Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.     

Quantitative analysis of ABCA1-dependent compartmentalization and trafficking of apolipoprotein A-I: implications for determining cellular kinetics of nascent high density lipoprotein biogenesis

The molecular mechanisms underlying the apoA-I/ABCA1 endocytic trafficking pathway in relation to high density lipoprotein (HDL) formation remain poorly understood. We have developed a quantitative cell surface biotinylation assay to determine the compartmentalization and trafficking of apoA-I between the plasma membrane (PM) and intracellular compartments (ICCs). Here we report that (125)I-apoA-I exhibited saturable association with the PM and ICCs in baby hamster kidney cells stably overexpressing ABCA1 and in fibroblasts. The PM was found to have a 2-fold higher capacity to accommodate apoA-I as compared with ICCs. Overexpressing various levels of ABCA1 in baby hamster kidney cells promoted the association of apoA-I with PM and ICCs compartments. The C-terminal deletion of apoA-I Delta(187-243) and reconstituted HDL particles exhibited reduced association of apoA-I with both the PM and ICCs. Interestingly, cell surface biotinylation with a cleavable biotin revealed that apoA-I induces ABCA1 endocytosis. Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). To better understand the role of the endocytotic pathway in the dynamics of the lipidation of apoA-I, a pulse-chase experiment was performed, and the dissociation (re-secretion) of (125)I-apoA-I from both PM and ICCs was monitored over a 6-h period. Unexpectedly, we found that the time required for 50% dissociation of (125)I-apoA-I from the PM was 4-fold slower than that from ICCs at 37 degrees C. Finally, treatment of the cells with phosphatidylcholine-specific phospholipase C, increased the dissociation of apoA-I from the PM. This study provides evidence that the lipidation of apoA-I occurs in two kinetically distinguishable compartments. The finding that apoA-I specifically mediates the continuous endocytic recycling of ABCA1, together with the kinetic data showing that apoA-I associated with ICCs is rapidly re-secreted, suggests that the endocytotic pathway plays a central role in the genesis of nascent HDL.     

Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis

It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated "high-capacity binding site" (HCBS). Glyburide drastically reduced (125)I-apoA-I binding to the HCBS, whereas (125)I-apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (Delta187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of (125)I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis.     

Molecular interactions between apoE and ABCA1: impact on apoE lipidation

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 (125)I-apoA-I to ABCA1 more efficiently than reconstituted HDL particles (IC(50) = 2.5 +/- 0.4 microg/ml vs. 12.3 +/- 1.3 microg/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 prebeta-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.     

Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.     

Binding, internalization and transport of apolipoprotein A-I by vascular endothelial cells

High density lipoproteins (HDL) and their main protein constituent, apolipoprotein A-I (apoA-I), exert potentially anti-atherogenic properties within the arterial wall. However, it is unknown how they are transported from the blood stream into the vascular wall. Here we investigated the interaction of apoA-I with endothelial cells. At 4 degrees C endothelial cells bound 125I-apoA-I with high affinity, Kd = 2.1 microg/ml and in a saturable manner (Bmax of 35 ng/mg cell protein). At 37 degrees C, the cell association of apoA-I revealed similar affinity as at 4 degrees C (Kd = 2.2 microg/ml) but the maximum specific cell association was much enhanced (Bmax = 360 ng/mg cell protein). Binding and cell association was competed by excess unlabeled apoA-I and HDL but not by albumin. Biotinylation experiments and electron microscopy studies showed that endothelial cells internalize labeled apoA-I. Only minor amounts of the internalized apoA-I were degraded. Cultivated in a Transwell system, the cells transported a fraction of 125I-apoA-I from the apical to the basolateral compartment in a competable and temperature-sensitive manner. Furthermore, after specific transport the originally prebeta-mobile and lipid-free apoA-I was recovered as particles which have electrophoretic alpha-mobility. We conclude that endothelial cells transcytose and lipidate lipid-free apoA-I.     

Role of the kidney in regulating the metabolism of HDL in rabbits: evidence that iodination alters the catabolism of apolipoprotein A-I by the kidney

To evaluate the factors that regulate HDL catabolism in vivo, we have measured the clearance of human apoA-I from rabbit plasma by following the isotopic decay of (125)I-apoA-I and the clearance of unlabeled apoA-I using a radioimmunometric assay (RIA). We show that the clearance of unlabeled apoA-I is 3-fold slower than that of (125)I-apoA-I. The mass clearance of iodinated apoA-I, as determined by RIA, is superimposable with the isotopic clearance of (125)I-apoA-I. The data demonstrate that iodination of tyrosine residues alters the apoA-I molecule in a manner that promotes an accelerated catabolism. The clearance from rabbit plasma of unmodified apoA-I on HDL(3) and a reconstituted HDL particle (LpA-I) were very similar and about 3-4-fold slower than that for (125)I-apoA-I on the lipoproteins. Therefore, HDL turnover in the rabbit is much slower than that estimated from tracer kinetic studies. To determine the role of the kidney in HDL metabolism, the kinetics of unmodified apoA-I and LpA-I were reevaluated in animals after a unilateral nephrectomy. Removal of one kidney was associated with a 40-50% reduction in creatinine clearance rates and a 34% decrease in the clearance rate of unlabeled apoA-I and LpA-I particles. In contrast, the clearance of (125)I-labeled molecules was much less affected by the removal of a kidney; FCR for (125)I-LpA-I was reduced by <10%. The data show that the kidneys are responsible for most (70%) of the catabolism of apoA-I and HDL in vivo, while (125)I-labeled apoA-I and HDL are rapidly catabolized by different tissues. Thus, the kidney is the major site for HDL catabolism in vivo. Modification of tyrosine residues on apoA-I may increase its plasma clearance rate by enhancing extra-renal degradation pathways.     

Initial interaction of apoA-I with ABCA1 impacts in vivo metabolic fate of nascent HDL

We investigated the in vivo metabolic fate of pre-beta HDL particles in human apolipoprotein A-I transgenic (hA-I (Tg)) mice. Pre-beta HDL tracers were assembled by incubation of [(125)I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-beta HDLs of increasing size (pre-beta1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-I (Tg)-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-beta2, -3, and -4 had similar plasma die-away rates, whereas pre-beta1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-beta HDL size increased. In plasma, pre-beta1 and -2 were rapidly (<5 min) remodeled into larger HDLs, whereas pre-beta3 and -4 were remodeled into smaller HDLs. Pre-beta HDLs were similarly remodeled in vitro with control or LCAT-immunodepleted plasma, but not when incubated with phospholipid transfer protein knockout plasma. Our results suggest that initial interaction of apoA-I with ABCA1 imparts a unique conformation that partially determines the in vivo metabolic fate of apoA-I, resulting in increased liver and decreased kidney catabolism as pre-beta HDL particle size increases.     

ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from murine hepatocytes

High levels of expression of the ATP binding cassette transporter A1 (ABCA1) in the liver and the need to over- or underexpress hepatic ABCA1 to impact plasma HDL levels in mice suggest a major role of the liver in HDL formation and in determining circulating HDL levels. Cultured murine hepatocytes were used to examine the role of hepatic ABCA1 in mediating the lipidation of apolipoprotein A-I (apoA-I) for HDL particle formation. Exogenous apoA-I stimulated cholesterol efflux to the medium from wild-type hepatocytes, but not from ABCA1-deficient (abca1(-/-)) hepatocytes. ApoA-I induced the formation of new HDL particles and enhanced the lipidation of endogenously secreted murine apoA-I in ABCA1-expressing but not abca1(-/-) hepatocytes. ABCA1-dependent cholesterol mobilization to apoA-I increased new cholesterol synthesis, indicating depletion of the regulatory pool of hepatocyte cholesterol during HDL formation. Secretion of triacylglycerol and apoB was decreased following apoA-I incubation with ABCA1-expressing but not abca1(-/-) hepatocytes. These results support a major role for hepatocyte ABCA1 in generating a critical pool of HDL precursor particles that enhance further HDL generation and passive cholesterol mobilization in the periphery. The results also suggest that diversion of hepatocyte cholesterol into the "reverse" cholesterol transport pathway diminishes cholesterol availability for apoB-containing lipoprotein secretion by the liver.     

Lipidation of apolipoprotein A-I by ATP-binding cassette transporter (ABC) A1 generates an interaction partner for ABCG1 but not for scavenger receptor BI

The ATP-binding cassette transporters ABCA1 and ABCG1 as well as scavenger receptor BI (SR-BI) mediate the efflux of lipids from macrophages to apolipoprotein A-I (apoA-I) and high density lipoproteins (HDL). We used RNA interference in RAW264.7 macrophages to study the interactions of ABCA1, ABCG1, and SR-BI with lipid-free apoA-I, native and reconstituted HDL with apoA-I:phosphatidylcholine ratios of either 1:40 (rHDL(1:40)) or 1:100 (rHDL(1:100)). Knock-down of ABCA1 inhibits the cellular binding at 4 degrees C of lipid-free apoA-I but not of HDL whereas suppression of ABCG1 or SR-BI reduces the binding of HDL but not lipid-free apoA-I. The degree of lipidation influences the interactions of rHDL with ABCG1 and SR-BI. Knock-down of ABCG1 inhibits more effectively the binding and cholesterol efflux capacities of lipid-poorer rHDL(1:40) whereas knock-down of SR-BI has a more profound effect on the binding and cholesterol efflux capacities of lipid-richer rHDL(1:100). Moreover, knock-down of ABCG1 but not SR-BI interferes with the association of lipid-free apoA-I during prolonged incubation at 37 degrees C. Finally, knock-down of ABCG1 inhibits the binding of initially lipid-free apoA-I which has been preconditioned by cells with high ABCA1 activity. The gained ability of initially lipid-free apoA-I to interact with ABCG1 is accompanied by its shift from electrophoretic pre-beta- to alpha-mobility. Taken together, these data suggest that the interaction of lipid-free apoA-I with ABCA1 generates a particle that immediately interacts with ABCG1 but not with SR-BI. Furthermore, the degree of lipidation influences the interaction of HDL with ABCG1 or SR-BI.     

Binding of partially reassembled high-density lipoprotein to isolated human small intestine epithelial cells. Effect of lipid composition

The binding of human high-density lipoprotein (HDL3), apolipoprotein A-I (apoA-I) and recombinants of apoA-I with cholesterol and/or dimyristoylphosphatidylcholine (DMPC) to the HDL receptor on isolated human small intestine epithelial cells was studied. ApoA-I competed for 125I-labelled HDL3 binding sites less effectively than HDL3, and a lower amount of 125I-labelled apoA-I than 125I-HDL3 was bound to cells. The apoA-I/DMPC recombinant competed for 125I-HDL3 binding sites nearly as well as HDL3, and 125I-apoA-I/DMPC recombinant bound to cells with at least the same efficiency as 125I-HDL3. The apoA-I/DMPC/cholesterol recombinant failed to compete for 125I-HDL3 binding sites, and the 125I-apoA-I/DMPC/cholesterol complex binding to cells was several-fold lower than that of other particles. All particles bound to cells with similar dissociation constants. Tetranitromethane-modified HDL3 failed to bind to high-affinity specific binding sites and compete with 125I-HDL3 for binding. The results obtained make it possible to assume that, while apoA-I may be a determinant of the HDL receptor, the lipid composition of the lipoprotein may affect its interaction with the receptor.     

Influence of apolipoprotein A-I and apolipoprotein A-II availability on nascent HDL heterogeneity

It is important to understand HDL heterogeneity because various subspecies possess different functionalities. To understand the origins of HDL heterogeneity arising from the existence of particles containing only apoA-I (LpA-I) and particles containing both apoA-I and apoA-II (LpA-I+A-II), we compared the abilities of both proteins to promote ABCA1-mediated efflux of cholesterol from HepG2 cells and form nascent HDL particles. When added separately, exogenous apoA-I and apoA-II were equally effective in promoting cholesterol efflux, although the resultant LpA-I and LpA-II particles had different sizes. When apoA-I and apoA-II were mixed together at initial molar ratios ranging from 1:1 to 16:1 to generate nascent LpA-I+A-II HDL particles, the particle size distribution altered, and the two proteins were incorporated into the nascent HDL in proportion to their initial ratio. Both proteins formed nascent HDL particles with equal efficiency, and the relative amounts of apoA-I and apoA-II incorporation were driven by mass action. The ratio of lipid-free apoA-I and apoA-II available at the surface of ABCA1-expressing cells is a major factor in determining the contents of these proteins in nascent HDL. Manipulation of this ratio provides a means of altering the relative distribution of LpA-I and LpA-I+A-II HDL particles.     

Cross-linking and lipid efflux properties of apoA-I mutants suggest direct association between apoA-I helices and ABCA1

To explore the functional interactions between apoA-I and ABCA1, we correlated the cross-linking properties of several apoA-I mutants with their ability to promote cholesterol efflux. In a competitive cross-linking assay, amino-terminal deletion and double amino- and carboxy-terminal deletion mutants of apoA-I competed effectively the cross-linking of WT (125)I-apoA-I to ABCA1, while the carboxy-terminal deletion mutant apoA-I[Delta(220-243)] competed poorly. Direct cross-linking of WT apoA-I, amino-terminal, and double deletion mutants of apoA-I to ABCA1 showed similar apparent K(d) values (49-74 nM), whereas the apparent K(d) values of the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] were increased 3-fold. Analysis of several internal deletions and point mutants of apoA-I showed that apoA-I[Delta(61-78)], apoA-I[Delta(89-99)], apoA-I[Delta(136-143)], apoA-I[Delta(144-165)], apoA-I[D102A/D103A], apoA-I[E125K/E128K/K133E/E139K], apoA-I[L141R], apoA-I[R160V/H162A], and WT apoA-I had similar ABCA1-mediated lipid efflux, and all competed efficiently the cross-linking of WT (125)I-apoA-I to ABCA1. WT apoA-I and ABCA1 could be cross-linked with a 3 A cross-linker. The WT apoA-I, amino, carboxy and double deletion mutants of apoA-I showed differences in the cross-linking to WT ABCA1 and the mutant ABCA1[W590S]. The findings are consistent with a direct association of different combinations of apoA-I helices with a complementary ABCA1 domain. Mutations that alter ABCA1/apoA-I association affect cholesterol efflux and inhibit biogenesis of HDL.     

Carboxyl terminus of apolipoprotein A-I (ApoA-I) is necessary for the transport of lipid-free ApoA-I but not prelipidated ApoA-I particles through aortic endothelial cells

High density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) must leave the circulation and pass the endothelium to exert their atheroprotective actions in the arterial wall. We previously demonstrated that the transendothelial transport of apoA-I involves ATP-binding cassette transporter (ABC) A1 and re-secretion of lipidated particles. Transendothelial transport of HDL is modulated by ABCG1 and the scavenger receptor BI (SR-BI). We hypothesize that apoA-I transport is started by the ABCA1-mediated generation of a lipidated particle which is then transported by ABCA1-independent pathways. To test this hypothesis we analyzed the endothelial binding and transport properties of initially lipid-free as well as prelipidated apoA-I mutants. Lipid-free apoA-I mutants with a defective carboxyl-terminal domain showed an 80% decreased specific binding and 90% decreased specific transport by aortic endothelial cells. After prior cell-free lipidation of the mutants, the resulting HDL-like particles were transported through endothelial cells by an ABCG1- and SR-BI-dependent process. ApoA-I mutants with deletions of either the amino terminus or both the amino and carboxyl termini showed dramatic increases in nonspecific binding but no specific binding or transport. Prior cell-free lipidation did not rescue these anomalies. Our findings of stringent structure-function relationships underline the specificity of transendothelial apoA-I transport and suggest that lipidation of initially lipid-free apoA-I is necessary but not sufficient for specific transendothelial transport. Our data also support the model of a two-step process for the transendothelial transport of apoA-I in which apoA-I is initially lipidated by ABCA1 and then further processed by ABCA1-independent mechanisms.     

Apolipoprotein A-I activates cellular cAMP signaling through the ABCA1 transporter

It has been suggested that the signal transduction pathway initiated by apoA-I activates key proteins involved in cellular lipid efflux. We investigated apoA-I-mediated cAMP signaling in cultured human fibroblasts induced with (22R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Treatment of stimulated fibroblasts with apoA-I for short periods of time (相似文献   

ABCA1 is required for normal central nervous system ApoE levels and for lipidation of astrocyte-secreted apoE

ABCA1 is an ATP-binding cassette protein that transports cellular cholesterol and phospholipids onto high density lipoproteins (HDL) in plasma. Lack of ABCA1 in humans and mice causes abnormal lipidation and increased catabolism of HDL, resulting in very low plasma apoA-I, apoA-II, and HDL. Herein, we have used Abca1-/- mice to ask whether ABCA1 is involved in lipidation of HDL in the central nervous system (CNS). ApoE is the most abundant CNS apolipoprotein and is present in HDL-like lipoproteins in CSF. We found that Abca1-/- mice have greatly decreased apoE levels in both the cortex (80% reduction) and the CSF (98% reduction). CSF from Abca1-/- mice had significantly reduced cholesterol as well as small apoE-containing lipoproteins, suggesting abnormal lipidation of apoE. Astrocytes, the primary producer of CNS apoE, were cultured from Abca1+/+, +/-, and -/- mice, and nascent lipoprotein particles were collected. Abca1-/- astrocytes secreted lipoprotein particles that had markedly decreased cholesterol and apoE and had smaller apoE-containing particles than particles from Abca1+/+ astrocytes. These findings demonstrate that ABCA1 plays a critical role in CNS apoE metabolism. Since apoE isoforms and levels strongly influence Alzheimer's disease pathology and risk, these data suggest that ABCA1 may be a novel therapeutic target.     

Characterization of oligomeric human ATP binding cassette transporter A1. Potential implications for determining the structure of nascent high density lipoprotein particles

The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nascent apoA-I-containing particles (LpA-I) were investigated. Using n-dodecylmaltoside and perfluoro-octanoic acid combined with non-denaturing gel, the majority of ABCA1 was found as a tetramer in ABCA1-induced human fibroblasts. Furthermore, using chemical cross-linking and SDS-PAGE, ABCA1 dimers but not the tetramers were found covalently linked. Oligomeric ABCA1 was present in isolated plasma membranes as well as in intracellular compartments. Interestingly, apoA-I was found to be associated with both dimeric and tetrameric, but not monomeric, forms of ABCA1. Neither apoA-I nor lipid molecules did affect ABCA1 oligomerization. Immunoprecipitation analysis showed that oligomeric ABCA1 did not contain other associated proteins. We next investigated the relationship between the oligomeric ABCA1 complex and the structure of LpA-I. Lipid-free apoA-I incubated with normal cells generated LpA-I with diameters between 9.5 and 20 nm. Subsequent isolation of LpA-I followed by cross-linking revealed the presence of four and eight apoA-I molecules per particle, whereas apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles and remained in the monomeric form. These results demonstrate that: 1) ABCA1 exists as an oligomeric complex; and 2) ABCA1 oligomerization was independent of apoA-I binding and lipid molecules. The findings that the majority of ABCA1 exists as a tetramer that binds apoA-I, together with the observation that LpA-I contains at least four molecules of apoA-I per particle, support the concept that the homotetrameric ABCA1 complex constitutes the minimum functional unit required for the biogenesis of high density lipoprotein particles.     

Binding characteristics of high density lipoprotein subclasses to porcine liver, adrenal and skeletal muscle plasma membranes

1. We compared binding characteristics of 125I-labeled high density lipoprotein (HDL) subclasses to porcine liver, adrenal and skeletal muscle plasma membranes. 2. HDL subclasses were discriminated by their buoyant densities (HDL2 and HDL3) or by their apolipoprotein (apo) content (Lp-AI (particles containing apoA-I but no apoA-II) and LpA-I/A-II (particles containing both apoA-I and apoA-II)). 3. HDL2 and HDL3 showed saturable binding to the three types of membrane preparations. 4. No differences were found in the Kds within one HDL subclass. 5. Kds and maximal binding of HDL2 were lower than these of HDL3. Unlabeled HDL2 and HDL3, but not LDL, effectively displaced 125I-HDL2 and 125I-HDL3. 6. Binding of HDL was independent of the concentration of NaCl and did not require calcium. 7. These results suggest a process mediated by a single specific receptor in porcine liver, adrenal and skeletal muscle plasma membranes. 8. We also studied binding characteristics of HDL subclasses Lp-AI and LpA-I/A-II to porcine liver membranes. LpA-I showed the highest Kd and maximal binding. 9. All types of HDL subclasses studied (i.e. HDL2, HDL3, LpA-I and LpA-I/A-II) effectively competed for binding of both Lp-AI and LpA-I/A-II, suggesting that the HDL subclasses studied bind to the same receptor by their apoA-I moiety.