Lipid-poor apolipoprotein A-I in Hep G2 cells: formation of lipid-rich particles by incubation with dimyristoylphosphatidylcholine

Apolipoprotein A-I is a major secretory product of the human hepatoma cell line, Hep G2; approx. 70% of apolipoprotein A-I was separated from the medium as lipid-poor apolipoprotein A-I in the d greater than 1.21 g/ml fraction while 30% was associated with high-density lipoproteins (HDL) of d 1.063-1.21 g/ml. The lipid-poor apolipoprotein A-I contains 50% proapolipoprotein A-I which is similar to the isoform distribution in Hep G2 preformed HDL. We tested the ability of lipid-poor apolipoprotein A-I from Hep G2 to form complexes with dimyristoylphosphatidylcholine (DMPC) vesicles at DMPC/apolipoprotein A-I molar ratios of 100:1 and 300:1. Lipid-poor apolipoprotein A-I was recovered in complex form while at a 300:1 ratio, 68.8 +/- 6.3% was recovered. On electron microscopy, the former complexes were small discs 16.9 nm +/- 4.5 S.D. in diameter while the latter were larger discs 21.4 +/- 4.4 nm diameter. Non-denaturing gradient gel electrophoresis of complexes formed at a 100:1 ratio had a peak in the region corresponding to 9.64 +/- 0.08 nm; these particles possessed two apolipoprotein A-I molecules. At the higher ratio, 300:1, two distinct complexes were identifiable, one which banded in the 9.7 nm region and the other in the 16.9-18.7 nm region. The former particles contained two molecules of apolipoprotein A-I and the latter, three molecules. This study demonstrates that lipid-poor apolipoprotein A-I which is rich in more basic isoforms forms discrete lipoprotein complexes similar to those formed by mature apolipoprotein A-I. It is further suggested that, under the appropriate conditions, precursor or nascent HDL may be assembled extracellularly.     

Human lymphedema fluid lipoproteins: particle size, cholesterol and apolipoprotein distributions, and electron microscopic structure

The concentration of cholesterol, apolipoproteins A-I, B, and E has been determined in lymphedema fluid from nine patients with chronic primary lymphedema. The concentrations were: 38.14 +/- 21.06 mg/dl for cholesterol, 15.6 +/- 6.17 mg/dl for apolipoprotein A-I, 7.5 +/- 2.8 mg/dl for apolipoprotein B, and 1.87 +/- 0.50 mg/dl for apolipoprotein E. These values represent 23%, 12%, 6%, and 38% of plasma concentrations, respectively. The ratio of esterified to unesterified cholesterol in lymphedema fluid was 1.46 +/- 0.45. Lipoproteins of lymphedema fluid were fractionated according to particle size by gradient gel electrophoresis and by exclusion chromatography. Gradient gel electrophoresis showed that a majority of high density lipoproteins (HDL) of lymphedema fluid were larger than ferritin (mol wt 440,000) and smaller than low density lipoproteins (LDL); several discrete subpopulations could be seen with the large HDL region. Fractionation by exclusion chromatography showed that more than 25% of apolipoprotein A-I and all of apolipoprotein E in lymphedema fluid was associated with particles larger than plasma HDL2. Apolipoprotein A-I also eluted in fractions that contained particles the size of or smaller than albumin. Isolation of lipoproteins by sequential ultracentrifugation showed that less than 25% of lymphedema fluid cholesterol was associated with apolipoprotein B. The majority of apolipoprotein A-containing lipoproteins of lymphedema fluid were less dense than those in plasma. Ultracentrifugally separated fractions of lipoproteins were examined by electron microscopy. The fraction d less than 1.019 g/ml contained little material, while fraction d 1.019-1.063 g/ml contained two types of particles: round particles 17-26 nm in diameter and square-packing particles 13-17 nm on a side. Fractions d 1.063-1.085 g/ml had extensive arrays of square-packing particles 13-14 nm in size. Fractions d 1.085-1.11 g/ml and fractions d 1.11-1.21 g/ml contained round HDL, 12-13 nm diameter and 10 nm diameter, respectively. Discoidal particles were observed infrequently.     

Molecular pathways in the transformation of model discoidal lipoprotein complexes induced by lecithin:cholesterol acyltransferase

Incubation (24 h, 37 degrees C) of discoidal complexes of phosphatidylcholine and apolipoprotein A-I (molar ratio 95 +/- 10 egg yolk phosphatidylcholine-apolipoprotein A-I; 10.5 X 4.0 nm, long X short dimension; designated, class 3 complexes) with the ultracentrifugal d greater than 1.21 g/ml fraction transformed the discoidal complexes to a small product with apparent mean hydrated and nonhydrated diameter of 7.8 and 6.6 nm, respectively. Formation of the small product was associated with marked reduction in phosphatidylcholine-apolipoprotein AI molar ratio of the complexes (on average from 95:1 to 45:1). Phospholipase A2 activity of lecithin:cholesterol acyltransferase participated in the depletion process, as evidenced by production of unesterified fatty acids. In the presence of the d greater than 1.21 g/ml fraction or partially purified lecithin:cholesterol acyltransferase and a source of unesterified cholesterol, the small product could be transformed to a core-containing (cholesteryl ester) round product with a hydrated and nonhydrated diameter of 8.6 and 7.5 nm, respectively. By means of cross-linking with dimethylsuberimidate, the protein moiety of the small product was shown to contain primarily two apolipoprotein A-I molecules per particle, while the large product contained three apolipoprotein A-I molecules per particle. The increase in number of apolipoprotein A-I molecules per particle during transformation of the small to the large product appeared to result from fusion of the small particles during core build-up and release of excess apolipoprotein A-I from the fusion product. The results obtained with the model complexes were consistent for the most part with recent observations (Chen, C., Applegate, K., King, W.C., Glomset, J.A., Norum, K.R. and Gjone, E. (1984) J. Lipid Res. 25, 269-282) on the transformation, by lecithin:cholesterol acyltransferase, of the small spherical high-density lipoproteins of patients with familial lecithin:cholesterol acyltransferase deficiency.     

Interaction of apolipoprotein A-II with recombinant HDL containing egg phosphatidylcholine, unesterified cholesterol and apolipoprotein A-I

The preparation of discoidal, recombinant HDL (r-HDL) containing various phospholipids, apolipoproteins and a range of concentrations of unesterified cholesterol has been reported by several investigators. The present study describes the preparation of r-HDL containing both apolipoprotein (apo) A-I and apo A-II. r-HDL with 100:1 (mol:mol) egg PC.apo A-I and 0 (Series I), 5 (Series II) or 10 (Series III) mol% unesterified cholesterol were prepared by the cholate dialysis method. The resulting complexes had a Stokes' radius of 4.7 nm and contained two molecules of apo A-I per particle. When the r-HDL (2.0 mg apo A-I) were supplemented with 1.0 mg of apo A-II, one of the apo A-I molecules was replaced by two molecules of apo A-II. This modification was not accompanied by a loss of phospholipid, nor by major change in particle size. The addition of 2.5 or 4.0 mg of apo A-II resulted in the displacement of both apo A-I molecules from a proportion of the r-HDL and the formation of smaller particles (Stokes' radius 3.9 nm), which contained half the original number of egg PC molecules and three molecules of apo A-II. The amount of apo A-I displaced was dependent on the concentration of unesterified cholesterol in the r-HDL: when 2.5 mg of apo A-II was added to the Series I, II and III r-HDL, 44, 60 and 70%, respectively, of the apo A-I was displaced. Addition of 4.0 mg of apo A-II did not promote further displacement of apo A-I from any of the r-HDL. By contrast, the association of apo A-II with r-HDL was independent of the concentration of unesterified cholesterol and was a linear function of the amount of apo A-II which had been added. It is concluded that (1), the structural integrity of egg PC.unesterified cholesterol.apo A-I r-HDL, which contain two molecules of apo A-I, is not affected when one of the apo A-I molecules is replaced by two molecules of apo A-II; (2), when both apo A-I molecules are replaced by apo A-II, small particles which contain three molecules of apo A-II are formed; and (3), the displacement of apo A-I from r-HDL is facilitated by the presence of unesterified cholesterol in the particles.     

Transformation of large discoidal complexes of apolipoprotein A-I and phosphatidylcholine by lecithin-cholesterol acyltransferase

Using a cholate-dialysis recombination procedure, complexes of apolipoprotein A-I and synthetic phosphatidylcholine (1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC] were prepared in mixtures at a relatively high molar ratio of 150:1 phosphatidylcholine/apolipoprotein A-I. Particle size distribution analysis by gradient gel electrophoresis of the recombinant mixtures indicated the presence of a series of discrete complexes that included species migrating at RF values observed for discoidal particles in nascent high-density lipoproteins (HDL) in plasma of lecithin-cholesterol acyltransferase-deficient subjects. One of these complex species, designated complex class 6, formed with either phosphatidylcholine, was isolated by gel filtration and characterized at follows: discoidal shape (mean diameter 20.8 nm (POPC) and 19.0 nm (DOPC]; molar ratio, phosphatidylcholine/apolipoprotein A-I, 155:1 (POPC) and 130:1 (DOPC); and both containing 4 molecules of apolipoprotein A-I per particle. Incubation of class 6 complexes with lecithin-cholesterol acyltransferase (EC 2.3.1.43) and a source of unesterified cholesterol (low-density lipoprotein (LDL] was shown by electron microscopy to result in a progressive transformation of the discoidal particles (0 h) to deformable (2.5 h) and to spherical particles (24 h). The spherical particles (diameter 13.6 nm (POPC) and 12.5 nm (DOPC) exhibit sizes at the upper boundary of the interval defining the human plasma (HDL2b)gge (12.9-9.8 nm). The spherical particles contain a cholesteryl ester core that reaches a limiting molar ratio of approx. 50-55:1 cholesteryl ester/apolipoprotein A-I. The deformable particles assume a rectangular shape under negative staining and, relative to the 24-h spherical product, are enriched in phosphatidylcholine. Chemical crosslinking (by dimethyl suberimidate) of the isolated transformation products shows the 24-h spherical particle to contain predominantly 4 apolipoprotein A-I molecules; products produced after intermediate periods of time appear to contain species with 3 and 4 apolipoproteins per particle. Our in vitro studies indicate a potential pathway in the origins of large, apolipoprotein A-I-containing plasma HDL particles. The deformable species observed during transformation were similar in size and shape to particles observed in interstitial fluid.     

Characterization of discoidal complexes of phosphatidylcholine, apolipoprotein A-I and cholesterol by gradient gel electrophoresis

Complexes of egg yolk phosphatidylcholine and apolipoprotein A-I were prepared by a detergent (sodium cholate)-dialysis method and characterized by gradient gel electrophoresis, gel filtration, electron microscopy and chemical analysis. Multicomponent electrophoretic patterns were obtained indicating formation of at least eight classes of discoidal complexes. The relative contribution of the different classes to the electrophoretic pattern was a function of the molar ratio of phosphatidylcholine:apolipoprotein A-I in the interaction mixture. Molar ratios of phosphatidylcholine:apolipoprotein A-I in isolated complexes were strongly and positively correlated with disc diameter obtained by electron microscopy. Incorporation of unesterified cholesterol into phosphatidylcholine/apolipoprotein A-I interaction mixtures also resulted in formation of unique complexes but with considerably different particle size distributions relative to those observed in the absence of cholesterol. One common consequence of cholesterol incorporation into interaction mixtures of 87.5:1 and 150:1 molar ratio of phosphatidylcholine:apolipoprotein A-I was the disappearance of a major complex class with diameter of 10.8 nm and the appearance of a major component with diameter of approximately 8.8 nm. Electrophoretic patterns of cholesterol-containing complexes showed a strong similarity to patterns recently published for high density lipoproteins from plasma of lecithin:cholesterol acyltransferase-deficient subjects, suggesting that the complexes formed in vitro by the detergent-dialysis method may serve as appropriate models for investigation of the origins of the HDL particle size distribution.     

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.     

Isolation and lipid-binding properties of rat apolipoprotein A-IV

Apolipoprotein A-IV was isolated from the d less than 1.21 g/ml fraction of rat serum by gel filtration followed by heparin-Sepharose affinity chromatography; this method also facilitated the preparation of apolipoprotein A-I and apolipoprotein E. The apolipoprotein A-IV preparation was characterized by SDS-gel electrophoresis, isoelectric focusing, amino acid analysis and immunodiffusion. The lipid-binding properties of this protein were studied. Apolipoprotein A-IV associated with dimyristoylphosphatidylcholine (DMPC) to form recombinants which contained two molecules of apolipoprotein A-IV and had a lipid/protein molar ratio of 110. The density of the DMPC/apolipoprotein A-IV particles was determined to be 1.08 g/ml and the particles were visualized by electron microscopy as discs which were 5.8 nm thick and 18.0 nm in diameter. The stability of the DMPC/apolipoprotein A-IV recombinants, as determined by resistance to denaturation, was comparable to the stability of DMPC/apolipoprotein A-I complexes. However, by competition studies it was found that apolipoprotein A-I competed for the binding to DMPC more effectively than did apolipoprotein A-IV. It is concluded that, while rat apolipoprotein A-IV resembles other apolipoproteins in its lipid-binding characteristics, it may be displaced from lipid complexes by apolipoprotein A-I.     

Expression of human apolipoprotein E but not that of apolipoprotein A-I by mouse C127 cells is associated with increased secretion of lipids in the form of vesicles and discs.

Transfected mouse mammary-derived cells (C127) expressing human apolipoprotein (apo) E (C127E) were used a) to determine whether the lipid-binding character of apoE is sufficient to promote its assembly with lipid to form lipoprotein-like particles when expressed by cells not normally expressing apolipoproteins; b) to characterize the secreted complexes in terms of morphology, size, and composition; and finally c) to determine whether apoE or apoA-I gene expression by these transfected cells has any effect on the levels and the profiles of the synthesized and secreted lipids. The findings of the present study demonstrate that: a) as determined by density gradient ultracentrifugation and gel filtration chromatography, about 20% of the secreted [35S]methionine-labeled apoE expressed by C127E cells is lipid-associated. b) Negative-stain electron microscopic analysis of the lipid-protein complexes recovered in the lipoprotein fractions (d less than 1.21 g/ml) revealed that approximately 13% of the total population of particles were discs (16 +/- 5 nm mean diameter and 4-6 nm thick), resembling nascent high density lipoproteins (HDL). The majority of the particles however (greater than 82%) appeared vesicular with varying diameters (48 +/- 40 nm mean diameter). The discoidal and the vesicular appearance of the particles secreted by C127E cells is consistent with the composition of lipids. These consisted mostly of surface lipids, phospholipids (45 +/- 18%), diacylglycerols (36 +/- 17%), and free cholesterol (17 +/- 7%) (by weight). c) Expression of apoE by C127E cells was associated with an increased release of [35S]methionine-labeled protein and [3H]glycerol-labeled lipid (3- to 5- and 4- to 8-fold, respectively) compared to nontransfected C127 cells. Expression of mutant apoE or normal apoA-I, however, was not associated with increased release of the major lipid classes compared to the parent C127 cells, strongly suggesting that this character of C127E cells is specific to apoE expression. The release of lipids by C127E cells could be reduced considerably by the addition of the metabolic inhibitors, colchicine or cycloheximide (10 and 1 microM, respectively), suggesting that lipid release by C127E cells is an active process requiring both protein synthesis and functional secretory mechanisms. Taken together the findings suggest that apoE expression by C127 cells promotes the formation of nascent discoidal lipoprotein-like particles and enhances the release of vesicular lipids, possibly by promoting shedding of cell plasma membrane fragments.     

Combined interaction of phospholipase C and apolipoprotein A-I with small unilamellar lecithin-cholesterol vesicles: influence of apolipoprotein A-I concentration and vesicle composition

We report the combined effects of phospholipase C (PLC), a pronucleating factor, and apolipoprotein A-I (apo A-I), an antinucleating factor, in solutions of model bile. Results indicate that apo A-I inhibits cholesterol nucleation from unilamellar lecithin vesicles by two mechanisms. Initially, inhibition is achieved by apo A-I shielding of hydrophobic diacylglycerol (DAG) moieties so as to prevent vesicle aggregation. Protection via shielding is temporary. It is lost when the DAG/apo A-I molar ratio exceeds a critical value. Subsequently, apo A-I forms small ( approximately 5-15 nm) complexes with lecithin and cholesterol that coexist with lipid-stabilized (400-800 nm) DAG oil droplets. This microstructural transition from vesicles to complexes avoids nucleation of cholesterol crystals and is a newly discovered mechanism by which apo A-I serves as an antinucleating agent in bile. The critical value at which a microstructural transition occurs depends on binding of apo A-I and so varies with the cholesterol mole fraction of vesicles. Aggregation of small, unilamellar, egg lecithin vesicles (SUVs) with varying cholesterol composition (0-60 mol %) was monitored for a range of apo A-I concentrations (2 to 89 microg/mL). Suppression of aggregation persists so long as the DAG-to-bound-apo A-I molar ratio is less than 100. A fluorescence assay involving dansylated lecithin shows that the suppression is an indirect effect of apo A-I rather than a direct inhibition of PLC enzyme activity. The DAG-to-total apo A-I molar ratio at which suppression is lost increases with cholesterol because of differences in apo A-I binding. Above this value, a microstructural transition to DAG droplets and lecithin/cholesterol A-I complexes occurs, as evidenced by sudden increases in turbidity and size and enhancement of Forster resonance energy transfer; structures are confirmed by cryo TEM.     

Chain length-function correlation of amphiphilic peptides. Synthesis and surface properties of a tetratetracontapeptide segment of apolipoprotein A-I

The segment corresponding to residues 121 to 164 of human plasma apolipoprotein A-I (apo-A-I) has been synthesized by the Merrifield solid phase method. The peptide binds to unilamellar phospholipid vesicles and to phospholipid-cholesterol mixed vesicles. The surface affinity of the peptide measured in this way indicated that the mechanism of binding is the same as that of apo A-I (144-165) and apo A-I itself. The peptide appears to be a globular monomer in a aqueous solution, with 17% alpha helix content. The peptide bound to vesicles activates lecithin:cholesterol acyltransferase: compared to apo A-I, the peptide is about 30% as efficient in the activation of cholesterol esterification and of phospholipid hydrolysis when the surface is saturated by the activator. For a variety of amphiphilic peptides and for apo A-I, the lecithin: cholesterol acyltransferase-activating ability correlates well with their alpha helix contents in 50% trifluoroethanol.     

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.     

Effect of high-density lipoproteins with varying ratios of apolipoprotein A-I to apolipoprotein A-II on steroidogenesis by cultured rat ovary granulosa cells

Plasma high-density lipoproteins (HDL) can provide rat ovary steroidogenic tissue with cholesterol for steroid hormone production, but the mechanism of cholesterol transfer is unknown. To test the importance of apolipoprotein A-I (the major HDL apolipoprotein) in HDL-cell interactions, we examined the ability of canine-human HDL hybrids containing various proportions of canine apolipoprotein A-I and human apolipoprotein A-II to stimulate steroidogenesis by cultured rat ovary granulosa cells. We observed that as the apolipoprotein A-II to apolipoprotein A-II ratio decreased, the ability of the hybrid particles to stimulate granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, granulosa cell progestin (progesterone and 20 alpha-dihydroprogesterone) production diminished. However, apolipoprotein A-I was not necessary for cholesterol transfer, since hybrids with less than 5% of their total apolipoprotein mass as apolipoprotein A-I stimulated progestin production 30% as effectively as canine HDL, which contained essentially only apolipoprotein A-I. These data indicate that the delivery of cholesterol from HDL into the rat ovary cell for steroidogenesis is not strictly dependent on the presence of a specific HDL apolipoprotein.     

High density lipoprotein particle size distribution in subjects with obstructive jaundice

High density lipoproteins (HDL) from 14 patients with obstructive jaundice were examined by gradient gel electrophoresis to determine the effect of obstruction on particle size distribution. HDL from 7 of these patients were fractionated by gel permeation chromatography and further characterized by electron microscopy, SDS gel electrophoresis, apolipoprotein A-I and apolipoprotein A-II immunoturbidimetry, and analysis of chemical composition. In addition, lecithin:cholesterol acyltransferase (LCAT) activity was measured and correlated with plasma apolipoprotein A-I concentration and particle size distribution. HDL were abnormal in all patients regardless of severity, cause, or duration of obstruction. The major HDL subfraction in normal subjects, HDL3a (radius 4.1-4.3 nm) was either absent or considerably diminished, and HDL2b (radius 5.3 nm) was also frequently absent. Very small particles comparable in size to normal HDL3c (radius 3.8 nm) were prominent. In patients with a bilirubin concentration greater than 250 mumol/l, normal HDL had totally disappeared and were replaced by large discoidal particles of radius 8.5 nm and small spherical particles of radius 3.6-3.7 nm. Both populations of particles were markedly depleted of cholesteryl ester and enriched in free cholesterol and phospholipid. The discoidal particles were rich in apolipoproteins E, A-I, A-II, and C, while the small spherical particles contained predominantly apolipoprotein A-I. LCAT activity was diminished in all subjects to 8-54% of normal, and was strongly positively correlated (r = 0.91 P less than 0.05) with plasma apolipoprotein A-I levels.     

Activation of phosphatidylcholine-sterol acyltransferase by human apolipoprotein E isoforms

The reaction catalysed by phosphatidylcholine-sterol acyltransferase (EC 2.3.1.43) is believed to be the major source of cholesteryl ester in human plasma; the enzyme requires a protein activator. Several human apolipoproteins were found to exhibit an activator function, the major one being apolipoprotein A-I. Human apolipoprotein E exists in the population mainly in three different genetic isoforms; apolipoprotein E-2, E-3 and E-4. These isopeptides were isolated from subjects homozygous for one of the isoforms, incorporated into phospholipid/cholesterol/[14C]cholesterol complexes by the cholate dialysis procedure and used to measure capacity to activate phosphatidylcholine-sterol acyltransferase in comparison to apolipoprotein A-I lipid substrate particles prepared by the same procedure. Acyltransferase activity was measured by the formation of [14C]cholesteryl ester from [14C]cholesterol using purified enzyme. With egg yolk phosphatidylcholine as acyl donor, apo E was 15-19% as efficient as apolipoprotein A-I for activation of the acyltransferase. Apo-E-stimulated cholesteryl ester formation by the enzyme was enhanced when 1-oleoyl-2-palmitoyl-glycerophosphocholine was used as a substrate phospholipid (45% of apo A-I/phosphatidylcholine control) and most pronounced with dimyristoylglycerophosphocholine (75% of apo A-I/phosphatidylcholine control). No significant difference in activation was found between apo E isoforms. It is concluded that apolipoprotein E activates phosphatidylcholine-sterol acyltransferase in vitro and that apolipoprotein E isoforms are similarly effective.     

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.     

Human high denisty apolipoprotein A-I-lysolecithin-lecithin and sphingomyelin complexes. A method for high yield recombinations to lipoprotein complexes of reproducible stoichiometry.

High denisty apolipoprotein A-1 (apoLp A-I) has been prepared in a chromatographically and immunochemically homogeneous form. This apoprotein forms trimeric and tetrameric aggregates in aqueous solutions at higher concentrations. ApoLp A-I has been recombined in almost quantitative yield in the presence of lysolecithin with phosphatidylcholine and sphingomyelin to particles of reproducible stoichiometry. Lysolecithin is not required for the interactions of lecithin and sphingomyelin with the apoprotein A-I or for the stability of these complexes. Dialysis removes most of the lysolecithin without the loss of lecithin and sphingomyelin. ApoLp A-I-lecithin particles have a molecular weight of 200 000 and contain 50 molecules lecithin and 25 of lysolecithin. ApoLp A-I-sphingomyelin complexes contain 50 sphingomyelin and 13 lysolecithin molecules. The former particles show up as discs of 100 A diameter, and the latter particles are 250 A in diameter. Their thickness was estimated as 25 A in the apoLp A-I lecithin and 60 A in the apoLp A-I-sphingomyelin particles. ApoLp A-I and lysolecithin form complexes whose densities depend on the lysolecithin concentration. Lysolecithin enhances the binding of phosphatidylcholine to apoLP A-I, yielding lipoprotein complexes with decreasing density. The yield of apoLp A-I-sphingomyelin-lysolecithin complexes is proportional to the lysolecithin concentration. The ratio of apoLp A-I to sphingomyelin in all these complexes remains constant.     

Identification and partial characterization of discrete apolipoprotein A-containing lipoprotein particles secreted by human hepatoma cell line HepG2

The purpose of this study was to identify the apolipoprotein A-containing lipoprotein particles produced by HepG2 cells. The apolipoprotein A-containing lipoproteins separated from apolipoprotein B-containing lipoproteins by affinity chromatography of culture medium on concanavalin A were fractionated on an immunosorber with monoclonal antibodies to apolipoprotein A-II. The retained fraction contained apolipoproteins A-I, A-II and E, while the unretained fraction contained apolipoproteins A-I and E. Both fractions were characterized by free cholesterol as the major and triglycerides and cholesterol esters as the minor neutral lipids. Further chromatography of both fractions on an immunosorber with monoclonal antibodies to apolipoprotein A-I showed that 1) apolipoprotein A-II only occurs in association with apolipoprotein A-I, 2) apolipoprotein A-IV is only present as part of a separate lipoprotein family (lipoprotein A-IV), and 3) apolipoprotein E-enriched lipoprotein A-I:A-II and lipoprotein A-I are the main apolipoprotein A-containing lipoproteins secreted by HepG2 cells.     

Conversion of apolipoprotein-specific high-density lipoprotein populations during incubation of human plasma

Incubation studies were performed on plasma obtained from subjects selected for relatively low levels of high-density lipoprotein cholesterol (HDL-C) (no greater than 30 mg/dl) and particle size distributions enriched in the HDL3 subclass. Incubation (12 h, 37 degrees C) of plasma in the presence or absence of lecithin: cholesterol acyltransferase activity produces marked alteration in size profiles of both major apolipoprotein-specific HDL3 populations (HDL3(AI w AII), HDL3 species containing both apolipoprotein A-I and apolipoprotein A-II, and HDL3(AI w/o AII), HDL3 species containing apolipoprotein A-I) as isolated by immunoaffinity chromatography. In the presence or absence of lecithin: cholesterol acyltransferase activity, plasma incubation results in a shift of HDL3(AI w AII) species (initial mean sizes of major components, approx. 8.8 and 8.0 nm) predominantly to larger particles (mean size, 9.8 nm). A less prominent shift to smaller particles (mean size, 7.8 nm) accompanies the conversion to larger particles only when the enzyme is active. Combined shifts to larger (mean size, 9.8 nm) and smaller (mean size, 7.4 nm) particles are observed for HDL3(AI w/o AII) particles (mean size, 8.3 nm) also only in the presence of enzyme activity. However, in the absence of enzyme activity, HDL3(AI w/o AII) species, unlike the HDL3(AI w AII) species, are converted to smaller (mean size 7.4 nm) rather than to larger particles. Like native HDL2b(AI w/o AII) particles, the larger HDL3(AI w/o AII) conversion products exhibit a protein moiety with molecular weight equivalent to four apolipoprotein A-I molecules per particle; small HDL3(AI w/o AII) products are comprised predominantly of particles with two apolipoprotein A-I per particle. Incubation-induced conversion of HDL3 particles in the presence of lecithin: cholesterol acyltransferase activity is associated with increased binding of both apolipoprotein-specific HDL populations to low-density lipoproteins (LDL). The present studies indicate that, in the absence of lecithin: cholesterol acyltransferase activity, the two HDL3 populations follow different conversion pathways, possibly due to apolipoprotein-specific activities of lipid transfer protein or conversion protein in plasma. Our studies also suggest that lecithin: cholesterol acyltransferase activity may play a role in the origins of large HDL2b(AI w/o AII) species in human plasma by participating in the conversion of HDL3(AI w/o AII) particles, initially with three apolipoprotein A-I, to larger particles with four apolipoprotein A-I per particle.     

Characterization of lipoproteins produced by the human liver cell line, Hep G2, under defined conditions

Confluent monolayers of the human hepatoblastoma-derived cell line, Hep G2, were incubated in serum-free medium. Conditioned medium was ultracentrifugally separated into d less than 1.063 g/ml and d 1.063-1.20 g/ml fractions since very little VLDL was observed. The d less than 1.063 g/ml fraction was examined by electron microscopy; it contained particles of 24.5 +/- 2.3 nm diameter, similar in size to plasma LDL; a similar size was demonstrated by nondenaturing gradient gel electrophoresis. These particles possessed apoB-100 only. The d less than 1.063 g/ml fraction had a lipid composition unlike that of plasma LDL; unesterified cholesterol was elevated, there was relatively little cholesteryl ester, and triglyceride was the major core lipid. The d 1.063-1.20 g/ml fraction was heterogeneous in size and morphology. Electron microscopy revealed discoidal particles (14.9 +/- 3.2 nm long axis and 4.5 +/- 0.2 nm short axis) as well as small spherical ones (7.6 +/- 1.4 nm diameter). Nondenaturing gradient gel electrophoresis consistently showed the presence of peaks at 13.4 11.9, 9.7, and 7.4 nm. The latter peak was conspicuous and probably corresponded to the small spherical structures seen by electron microscopy. Unlike plasma HDL, Hep G2 d 1.063-1.20 g/ml lipoproteins contained little or no stainable material in the (HDL3a)gge region by gradient gel electrophoresis. Hep G2 d 1.063-1.20 g/ml lipoproteins differed significantly in composition from their plasma counterparts; unesterified cholesterol and phospholipid were elevated and the mole ratio of unesterified cholesterol to phospholipid was 0.8. Cholesteryl ester content was extremely low. ApoA-I was the major apolipoprotein, while apoE was the next most abundant protein; small quantities of apoA-II and apoCs were also present. Immunoblot analysis of the d 1.063-1.20 g/ml fraction after gradient gel electrophoresis showed that apoE was localized in the larger pore region of the gel (apparent diameter greater than 12.2 nm); the apoA-I distribution in this fraction was very broad (7.1-12.2 nm), and included a distinct band at 7.4 nm. Immunoblotting after gradient gel electrophoresis of concentrated medium revealed that a significant fraction of apoA-I in the uncentrifuged medium was in a lipid-poor or lipid-free form. This cell line may be a useful model for investigating the metabolism of newly formed HDL.