The effects of mutations in helices 4 and 6 of ApoA-I on scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux suggest that formation of a productive complex between reconstituted high density lipoprotein and SR-BI is required for efficient lipid transport

We have studied the effects of mutations in apoA-I on reconstituted high density lipoprotein (HDL) particle (rHDL(apoA-I)) binding to and cholesterol efflux from wild-type (WT) and mutant forms of the HDL receptor SR-BI expressed by ldlA-7 cells. Mutations in helix 4 or helix 6 of the apoA-I reduced efflux by 79 and 51%, respectively, without substantially altering receptor binding (apparent K(d) values of 1.1-4.4 microg of protein/ml). SR-BI with an M158R mutation bound poorly to rHDL with WT and helix 4 mutant apoA-I; the helix 6 mutant restored tight binding to SR-BI(M158R) (K(d) values of 48, 60, and 7 microg of protein/ml, respectively). SR-BI(M158R)-mediated cholesterol efflux rates, normalized for binding, were high for all three rHDLs (71-111% of control). In contrast, absolute (12-19%) and binding-corrected (24-47%) efflux rates for all three rHDLs mediated by SR-BI with Q402R/Q418R mutations were very low. We propose that formation of a productive complex between apoA-I in rHDL and SR-BI, in which the lipoprotein and the receptor must either be precisely aligned or have the capacity to undergo appropriate conformational changes, is required for efficient SR-BI-mediated cholesterol efflux. Some mutations in apoA-I and/or SR-BI can result in high affinity, but non-productive, binding that does not permit efficient cholesterol efflux.     

Reconstituted discoidal ApoE-phospholipid particles are ligands for the scavenger receptor BI. The amino-terminal 1-165 domain of ApoE suffices for receptor binding

The high density lipoprotein receptor, scavenger receptor class B type I (SR-BI), recognizes lipid-bound apolipoprotein A-I (apoA-I) and other apolipoproteins. Here, we have used large scale cultures of apoE-expressing cells to purify apoE and prepare apoE containing reconstituted discoidal 1-palmitoyl-2-oleoyl-l-phosphatidylcholine (POPC)-apoE particles. These particles have been used to examine their binding to wild-type and mutant forms of SR-BI expressed in transfected ldlA-7 cells. Specific binding to SR-BI was determined by subtracting from the total binding, nonspecific values measured using either control untransfected ldlA-7 cells or by inhibiting SR-BI-mediated binding with a high titer antireceptor-blocking antibody. POPC-apoE particles generated using apoE2, apoE3, apoE4, or the carboxyl-terminally truncated forms apoE165, apoE202, apoE229, and apoE259 all bound tightly to wild-type SR-BI with similar affinities (K(d) = 35-45 microg/ml). Binding was nearly abolished in a cell line expressing the ldlA (Q402R/Q418R) double mutant form of SR-BI that is unable to bind native high density lipoprotein but binds low density lipoprotein normally. The findings establish that apoE is a ligand for SR-BI and that the receptor binding domain is located in the amino-terminal 1-165-region of the protein. SR-BI-apoE interactions may contribute to cholesterol homeostasis in tissues and cells expressing SR-BI that are accessible to apoE-containing lipoproteins.     

Selective uptake of HDL cholesteryl esters and cholesterol efflux from mouse peritoneal macrophages independent of SR-BI

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl esters (CEs) and facilitates the efflux of unesterified cholesterol. SR-BI expression in macrophages presumably plays a role in atherosclerosis. The role of SR-BI for selective CE uptake and cholesterol efflux in macrophages was explored. Macrophages and HDL originated from wild-type (WT) or SR-BI knockout (KO; homozygous) mice. For uptake, macrophages were incubated in medium containing 125I-/3H-labeled HDL. For lipid removal, [3H]cholesterol efflux was analyzed using HDL as acceptor. Selective uptake of HDL CE ([3H]cholesteryl oleyl ether - 125I-tyramine cellobiose) was similar in WT and SR-BI KO macrophages. Radiolabeled SR-BI KO-HDL yielded a lower rate of selective uptake compared with WT-HDL in WT and SR-BI KO macrophages. Cholesterol efflux was similar in WT and SR-BI KO cells using HDL as acceptor. SR-BI KO-HDL more efficiently promoted cholesterol removal compared with WT-HDL from both types of macrophages. Macrophages selectively take up HDL CE independently of SR-BI. Additionally, in macrophages, there is substantial cholesterol efflux that is not mediated by SR-BI. Therefore, SR-BI-independent mechanisms mediate selective CE uptake and cholesterol removal. SR-BI KO-HDL is an inferior donor for selective CE uptake compared with WT-HDL, whereas SR-BI KO-HDL more efficiently promotes cholesterol efflux.     

Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor

The murine scavenger receptor class B, type I (mSR-BI) is a receptor for high density lipoprotein (HDL), low density lipoprotein (LDL), and acetylated LDL (AcLDL). It mediates selective uptake of lipoprotein lipid and stimulates efflux of [(3)H]cholesterol to lipoproteins. SR-BI-mediated [(3)H]cholesterol efflux was proposed to be independent of ligand binding. In this study, using anti-mSR-BI antibody KKB-1 and two mSR-BI mutants with altered ligand binding properties, we demonstrated that SR-BI-mediated [(3)H]cholesterol efflux to lipoproteins was correlated with ligand binding and lipid uptake activities of the receptor. The KKB-1 antibody, which blocked lipoprotein binding without substantially altering the cholesterol oxidase-accessible cellular [(3)H]cholesterol, also blocked [(3)H]cholesterol efflux to HDL and LDL. One of the SR-BI mutants, which has a double substitution of arginines for glutamines at positions 402 and 418 (Q402R/Q418R), exhibited a high level of LDL binding and lipid uptake from LDL, but lost most of the corresponding HDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to LDL, but not to HDL. Another mutant, M158R, with an arginine in place of methionine at position 158, exhibited reduced HDL and LDL receptor activities, but apparently normal AcLDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to AcLDL, but not to HDL or LDL. These results suggest that SR-BI-stimulated [(3)H]cholesterol efflux to lipoproteins critically depends on ligand binding to this receptor and raise the possibility that the mechanisms of selective lipid uptake and [(3)H]cholesterol efflux may be intimately related.     

SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL

This study compares the roles of ABCG1 and scavenger receptor class B type I (SR-BI) singly or together in promoting net cellular cholesterol efflux to plasma HDL containing active LCAT. In transfected cells, SR-BI promoted free cholesterol efflux to HDL, but this was offset by an increased uptake of HDL cholesteryl ester (CE) into cells, resulting in no net efflux. Coexpression of SR-BI with ABCG1 inhibited the ABCG1-mediated net cholesterol efflux to HDL, apparently by promoting the reuptake of CE from medium. However, ABCG1-mediated cholesterol efflux was not altered in cholesterol-loaded, SR-BI-deficient (SR-BI(-/-)) macrophages. Briefly cultured macrophages collected from SR-BI(-/-) mice loaded with acetylated LDL in the peritoneal cavity did exhibit reduced efflux to HDL. However, this was attributable to reduced expression of ABCG1 and ABCA1, likely reflecting increased macrophage cholesterol efflux to apolipoprotein E-enriched HDL during loading in SR-BI(-/-) mice. In conclusion, cellular SR-BI does not promote net cholesterol efflux from cells to plasma HDL containing active LCAT as a result of the reuptake of HDL-CE into cells. Previous findings of increased atherosclerosis in mice transplanted with SR-BI(-/-) bone marrow probably cannot be explained by a defect in macrophage cholesterol efflux.     

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.     

The lipidation status of acute-phase protein serum amyloid A determines cholesterol mobilization via scavenger receptor class B, type I

During the acute-phase reaction, SAA (serum amyloid A) replaces apoA-I (apolipoprotein A-I) as the major HDL (high-density lipoprotein)-associated apolipoprotein. A remarkable portion of SAA exists in a lipid-free/lipid-poor form and promotes ABCA1 (ATP-binding cassette transporter A1)-dependent cellular cholesterol efflux. In contrast with lipid-free apoA-I and apoE, lipid-free SAA was recently reported to mobilize SR-BI (scavenger receptor class B, type I)-dependent cellular cholesterol efflux [Van der Westhuyzen, Cai, de Beer and de Beer (2005) J. Biol. Chem. 280, 35890-35895]. This unique property could strongly affect cellular cholesterol mobilization during inflammation. However, in the present study, we show that overexpression of SR-BI in HEK-293 cells (human embryonic kidney cells) (devoid of ABCA1) failed to mobilize cholesterol to lipid-free or lipid-poor SAA. Only reconstituted vesicles containing phospholipids and SAA promoted SR-BI-mediated cholesterol efflux. Cholesterol efflux from HEK-293 and HEK-293[SR-BI] cells to lipid-free and lipid-poor SAA was minimal, while efficient efflux was observed from fibroblasts and CHO cells (Chinese-hamster ovary cells) both expressing functional ABCA1. Overexpression of SR-BI in CHO cells strongly attenuated cholesterol efflux to lipid-free SAA even in the presence of an SR-BI-blocking IgG. This implies that SR-BI attenuates ABCA1-mediated cholesterol efflux in a way that is not dependent on SR-BI-mediated re-uptake of cholesterol. The present in vitro experiments demonstrate that the lipidation status of SAA is a critical factor governing cholesterol acceptor properties of this amphipathic apolipoprotein. In addition, we demonstrate that SAA mediates cellular cholesterol efflux via the ABCA1 and/or SR-BI pathway in a similar way to apoA-I.     

Substitutions of glutamate 110 and 111 in the middle helix 4 of human apolipoprotein A-I (apoA-I) by alanine affect the structure and in vitro functions of apoA-I and induce severe hypertriglyceridemia in apoA-I-deficient mice

Hypertriglyceridemia is a common pathological condition in humans of mostly unknown etiology. Here we report induction of dyslipidemia characterized by severe hypertriglyceridemia as a result of point mutations in human apolipoprotein A-I (apoA-I). Adenovirus-mediated gene transfer in apoA-I-deficient (apoA-I(-)(/)(-)) mice showed that mice expressing an apoA-I[E110A/E111A] mutant had comparable hepatic mRNA levels with WT controls but greatly increased plasma triglyceride and elevated plasma cholesterol levels. In addition, they had decreased apoE and apoCII levels and increased apoB48 levels in very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL). Fast protein liquid chromatography (FPLC) analysis of plasma showed that most of cholesterol and approximately 15% of the mutant apoA-I were distributed in the VLDL and IDL regions and all the triglycerides in the VLDL region. Hypertriglyceridemia was corrected by coinfection of mice with recombinant adenoviruses expressing the mutant apoA-I and human lipoprotein lipase. Physicochemical studies indicated that the apoA-I mutation decreased the alpha-helical content, the stability, and the unfolding cooperativity of both lipid-free and lipid-bound apoA-I. In vitro functional analyses showed that reconstituted HDL (rHDL) particles containing the mutant apoA-I had 53% of scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux capacity and 37% capacity to activate lecithin:cholesterol acyltransferase (LCAT) as compared to the WT control. The mutant lipid-free apoA-I had normal capacity to promote ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. The findings indicate that subtle structural alterations in apoA-I may alter the stability and functions of apoA-I and high-density lipoprotein (HDL) and may cause hypertriglyceridemia.     

Cellular SR-BI and ABCA1-mediated cholesterol efflux are gender-specific in healthy subjects

We evaluated the impact of gender differences in both the quantitative and qualitative features of HDL subspecies on cellular free cholesterol efflux through the scavenger receptor class B type I (SR-BI), ABCA1, and ABCG1 pathways. For that purpose, healthy subjects (30 men and 26 women) matched for age, body mass index, triglyceride, apolipoprotein A-I, and high density lipoprotein-cholesterol (HDL-C) levels were recruited. We observed a significant increase (+14%; P < 0.03) in the capacity of whole sera from women to mediate cellular free cholesterol efflux via the SR-BI-dependent pathway compared with sera from men. Such enhanced efflux capacity resulted from a significant increase in plasma levels of large cholesteryl ester-rich HDL2 particles (+20%; P < 0.04) as well as from an enhanced capacity (+14%; P < 0.03) of these particles to mediate cellular free cholesterol efflux via SR-BI. By contrast, plasma from men displayed an enhanced free cholesterol efflux capacity (+31%; P < 0.001) via the ABCA1 transporter pathway compared with that from women, which resulted from a 2.4-fold increase in the plasma level of prebeta particles (P < 0.008). Moreover, in women, SR-BI-mediated cellular free cholesterol efflux was significantly correlated with plasma HDL-C (r = 0.72, P < 0.0001), whereas this relationship was not observed in men. In conclusion, HDL-C level may not represent the absolute indicator of the efficiency of the initial step of the reverse cholesterol transport.     

High glucose stimulates macrophage SR-BI expression and induces a switch in its activity from cholesterol efflux to cholesterol influx

Aims

Diabetes is associated with atherogenesis and macrophage-foam cell formation, due in part to a decrease in HDL-mediated cholesterol efflux from macrophages. This study examined the expression of proteins involved in cholesterol transport, i.e. ABCA1 and SR-BI, under diabetic conditions.

Methods and results

ABCA1 expression was similar, whereas SR-BI expression (mRNA and protein) was significantly increased in mouse peritoneal macrophages (MPM) harvested from C57Bl/6 diabetic mice, compared to MPM from control non-diabetic mice. Similar results were obtained in vitro in J-774A.1 macrophage-like cell line incubated with high (30 mM) vs. low (5 mM) glucose concentrations. Accordingly, association and internalization of HDL to MPM from diabetic mice, or to J-774A.1 macrophages grown under diabetic conditions was significantly higher compared to control cells. Unexpectedly, however, increased macrophage SR-BI expression was associated with a substantial reduction in HDL-mediated cholesterol efflux from the macrophages. Moreover, total cellular cholesterol content was increased by 28% in macrophages incubated with HDL under high glucose concentrations, compared to low glucose concentrations. This effect was abolished by a rabbit polyclonal anti-SR-BI, which blocks binding to the receptor, or alternatively by using BLT1, a specific inhibitor of lipid transport via the SR-BI.

Conclusions

Diabetes stimulates the expression of SR-BI in macrophages and leads to a shift in its activity from HDL-mediated cholesterol efflux to HDL-mediated cholesterol influx. These effects may lead to increased foam cell formation and atherosclerosis development.     

Severely altered cholesterol homeostasis in macrophages lacking apoE and SR-BI

Mice deficient in scavenger receptor class B type I (SR-BI) and apolipoprotein E (apoE) [double knockout (DKO) mice] develop dyslipidemia, accelerated atherosclerosis, and myocardial infarction, and die prematurely. We examined effects of apoE and SR-BI deficiency on macrophage cholesterol homeostasis. DKO macrophages had increased total cholesterol (TC) stores (220-380 microg/mg protein) compared with apoE-/- cells (40 microg/mg), showed significant lysosomal lipid engorgement, and increased their TC by 34% after exposure to HDL. DKO macrophages from apoE-/- mice reconstituted with DKO bone marrow showed less cholesterol accumulation (89 microg/mg), suggesting that the dyslipidemia of DKO mice explains part of the cellular cholesterol defect. However, analyses of DKO and apoE-/- macrophages from transplanted apoE-/- mice revealed a role for macrophage SR-BI, inasmuch as the TC in DKO macrophages increased by 10% in the presence of HDL, whereas apoE-/- macrophage TC decreased by 33%. After incubation with HDL, the free cholesterol (FC) increased by 29% in DKO macrophages, and decreased by 8% in apoE-/- cells, and only DKO cells had FC in large peri-nuclear pools. Similar trends were observed with apoA-I as an acceptor. Thus, the abnormal cholesterol homeostasis of DKO macrophages is due to the plasma lipid environment of DKO mice and to altered trafficking of macrophage cholesterol. Both factors are likely to contribute to the accelerated atherosclerosis in DKO mice.     

LDL particle subspecies are distinct in their capacity to mediate free cholesterol efflux via the SR-BI/Cla-1 receptor

The human scavenger receptor SR-BI/Cla-1 promotes efflux of free cholesterol from cells to both high-density and low-density lipoproteins (HDL, LDL). SR-BI/Cla-1-mediated cholesterol efflux to HDL is dependent on particle size, lipid content and apolipoprotein conformation; in contrast, the capacity of LDL subspecies to accept cellular cholesterol via this receptor is indeterminate. Cholesterol efflux assays were performed with CHO cells stably transfected with Cla-1 cDNA. Expression of Cla-1 in CHO cells induced elevation in total cholesterol efflux to plasma, LDL and HDL. Such Cla-1-specific efflux was abrogated by addition of anti-Cla-1 antibody. LDL were fractionated into five subspecies either on the basis of hydrated density or size. Among LDL subfractions, small dense LDL (sdLDL) were 1.5-to 3-fold less active acceptors for Cla-1-mediated cellular cholesterol efflux. Equally, sdLDL markedly reduced Cla-1-specific cholesterol efflux to large buoyant LDL in a dose-dependent manner. Conversely, sdLDL did not influence efflux to HDL(2). These findings provide evidence that LDL particles are heterogeneous in their capacity to promote Cla-1-mediated cholesterol efflux. Relative to HDL(2), large buoyant LDL may constitute physiologically-relevant acceptors for cholesterol efflux via Cla-1.     

Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI

To study the mechanisms of hepatic HDL formation, we investigated the roles of ABCA1, ABCG1, and SR-BI in nascent HDL formation in primary hepatocytes isolated from mice deficient in ABCA1, ABCG1, or SR-BI and from wild-type (WT) mice. Under basal conditions, in WT hepatocytes, cholesterol efflux to exogenous apoA-I was accompanied by conversion of apoA-I to HDL-sized particles. LXR activation by T0901317 markedly enhanced the formation of larger HDL-sized particles as well as cellular cholesterol efflux to apoA-I. Glyburide treatment completely abolished the formation of 7.4 nm diameter and greater particles but led to the formation of novel 7.2 nm-sized particles. However, cells lacking ABCA1 failed to form such particles. ABCG1-deficient cells showed similar capacity to efflux cholesterol to apoA-I and to form nascent HDL particles compared with WT cells. Cholesterol efflux to apoA-I and nascent HDL formation were slightly but significantly enhanced in SR-BI-deficient cells compared with WT cells under basal but not LXR activated conditions. As in WT but not in ABCA1-deficient hepatocytes, 7.2 nm-sized particles generated by glyburide treatment were also detected in ABCG1-deficient and SR-BI-deficient hepatocytes. Our data indicate that hepatic nascent HDL formation is highly dependent on ABCA1 but not on ABCG1 or SR-BI.     

Apolipoprotein A-I-stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.     

Synthesis and secretion of apoE in thioglycolate-elicited mouse peritoneal macrophages: effect of cholesterol efflux

ApoE synthesis and secretion, as a function of cellular cholesterol content and cholesterol efflux, was studied in thioglycolate-elicited mouse peritoneal macrophages. As expected, loading elicited macrophages with cholesterol induced a 5-fold increase in apoE secretion and a 2.5-fold increase in cellular apoE content over a 5-h period. Treatment of cholesterol-loaded cells with HDL3 further increased apoE secretion 1.7-fold and decreased cellular cholesterol by 20%. Treatment of cholesterol-loaded cells with HDL3 and SAH 58.035 (an ACAT inhibitor) increased apoE secretion 2.4-fold and decreased cellular cholesterol content by 35%. Treatment of the cells with the ACAT inhibitor alone suppressed apoE secretion by 40% but did not change cellular cholesterol content. Northern blot analysis of RNA indicated that cholesterol loading increased apoE mRNA 2-fold. ApoE mRNA levels were not further affected by treatment with HDL3 and/or the ACAT inhibitor. Cholesterol-loaded cells, in the absence of HDL3, secreted apoE into the media in two fractions as determined by column chromatography: a large molecular weight complex, (larger than HDL), and an essentially lipid-free protein. In the presence of HDL3, the cells secreted apoE in three fractions: a large molecular weight complex, an essentially lipid-free protein, and over 50% of apoE associated with HDL. In the process, HDL3 became larger and eluted in a position identical to that of HDL2. A small amount of HDL3-derived material was also transformed to an LDL-size particle. Incubation of HDL3 in the absence of cholesterol-loaded cells did not produce these changes. It is concluded that cholesterol-loading increases apoE mRNA content and apoE synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure

Scavenger receptor (SR)-BI catalyzes the selective uptake of cholesteryl ester (CE) from high density lipoprotein (HDL) by a two-step process that involves the following: 1) binding of HDL to the receptor and 2) diffusion of the CE molecules into the cell plasma membrane. We examined the effects of the size of discoidal HDL particles containing wild-type (WT) apoA-I on selective uptake of CE and efflux of cellular free (unesterified) cholesterol (FC) from COS-7 cells expressing SR-BI to determine the following: 1) the influence of apoA-I conformation on the lipid transfer process, and 2) the contribution of receptor binding-dependent processes to the overall efflux of cellular FC. Large (10 nm diameter) reconstituted HDL bound to SR-BI better (B(max) approximately 420 versus 220 ng of apoA-I/mg cell protein), delivered more CE, and promoted more FC efflux than small ( approximately 8 nm) particles. When normalized to the number of reconstituted HDL particles bound to the receptor, the efficiencies of either CE uptake or FC efflux with these particles were the same indicating that altering the conformation of WT apoA-I modulates binding to the receptor (step 1) but does not change the efficiency of the subsequent lipid transfer (step 2); this implies that binding induces an optimal alignment of the WT apoA-I.SR-BI complex so that the efficiency of lipid transfer is always the same. FC efflux to HDL is affected both by binding of HDL to SR-BI and by the ability of the receptor to perturb the packing of FC molecules in the cell plasma membrane.     

Apolipoprotein E-dependent cholesterol efflux from macrophages: kinetic study and divergent mechanisms for endogenous versus exogenous apolipoprotein E.

In these studies, we have utilized a J774 macrophage model in order to compare phospholipid and cholesterol efflux kinetics in macrophage cells that do not express endogenous apoE to cells transfected to express physiologic levels of human apoE. This model was also used to compare the effect of exogenously added versus endogenously expressed apoE on cholesterol efflux kinetics from macrophages. ApoE expression increased free cholesterol and phospholipid efflux into the medium, but did not change the free cholesterol/phospholipid molar ratio of secreted lipids. Kinetic examination showed that free cholesterol and phospholipid appeared simultaneously in the medium, and that cholesterol loading widened the difference in the rate of cholesterol efflux between apoE-expressing and non-expressing macrophages. Addition of exogenous lipid-free apoE added to non-expressing cells, at a >2-fold higher medium concentration than that produced by endogenous expression, produced less cholesterol efflux than that observed from apoE-expressing cells. The addition of phosphatidylcholine liposomes substantially increased cholesterol efflux from apoE-expressing and non-expressing J774 cells. Addition of these liposomes eliminated the enhanced cholesterol efflux produced by addition of exogenous apoE. On the other hand, even in the presence of phosphatidylcholine liposomes, cholesterol efflux rates remained significantly higher from apoE-expressing macrophages than non-expressing cells. Similar results were obtained when efflux was studied in the presence of cyclodextrin. These results suggest that endogenous expression of apoE by macrophages alters cell cholesterol balance via mechanisms distinct from those utilized by the extracellular addition of apoE, and may involve intracellular or pericellular mechanisms.     

Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux

Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1’s ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the “double super helix.” Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.     

Detergent-mediated phospholipidation of plasma lipoproteins increases HDL cholesterophilicity and cholesterol efflux via SR-BI

Cellular cholesterol efflux is an early, obligatory step in reverse cholesterol transport, the putative antiatherogenic mechanism by which human plasma high-density lipoproteins (HDL) transport cholesterol from peripheral tissue to the liver for recycling or disposal. HDL-phospholipid content is the essential cholesterol-binding component of lipoproteins and therefore a major determinant of cholesterol efflux. Thus, increased phospholipidation of lipoproteins, particularly HDL, is one strategy for increasing cholesterol efflux. This study validates a simple, new detergent perturbation method for the phospholipidation of plasma lipoproteins; we have quantified the cholesterophilicity of human plasma lipoproteins and the effects of lipoprotein phospholipidation on cholesterophilicity and cellular cholesterol efflux mediated by the class B type I scavenger receptor (SR-BI). We determined that low-density lipoproteins (LDL) are more cholesterophilic than HDL and that LDL has a higher affinity for phospholipids than HDL whereas HDL has a higher phospholipid capacity than LDL. Phospholipidation of total human plasma lipoproteins enhances cholesterol efflux, an effect that occurs largely through the preferential phospholipidation of HDL. We conclude that increasing HDL phospholipid increases its cholesterophilicity, thereby making it a better acceptor of cellular cholesterol efflux. Phospholipidation of lipoproteins by detergent perturbation is a simple way to increase HDL cholesterophilicity and cholesterol efflux in a way that may be clinically useful.     

The C-terminal lipid-binding domain of apolipoprotein E is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high-density lipoproteins

This study was undertaken to identify the alpha-helical domains of human apoE that mediate cellular cholesterol efflux and HDL assembly via ATP-binding cassette transporter A1 (ABCA1). The C-terminal (CT) domain (residues 222-299) of apoE was found to stimulate ABCA1-dependent cholesterol efflux in a manner similar to that of intact apoE2, -E3, and -E4 in studies using J774 macrophages and HeLa cells. The N-terminal (NT) four-helix bundle domain (residues 1-191) was a relatively poor mediator of cholesterol efflux. On a per molecule basis, the CT domain stimulated cholesterol efflux with the same efficiency (Km approximately 0.2 microM) as intact apoA-I and apoE. Gel filtration chromatography of conditioned medium from ABCA1-expressing J774 cells revealed that, like the intact apoE isoforms, the CT domain promoted the assembly of HDL particles with diameters of 8 and 13 nm. Removal of the CT domain abolished the formation of HDL-sized particles, and only larger particles eluting in the void volume were formed. Studies with CT truncation mutants of apoE3 and peptides indicated that hydrophobic helical segments governed the efficiency of cellular cholesterol efflux and that conjoined class A and G amphipathic alpha-helices were required for optimal efflux activity. Collectively, the data suggest that the CT lipid-binding domain of apoE encompassing amino acids 222-299 is necessary and sufficient for mediating ABCA1 lipid efflux and HDL particle assembly.