A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited ␣-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated ␣-LpA-I-like particles), whereas lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles. These results demonstrate that: 1) the physical interaction of apoA-I with ABCA1 does not depend on membrane phosphatidylcholine or sphingomyelin; 2) the association of apoA-I with lipids reduces its ability to interact with ABCA1; and 3) the lipid translocase activity of ABCA1 generates ␣-LpA-I-like particles. This process plays in vivo a key role in HDL biogenesis.
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. Lipidfree apoE3 inhibited the binding of 125 I-apoA-I to ABCA1 more efficiently than reconstituted HDL particles (IC 50 ؍ 2.5 ؎ 0.4 g/ml vs. 12.3 ؎ 1.3 g/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 pre  -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 densitysized 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 (1). Thus, apoE is believed to play a significant role in the onset and development of coronary artery atherosclerosis (2) and the pathophysiology of Alzheimer's disease (3). 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 (4, 5) and conversely by the protection against or regression of atherosclerosis in apoE-deficient animals supplemented with apoE (6, 7). 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 (8). 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 (9). ApoE4 is associated with high plasma cholesterol level and increased risk for both coronary heart disease and Alzheimer's disease (2, 10).Although it is clear that apoE plays an important ro...
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