Interaction of plasma-derived lipid transfer protein with macrophages in culture

This study investigates the ability of human plasma-derived lipid transfer protein to facilitate lipid transfer to and from intact viable cells in culture. Mouse peritoneal macrophages or J774 macrophages were preincubated with acetylated low density lipoprotein and [3H]oleate/albumin to promote the intracellular synthesis and accumulation of cholesteryl [3H]oleate and 3H-labeled triglyceride. The addition of partially purified lipid transfer protein to cultures of lipid-loaded macrophages resulted in a time and concentration-dependent transfer of radiolabeled cholesteryl ester and triglyceride from macrophages to the medium. At 48 hr, lipid transfer protein facilitated the net transfer of 16 and 11% of cellular cholesteryl ester and triglyceride radioactivity, respectively, to the medium; transfer in the absence of the lipid transfer protein was less than 2%. The transfer of cholesteryl ester radioactivity was accompanied by a similar decrease in cellular cholesteryl ester mass indicating a net transfer event. Lipid transfer from cells was not dependent on the presence of a lipoprotein acceptor in the medium; however, low and high density lipoproteins present at 200 micrograms cholesterol/ml did significantly stimulate the transfer protein-facilitated efflux of these lipids. Lipid transfer protein did not appear capable of transferring radiolabeled lipid from low density or high density lipoprotein to macrophages. Radiolabeled cholesteryl ester and triglyceride transferred from cells to the medium by lipid transfer protein were associated with large molecular weight (greater than 2 x 10(6)) components in the medium with an average density greater than 1.21 g/ml; these lipids were not associated with lipid transfer protein itself. However, these radiolabeled lipids were readily incorporated into low or high density lipoproteins when these lipoproteins were added to the medium either during or after its incubation with cells. It is concluded that lipid transfer protein can facilitate the net efflux of cholesteryl esters from intact, living macrophages. These studies suggest a novel and potentially antiatherogenic role for lipid transfer protein.     

Specificity of lipid transfer protein for molecular species of cholesteryl ester

The capacity of the plasma-derived lipid transfer protein to facilitate the transfer of various cholesteryl ester species has been investigated. Four different molecular species of cholesteryl ester were incorporated into either reconstituted high density lipoproteins or phosphatidylcholine liposomes, and the resulting particles were used as donors in standardized lipid transfer assays. With reconstituted high density lipoproteins as substrate, the rate of transfer of cholesteryl esters was cholesteryl oleate greater than cholesteryl linoleate greater than cholesteryl arachidonate greater than cholesteryl palmitate. The transfer rate for cholesteryl oleate was 154% of that for cholesteryl palmitate. Liposome substrates gave similar results. It is concluded that lipid transfer protein transfers all major species of cholesteryl ester found in plasma; however, the relative rates of transfer were significantly affected by acyl chain composition. The transfer rates appeared to reflect substrate specificity rather than substrate availability within the donor particle.     

Free cholesterol is a potent regulator of lipid transfer protein function

This study investigates the effect of altered lipoprotein free cholesterol (FC) content on the transfer of cholesteryl ester (CE) and triglyceride (TG) from very low- (VLDL), low- (LDL), and high-(HDL) density lipoproteins by the plasma-derived lipid transfer protein (LTP). The FC content of VLDL and HDL was selectively altered by incubating these lipoproteins with FC/phospholipid dispersions of varying composition. FC-modified lipoproteins were then equilibrated with [3H] TG, [14C]CE-labeled lipoproteins of another class to facilitate the subsequent modification of the radiolabeled donor lipoproteins. LTP was added and the extent of radiolabeled TG and CE transfer determined after 1 h. With either LDL or VLDL as lipid donor, an increase in the FC content of these lipoproteins caused a concentration-dependent inhibition (up to 50%) of CE transfer from these particles, without any significant effect on TG transfer. In contrast, with HDL as donor, increasing the HDL FC content had little effect on CE transfer from HDL, but markedly stimulated (up to 2.5-fold) the transfer of TG. This differential effect of FC on the unidirectional transfer of radiolabeled lipids from VLDL and HDL led to marked effects on LTP-facilitated net mass transfer of lipids. During long-term incubation of a constant amount of LTP with FC-modified VLDL and HDL, the extent of net mass transfer was linearly related to lipoprotein FC content; a 4-fold increase in FC content resulted in a 3-fold stimulation of the CE mass transferred to VLDL, which was coupled to an equimolar, reciprocal transfer of TG mass to HDL. Since lipid transfer between lipoproteins is integral to the process of reverse cholesterol transport, we conclude that lipoprotein FC levels are a potent, positive regulator of the pathways involved in sterol clearance. FC may modulate lipid transfer by altering the availability of CE and TG to LTP at the lipoprotein surface.     

A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB) lipoproteins. Its activity is classically measured by incubating purified MTP or cellular homogenates with donor vesicles containing radiolabeled lipids, precipitating the donor vesicles, and measuring the radioactivity transferred to acceptor vesicles. Here, we describe a simple, rapid, and sensitive fluorescence assay for MTP. In this assay, purified MTP or cellular homogenates are incubated with small unilamellar donor vesicles containing quenched fluorescent lipids (triacylglycerols, cholesteryl esters, and phospholipids) and different types of acceptor vesicles made up of phosphatidylcholine or phosphatidylcholine and triacylglycerols. Increases in fluorescence attributable to MTP-mediated lipid transfer are measured after 30 min. MTP's lipid transfer activity could be assayed using apoB lipoproteins but not with high density lipoproteins as acceptors. The assay was used to measure MTP activity in cell and tissue homogenates. Furthermore, the assay was useful in studying the inhibition of the cellular as well as purified MTP by its antagonists. This new method is amenable to automation and can be easily adopted for large-scale, high-throughput screening.     

Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.     

Reconstitution of the lipoprotein cholesteryl ester transfer process using isolated rat ovary plasma membranes.

Steroidogenic cells are able to utilize lipoprotein-derived cholesteryl esters for steroidogenesis without internalizing intact lipoproteins. In the current report, we provide evidence that an early step in this process may be the selective extraction of cholesteryl esters at the cell (plasma membrane) surface. We have used a highly purified plasma membrane preparation from rat luteinized ovaries for incubation with rat- and human-derived high density (HDL) and low density (LDL) lipoproteins. The lipoproteins were modified with residualizing [125I]apoprotein or [3H]cholesteryl ester markers. Following trypsin treatment to remove intact surface-bound apoprotein particles, the membranes were analyzed for transferred radioactive labels. The results show that all the lipoproteins tested could serve as cholesteryl ester donors. Although far more [3H]cholesteryl ester than [125I]apoprotein radioactivity was transferred to plasma membranes in each case, and varied with the ligand used, the total (net) mass of cholesteryl ester transferred was comparable with the different lipoproteins. These data were confirmed using direct chemical methodology. Transfer was found to be specific for cholesteryl esters or ethers and did not involve other lipoprotein core lipids tested. Endomembranes from the same tissue could not substitute for plasma membranes as the primary cholesteryl ester acceptor. These results provide evidence that a reconstituted lipoprotein-plasma membrane system can simulate the cholesteryl ester extraction process described in situ and suggest uses for this methodology in future experiments designed to understand the transfer process.     

Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro.

The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.     

The transfer of lipids from protein-free lipoprotein models to human fibroblasts in culture

Lipid microemulsions were prepared by sonication of mixtures of cholesteryl ester, triacylglycerol, phosphatidylcholine and cholesterol in aqueous dispersions and were purified by gel filtration. The resulting emulsion particles were characterized by differential scanning calorimetry, electron microscopy and analytical gel filtration and were shown to have the size and general organization of low-density lipoprotein. The lipid microemulsions were used as protein-free plasma lipoprotein models for studies of the receptor-independent transfer of lipids to human fibroblasts in culture. The transfer rate of [3H]cholesterol increased with the donor concentration and with the molar ratio between cholesterol and phosphatidylcholine in the donor particles. A maximal transfer value of 1 nmol per mg protein per h was obtained for cholesterol/phosphatidylcholine 1:1 particles. There was a profound temperature effect on the cholesterol transfer. The effect of altering the core lipid of the emulsion particles on the [3H]cholesterol transfer rate was small giving a somewhat higher rate with cholesteryl oleate and cholesteryl stearate than with cholesteryl linoleate. Addition of trioleoylglycerol to the cholesteryl ester core had no effect on the transfer rate. The transfer rate of palmitoyl[14C]oleoylphosphatidylcholine was found to be about 1/5 of that obtained for [3H]cholesterol. About 50% of the cell-associated [14C]cholesteryl oleate was found in the trypsin-releasable pool, while 25% was internalized by the cells at a rate of 0.06 nmol X mg-1 X h-1. Trioleoylglycerol was internalized at the same rate as the cholesteryl ester. Our data suggest that the lipoprotein lipid composition may play a role in the receptor-independent cellular uptake of cholesterol.     

Cholesteryl oleyl and linoleyl ethers do not trace their ester counterparts in animals with plasma cholesteryl ester transfer activity

Cholesteryl ethers are nonhydrolyzable tracers of cholesteryl esters. We report here that the ethers are not legitimate tracers of esters in systems involving plasma cholesteryl ester transfer activity. On intravenous injection of doubly labeled high density lipoproteins into rabbits, cholesteryl ester tracer was more rapidly transferred to other lipoprotein fractions than was cholesteryl ether tracer. In direct assays in vitro, the rate of transfer of esters was about two times that of the ether. This difference was not due to tracer impurity or lability of 3H, did not depend on the nature of the donor or acceptor lipoprotein, and was similar for cholesteryl ester transfer activities of both human and rabbit origin.     

Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.     

Preferential enrichment of large-sized very low density lipoprotein populations with transferred cholesteryl esters

The effect of lipid transfer proteins on the exchange and transfer of cholesteryl esters from rat plasma HDL2 to human very low (VLDL) and low density (LDL) lipoprotein populations was studied. The use of a combination of radiochemical and chemical methods allowed separate assessment of [3H]cholesteryl ester exchange and of cholesteryl ester transfer. VLDL-I was the preferred acceptor for transferred cholesteryl esters, followed by VLDL-II and VLDL-III. LDL did not acquire cholesteryl esters. The contribution of exchange of [3H]cholesteryl esters to total transfer was highest for LDL and decreased in reverse order along the VLDL density range. Inactivation of lecithin: cholesterol acyltransferase (LCAT) and heating the HDL2 for 60 min at 56 degrees C accelerated transfer and exchange of [3H]cholesteryl esters. Addition of lipid transfer proteins increased cholesterol esterification in all systems. The data demonstrate that large-sized, triglyceride-rich VLDL particles are preferred acceptors for transferred cholesteryl esters. It is suggested that enrichment of very low density lipoproteins with cholesteryl esters reflects the triglyceride content of the particles.     

Plasma cholesteryl ester transfer protein has binding sites for neutral lipids and phospholipids

The plasma cholesteryl ester-transfer protein (CETP, Mr 74,000) promotes exchange of both neutral lipids and phospholipids (phosphatidylcholine, PC) between lipoproteins. To investigate the mechanism of facilitated lipid transfer, CETP was incubated with unilamellar egg PC vesicles containing small amounts of cholesteryl ester (CE) or triglyceride, and then analyzed by gel filtration chromatography. There was rapid transfer of radiolabeled CE or triglyceride and PC from vesicles to CETP. The CETP with bound lipids was isolated and incubated with low density lipoproteins (LDL), resulting in transfer of the lipids to LDL. The CETP bound up to 0.9 mol of CE or 0.2 mol of triglyceride and 11 mol of PC/mol of CETP. para-Chloromercuriphenylsulfonate, an inhibitor of CE and triglyceride transfer, was found to decrease the binding of radiolabeled CE and triglyceride by CETP. Under various conditions the CETP eluted either as an apparent monomer with bound lipid (Mr 75,000-93,000), or in complexes with vesicles. The distribution of CETP between these two states was influenced by the presence of apoA-I or albumin, incubation time, vesicle/CETP ratio, and buffer pH and ionic strength. The results indicate that the CETP has binding sites for CE, triglyceride, and PC which readily equilibrate with lipoprotein lipids and suggest that CETP can act as a carrier of lipid between lipoproteins.     

Concentration of neutral lipids in the phospholipid surface of substrate particles determines lipid transfer protein activity

To better understand the mechanism of lipid transfer protein (LTP) action and the effects of altered lipoprotein composition on its activity, we evaluated the dependence of LTP activity on the concentrations of cholesteryl ester (CE) and/or triglyceride (TG) in the phospholipid bilayer of substrate particles. Phosphatidylcholine (PC)-cholesterol liposomes containing up to 2 mole% TG and/or CE were prepared by cholate dialysis and used as either the donor of lipids to, or the acceptor of lipids from, low density lipoproteins (LDL). CE or TG transfer from liposomes of varying neutral lipid content to LDL showed saturation kinetics with an apparent Km of less than or equal to 0.2 mole%. Throughout this concentration-dependent response. PC transfer, which depended on the same LTP-donor particle binding interactions as those required for neutral lipid transfer, was essentially unchanged. Lipid transfer in the reverse direction (from LDL to liposomes of varying neutral lipid content) followed the same kinetics showing that transfer between the two particles is tightly coupled and bidirectional. When liposomes contained both TG and CE, these lipids competed for transfer in a manner analogous to that previously noted with lipoprotein substrates. In conclusion, CE and TG transfer activities are determined by the concentration of these lipids in the phospholipid surface of donor and acceptor particles. At low TG and CE concentrations, LTP bound to the liposome surface as indicated by PC transfer, but only a portion of these interactions actually facilitated a neutral lipid transfer event. Thus, the overall rate of neutral lipid transfer, and the competition between TG and CE for transfer, depend on the concentrations of these lipids in the phospholipid layer.     

Familial cholesteryl ester transfer protein deficiency is associated with triglyceride-rich low density lipoproteins containing cholesteryl esters of probable intracellular origin

The net transfer of core lipids between lipoproteins is facilitated by cholesteryl ester transfer protein (CETP). We have recently documented CETP deficiency in a family with hyperalphalipoproteinemia, due to a CETP gene splicing defect. The purpose of the present study was to characterize the plasma lipoproteins within the low density lipoprotein (LDL) density range and also the cholesteryl ester fatty acid distribution amongst lipoproteins in CETP-deficient subjects. In CETP deficiency, the conventional LDL density range contained both an apoE-rich enlarged high density lipoprotein (HDL) (resembling HDLc), and also apoB-containing lipoproteins. Native gradient gel electrophoresis revealed clear speciation of LDL subclasses, including a distinct population larger in size than normal LDL. Anti-apoB affinity-purified LDL from the CETP-deficient subjects were shown to contain an elevated triglyceride to cholesteryl ester ratio, and also a high ratio of cholesteryl oleate to cholesteryl linoleate, compared to their own HDL or to LDL from normal subjects. Addition of purified CETP to CETP-deficient plasma results in equilibration of very low density lipoprotein (VLDL) cholesteryl esters with those of HDL. These data suggest that, in CETP-deficient humans, the cholesteryl esters of VLDL and its catabolic product, LDL, originate predominantly from intracellular acyl-CoA:cholesterol acyltransferase (ACAT). The CETP plays a role in the normal formation of LDL, removing triglyceride and transferring LCAT-derived cholesteryl esters into LDL precursors.     

Affinity of lipid transfer protein for lipid and lipoprotein particles as influenced by lecithin-cholesterol acyltransferase

The effects of lecithin-cholesterol acyltransferase (LCAT) on the transfer of cholesterol esters mediated by lipid transfer protein (LTP) and its affinity for lipid and lipoprotein particles were investigated. When the single bilayer vesicle preparations (containing phosphatidylcholine, cholesterol, cholesteryl ester, and apolipoprotein- (apo) A-I at the molar ratio of 90:30:1.2:0.18) or high density lipoprotein 3 (HDL3) were used as the cholesteryl ester donor and low density lipoproteins (LDL) as the acceptor, the transfer activity of LTP was enhanced by the addition of low concentrations of LCAT. In contrast, no enhancement of cholesteryl ester transfer was observed upon addition of LCAT to either the discoidal bilayer particle preparations (containing phosphatidylcholine, cholesterol, cholesteryl ester, and apo-A-I at the molar ratio of 90:30:1.2:1.0) or high density lipoprotein 2 (HDL2). Although both apo-A-I and apo-A-II promoted the transfer of cholesteryl ester from vesicles to LDL, the additional enhancement of the transfer by LCAT was observed only with the vesicles containing apo-A-I. Gel permeation chromatography of LTP/vesicle and LTP/HDL3 mixtures in the presence and absence of LCAT showed that the affinity of LTP for both the vesicles and HDL3 increased upon addition of LCAT. In contrast, neither HDL2 nor discoidal bilayer particles showed any significant enhancement of LTP binding upon addition of LCAT. By using LCAT covalently bound to Sepharose 4B, a maximal interaction between LTP and bound LCAT was shown to occur at the ionic strength of 0.16. Deviation from this ionic strength reduced the extent of the interaction. At the ionic strength of 0.01 and 0.5, the elution volume of LTP was identical to that of bovine serum albumin.     

Lipoprotein interconversions in an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph

Hemolymph lipoproteins (lipophorins) of adult Manduca sexta are disinct from larval forms in density, lipid content, composition, and the presence of a third, low molecular weight apoprotein. Generally, only one lipoprotein species exists in M. sexta hemolymph during any given life stage. Progression through the life cycle results in alterations of existing lipoproteins to produce new forms, without new protein synthesis. The observed alterations in lipoprotein density could result from facilitated lipid transfer in insect hemolymph. An in vitro assay of facilitated lipid transfer was developed which employs a high density lipophorin from the wandering larva (density = 1.18 g/ml) as acceptor and adult low density lipophorin (density = 1.03 g/ml) as donor. Adult lipophorin-deficient hemolymph was shown to catalyze a time-dependent equilibration of the starting lipoproteins to produce a new intermediate lipophorin, Lp-I. Hydrodynamic experiments on the donor, acceptor, and product lipoproteins excluded fusion as the mechanism whereby Lp-I is produced. Thus, it is concluded that Lp-I results from facilitated net lipid transfer from low to high density lipoprotein. Furthermore, experiments conducted with radioiodinated donor and radioiodinated acceptor lipoproteins demonstrated that apoprotein exchange does not occur during the lipid transfer reaction. When donor lipoprotein was labeled in the lipid moiety with carbon-14, evidence of diacylglycerol and phospholipid exchange was obtained. Partial characterization of the lipid transfer factor revealed a relationship between incubation time, donor concentration, acceptor concentration, lipophorin-deficient hemolymph concentration, and transfer activity, as measured by Lp-I production. It is concluded that lipophorin-deficient hemolymph contains one or more factor(s) that catalyze net lipid transfer as well as diacylglycerol and phospholipid exchange between lipophorins to produce a single form at equilibrium.     

Action of a microbial glycerophospholipid:cholesterol acyltransferase on plasma from normal and LCAT-deficient subjects

The action of a bacterial acyltransferase similar in overall reaction mechanism to the plasma enzyme lecithin:cholesterol acyltransferase (LCAT) has been studied using normal plasma and lipoproteins and plasma from LCAT-deficient patients. The microbial enzyme (GCAT) catalyzed acyl transfer using phosphatidylcholine and cholesterol in all of the lipoprotein fractions, presumably because it has no apolipoprotein cofactor. In addition, the enzyme was capable of hydrolyzing cholesteryl ester in lipoproteins but not in small unilamellar vesicles nor in micellar dispersions containing low amounts of Triton X-100. This suggests that cholesteryl ester is exposed on the surface of lipoprotein particles or that it may be transferred there quickly from the interior. Although considerable interconversion of radiolabeled cholesterol and cholesteryl ester could be demonstrated upon treatment of normal plasma or lipoproteins with the enzyme, there was little change in the actual amount of either steroid. This indicates that the rate of cholesteryl ester formation is very similar to the rate of hydrolysis. The relative proportions of cholesterol and cholesteryl ester in normal plasma are therefore near the equilibrium ratio for the reaction carried out by GCAT, or the ratio is controlled by the properties of the lipoproteins themselves. During reaction with the microbial acyltransferase, the ratio of cholesterol to cholesteryl ester in plasma from LCAT-deficient patients was reduced substantially, suggesting that the enzyme may have some practical applications.     

Facilitated transfer of cholesteryl ester between rough and smooth microsomal membranes by plasma lipid transfer protein

The accessibility of intracellular membrane cholesteryl esters to removal was tested with plasma lipid transfer protein as a tool. Incubation of a mixture of non-radioactive smooth microsomes + rough microsomes prelabeled with cholesteryl ester resulted in slight movement (2-4%) of radioactive cholesteryl ester into smooth microsomes. With the addition of increasing amounts of plasma lipid transfer protein to the mixture, the % transfer of cholesteryl ester into smooth microsomes progressively increased until a plateau was reached at 14%. Movement of cholesteryl ester in the reverse direction was examined with non-radioactive rough microsomes as an acceptor and smooth microsomes prelabeled with cholesteryl ester as a donor. The pattern of the % cholesteryl ester transferred in the reverse and forward direction was almost identical in the presence of plasma lipid transfer protein, showing bidirectional movement of cholesteryl ester between membranes.     

Fluorescence assay of the specificity of human plasma and bovine liver phospholipid transfer proteins

The specificities of a human plasma and bovine liver phospholipid transfer protein were studied using a fluorescence assay based on the transfer of pyrenyl phospholipids. This method was used previously to determine the mechanism of spontaneous transfer of phospholipids between model lipoproteins (Massey, J.B., Gotto, A.M., Jr. and Pownall, H.J. (1982) Biochemistry 21, 3630-3636). The pyrenyl phospholipids varied in the headgroup moiety; pyrenyl phosphatidylcholines contained different fatty acyl chains in the sn-1 position. Model high-density lipoproteins (R-HDL) consisting of apolipoprotein A-I and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were used as donor and acceptor particles. As previously shown, the bovine liver protein mediated the transfer of only phosphatidylcholine. In contrast, the human plasma protein transferred all species studied which included a phosphatidylserine, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, galactosylcerebroside, and a diacylglycerol. The activity of these transfer proteins was only slightly affected by changes in the acyl chain composition of the transferring lipid. Pyrenyl and radioactive ([3H]POPC) phospholipids were transferred with equal rates by the human transfer protein, suggesting that this protein has similar binding characteristics for pyrenyl and natural phospholipids. Spontaneous phospholipid transfer occurs by the aqueous diffusion of monomeric lipid where the rate is highly dependent on fatty acyl chain composition. In this study, no correlation between the rate of spontaneous transfer and protein-mediated transfer was found. The apparent Km values for R-HDL and low-density lipoprotein (LDL), when used as acceptors, were similar when based on the number of acceptor particles. The apparent Vmax for the bovine liver protein was identical for R-HDL and LDL but for the plasma protein Vmax was slightly higher for R-HDL. These results suggest that, like the bovine liver protein, the plasma protein functions as a phospholipid-binding carrier that exchanges phospholipids between membrane surfaces. The assay of lipid transfer proteins by pyrenyl-labeled lipids is faster and easier to perform than other current methods, which require separation of donor and acceptor particles, and is suitable for studies on the function and mechanism of action of lipid transfer proteins.     

The surface cholesteryl ester content of donor and acceptor particles regulates CETP: a liposome-based approach to assess the substrate properties of lipoproteins

Cholesteryl ester transfer protein (CETP) activity is regulated, in part, by lipoprotein composition. We previously demonstrated that CETP activity follows saturation kinetics as cholesteryl ester (CE) levels in the phospholipid surface of donor particles are increased. We propose here that the plateau of CETP activity occurs because the surface concentration of CE in the acceptor becomes rate limiting. This hypothesis was tested in CETP assays between synthetic liposomes whose CE content was varied independently. As donor CE increased, CETP activity followed saturable kinetics, but the slope of the first-order portion of the curve and the maximum achievable CE transfer rate were linearly related to the acceptor's surface CE concentration. These findings, plus studies with free cholesterol-modified LDL, strongly suggest that CE-rich donor liposomes can measure the CETP-accessible CE in acceptor lipoproteins. CETP activity from CE-rich liposomes to multiple control LDLs ranged 1.8-fold despite equivalent CETP binding capacity, suggesting that LDLs vary widely in their capacity to present CE to CETP. Thus, CETP activity depends on the surface availability of substrate lipids in the donor and acceptor. Donor liposomes with high CE content can be used to assess how subtle changes in composition alter the substrate potential of plasma lipoproteins.