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• W2049660003 abstract "Apolipoprotein E (apoE)/ABCA1 interactions were investigated in human intact fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Here, we show that purified human plasma apoE3 forms a complex with ABCA1 in normal fibroblasts. Lipid-free apoE3 inhibited the binding of 125I-apoA-I to ABCA1 more efficiently than reconstituted HDL particles (IC50 = 2.5 ± 0.4 μg/ml vs. 12.3 ± 1.3 μg/ml). ApoE isoforms showed similar binding for ABCA1 and exhibited identical kinetics in their abilities to induce ABCA1-dependent cholesterol efflux. Mutation of ABCA1 associated with Tangier disease (C1477R) abolished both apoE3 binding and apoE3-mediated cholesterol efflux. Analysis of apoE3-containing particles generated during the incubation of lipid-free apoE3 with stimulated normal cells showed nascent apoE3/cholesterol/phospholipid complexes that exhibited preβ-electrophoretic mobility with a particle size ranging from 9 to 15 nm, whereas lipid-free apoE3 incubated with ABCA1 mutant (C1477R) cells was unable to form such particles.These results demonstrate that 1) apoE association with lipids reduced its ability to interact with ABCA1; 2) apoE isoforms did not affect apoE binding to ABCA1; 3) apoE-mediated ABCA1-dependent cholesterol efflux was not affected by apoE isoforms in fibroblasts; and 4) the lipid translocase activity of ABCA1 generates apoE-containing high density-sized lipoprotein particles. Thus, ABCA1 is essential for the biogenesis of high density-sized lipoprotein containing only apoE particles in vivo. Apolipoprotein E (apoE)/ABCA1 interactions were investigated in human intact fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Here, we show that purified human plasma apoE3 forms a complex with ABCA1 in normal fibroblasts. Lipid-free apoE3 inhibited the binding of 125I-apoA-I to ABCA1 more efficiently than reconstituted HDL particles (IC50 = 2.5 ± 0.4 μg/ml vs. 12.3 ± 1.3 μg/ml). ApoE isoforms showed similar binding for ABCA1 and exhibited identical kinetics in their abilities to induce ABCA1-dependent cholesterol efflux. Mutation of ABCA1 associated with Tangier disease (C1477R) abolished both apoE3 binding and apoE3-mediated cholesterol efflux. Analysis of apoE3-containing particles generated during the incubation of lipid-free apoE3 with stimulated normal cells showed nascent apoE3/cholesterol/phospholipid complexes that exhibited preβ-electrophoretic mobility with a particle size ranging from 9 to 15 nm, whereas lipid-free apoE3 incubated with ABCA1 mutant (C1477R) cells was unable to form such particles. These results demonstrate that 1) apoE association with lipids reduced its ability to interact with ABCA1; 2) apoE isoforms did not affect apoE binding to ABCA1; 3) apoE-mediated ABCA1-dependent cholesterol efflux was not affected by apoE isoforms in fibroblasts; and 4) the lipid translocase activity of ABCA1 generates apoE-containing high density-sized lipoprotein particles. Thus, ABCA1 is essential for the biogenesis of high density-sized lipoprotein containing only apoE particles in vivo. Human apolipoprotein E (apoE) is an arginine-rich glycoprotein (34,200 Da) that plays a pivotal role in lipoprotein metabolism and neurobiology through its interactions with heparan sulfate proteoglycans and the LDL receptor family (1Mahley R.W. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology.Science. 1988; 240: 622-630Google Scholar). Thus, apoE is believed to play a significant role in the onset and development of coronary artery atherosclerosis (2Davignon J. Gregg R.E. Sing C.F. Apolipoprotein E polymorphism and atherosclerosis.Arteriosclerosis. 1988; 8: 1-21Google Scholar) and the pathophysiology of Alzheimer's disease (3Poirier J. Minnich A. Davignon J. Apolipoprotein E, synaptic plasticity and Alzheimer's disease.Ann. Med. 1995; 27: 663-670Google Scholar). The importance of apoE in the pathogenesis of atherosclerosis has been strikingly demonstrated by the presence of spontaneous atherosclerosis in experimental animals made deficient in apoE (4Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.Science. 1992; 258: 468-471Google Scholar, 5Linton M.F. Atkinson J.B. Fazio S. Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation.Science. 1995; 267: 1034-1037Google Scholar) and conversely by the protection against or regression of atherosclerosis in apoE-deficient animals supplemented with apoE (6Boisvert W.A. Spangenberg J. Curtiss L.K. Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation.J. Clin. Invest. 1995; 96: 1118-1124Google Scholar, 7Bellosta S. Mahley R.W. Sanan D.A. Murata J. Newland D.L. Taylor J.M. Pitas R.E. Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice.J. Clin. Invest. 1995; 96: 2170-2179Google Scholar). ApoE exists in three isoforms, apoE2, apoE3, and apoE4, each differing by cysteine and arginine at positions 112 and 158. ApoE3, the most common form, contains cysteine and arginine at these positions, respectively, whereas apoE2 contains cysteine and apoE4 contains arginine at both sites (8Weisgraber K.H. Rall Jr., S.C. Mahley R.W. Human E apoprotein heterogeneity. Cysteine-arginine interchanges in the amino acid sequence of the apo-E isoforms.J. Biol. Chem. 1981; 256: 9077-9083Google Scholar). These differences have profound effects on the biological functions of apoE. Both apoE3 and apoE4 bind to the LDL receptor with high affinity, whereas apoE2 exhibits defective binding to the LDL receptor and is associated with type III hyperlipoproteinemia (9Mahley R.W. Huang Y. Rall Jr., S.C. Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia). Questions, quandaries, and paradoxes.J. Lipid Res. 1999; 40: 1933-1949Google Scholar). ApoE4 is associated with high plasma cholesterol level and increased risk for both coronary heart disease and Alzheimer's disease (2Davignon J. Gregg R.E. Sing C.F. Apolipoprotein E polymorphism and atherosclerosis.Arteriosclerosis. 1988; 8: 1-21Google Scholar, 10Weisgraber K.H. Mahley R.W. Human apolipoprotein E: the Alzheimer's disease connection.FASEB J. 1996; 10: 1485-1494Google Scholar). Although it is clear that apoE plays an important role in the reverse cholesterol transport (RCT) process, structural determinants of molecular interactions between key proteins involved in RCT and apoE have not yet been elucidated. However, there is ample evidence that apoE can directly affect the ability of HDL particles to mediate cellular lipid efflux. For example, it has been documented that the capacity of apoE-depleted HDL of human or HDL of apoE-deficient mice to promote cholesterol efflux from mouse peritoneal macrophages is decreased and can be restored to normal by the addition of apoE (11Basu S.K. Ho Y.K. Brown M.S. Bilheimer D.W. Anderson R.G. Goldstein J.L. Biochemical and genetic studies of the apoprotein E secreted by mouse macrophages and human monocytes.J. Biol. Chem. 1982; 257: 9788-9795Google Scholar, 12Hayek T. Oiknine J. Brook J.G. Aviram M. Role of HDL apolipoprotein E in cellular cholesterol efflux: studies in apo E knockout transgenic mice.Biochem. Biophys. Res. Commun. 1994; 205: 1072-1078Google Scholar). A recent study from our laboratory documented that human plasma high density-sized lipoprotein containing only apoE particles (HDL-LpE) is very effective in removing acetyl-LDL-derived [3H]cholesterol from J744 macrophages (13Krimbou L. Marcil M. Chiba H. Genest Jr., J. Structural and functional properties of human plasma high density-sized lipoprotein containing only apoE particles.J. Lipid Res. 2003; 44: 884-892Google Scholar). Phenotype-specific differences have also been shown in the ability of apoE to promote cholesterol efflux from cultured cells. γ-LpE from apoE3/3 individuals stimulated 7- to 13-fold more cholesterol efflux from cultured fibroblasts than the same fraction from apoE2/2 or apoE4/4 individuals (14Huang Y. von Eckardstein A. Wu S. Assmann G. Effects of the apolipoprotein E polymorphism on uptake and transfer of cell-derived cholesterol in plasma.J. Clin. Invest. 1995; 96: 2693-2701Google Scholar). Moreover, it was documented that apoE promotes lipid release from astrocytes and neurons in an isoform-dependent manner (15Michikawa M. Fan Q.W. Isobe I. Yanagisawa K. Apolipoprotein E exhibits isoform-specific promotion of lipid efflux from astrocytes and neurons in culture.J. Neurochem. 2000; 74: 1008-1016Google Scholar). The discovery that the low HDL levels associated with Tangier disease (TD) and familial HDL deficiency are attributable to mutations in the ABCA1 gene (16Brooks-Wilson A. Marcil M. Clee S.M. Zhang L-H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O.F. Loubser O. Francis Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J.D. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J.P. Genest Jr., J. Hayden M.R. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.Nat. Genet. 1999; 22: 336-345Google Scholar, 17Marcil M. Brooks-Wilson A. Clee S.M. Roomp K. Zhang L-H. Yu L. Collins J.A. van Dam M. Molhuizen H.O.F. Loubster O. Francis Ouellette B.F. Sensen C.W. Fichter K. Mott S. Denis M. Boucher B. Pimstone S. Genest Jr., J. Kastelein J.J.P. Hayden M.R. Mutations in the ABC1 gene in familial HDL deficiency with defective cholesterol efflux.Lancet. 1999; 354: 1341-1346Google Scholar) has revealed that this transporter is crucial for HDL biogenesis, because it mediates the apolipoprotein-dependent transfer of intracellular cholesterol and phospholipids to lipid-free apolipoproteins (18Brewer Jr., H.B. Santamarina-Fojo S. New insights into the role of the adenosine triphosphate-binding cassette transporters in high-density lipoprotein metabolism and reverse cholesterol transport.Am. J. Cardiol. 2003; 91: 3E-11EGoogle Scholar, 19Attie A.D. Kastelein J.P. Hayden M.R. Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibility to atherosclerosis.J. Lipid Res. 2001; 42: 1717-1726Google Scholar, 20Marcil M. Bissonnette R. Vincent J. Krimbou L. Genest J. Cellular phospholipid and cholesterol efflux in high-density lipoprotein deficiency.Circulation. 2003; 107: 1366-1371Google Scholar). To better define the role of ABCA1 in the biogenesis of LpE particles, experiments were directed in the present study at defining the mechanism by which apoE is lipidated by ABCA1 and how the interactions of apoE and ABCA1 can be affected by apoE association with lipids, by apoE isoforms, or by naturally occurring mutation of ABCA1. For the present study, we selected fibroblasts from three normal control subjects and one patient with TD (compound heterozygous carrying the mutations C1477R and the splice site G→C in exon 24) as previously described (16Brooks-Wilson A. Marcil M. Clee S.M. Zhang L-H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O.F. Loubser O. Francis Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J.D. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J.P. Genest Jr., J. Hayden M.R. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.Nat. Genet. 1999; 22: 336-345Google Scholar, 20Marcil M. Bissonnette R. Vincent J. Krimbou L. Genest J. Cellular phospholipid and cholesterol efflux in high-density lipoprotein deficiency.Circulation. 2003; 107: 1366-1371Google Scholar). The protocol for the study was reviewed and accepted by the Research Ethics Board of the McGill University Health Centre. Separate consent forms for blood sampling, DNA isolation, and skin biopsy were provided. Human skin fibroblasts were obtained from 3.0 mm punch biopsies of the forearm of the patient and healthy control subjects and cultured in DMEM supplemented with 0.1% nonessential amino acids, penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% FBS. Purified human plasma apoE isoforms were a gift from Dr. Karl H. Weisgraber (Gladstone Institutes of Cardiovascular Disease, San Francisco, CA). Before use, either lyophilized apoE or apoA-I (Biodesign) was resolubilized in 4 M guanidine HCl and dialyzed extensively against Tris buffer (10 mM Tris, 150 mM NaCl, and 1.0 mM EDTA, pH 8.2) as described previously (21Gillotte K.L. Davidson W.S. Lund-Katz S. Rothblat G.H. Phillips M.C. Removal of cellular cholesterol by pre-beta-HDL involves plasma membrane microsolubilization.J. Lipid Res. 1998; 39: 1918-1928Google Scholar). Cholesterol efflux was determined as previously described (20Marcil M. Bissonnette R. Vincent J. Krimbou L. Genest J. Cellular phospholipid and cholesterol efflux in high-density lipoprotein deficiency.Circulation. 2003; 107: 1366-1371Google Scholar) with minor modifications. Briefly, 50,000 cells were seeded in 12-well plates. At midconfluence, the cells were labeled with 0.2 μCi/ml [3H]cholesterol (Perkin Elmer) for 48 h. At confluence, cells were cholesterol-loaded (20 μg/ml) for 24 h. During a 24 h equilibration period, cells were stimulated or not with 2.5 μg/ml 22(R)-hydroxycholesterol and 5 μM 9-cis-retinoic acid for 20 h. Cholesterol efflux was determined for the indicated concentration and time as follows: 3H cpm in medium/(3H cpm in medium + 3H cpm in cells); the results were expressed as percentage of total radiolabeled cholesterol. Cells from a normal and a TD subject were grown to confluence in 100 mm diameter dishes, labeled with 15 μCi/ml [14C]cholesterol (Perkin Elmer) for 48 h, and then cholesterol-loaded, equilibrated, and stimulated as described above. Cellular [14C]cholesterol labeling was used for the characterization of apoE-containing particles generated during the incubation of lipid-free apoE3 with stimulated cells. Cell phospholipids were labeled with [32P]orthophosphate as follows: fibroblasts from a control and a TD subject were grown to confluence in 100 mm diameter dishes and incubated for 72 h with 300 μCi of [32P]orthophosphate mixed with DMEM. After the first 24 h of incubation with [32P]orthophosphate, cells were cholesterol-loaded (20 μg/ml) for 24 h, and then during a 24 h equilibration period, the cells were stimulated as described above before incubation with apoE3. Complexes comprising apoE3, POPC, and cholesterol were prepared using the sodium cholate dialysis method described by Jonas, Steinmetz, and Churgay (22Jonas A. Steinmetz A. Churgay L. The number of amphipathic alpha-helical segments of apolipoproteins A-I, E, and A-IV determines the size and functional properties of their reconstituted lipoprotein particles.J. Biol. Chem. 1993; 268: 1596-1602Google Scholar). An apoE3/POPC/cholesterol molar ratio of 1:100:5 was used in this experiment. Reconstituted HDL particles [r(LpE3)] were further concentrated by ultrafiltration (spiral ultrafiltration cartridge, MWCO 50,000; Amicon) to discard any lipid-free apoE3 or proteolytic peptides. ApoE3/lipid complex formation was verified by analysis with native polyacrylamide gradient (8–25%) gel electrophoresis. The competitive binding assay was performed as previously described (23Denis M. Haidar B. Marcil M. Bouvier M. Krimbou L. Genest Jr., J. Molecular and cellular physiology of apolipoprotein A-I lipidation by the ATP-binding cassette transporter A1 (ABCA1).J. Biol. Chem. 2004; 279: 7384-7394Google Scholar). Briefly, apoA-I isolated from human plasma (Biodesign) was iodinated with 125I by IODO-GEN® (Pierce) to a specific activity of 800 cpm/ng apoA-I. Cells were grown on 24-well plates and stimulated with 2.5 μg/ml 22(R)-hydroxycholesterol and 5 μM 9-cis-retinoic acid for 20 h. Cells were then incubated for 2 h at 37°C with 1 μg/ml 125I-apoA-I in DMEM/BSA (1 mg/ml) in the presence or absence of increasing amounts of the unlabeled competitor [apoA-I, apoE3, and r(LpE3)]. The cells were then washed rapidly two times with ice-cold PBS/BSA and two times with cold PBS and lysed with 0.1 N NaOH. The amount of bound iodinated ligand was determined by γ counting, and the protein content was measured using a modified Lowry method as described previously (24Lowry O.H. Rosebrough N.F. Fan A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar). Chemical cross-linking was performed as described by Wang et al. (25Wang N. Silver D.L. Costet P. Tall A.R. Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1.J. Biol. Chem. 2000; 275: 33053-33058Google Scholar) with a minor modification. Fibroblasts were grown to confluence in 100 mm diameter dishes and then stimulated or not with 2.5 μg/ml 22(R)-hydroxycholesterol and 5 μM 9-cis-retinoic acid for 20 h in DMEM/BSA. Cells were incubated in the presence or absence of 3 μg/ml apoE2, apoE3, apoE4, or apoA-I in DMEM/BSA for 1 h at 37°C. Cells were then placed on ice for 15 min and washed three times with PBS. Dithiobis(succinimidylpropionate) (DSP; cross-linker agent) was dissolved immediately before use in DMSO and diluted to 250 μM with PBS. Eight milliliters of DSP solution was added in each well. Cells were then incubated at room temperature for 1 h; the medium was removed, and the cells were washed twice with PBS. Cells were lysed at 4°C with immunoprecipitation buffer containing 20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, and 1% Triton X-100 (Invitrogen), and the suspension was allowed to stand for 30 min at 4°C in the presence of a protease inhibitor cocktail (Roche Diagnostics). ApoE/ABCA1 and apoA-I/ABCA1 complexes were immunoprecipitated with an affinity-purified polyclonal anti-ABCA1 antibody (Novus Biologicals) as described (26Haidar B. Denis M. Krimbou L. Marcil M. Genest Jr., J. cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts.J. Lipid Res. 2002; 43: 2087-2094Google Scholar) or used for solid-phase binding assay. Immunoprecipitated complexes were separated on SDS gels as previously described (26Haidar B. Denis M. Krimbou L. Marcil M. Genest Jr., J. cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts.J. Lipid Res. 2002; 43: 2087-2094Google Scholar). ApoE or apoA-I associated with ABCA1 was detected by affinity-purified human anti-apoE or anti-apoA-I antibodies (12171-21E and 12171-21A, respectively; Genzyme Corp.). Fibroblasts were grown to confluence in 100 mm diameter dishes and then stimulated or not for 20 h. Cells were incubated with either apoE or apoA-I (3 μg/ml), and then chemical cross-linking was performed as described above. The cells were swollen on ice for 10 min and then homogenized with 20 strokes in a tight-fitting Dounce homogenizer. After centrifugation at 1,000 g and 4°C for 3 min to remove unbroken cells and nuclei, the supernatant was recentrifuged at 100,000 g and 4°C for 60 min. The resulting supernatant was discarded, and the final microsomal fraction pellet was resuspended in 250 μl of immunoprecipitation buffer. Total microsomal fraction containing apoE/ABCA1 and apoA-I/ABCA1 complexes were used for solid-phase binding assays. To quantitate apoE isoform binding to ABCA1, we used solid-phase binding assays. Ninety-six-well microtiter plates (Nunc Immunosorb modules) were coated overnight at 4°C with either 5 μg/ml anti-ABCA1 antibody or albumin in PBS. Unbound proteins were washed from the wells, and nonspecific binding sites were blocked by incubation with 5% BSA and 0.05% Tween 20 in PBS for 1 h at room temperature. Direct binding assay was performed by adding either apoE isoforms or apoA-I associated with ABCA1 from both stimulated and unstimulated cells prepared as described above. Isolated microsomal fraction (120 ng) was added to immobilized anti-ABCA1 antibody or BSA-coated plates in Tris-buffered saline, pH 7.5, containing 1% BSA, 0.01% Tween 20, and 1 mM CaCl2. Then, the plates were incubated for 16 h at 4°C. Unbound proteins were aspirated, and the wells were washed three times with PBS. To detect bound apoE or apoA-I, the plates were incubated with 125I-labeled affinity-purified polyclonal anti-human apoE or apoA-I antibody for 1 h at 37°C. The wells were then washed three times with PBS, and bound radioactivity was removed with 10% SDS and counted. To detect nonspecific binding, all assays were done simultaneously on plates coated with BSA alone as described above. The background binding to BSA was subtracted from all samples and represented less than 5% of total binding. ApoE3-containing particles were separated by two-dimensional polyacrylamide nondenaturing gradient gel electrophoresis (2D-PAGGE) as previously described (27Krimbou L. Tremblay M. Davignon J. Cohn J.S. Characterization of human plasma apolipoprotein E-containing lipoproteins in the high density lipoprotein size range: focus on pre-beta1-LpE, pre-beta2-LpE, and alpha-LpE.J. Lipid Res. 1997; 38: 35-48Google Scholar, 28Krimbou L. Marcil M. Davignon J. Genest Jr., J. Interaction of lecithin:cholesterol acyltransferase (LCAT).alpha 2-macroglobulin complex with low density lipoprotein receptor-related protein (LRP). Evidence for an alpha 2-macroglobulin/LRP receptor-mediated system participating in LCAT clearance.J. Biol. Chem. 2001; 276: 33241-33248Google Scholar). Briefly, samples (30–100 μl) were separated in the first dimension (according to their charge) by 0.75% agarose gel electrophoresis (100 V, 3 h, 4°C) and in the second dimension (according to their size) by 5–23% polyacrylamide concave gradient gel electrophoresis (125 V, 24 h, 4°C). An iodinated high molecular weight protein mixture (7.1–17.0 nm; Amersham) was run as a standard on each gel. Electrophoretically separated samples were electrotransferred (30 V, 24 h, 4°C) onto nitrocellulose membranes (Hybond ECL; Amersham). ApoE3-containing particles were detected by incubating the membranes with immunopurified polyclonal anti-apoE antibody labeled with 125I. The presence of either [14C]cholesterol or [32P]phospholipids was detected directly by autoradiography using Kodak XAR-2 film. In agreement with previous studies (29Denis M. Bissonnette R. Haidar B. Krimbou L. Bouvier M. Genest J. Expression, regulation, and activity of ABCA1 in human cell lines.Mol. Genet. Metab. 2003; 78: 265-274Google Scholar, 30Wellington C.L. Yang Y.Z. Zhou S. Clee S.M. Tan B. Hirano K. Zwarts K. Kwok A. Gelfer A. Marcil M. Newman S. Roomp K. Singaraja R. Collins J.A. Zhang L-H. Groen A.K. Hovingh K. Brownlie A. Tafuri S. Genest Jr., J. Kastelein J.J.P. Hayden M.R. Truncation mutations in ABCA1 suppress normal upregulation of full-length ABCA1 by 9-cis-retinoic acid and 22-R-hydroxycholesterol.J. Lipid Res. 2002; 43: 1939-1949Google Scholar), pretreatment of fibroblasts with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells), as described in Materials and Methods, increased the level of ABCA1 protein by ∼4-fold (data not shown). This induction of ABCA1 increased apoE3-mediated cholesterol efflux by ∼2.5-fold and apoE binding to ABCA1 by 3-fold (see data below). It is well established that oxysterols, including 22(S)-hydroxycholesterol, are high-affinity endogenous ligands for liver X receptor, the nuclear receptor that upon dimerization with retinoid X receptor induces ABCA1 gene transcription in macrophages and other cells (31Schmitz G. Langmann T. Structure, function and regulation of the ABC1 gene product.Curr. Opin. Lipidol. 2001; 12: 129-140Google Scholar, 32Santamarina-Fojo S. Remaley A.T. Neufeld E.B. Brewer Jr., H.B. Regulation and intracellular trafficking of the ABCA1 transporter.J. Lipid Res. 2001; 42: 1339-1345Google Scholar). In the present study, we have examined the binding of apoE3 to ABCA1 in normal cultured stimulated cells. As shown in Fig. 1A(left panels), a marked and consistent association of apoE3 with ABCA1 was detected using chemical cross-linking, as described in Materials and Methods. ApoA-I was used as a control for this experiment (Fig. 1A, right panels). It is well established that the conformation of apolipoproteins within HDL particles is affected by their association with lipid molecules. Therefore, it was of interest to determine whether apoE conformation/organization within particles would affect its interaction with ABCA1. Competition assays were performed to determine the ability of lipid-free apoE3, as well as discoidal r(LpE3) with a molecular diameter of 13 nm, to compete for the binding of 125I-apoA-I to ABCA1 in stimulated cells. As shown in Fig. 1B, both lipid-free apoE3 and apoA-I have similar capacities to compete for binding to ABCA1. In contrast, lipid-free apoE3 inhibited the binding of 125I-apoA-I to ABCA1 more efficiently than did r(LpE3) (IC50 = 2.5 ± 0.4 μg/ml vs. 12.3 ± 1.3 μg/ml). Control experiments were conducted to examine whether the apparent decrease in cell binding of the labeled apoA-I may be attributable to 125I-apoA-I binding to different competitor particles instead of the cells. An experiment was carried out in which apoE3, r(LpE3), and r(LpA-I) particles were incubated with 125I-apoA-I under conditions similar to those used for the apoA-I binding assay, and the samples were separated by fast protein liquid chromatography. No significant amount of 125I-apoA-I was associated with apoE3, r(LpE3), or r(LpA-I) (data not shown), supporting our results shown in Fig. 1B. Because phenotype-specific differences have been shown in the ability of apoE to interact with many proteins (9Mahley R.W. Huang Y. Rall Jr., S.C. Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia). Questions, quandaries, and paradoxes.J. Lipid Res. 1999; 40: 1933-1949Google Scholar, 33Strittmatter W.J. Saunders A.M. Goedert M. Weisgraber K.H. Dong L.M. Jakes R. Huang D.Y. Pericak-Vance M. Schmechel D. Roses A.D. Isoform-specific interactions of apolipoprotein E with microtubule-associated protein tau: implications for Alzheimer disease.Proc. Natl. Acad. Sci. USA. 1994; 91: 11183-11186Google Scholar, 34Schmechel D.E. Saunders A.M. Strittmatter W.J. Crain B.J. Hulette C.M. Joo S.H. Pericak-Vance M.A. Goldgaber D. Roses A.D. Increased amyloid beta-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease.Proc. Natl. Acad. Sci. USA. 1993; 90: 9649-9653Google Scholar, 35Krimbou L. Tremblay M. Davignon J. Cohn J.S. Association of apolipoprotein E with alpha2-macroglobulin in human plasma.J. Lipid Res. 1998; 39: 2373-2386Google Scholar), we posed the question of whether the interaction of apoE with ABCA1 could be affected by apoE isoforms. Cross-linking experiments were carried out with purified plasma apoE2, apoE3, or apoE4. As seen in Fig. 2(upper panel), all three naturally occurring isoforms of apoE can cross-link to ABCA1 in stimulated cells. To quantitate the relative binding of apoE isoforms to ABCA1, we used the solid-phase binding assay described in Materials and Methods. First, apoE2, apoE3, apoE4, or apoA-I was cross-linked to ABCA1 in stimulated and unstimulated cells, and total microsomal fractions were isolated. ApoE isoforms or apoA-I associated with ABCA1-containing microsomal fraction (120 ng of protein) was immunoprecipitated with an affinity-purified anti-ABCA1 antibody, which had been immobilized by passive adsorption to microtiter plates. Either apoE or apoA-I associated with ABCA1 was quantitated as described in Materials and Methods. In separate experiments, the saturation of anti-ABCA1 antibody was determined by incubating increasing amounts of total microsomal fraction containing apoE/ABCA1 complex with immobilized anti-ABCA1 antibody, and apoE associated with ABCA1 was detected by anti-apoE antibody. In the present binding assay, saturation was reached at 300 ng of protein. Figure 2 (lower panel) shows that under the same conditions, the binding of apoE2, apoE3, and apoE4 to ABCA1 is similar. As expected, both apoE isoforms and apoA-I bound to ABCA1 approximately three times more in stimulated than in unstimulated cells. ApoA-I binding to ABCA1 was used as a control in this assay. Phenotype-specific differences have been shown in the ability of apoE to promote cholesterol efflux from cultured cells (14Huang Y. von Eckardstein A. Wu S. Assmann G. Effects of the apolipoprotein E polymorphism on uptake and transfer of cell-derived cholesterol in plasma.J. Clin. Invest. 1995; 96: 2693-2701Google Scholar, 15Michikawa M. Fan Q.W. Isobe I. Yanagisawa K. Apolipoprotein E exhibits isoform-specific promotion of lipid efflux from astrocytes and neurons in culture.J. Neurochem. 2000; 74: 1008-1016Google Scholar, 36Gong J.S. Kobayashi M. Hayashi H. Zou K. Sawamura N. Fujita S.C. Yanagisawa K. Michikawa M. Apolipoprotein E (ApoE) isoform-dependent lipid release from astrocytes prepared from human ApoE3 and ApoE4 knock-in mice.J. Biol. Chem. 2002; 277: 29919-29926Google Scholar). We examined the effect of apoE isoform on the ability of apoE to mediate ABCA1-dependent cellular cholesterol efflux. As shown in Fig. 3(upper panel), the three apoE isoforms mediate cellular cholesterol efflux approximately 2.5 times more in stimulated than in unstimulated cells in a dose-dependent manner. At the same time, apoE2, apoE3, and apoE4 showed similar abilities to induce cellular cholesterol efflux in a time-dependent manner. Furthermore, in our stimulated cell culture system, apoE-mediated cholesterol efflux reached saturation after a 16 h incubation (Fig. 3, lower panel). To determine whether naturally occurring mutants of ABCA1 might affect apoE3 binding, cross-linking of apoE3 to mutant ABCA1 (C1477R) was examined. As shown in Fig. 4, C1477R mutant abolished b" @default.
• W2049660003 created "2016-06-24" @default.
• W2049660003 creator A5011375774 @default.
• W2049660003 creator A5012432729 @default.
• W2049660003 creator A5022102417 @default.
• W2049660003 creator A5053442157 @default.
• W2049660003 creator A5069976631 @default.
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• W2049660003 date "2004-05-01" @default.
• W2049660003 modified "2023-09-29" @default.
• W2049660003 title "Molecular interactions between apoE and ABCA1" @default.
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