Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Interaction of cholesterol, phospholipid and apoprotein in high density lipoprotein recombinants.

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of 3H cholesterol/14C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27 degrees C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1, or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. 160 X 55 A. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Membrane cholesterol uptake by recombinant lipoproteins

Recombinant lipoproteins, prepared with apo A-I isolated from human high density lipoprotein (HDL) and various phospholipids (PLs), were compared with respect to their ability to remove cholesterol (Chol) from labelled erythrocyte ghost membranes. It was found that uptake of Chol was essentially complete following an 8 h incubation at 37 degrees C. Quantitation of the amount of cholesterol taken up showed that recombinants prepared from bovine brain sphingomyelin (BBSM) or dipalmitoyl phosphatidylcholine (DPPC) acquired the highest proportion of Chol (80-140 mol/mol protein), whereas shorter chain phospholipids like dimyristoyl phosphatidylcholine (DMPC) acquired little or no membrane Chol. Chemical analysis of the incubation products indicated that this latter result was due to loss of PL, presumably to the membrane, with consequent disruption of the recombinant particle. Results with DPPC:A-I recombinants of differing PL/protein ratios and sizes showed that Chol uptake was fairly constant at 0.70 mol Chol/mol PL. It is concluded that discoidal, phospholipid-rich recombinant lipoproteins can effectively take up substantial amounts of Chol from physiological membranes, provided that the PLs utilized form micellar complexes which are capable of retaining their structural integrity during the incubation with the membranes.     

Displacement of the human apoprotein A-I by the human apoprotein A-II from complexes of (apoprotein A-I)-phosphatidylcholine-cholesterol

Reassembly experiments, involving isolated human apoproteins A-I and A-II and (dimyristoylglycerophosphocholine)-cholesterol vesicles were performed with apoprotein mixtures at apoprotein A-I/A-II molar ratios varying between 0 and 3. The apoproteins were incubated at 24 degrees C. 28 degrees C and 32 degrees C with either pure dimyristoyl-glycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 2, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after labeling the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A-I and A-II were quantified separately by immunonephelometry. The content of apoprotein A-I was also monitored by measuring the intrinsic tryptophan fluorescence. The results suggest that apoprotein A-II has a greater affinity than apoprotein A-I for the phospholipid-cholesterol vesicles and that apoprotein A-II is able to quantitatively displace apoprotein A-I from the lipid-protein complexes. The content of apoprotein A-II in the complexes increases proportionally to the concentration of apoprotein A-II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A-II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A-I is displaced by 2 mol human apoprotein A-II.     

Effects of apolipoproteins on the kinetics of cholesterol exchange

The effects of apolipoproteins on the kinetics of cholesterol exchange have been investigated by monitoring the transfer of [14C]cholesterol from donor phospholipid/cholesterol complexes containing human apolipoproteins A, B, or C. Negatively charged discoidal and vesicular particles containing purified apolipoproteins complexed with lipid (75 mol % egg PC, 15 mol % dicetyl phosphate, and 10 mol % cholesterol) and a trace of [14C]cholesterol were incubated with a 10-fold excess of neural, acceptor, small unilamellar vesicles (SUV; 90 mol % egg PC and 10 mol % cholesterol). The donor and acceptor particles were separated by chromatography on DEAE-Sepharose, and the rate of movement of labeled cholesterol was analyzed as a first-order exchange process. The kinetics of exchange of cholesterol from both vesicular and discoidal complexes that contain apoproteins are consistent with an aqueous diffusion mechanism, as has been established previously for PC/cholesterol SUV. The addition of 2-3 molecules of apo A-I to a donor SUV does not significantly alter the half-time (t1/2), which is 80 +/- 9 min at 37 degrees C. However, addition of 5-12 apo A-I molecules progressively decreases t1/2 from 65 +/- 2 to 45 +/- 4 min. This enhancement in the rate of desorption of cholesterol molecules is presumed to arise from the creation of packing defects at boundaries around the apoprotein molecules, which are intercalated among the phospholipid and cholesterol molecules in the surface of the donor SUV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors influencing interaction of human plasma low-density lipoproteins with discoidal complexes of apolipoprotein A-I and phosphatidylcholine

The effect of plasma components on the particle size distribution and chemical composition of human plasma low-density lipoproteins (LDL) during interaction with discoidal complexes of human apolipoprotein A-I and phosphatidylcholine (PC) was investigated. Incubation (37 degrees C, 1 h and 6 h) of LDL with discoidal complexes in the presence of the plasma ultracentrifugal d greater than 1.20 g/ml fraction (activity of lecithin-cholesterol acyltransferase inhibited) produces an increase in LDL apparent particle diameter two-to six-fold greater than that observed in the absence of the plasma d greater than 1.20 g/ml fraction. In incubation mixtures of LDL and discoidal complexes, both in the presence and absence of the plasma d greater than 1.20 g/ml fraction, the extent of LDL apparent particle diameter increase is: (1) approximately three-fold greater at 6 h than at 1 h, and (2) markedly greater for LDL with initially small (22.4-24.0 nm) major components than for LDL with initially large (26.2-26.8 nm) major components. The facilitation factor in the plasma d greater than 1.20 g/ml fraction is not plasma phospholipid transfer protein. Purified human serum albumin produces an apparent particle diameter increase comparable to the plasma d greater than 1.20 g/ml fraction. The discoidal complex-induced increase in LDL apparent particle diameter value by albumin is associated with an increase in phospholipid uptake by LDL and a decreased loss of LDL unesterified cholesterol. In preliminary experiments, high-density lipoproteins (HDL) reverse the apparent particle diameter increase originally induced by discoidal complexes. The presence of HDL (HDL phospholipid/LDL phospholipid molar ratio of 10:1) in the incubation (6 h) mixture of LDL and discoidal complexes also attenuates LDL apparent particle diameter increase. In vivo, the plasma LDL/HDL ratio may be a controlling factor in determining the extent to which phospholipid uptake and the associated change in LDL particle size distribution occurs.     

Incorporation of cholesterol into apolipoprotein A-I-dimyristoylphosphatidylcholine recombinants

Apolipoprotein A-I (apoA-I) spontaneously associates with dimyristoylphosphatidylcholine (DMPC) liposomes to form discoidal high-density lipoprotein (HDL) recombinants. The uptake of cholesterol by this model HDL was studied by incubation with Celite-dispersed cholesterol. Separation of the resulting complexes by gradient centrifugation and gel filtration showed a heterogeneous distribution of particle size and composition as a consequence of the disruption and rearrangement of the recombinants. Quantitation of the amount of cholesterol taken up gave values between about 28 and 40 mol% cholesterol for the fractions within the protein peaks; the fractions with the lowest DMPC/apoA-I ratios had the lowest cholesterol contents. In another set of experiments, the association of apoA-I with DMPC-cholesterol liposomes was shown to result in complexes with characteristics similar to those obtained by the cholesterol-uptake experiments. Low concentrations of cholesterol in the liposomes enhanced the rate of lipid-protein association, but larger amounts decreased the yield of complexes by making the process thermodynamically and kinetically unfavorable. The enthalpy of recombinant formation increased with decreasing lipid/protein ratio and increasing cholesterol content, and became endothermic at about 23 mol% cholesterol. The effect of cholesterol on the thermal properties of HDL recombinants suggests that cholesterol is partially excluded from the boundary region adjacent to apoA-I. It is concluded that discoidal HDL recombinants, as a model for 'nascent' HDL, can acquire substantial amounts of cholesterol, which may be of great physiological importance for the reverse cholesterol transport and prevention of atherosclerosis.     

Interaction of human-plasma low-density lipoproteins with discoidal complexes of apolipoprotein A-I and phosphatidylcholine, and characterization of the interaction products

Interaction of human low-density lipoproteins (LDL) with discoidal complexes comprised of egg yolk phosphatidylcholine and human apolipoprotein A-I (molar ratio, 88:1, respectively) was investigated. The multicomponent gradient gel electrophoretic pattern of LDL is transformed to one that includes a predominant component with an apparent particle diameter larger than that of the initial major LDL but still in the size range of normal LDL. The apparent particle diameter increase (range, 0.2-3.5 nm) is proportional to the increase (range, 6-40%) in LDL phospholipid/protein weight ratio following incubation (37 degrees C; 6 and 24 h); the smaller the initial LDL diameter, the greater the apparent particle diameter increase and percentage of phospholipid uptake. The LDL unesterified cholesterol/protein weight ratio decreases (range, 33-39%), but does not correlate with the increase in apparent particle diameter value. Interaction products are round particles with intact apolipoprotein B and show no evidence of phospholipid degradation. The products appear more dense than expected from the size vs. density relationship observed for nonincubated LDL subspecies. In addition to products in the normal LDL size range, larger components (apparent particle diameter range, 29.0-41.2 nm) also form and may be association complexes of phospholipid-modified LDL. Our results indicate that phospholipid uptake by LDL may contribute to the particle size polydispersity observed in plasma LDL.     

Interaction of plasma apolipoproteins with lipid monolayers

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidyl[¹⁴C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipid by apolipoprotein C-III.The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface.     

Reaction of lecithin: cholesterol acyltransferase with micellar substrates. Effect of particle sizes

Micellar, discoidal complexes were prepared from L-alpha-dipalmitoylphosphatidylcholine (DPPC) or egg phosphatidylcholine (egg-PC), cholesterol, and human apolipoprotein A-I by the cholate dialysis method. Reaction mixtures containing from 70:7:1 to 500:50:1, PC/cholesterol/apolipoprotein A-I (mol/mol) were fractionated by gel-filtration into various complex fractions. The isolated DPPC complexes ranged in size from 103 to 380 A in diameter, and in composition from 70:7:1 to 470:45:1, PC/cholesterol/apolipoprotein A-I (mol/mol), respectively. In contrast, the isolated egg-PC complexes only ranged in size from 105 to 214 A in diameter, and in composition from 65:5:1 to 153:17:1, PC/cholesterol/apolipoprotein A-I (mol/mol), respectively. Measurements of fluorescence wavelength maxima and fluorescence polarization of tryptophan residues of apolipoprotein A-I, in both series of complexes, revealed uniform spectral properties for all the egg-PC containing complexes. The DPPC complexes, on the other hand, had maxima in the fluorescence parameters for complexes with diameters around 200 A. When reacted with purified human lecithin:cholesterol acyltransferase, either at constant apolipoprotein A-I or at constant lipid concentration, all egg-PC complexes had very similar reaction rates, but the DPPC complex series exhibited major differences in reactivity. Minima in reaction rates occurred for DPPC complexes around 200 A in diameter, and optimal rates were observed with the small discoidal complexes (110 A in diameter). These reaction rates correlate well with the apolipoprotein A-I fluorescence properties and indicate that the apolipoprotein structure, reflected at the interface with phosphatidylcholine, may be the most important factor in determining complex reactivity with lecithin:cholesterol acyltransferase.     

Interaction of model discoidal complexes of phosphatidylcholine and apolipoprotein A-I with plasma components. Physical and chemical properties of the transformed complexes

Conversion of model discoidal complexes of egg yolk phosphatidylcholine and apolipoprotein A-I, upon interaction with a source of lecithin:cholesterol acyltransferase (plasma d greater than or equal to 1.21 g/ml fraction or partially purified enzyme) and with different sources of substrate unesterified cholesterol (LDL, VLDL or cholesterol incorporated into complexes), was investigated by gradient gel electrophoresis, gel filtration, equilibrium density gradient ultracentrifugation, electron microscopy and chemical analysis. When the incubation mixture contained an inhibitor of lecithin:cholesterol acyltransferase, discoidal complexes with mean long dimension of approximately 10.5 +/- 1.9 nm were converted (within 1 h) predominantly to small round particles and were partially depleted of their phospholipid content. Upon electrophoresis the small particles showed peak maxima within the migration intervals of the human plasma ( HDL3b ) gge and ( HDL3c ) gge subpopulations with associated particle size ranges of 7.8-8.2 and 7.2-7.8 nm, respectively. Within 1 h, in the presence of activated enzyme, the complexes were again converted in major part to the small particles. However, further incubation resulted in an apparent single-step conversion to a larger major product with peak maximum occurring within the migration intervals of the ( HDL2a ) gge and the ( HDL3a ) gge subpopulations (particle size ranges 8.8-9.8 and 8.2-8.8 nm, respectively). Formation of an apolar core was indicated by detection of cholesteryl esters in the conversion product. The form in which the substrate unesterified cholesterol was introduced did not markedly influence the size properties of the final conversion product. With VLDL as source of substrate, considerable incorporation of triacylglycerol occurred in company with a lower level of cholesteryl esters, suggesting transfer of these lipids during formation of the apolar core. Incubation of complexes with a partially purified (3000-fold) preparation of lecithin:cholesterol acyltransferase yielded a product similar in properties to that when the d greater than or equal to 1.21 g/ml fraction was used. Our model discoidal complexes and their conversion products exhibit properties very similar to those of potential precursors to HDL as well as of mature HDL particles. Their further investigation shows promise of providing detailed insight into the possible origin and heterogeneity of human plasma HDL.     

Activation of lecithin cholesterol acyltransferase by human apolipoprotein E in discoidal complexes with lipids

In a continued investigation of lecithin cholesterol acyltransferase reaction with micellar discoidal complexes of phosphatidylcholine, cholesterol, and various water soluble apolipoproteins, we prepared complexes containing human apo-E by the cholate dialysis method. These complexes were systematically compared to apo-A-I complexes synthesized under the same reaction conditions. Apo-E complexes (134 A in diameter) were slightly larger than apo-A-I complexes (110 A) but were very similar in terms of their protein and lipid content (2.4:0.10:1.0, egg phosphatidylcholine/cholesterol/apolipoprotein, w/w) and in the percentage of apolipoprotein in alpha-helical structure (72-74%). Concentration and temperature-dependence experiments on the velocity of the lecithin cholesterol acyltransferase reaction revealed differences in apparent Km values and small differences in apparent Vmax but very similar activation energies (18-20 kcal/mol). These observations suggest that differences in lecithin cholesterol acyltransferase activation by apo-A-I and apo-E are primarily a result of different affinities of the enzyme for the particles but that the rate-limiting step of the reaction is comparable for both complexes. Apo-E was found to be 18% as effective as apo-A-I in activating purified human lecithin cholesterol acyltransferase. Addition of free apo-A-I to apo-E complexes resulted in the exchange of bound for free apolipoprotein causing a slight increase in the reactivity with the enzyme when the incubation mixture was assayed. When the unbound apolipoproteins were removed by ultracentrifugation reisolated complexes containing both apo-E and apo-A-I demonstrated an even greater increase in reactivity with the enzyme.     

Apoprotein E mediates the interaction of beta-VLDL with macrophages

beta-Very low density lipoproteins (beta-VLDL) isolated from cholesterol-fed rhesus monkeys stimulated cholesteryl ester synthesis and accumulation in mouse peritoneal macrophages. The apoprotein specificity and requirement for the cell surface uptake of beta-VLDL was investigated by treating the beta-VLDL with trypsin (beta-VLDL (T], incubating the beta-VLDL (T) with other lipoproteins or apoproteins, reisolating the beta-VLDL (T) and measuring its biological activity which, for this study, is defined as the ability of the lipoprotein to stimulate cholesterol esterification in the macrophages. Trypsin treatment of beta-VLDL abolished its biological activity. Apoprotein analysis of the beta-VLDL (T) demonstrated the absence of intact apoproteins B-100, B-48, and E. The J774 macrophage-like cell line and mouse peritoneal macrophages responded similarly with respect to cholesterol esterification following incubation with inactive and treated beta-VLDL. The J774 macrophage-like cell line was used to establish the conditions necessary for the restoration of biologic activity to the trypsinized beta-VLDL. The loss of biological activity of beta-VLDL (T) could be reversed by restoring apoprotein E-containing LDL from hyperlipemic monkeys or purified apoprotein E. Apoprotein A-I had no such effect. The restored biological activity of the beta-VLDL (T) was proportional to the amount of apoprotein E acquired by the lipoprotein. beta-VLDL particles composed of apoprotein E and either intact or degraded apoprotein B-100 had comparable biological activity. Thus, intact apoprotein E, without intact apoprotein B, is a sufficient mediator for the biological activity and metabolism of beta-VLDL by macrophages and plays a major role in receptor-lipoprotein interaction.     

Fluorescence depolarization studies and phase transition in human apoprotein . phospholipid complexes.

The microviscosity of unilamellar vesicles of dimyristoyl-3-sn-phosphatidylcholine and that of phosphatidylcholine . apoprotein complexes was followed by fluorescence depolarization after labeling with 1,6-diphenyl-1,3,5-hexatriene. The transition temperature from gel-crystalline to liquid-crystalline phase in 24 degrees C for the dimyristoyl-phosphatidylcholine vesicles and is shifted to around 30 degrees C in the complexes between phosphatidylcholine and apoA-I, apoA-II, apoC-I, apoC-III proteins while the cooperativity of the transition is decreased. At temperatures below the transition of the phospholipid, the microviscosity of the complexes of phosphatidylcholine with apoA-I, apoA-II and apoC-I proteins is lower than that of the phosphatidylcholine, while the opposite effect is observed above 30 degrees C. The phosphatidylcholine . apoprotein complexes isolated on a Sepharose 6B column have a molecular weight around 100 000 and a phosphatidylcholine/apoprotein ratio of 2--2.6 (w/w). The microviscosity measurments at 35 degrees C performed after elution of the column enable the complex to be detected. The size and microviscosity of the apoprotein . phosphatidylcholine complex is compatible with a model where the vesicular structure has disappeared and the amino acid side chains present hydrophobic interaction with the phosphatidylcholine acyl chains.     

Role of apolipoproteins in cellular cholesterol efflux

The effects of serum apolipoproteins, particle size and concentration on the effectiveness of phosphatidylcholine (PC)-containing acceptor particles in causing release of cholesterol from cells growing in culture have been investigated. The acceptor particles were prepared by detergent-dialysis procedures and were either egg PC small unilamellar vesicles (SUV) or discoidal complexes of egg PC with apoproteins from human high-density lipoprotein (HDL). Gel filtration chromatography was employed to isolate particles of defined composition and size. The half-times (t 1/2) for the unidirectional efflux of cholesterol from cells prelabeled with [3H]cholesterol were measured as a function of acceptor PC concentration in the extracellular medium. HDL apolipoprotein-egg PC discoidal complexes at 100 micrograms PC/ml gave the following t 1/2 values when incubated with rat Fu5AH hepatoma, human HepG2 hepatoma, human GM3468 skin fibroblast, L-cell and mouse J774 macrophage-tumor cells: 11 +/- 2, 22 +/- 5, 84 +/- 18, 17 +/- 2 and 32 +/- 6 h, respectively. Equivalent experiments using purified apolipoprotein A-I or the total apolipoprotein C fraction to form the egg PC complexes showed that the t 1/2 values for the hepatoma cells were unaltered. However, with the fibroblasts, L-cells and J774 macrophages, the apolipoprotein C complexes gave significantly longer t 1/2 than complexes of egg PC with either apolipoprotein A-I or HDL apolipoprotein which gave the same t 1/2. An analysis based on the theory of fast coagulation of colloid particles to describe collisions between desorbed cholesterol molecules and acceptor particles predicts that the dependence of t 1/2 for cholesterol efflux from a given cell to different acceptors should be normalized when the extracellular level of acceptors is expressed in terms of the product of the radius of the particle times the number concentration of acceptor particles. The decrease in t 1/2 for cholesterol efflux from fibroblasts when the egg PC acceptor was changed from an SUV to an apolipoprotein HDL discoidal complex is consistent with the above concepts. The primary effect of the apolipoproteins in promoting cellular cholesterol efflux seems to be the solubilization of PC so that the PC is present in the extracellular medium as many small particles.     

Incorporation of cholesterol into high density lipoprotein recombinants.

Apo-A-1, the principal apoprotein of high density lipoprotein, was incubated with cholesterol containing liposomes of dimyristoyl lecithin, lecithin from high density lipoprotein or sphingomyelin. Conditions were chosen to give 100% conversion of cholesterol-free liposomes into recombinants which were isolated by density gradient ultracentrifugation. For all phospholipids, there was a progressive decrease in incorporation of lipid into recombinants with increasing cholesterol/phospholipid ratio. The cholesterol/phospholipid ratio of recombinants was ～ 45% of unreacted liposomes, for all initial cholesterol/phospholipid ratios. The reduced cholesterol content suggests exclusion of cholesterol from a fraction of recombinant phospholipid, probably a boundary layer in contact with apo A-1.     

Activation of lecithin: cholesterol acyltransferase by human apolipoprotein A-IV

Human plasma apoproteins (apo) A-I and A-IV both activate the enzyme lecithin:cholesterol acyltransferase (EC 2.3.1.43). Lecithin:cholesterol acyltransferase activity was measured by the conversion of [4-14C] cholesterol to [4-14C]cholesteryl ester using artificial phospholipid/cholesterol/[4-14C]cholesterol/apoprotein substrates. The substrate was prepared by the addition of apoprotein to a sonicated aqueous dispersion of phospholipid/cholesterol/[4-14C]cholesterol. The activation of lecithin:cholesterol acyltransferase by apo-A-I and -A-IV differed, depending upon the nature of the hydrocarbon chains of the sn-L-alpha-phosphatidylcholine acyl donor. Apo-A-I was a more potent activator than apo-A-IV with egg yolk lecithin, L-alpha-dioleoylphosphatidylcholine, and L-alpha-phosphatidylcholine substituted with one saturated and one unsaturated fatty acid regardless of the substitution position. When L-alpha-phosphatidylcholine esterified with two saturated fatty acids was used as acyl donor, apo-A-IV was more active than apo-A-I in stimulating the lecithin:cholesterol acyltransferase reaction. Complexes of phosphatidylcholines substituted with two saturated fatty acids served as substrate for lecithin:cholesterol acyltransferase even in the absence of any activator protein. Essentially the same results were obtained when substrate complexes (phospholipid-cholesterol-[4-14C]cholesterol-apoprotein) were prepared by a detergent dialysis procedure. Apo-A-IV-L-alpha-dimyristoylphosphatidylcholine complexes thus prepared were shown to be homogeneous particles by column chromatography and density gradient ultracentrifugation. It is concluded that apo-A-IV is able to facilitate the lecithin:cholesterol acyltransferase reaction in vitro.     

Direct Measurement of the Structure of Reconstituted High-Density Lipoproteins by Cryo-EM

Early forms of high-density lipoproteins (HDL), nascent HDL, are formed by the interaction of apolipoprotein AI with macrophage and hepatic ATP-binding cassette transporter member 1. Various plasma activities convert nascent to mature HDL, comprising phosphatidylcholine (PC) and cholesterol, which are selectively removed by hepatic receptors. This process is important in reducing the cholesterol burden of arterial wall macrophages, an important cell type in all stages of atherosclerosis. Interaction of apolipoprotein AI with dimyristoyl (DM)PC forms reconstituted (r)HDL, which is a good model of nascent HDL. rHDL have been used as an antiathersclerosis therapy that enhances reverse cholesterol transport in humans and animal models. Thus, identification of the structure of rHDL would inform about that of nascent HDL and how rHDL improves reverse cholesterol transport in an atheroprotective way. Early studies of rHDL suggested a discoidal structure, which included pairs of antiparallel helices of apolipoprotein AI circumscribing a phospholipid bilayer. Another rHDL model based on small angle neutron scattering supported a double superhelical structure. Herein, we report a cryo-electron microscopy-based model of a large rHDL formed spontaneously from apolipoprotein AI, cholesterol, and excess DMPC and isolated to near homogeneity. After reconstruction we obtained an rHDL structure comprising DMPC, cholesterol, and apolipoprotein AI (423:74:1 mol/mol) forming a discoidal particle 360 Å in diameter and 45 Å thick; these dimensions are consistent with the stoichiometry of the particles. Given that cryo-electron microscopy directly observes projections of individual rHDL particles in different orientations, we can unambiguously state that rHDL particles are protein bounded discoidal bilayers.     

Reconstitution of lipoproteins. II. Lipid-protein interaction between dimyristoyl-lecithin and dimyristoyl-lecithin:cholesterol vesicles and purified apolipoprotein C-I and C-III2 studied by isomeric spin-labelled lecithins

The reconstitution of purified apolipoprotein C-I and C-III2 with sn-3-dimyristoyl-lecithin and sn-3-dimyristoyl-lecithin:cholesterol (10:1) vesicles was studied by electron spin resonance spectroscopy using isomeric 5'-, 12'-, and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probes. Results obtained from the temperature-induced changes of lipoprotein recombinants showed the hydrophilic nature of the lipid-protein interactions. The temperature-induced phospholipid phase transition, as measured by 5'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probe in recombinants containing apoprotein C-1 or apoprotein C-iii2, is very broad and has a small cooperative unit indicative of extensive lipid-protein interactions occurring at the head group region of the phospholipid bilayer. When 12"- and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithins are used as probes in the same system, similar sharper and more cooperative lipid phase changes are detected. These results indicate a surface location for both apoprotein C-I and apoprotein C-III2 with respect to the phospholipid bilayer in lipoprotein recombinants with and without cholesterol.     

Interaction of apoliprotein C-III with phosphatidylcholine vesicles. Dependence of aproprotein-phospholipid complex formation on vesicle structure.

We have studied the interaction of an apolipoprotein from human very low density lipoproteins (apoC-III) with egg yolk phosphatidylcholine in the form of single- and multi-bilayer vesicles. The reactivity of single-bilayer vesicles with apoC-III appears to be greater than that of the multi-bilayer vesicles according to several thermodynamic and spectrosconic criteria. In the complexes formed by the association of apoC-III with single-bilayer vesicles, the alpha-helical content of the peptide backbone and the apolarity of the environment around the tryptophan residues are greater than that observed in the complexes formed with the multibilayer vesicles. A higher yield and more homogeneous density distribution of lipid-apoprotein complexes results from the interaction of apoC-III with the single-bilayer vesicles relative to those obtained with the multi-bilayer vesicles. The enthalpy of association of apoC-III with phospholipid was greater for the single-shelled vesicles (25 kcal/mol apoC-III) than for the multi-shelled ones (18 kcal/mol apoC-III). The difference in reactivity of these two types of liposomes is not due to a difference in their fluidities since their fatty acid compositions are identical, but may be due to a difference in their areas of sterically accessible phospholipid, their permeabilities to the apoprotein, their radii of curvation, or a combination of these factors.