochemistry 1991, 30:603-608.
15.
Biochem 1985, 15:627-630.
16.
Ryan RO, Prasad SV, Henriksen EJ, Wells MA, Law JH: Lipoprotein Interconversions in an Insect, Manduca sexta. Evidence for a Lipid Transfer Factor in the Hemolymph. J Biol Chem 1986, 261:563-568.
17.
Ryan RO, Wells MA, Law JH: Lipid Transfer Protein from Manduca sexta Hemolymph. Biochem Biophys Res Common 1986, 136:260-265.
18.
Ryan RO, Senthilathipan KR, Wells MA, Law JH: Facilitated Diacylglycerol Exchange Between Insect Hemolymph Lipophorins. Properties of Manduca sexta Lipid Transfer Particle. J Biol Chem 1988, 263:14140-14145.
19.
20.
Ryan RO, Howe A, Scraba DG: Studies of the Morphology and Structure of the Plasma Lipid Transfer Particle from the Tobacco Hornworm, Manduca sexta. J Lipid Res 1990, 31:871-879. Takeuchi N, Chino H: Lipid Transfer Particle in the Hemolymph of the American Cockroach: Evidence for its Capacity to Transfer Hydrocarbons Between Lipophorin Particles. J Lipid Res 1993, 34:543-551.
21.
Van Heusden MC, Law JH: An Insect Lipid Transfer Particle Promotes Lipid Loading from Fat Body to Lipoprotein. J Biol Chem 1989, 264:17287-17292.
22.
Liu H, Ryan RO: Role of Lipid Transfer Particle in Transformation of Lipophorin in Manduca sexta Oocytes. Biochim Biophys Acta 1991, 1085:112-118.
23.
Tall AR: Plasma Cholesteryl Ester Transfer Protein. J Lipid Res 1993, 34:1255-1274.
24.
Silver ET, Scraba DG, Ryan RO: Lipid Transfer Particle-Induced Transformation of Human High Density Lipoprotein into Apolipoprotein A-I-Deficient Low Density Particles. J Biol Chem 1990, 265:22487-22492.
25.
Tall AR, Small DM: Solublization of Phospholipid Membranes by Human Plasma High Density Lipoproteins. Nature 1977, 265:] 63-164.
26.
Tall AR, Deckelbaum RJ, Small DM, Shipley GG: Thermal Behaviour of Human Plasma High Density Lipoprotein. Biochim Biophys Acta 1977, 487:145-153.
27.
Ryan RO, Yokoyama S, Liu H, Czarnecka H, Oikawa K, Kay CM: Human Apolipoprotein A-I Liberated From High Den-
40. •
H~rd K, Van Doom JM, Thomas-Oates JE, Kamerling JP, Van der Horst DJ: Structure of the Asn-Linked Oligosaccharides of Apolipophorin III From the Insect Locusta ml. gratorla. Carbohydrate-Linked 2-aminoethylphosphonate as a Constituent of a Glycoprolein. Biochemistry 1993, 32:766-775. The structure of the carbohydrate moieties of L. migratoria apoLp-III are reported. A remarkable feature is the presence of 2-aminoethylphosphonate bound to mannose and/or N-acetylglucosamine moieties. This is the first example of a glycoprotein containing 2-aminoethylphosphonate, which explains the previously observed lack of interaction of this protein with various lectins. 41. •
Weers PMM, Kay CM, Oikawa K, Wientzek M, Van der Horst DJ, Ryan RO: Factors Affecting lhe Stability and Conforma.. lion of Locusta migratorla Apollpophorin III. Biochemistry 1994 33:3617-3624. It is demonstrated that enzymatic deglycosylation of L. migratoria apoLpIII induces a time-dependent loss of helical structure with complete denaturation occurring over several days. The data suggest that the carbohydrate moiety of the protein functions in stabilization of the helix bundle. Also, spectroscopic data are presented which support a major reorientation of the protein upon interaction with a lipid surface. 42. •
Ryan RO, Oikawa K, Kay C: Conformational, Thermodynamic and Stability Properties of Manduca sexta Apolipophorin III. J Biol Chem 1993, 268:1525-1530. The structural stability of lipid-free M. sexta apoLp-III is determined. It is reported that apoLp-IH adopts a loosely folded structure that is sensitive to
505
506
Lipids environmental perturbation. It is postulated that salt bridge formation between oppositely charged amino acid side chains serves as a stabilizing force for the protein in the lipid-free helix bundle conformation. 43.
Wetterau JR, Aggerbeck LP, Rail, SC Jr, Weisgraber KH: Human apolipoprotein E3 in Aqueous Solution I. Evidence for Two Structural Domains. J Bio/ Chem 1988, 263:6240-6248.
44.
Marqusee S, Baldwin RL: Helix Stabilization of Glu-...Lys÷ Salt Bridges in Short Peptides of de novo Design. Proc Natl Acad
45. •
47.
48.
49.
Sci U S A 1987, 84:8898-8902.
50.
ZhangY, Lewis RNAH, McEIhaney RN, Ryan RO: Calorimetric and Spectroscopic Studies of the Interaction of Manduca sexta Apolipophorin III with Zwitlerionic, Anionic and Nonionic Lipids. Biochemistry 1993, 32:3942-3952.
51.
Differential scanning calorimetry and NMR spectroscopy are employed to evaluate the interaction of M. sexta apoLp-III with a series of lipids. The data indicate a strong interaction of apoLp-III with anionic lipids consistent with the concept that the binding of apoLp-III to lipid bilayers is mediated through ionic interactions at the lipid surface. Initial interaction of the protein is proposed to be followed by a conformational change to expose its hydrophobic interior which results in bilayer disruption. Wientzek M, Kay CM, Oikawa K, Ryan RO: Binding of Insect Apolipophorin III to Dimyristoylphosphalidylcholine Vesicles: Evidence for a Conformational Change. J Biol Chem 1994, 269:4605-4612. The interaction of M. sexta apoLp-III with DMPC multilamellar vesicles revealed the formation of a uniform population of disk-like particles whose composition and properties are consistent with apoLp-III interaction with DMPC fatty acyl chains around the perimeter of the bilayer disk. Calculations based on the particle dimensions, apoLp-III size and lipid content suggest apoLp-III may open about a hinge region as predicted by Holden and coworkers (see ref [39]). Fluorescence data are reported which also suggest a conformational change in the protein upon lipid interaction.
52.
53.
46. •
54.
Stryer /: Excited-State Proton-Transfer Reactions. A Deuterium Isotope Effect on Fluorescence. J Am Chem Soc 1966, 88:5708-5712. Lux SE, Hirz R, Shrager I, Gotto AM: The Influence of Lipid on the Conformation of Human Plasma High Density Apolipoproteins. J giol Chem 1972, 247:2598-2606. Das, S, Rand, RP: Modification by Diacylglycerol of the Structure and Interaction of Various Phospholipid Bilayer Systems. Biochemistry 1986, 25:2282-2289. Wang J, Liu H, Sykes BD, Ryan RO: 31p-NMR Study of the Phospholipid Moiety of Lipophorin Subspecies. Biochemistry 1992, 31:8706-8712. Suits AG, Chait A, Aviram M, Heinecke JW: Phagocytosis of Aggregated Lipoproteins by Macrophages: kow Density Lipoprotein Receptor-Dependent Foam Cell Formation. Proc Nat/ Acad Sci USA 1989, 86:2713-2717. Liu H, Scraba DG, Ryan RO: Prevention of Phospholipase-C Induced Aggregation of Low Density Lipoprotein by Amphipathic Apolipoproteins. FEBS Lett 1993, 316:27-33. Soulages JL, Wells MA: Effect of Diacylglycerol Content on Some Physicochemical Propedies of the Insect Lipoproteln, Lipophorin. Correlation with the Binding of Apolipophorin III. Biochemistry 1994, 33:2356-2362. Wilson C, Wardell MR, Weisgraber KH, Mahley RW, Agard DA: Three Dimensional Structure of the LDL ReceptorBinding Domain of Human Apolipoprotein E. Science 1991, 252:1817-1822.
RO l,Lyan, Lipid and Lipoprotein Research Group, 328 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.