Apolipoprotein E distribution among human plasma lipoproteins: role of the cysteine-arginine interchange at residue 112

Human apolipoprotein (apo) E occurs as three common isoforms (apoE4, E3, and E2), all of which influence plasma cholesterol levels. Although both apoE4 and E3 bind with equal effectiveness to the low density lipoprotein receptor, they associate preferentially with different classes of plasma lipoproteins: apoE4 with very low density lipoproteins, apoE3 with high density lipoproteins. The primary structure of apoE3 differs from that of apoE4 at only a single site; apoE3 has its sole cysteine residue at position 112, while apoE4 contains arginine at position 112 and completely lacks cysteine. The present study investigated how this structural difference between apoE4 and E3 determines their distribution among plasma lipoproteins, and analyzed the role of the disulfide-linked heterodimer apoE-A-II (which apoE4 cannot form) in determining the distribution. Human plasma was incubated with 125I-labeled apoE, and lipoproteins were separated by agarose chromatography. Both apoE3 that had been reduced and alkylated with iodoacetamide and apoE3-A-II distributed with high density lipoproteins, indicating that a combination of an inherent property of the monomeric apoE3 structure and apoE-A-II formation account for distribution of apoE3 to the high density lipoproteins. Cysteamine modification of apoE3 resulted in an apoE4-like distribution, demonstrating that a positive charge at position 112 determined the apoE4 distribution and that the effect was not exclusively due to the presence of arginine at this position.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of apolipoprotein(AII-E2-AII) complex in human plasma lipoprotein.

A new apolipoprotein complex designated as the apo(AII-E2-AII) complex was identified in the lipoprotein fractions of human plasma with apoE phenotypes containing apoE2 (E4/E2, E3/E2, and E2/E2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by an immunoblotting assay using anti-apoE or anti-apoAII antibodies, established that the apo(AII-E2-AII) complex, with a molecular weight of 58,000, was identical to the complex consisting of apoE and apoAII, and that it also dissociated following reduction with beta-mercaptoethanol. This new complex was also demonstrated to be distinct from the apo(E-AII) complex and apoE monomer by isoelectric focusing, in the samples that were not treated with beta-mercaptoethanol. In apoE phenotype E3/E2, the apo(AII-E2-AII) complex was primarily included in the high-density lipoprotein (HDL, 1.063 < d < 1.21 g/ml) fraction, but was also observed in a small quantity in the very-low-density lipoprotein (VLDL, d < 1.006 g/ml) fraction. For further characterization, the apo(AII-E2-AII) complex was isolated by preparative SDS-PAGE, and no contamination of apo(E-AII) complex and apoE monomer was detected by immunoblotting assay using an anti-apoE antibody. It was confirmed by an enzyme-linked immunosorbent assay (ELISA) system that a molecular ratio between apoAII monomer and apoE in the isolated apo(AII-E2-AII) complex was approx. 2, when the apo(E-AII) complex was used as a standard with the ratio of 1:1. It indicates that the apo(AII-E2-AII) complex is formed from two molecules of apoAII monomer and one molecule of apoE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of free apolipoproteins with macrophages. Formation of high density lipoprotein-like lipoproteins and reduction of cellular cholesterol

Mouse peritoneal macrophages, loaded with cholesteryl ester by incubating with acetylated human low density lipoprotein containing [3H]cholesteryl oleate, were exposed to purified human apolipoproteins (apo) A-I, A-II, C-III, or E in aqueous solutions. Unesterified cholesterol was released into the medium in the presence of apoA-I, -A-II, or -E, accompanied by the decrease in intracellular cholesteryl ester. ApoC-III had no such effects. Apparent Km values of the cholesterol release were estimated as 0.11, 0.14, and 0.24 microM, and Vmax values 35, 11, and 14 micrograms of cholesterol/mg of cell protein/6 h, for apoA-I, -A-II, and -E, respectively. The products formed with apoA-I, -A-II, or -E in the media were analyzed by density gradient ultracentrifugation when the cells were preloaded with [3H]cholesteryl oleate-acetylated low density lipoproteins and [3H]choline. Free [3H]cholesterol, [3H]phosphatidylcholine, and [3H]sphingomyelin were detected coincidentally as a symmetric peak at the density of 1.1 in each case. In the complex of lipids and apoA-I or apoA-II, the weight ratios of apolipoprotein/cholesterol/phosphatidylcholine/sphingomyelin/lysophosphatidyl- choline were estimated as 2.2:1:0.6:0.2:0.07 and 4.0:1:0.5:0.3:0.07, respectively. Both of the products formed with apoA-I and -A-II migrated slower than plasma high density lipoprotein in electrophoresis on agarose gel. Because the Km values are as low as 1:340-400, 1:140-160, and 1:6-8 of plasma concentrations of apoA-I, -A-II, and -E, respectively, the results have physiological relevance for a function of the free apolipoproteins in interstitial fluid to form high density lipoprotein and to reduce cellular cholesterol.     

A novel cell line (Caco-2) for the study of intestinal lipoprotein synthesis

Lipoprotein synthesis by the colonic adenocarcinoma cell line Caco-2 was investigated to assess the utility of this cell line as a model for the in vitro study of human intestinal lipid metabolism. Electron micrographic analysis of conditioned medium revealed that under basal conditions of culture post-confluent Caco-2 cells synthesize and secrete lipoprotein particles. Lipoproteins of density (d) less than 1.063 g/ml consist of a heterogeneous population of particles (diameter from 10 to 90 nm). This fraction consists of very low density lipoproteins (d less than 1.006 g/ml) and low density lipoproteins (d = 1.019-1.063 g/ml). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled Caco-2 lipoproteins revealed that very low density lipoproteins contain apolipoprotein E (apoE) and C apolipoproteins, while low density lipoproteins contained apoB-100, apoE, apoA-I, and C apolipoproteins. The 1.063-1.21 g/ml density fraction contained two morphological entities, discoidal (diameter 15.6 +/- 3.9 nm) and round high density lipoprotein particles (diameter 10.2 +/- 2.3 nm). The high density lipoproteins contained apoA-I, apoB-100, apoB-48, apoE, and the C apolipoproteins. Using isoelectric focusing polyacrylamide gel electrophoresis newly secreted apoA-I was identified as pro-apoA-I. ApoE and apoC-III released by Caco-2 cells were highly sialylated. mRNA species for apoA-I, apoC-III, and apoE, but not apoA-IV were identified by Northern blot analysis. ApoA-I, apoB, and apoE were visualized in Caco-2 cells by immunolocalization analysis. This intestinal cell line may be useful for in vitro studies of nutritional and hormonal regulation of lipoprotein synthesis.     

Human plasma lipoprotein [a]. Structural properties

When lipoprotein [a] was isolated in the presence of the proteolytic inhibitor Trasylol, its apoprotein exhibited one dominant band corresponding to a molecular weight of about 1.2 million when analyzed by electrophoresis on 3.25% sodium dodecyl sulfate-polyacrylamide gels. After chemical reduction, this band was missing but was replaced by two bands, one corresponding to a molecular weight of about 490,000 and the other to a molecular weight of about 645,000. Before treatment with reducing agents, the apolipoprotein [a] and apolipoprotein B immunoreactivities were detectable in the same electrophoretic band, but after reduction the apolipoprotein [a] was demonstrated to be separate from the apolipoprotein B. These results suggest that the apoprotein of lipoprotein [a] is composed of two subunits which are similar in molecular weight and are held together by one or more disulfide bonds. One subunit possesses apolipoprotein [a] and the other apolipoprotein B immunoreactivity. The secondary structure of the apoprotein components within lipoprotein [a] has been studied by circular dichroism and found to differ significantly from the secondary structure of the apoproteins in low density lipoproteins and high density lipoproteins. About 30% alpha-helical structure was measured in lipoprotein [a] compared to 48% in low density lipoproteins and 70% in high density lipoproteins. Lipoprotein [a] exhibited a much higher percentage of disordered structure than either of the other two lipoproteins.     

Apolipoprotein E3-Leiden contains a seven-amino acid insertion that is a tandem repeat of residues 121-127

Apolipoprotein (apo) E3-Leiden is a variant of apoE that is associated with dominant expression of type III hyperlipoproteinemia and that is defective in binding to the low density lipoprotein receptor. Therefore, the structure of apoE3-Leiden was investigated. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis apoE3-Leiden and its 22-kDa amino-terminal thrombolytic fragment migrated with a higher than normal apparent molecular weight. The structural abnormality of apoE3-Leiden was determined by sequencing its CNBr-, tryptic-, and Staphylococcus aureus V8 protease-generated peptides. In contrast to normal apoE3, which has a cysteine at residue 112, apoE3-Leiden does not contain any cysteine and has an arginine at position 112 (as does apoE4, which also completely lacks cysteine). The basis for the molecular weight difference was determined to be a seven-amino acid insertion that is a tandem repeat of residues 121-127 of normal apoE3, i.e. Glu-Val-Gln-Ala-Met-Leu-Gly, resulting in apoE3-Leiden having 306 amino acids rather than 299. The negatively charged glutamyl residues within the insertion compensates for the arginine substitution at residue 112; thus apoE3-Leiden focuses in the E3 position. The low density lipoprotein receptor binding activities of both intact apoE3-Leiden and its 22-kDa thrombolytic fragment were determined in an in vitro assay. Although apoE3-Leiden had only about 25% of normal binding activity, its 22-kDa thrombolytic fragment had nearly normal binding, suggesting that the carboxyl-terminal domain of apoE3-Leiden modulates the receptor binding function of its amino-terminal domain.     

Regulation of apoprotein synthesis and secretion in the human hepatoma Hep G2. The effect of exogenous lipoprotein

The regulation of lipoprotein assembly and secretion at a molecular level is incompletely understood. To begin to identify the determinants of apoprotein synthesis and distribution among lipoprotein classes, we have examined the effects of chylomicron remnants which deliver triglyceride and cholesterol, and beta very low density lipoprotein (beta VLDL), which deliver primarily cholesterol, on apolipoprotein synthesis and secretion by the human hepatoma Hep G2. Hep G2 cells were incubated with remnants or beta VLDL for 24 h, the medium was changed and the cells then incubated with [35S]methionine. The secreted lipoproteins were separated by gradient ultracentrifugation and the radiolabeled apoproteins were isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and counted. Remnants caused a 14-fold, and beta VLDL a 7-fold, increase in VLDL apoprotein (apo) secretion; the apoB/apoE ratio in this class was unchanged. Preincubation with either of the lipoproteins also stimulated low density lipoprotein apoB secretion. Preincubation with beta VLDL, but not with remnants, significantly increased apoE and apoA-I secreted in high density lipoprotein (HDL). In addition, the apoE/apoA-I ratio precipitated from the HDL of beta VLDL-treated cells by anti-apoE was 2.2-fold higher than that precipitated by anti-apoA-I. There was no difference in the ratios precipitated from control HDL. This was due to the secretion of a lipoprotein, subsequently isolated by immunoaffinity chromatography, that contained predominantly apoE. When Hep G2 cells were preincubated with oleic acid alone, total apoprotein secretion was not altered. However, cholesterol-rich liposomes stimulated secretion of newly synthesized apoE, but not apoB, while apoA-I secretion was variably affected. Cholesterol-poor liposomes had no effect. Thus, lipid supply is a determinant of apoprotein synthesis and secretion, and cholesterol may be of particular importance in initiating apoprotein synthesis.     

ELISA quantitation of apolipoproteins in plasma lipoprotein fractions: ApoE in apoB-containing lipoproteins (Lp B:E) and apoB in apoE-containing lipoproteins (Lp E:B)

Growing clinical evidence suggests that metabolic behavior and atherogenic potential vary within lipoprotein subclasses that can be defined by apolipoprotein variation. Variant constituency of apolipoproteins B and E (apoB and apoE) may be particularly important because of the central roles of these apolipoproteins in the endogeneous lipid delivery cascade. ApoB is the sole protein of low-density lipoprotein (LDL), and like LDL cholesterol, the plasma apoB level has been positively correlated with risk for atherosclerotic disease. ApoE is a major functional lipoprotein in the triglyceride-rich lipoproteins, and may be crucial in the conversion of very low density lipoprotein (VLDL) to LDL. Based on work by others that enabled the quantititation of apoB-containing particles by content of up to two other types of apolipoprotein, we have developed a method for determining the amount of apoE in apoB-containing lipoproteins (Lp B:E) and the amount of apoB in apoE-containing lipoproteins (Lp E:B). From the Lp B:E and Lp E:B concentrations, the molar ratio of apoE to apoB in lipoproteins containing apoB and/or apoE in plasma can be determined. The methodology is fast, specific, and sensitive and should prove extremely useful in further categorizing lipoproteins and characterizing their behavior. In applying this method to clinical groupings of normo- and hyperlipidemia, we found that the plasma triglyceride level correlated with the apoE and Lp B:E concentrations in plasma, while the total cholesterol level correlated with the apoB and Lp E:B levels.     

Apolipoprotein E metabolism in normolipoproteinemic human subjects

Human apolipoprotein E (apoE) is a constituent of plasma very low density and high density lipoproteins and is important in modulating the catabolism of remnants of triglyceride-rich lipoproteins. There are three common isoforms of apoE, designated apoE-2, E-3, and E-4, which are coded by three separate alleles (epsilon 2, epsilon 3, and epsilon 4) at a single genetic locus and inherited in the population in a co-dominant fashion. ApoE-3 is the predominant apoE isoform in the normolipidemic population, and epsilon 3 has been proposed to be the normal allele. ApoE-3 metabolism was studied in nine normolipidemic subjects homozygous for the epsilon 3 allele. In these subjects, the plasma apoE-3 concentration was 4.8 +/- 1.2 mg/dl (mean +/- SD), the plasma apoE-3 residence time was 0.73 +/- 0.18 days, and the plasma apoE-3 production rate was 3.4 +/- 1.5 mg/kg-day. The apoE in males, when compared to females, tended to have a shorter residence time (0.63 +/- 0.15 days versus 0.83 +/- 0.16), a higher production rate (4.20 +/- 1.73 mg/kg-days versus 2.60 +/- 0.78), but a similar plasma concentration (5.1 +/- 1.5 mg/dl versus 4.5 +/- 0.8). ApoE-3 had a more rapid catabolism from plasma than other apolipoproteins previously studied (apolipoproteins A-I, A-II, A-IV, B-100, C-II, and C-III) except for apolipoprotein B-48. The catabolism of apoE-3 in the individual lipoprotein subfractions was also examined and apoE was shown to be catabolized most rapidly from the VLDL and slowest from the HDL. The results of the kinetic analysis of apoE metabolism are consistent with apoE being important in the catabolism of triglyceride-rich lipoproteins and with HDL serving as a reservoir for apoE to reassociate with newly secreted triglyceride-rich lipoproteins.     

Apolipoprotein E phenotypes in Finnish youths: a cross-sectional and 6-year follow-up study

Apolipoprotein E (apoE) polymorphism is a genetic determinant of plasma lipid levels and of coronary heart disease (CHD) risk. We determined the apoE phenotypes and plasma lipid levels in 1577 youths aged 3 to 18 years in 1980. The subjects were randomly selected from five areas of Finland. ApoE phenotyping was performed directly from plasma by isoelectric focusing and immunoblotting. The apoE allele frequencies in the population sample were epsilon 2 = 0.039, epsilon 3 = 0.767, and epsilon 4 = 0.194. There were no differences in the apoE phenotype distribution between East and West Finland or between sexes. The concentrations of serum total cholesterol, low density lipoprotein cholesterol, and apolipoprotein B increased with apoE phenotype in the order of E2/2, E3/2, E4/2, E3/3, E4/3, and E4/4. This increase was already seen in 3-year-old children; it was observed in both sexes, but was clearer in males than in females. The mean levels of high density lipoprotein (HDL) cholesterol, apolipoprotein A-I, triglyceride, Lp[a] lipoprotein, and the activity of lecithin:cholesterol acyltransferase did not differ between the apoE phenotypes. The observed differences in serum cholesterol remained fairly stable during the 6-year follow-up from 1980 to 1986, while the mean serum cholesterol concentration in the whole study population decreased by 6.3%. This study confirms the reported higher frequency of the epsilon 4 allele in Finns as compared to most other populations; this may contribute to the high rates of CHD in Finland as compared to most other populations. The results do not, however, explain the higher rate of CHD in East Finland in comparison to the western part of the country.     

Characterization of apolipoprotein A-IV complexes and A-IV isoforms in human lymph and plasma lipoproteins

We have isolated and characterised A-IV apolipoprotein (apo-A-IV) from human lymph and plasma by immunoabsorbance chromatography and two-dimensional electrophoresis. Two different apo-A-IV-containing lipoproteins were isolated from four different sources, human lymph triglyceride-rich fraction (TRL), lymph lipoprotein-deficient fraction (LDF), plasma high-density lipoprotein (HDL), and plasma lipoprotein-deficient fraction (LDF). The lipoprotein complexes obtained from lymph TRL and plasma HDL were similar and contained apo-A-IV, apo-A-I, and small molecular weight peptides (apo-C or -A-II). The second lipoprotein complex was isolated from lymph LDF and plasma LDF, and contained apo-A-IV, apo-A-I, and a peptide of Mr = 59,000. The lipid composition of the lipoprotein complexes varied according to the source: triglyceride predominating in lymph TRL and phospholipid and cholesteryl ester from the other sources. Free cholesterol was conspicuously present in very small amounts. Using two-dimensional electrophoresis and immunoblotting techniques, eleven isoproteins of apo-A-IV were identified (pI-4.98, 5.06, 5.10, 5.15, 5.20, 5.22, 5.25, 5.30, 5.34, 5.42, and 5.48). The isoprotein pattern of lymph TRL and plasma HDL was similar, but that of lymph and plasma LDF were different patterns. These results suggest that apo-A-IV associated with d less than 1.21 lipoproteins and apo-A-IV present in LDF may be in metabolically separate lipoproteins and may have different physiological roles.     

Apolipoprotein E and lipoprotein lipase secreted from human monocyte-derived macrophages modulate very low density lipoprotein uptake

We investigated the roles of lipoprotein lipase and apolipoprotein E (apoE) secreted from human monocyte-derived macrophages in the uptake of very low density lipoproteins (VLDL). ApoCII-deficient VLDL were isolated from a patient with apoCII deficiency. The lipolytic conversion to higher density and the degradation of the apoCII-deficient VLDL by macrophages were very slight, whereas the addition of apoCII enhanced both their conversion and degradation. This suggests that the lipolysis and subsequent conversion of VLDL to lipoproteins of higher density are essential for the VLDL uptake by macrophages. VLDL incubated with macrophages obtained from subjects with E3/3 phenotype (E3/3-macrophages) showed a 17-fold greater affinity in inhibiting the binding of 2 micrograms/ml 125I-low density lipoprotein (LDL) to fibroblasts than native VLDL, whereas the incubation of VLDL with macrophages obtained from a subject with E2/2 phenotype (E2/2-macrophages) did not cause any increase in their affinity. Furthermore, 3 micrograms/ml 125I-VLDL obtained from a subject with E3/3 phenotype were degraded by E3/3-macrophages to a greater extent than by E2/2-macrophages (2-fold), indicating that VLDL uptake is influenced by the phenotype of apoE secreted by macrophages. From these results, we conclude that both lipolysis by lipoprotein lipase and incorporation of apoE secreted from macrophages alter the affinity of VLDL for the LDL receptors on the cells, resulting in facilitation of their receptor-mediated endocytosis.     

Chemical characterization of the serum very low density and high density lipoproteins from bullfrog, Rana catesbeiana.

The chemical properties of very low density and high density lipoproteins of adult bullfrog serum were determined. This serum contained extremely low levels of both very low density lipoprotein (10-30 mg/100 ml) and high density lipoprotein (5-10 mg/100 ml). The constituents of very low density lipoprotein, on a weight percentage basis, were found to be 48.1% triglyceride, 17.3% cholesterol ester, 8.8% cholesterol, 11.6% phospholipid, and 12% protein. These constituents were also present in high density lipoprotein with weight percentage values of 3.7%, 19.3%, 11.9%, 25.2%, and 36.8%, respectively. The fatty acid compositions of the triglycerides, cholesterol esters, and phosphatidylcholine were quite similar in the very low density lipoprotein and high density lipoprotein. However, shingomyelin fatty acid composition was appreciably different in the two lipoproteins. Disc gel electrophoresis in sodium dodecyl sulfate-polyacrylamide gels produced patterns with one major (approximate molecular weight, 7,000) and several minor bands for the apoprotein of very low density lipoprotein and one major (approximate molecular weight, 28,000) and several minor bands for that of high density lipoprotein.     

Capillary isotachophoresis study of lipoprotein network sensitive to apolipoprotein E phenotype. 1. ApoE distribution between lipoproteins

Sixteen patients differing widely in plasma triglyceride content were divided into three groups by their apolipoprotein E (apoE) phenotype—E33 homozygotes, E23, and E34 heterozygotes. The plasma lipid and apoE distribution between individual lipoproteins was followed by capillary isotachophoresis (CITP) of plasma samples pre-stained with lipid fluorescent probe NBD-C6-ceramide and by fluorescein-labeled apoE, respectively. Among 12 peaks visualized by ceramide staining, an individual peak with very low density lipoproteins (VLDL) was identified. The VLDL cholesterol and apoE content determined by CITP directly in whole plasma were significantly related to their content as determined by conventional analysis with isolated VLDL. The ceramide distribution among lipoprotein pools was insensitive to apoE phenotype (49–53 : 7–11 : 39–43% for HDL, VLDL, and IDL/LDL, respectively) while the preferential binding of apoE to VLDL was observed in E34 patients compared to E33 (62 : 19 : 20 vs. 70 : 9 : 22%). In a study of apoE/F displacement from lipoproteins at plasma titration by apoC-III in vitro, apoE was found to bind more tightly to VLDL from E34 compared to E33 patients as evidenced by both the increased non-displaceable apoE pool, the increased VLDL sorbtion capacity for apoE, and the decreased displacement parameter in a “container” model of lipoprotein binding. Two different types of apoE package in a whole lipoprotein profile were observed. ApoE structure in a particular lipoprotein may underlie the phenotype-sensitive apoE distribution and apoC-III interference in hypertriglyceridemia.     

The secretion of apolipoprotein E by human monocyte-derived macrophages

The secretion of newly synthesized apolipoprotein E (apo E) by human monocyte-derived macrophages (HMD macrophages) was measured in the medium of cells which had been incubated for 24 h with or without either native or acetylated low-density lipoproteins (LDL or AcLDL, respectively), and subsequently with [35S]methionine in the presence of high-density lipoproteins (HDL, 350 micrograms/ml) for 24 h, by isolating the lipoprotein fraction by centrifugation for 48 h at a density adjusted with KBr to 1.21 g/ml (d = 1.21). The d less than 1.21 medium was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or precipitation with trichloroacetic acid (TCA). Fluorography of the gels demonstrated that the d less than 1.21 fraction of the medium contained one major labeled band, which migrated with an apparent molecular weight of approximately 35,000. Immunoprecipitation of the d less than 1.21 fraction showed that the labeled band was precipitated by anti-apo E, but not by anti-HDL. As the apo E band appeared to be the only labeled band in the d less than 1.21 fraction, the amount of apo E secreted by the cells was quantitated by scintillation counting of the TCA-precipitable radioactivity in the d less than 1.21 fraction as compared with that in the whole medium. The proportion of secreted apo E to the total secreted protein was similar whether the cells had been in culture for 3 or 16 days, but was increased if the cells had been incubated with LDL or AcLDL. The proportion of apo E of the secreted proteins was always more than 6% and was as much as 16% when the cells were preincubated with lipoproteins, suggesting that the increased cholesterol influx induced apo E secretion.     

Structural and functional properties of human plasma high density-sized lipoprotein containing only apoE particles

To investigate the metabolism of HDL-apolipoprotein E (apoE) particles in human plasma, we isolated a fraction of plasma HDL-apoEs that lack apoA-I (HDL-LpE) from subjects with apoE3/3 phenotype by immunoaffinity. Plasma HDL-LpE had a particle size ranging from 9 nm to 18.5 nm in diameter and was characterized by two-dimensional nondenaturing gradient gel electrophoresis as having either gamma-, prebeta1-, prebeta2-, or alpha-electrophoretic mobility. HDL-LpE was also present in the medium of cultured human hepatoma cell lines and monocyte-derived macrophages. The majority of apoE3 was found as a monomeric form in HDL-LpE and floated at density d > 1.21 g/ml. Plasma levels of HDL-LpE in normolipidemic, CETP-deficient, and ABCA1-deficient subjects were 0.72 +/- 0.15 mg/dl (n = 12), 1.77 +/- 0.75 mg/dl (n = 3), and 0.55 +/- 0.11 mg/dl (n = 3), respectively. The ratio of HDL-apoE containing apoA-I to HDL-LpE was significantly higher 4 h after a fat load, representing a 35 +/- 9% increase (n = 3). Isolated plasma HDL-LpE3 was as effective as apoE3, reconstituted HDL particles, or apoA-I in promoting cellular cholesterol efflux. These results demonstrate that 1) plasma HDL-LpE may have hepatogenous and macrophagic origins; 2) HDL-LpE was preserved even with large reductions in apoA-I-containing lipoproteins; 3) HDL-LpE was active in the transfer of apoE to triglyceride-rich lipoproteins, and 4) HDL-LpEs efficiently take up cell-derived cholesterol.     

Apolipoprotein E2-Dunedin (228 Arg replaced by Cys): an apolipoprotein E2 variant with normal receptor-binding activity

Homozygosity for the apolipoprotein (apo) E variant apoE2(158 Arg----Cys) invariably gives rise to dysbetalipoproteinemia, and when associated with obesity or a gene for hyperlipidemia, results in type III hyperlipoproteinemia. The association of the E2/2 phenotype with type IV/V hyperlipoproteinemia rather than type III hyperlipoproteinemia in identical twin brothers led us to investigate the primary structure of their apoE. Lipoprotein electrophoresis on agarose gels confirmed the presence of increased very low density lipoproteins (VLDL) and chylomicrons but little, if any, beta-VLDL, indicating that these subjects did not have dysbetalipoproteinemia. When the apoE from these twins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a system that can distinguish apoE2(158 Arg----Cys) from all other known apoE variants, it gave rise to two components. One had the unique mobility of apoE2(158 Arg----Cys), and one migrated in the position of the other variants of apoE (and normal apoE3), indicating that the brothers were heterozygous for apoE2(158 Arg----Cys) and a second apoE2 isoform. Cysteamine modification and isoelectric focusing showed that, like apoE2(158 Arg----Cys), the second apoE2 isoform also contained two cysteine residues. The structural mutation in the second apoE2 isoform was determined by peptide sequencing. Like normal apoE3, this variant had arginine at position 158, but differed from apoE3 by the substitution of cysteine for arginine at position 228. Total apoE isolated from the brothers had the same receptor-binding activity in a competitive binding assay as a 1:1 mixture of normal apoE3 and apoE2(158 Arg----Cys).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.     

Unique lipoproteins secreted by primary astrocytes from wild type, apoE (-/-), and human apoE transgenic mice.

Composition of central nervous system lipoproteins affects the metabolism of lipoprotein constituents within the brain. The epsilon4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer's disease via an unknown mechanism(s). As glia are the primary central nervous system cell type that synthesize apoE, we characterized lipoproteins secreted by astrocytes from wild type (WT), apoE (-/-), and apoE transgenic mice expressing human apoE3 or apoE4 in a mouse apoE (-/-) background. Nondenaturing size exclusion chromatography demonstrates that WT, apoE3, and apoE4 astrocytes secrete particles the size of plasma high density lipoprotein (HDL) composed of phospholipid, free cholesterol, and protein, primarily apoE and apoJ. However, the lipid:apoE ratio of particles containing human apoE is significantly lower than WT. ApoE localizes across HDL-like particle sizes. ApoJ localizes to the smallest HDL-like particles. ApoE (-/-) astrocytes secrete little phospholipid or free cholesterol despite comparable apoJ expression, suggesting that apoE is required for normal secretion of astrocyte lipoproteins. Further, particles were not detected in apoE (-/-) samples by electron microscopy. Nondenaturing immunoprecipitation experiments indicate that apoE and apoJ reside predominantly on distinct particles. These studies suggest that apoE expression influences the unique structure of astrocyte lipoproteins, a process further modified by apoE species.