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Apolipoprotein (apo)A-I is an organizing scaffold protein that is critical to high-density lipoprotein (HDL) structure and metabolism, probably mediating many of its cardioprotective properties. However, HDL biogenesis is poorly understood, as lipid-free apoA-I has been notoriously resistant to high-resolution structural study. Published models from low-resolution techniques share certain features but vary considerably in shape and secondary structure. To tackle this central issue in lipoprotein biology, we assembled a team of structural biologists specializing in apolipoproteins and set out to build a consensus model of monomeric lipid-free human apoA-I. Combining novel and published cross-link constraints, small-angle X-ray scattering (SAXS), hydrogen-deuterium exchange (HDX) and crystallography data, we propose a time-averaged model consistent with much of the experimental data published over the last 40 years. The model provides a long-sought platform for understanding and testing details of HDL biogenesis, structure and function.

Apolipoprotein A-I (apoA-I) undergoes a large conformational reorganization during remodeling of high-density lipoprotein (HDL) particles. To detect structural transition of apoA-I upon HDL formation, we developed novel monoclonal antibodies (mAbs). Splenocytes from BALB/c mice immunized with a recombinant human apoA-I, with or without conjugation with keyhole limpet hemocyanin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After the HAT-selection and cloning, we established nine hybridoma clones secreting anti-apoA-I mAbs in which four mAbs recognize epitopes on the N-terminal half of apoA-I while the other five mAbs recognize the central region. ELISA and bio-layer interferometry measurements demonstrated that mAbs whose epitopes are within residues 1-43 or 44-65 obviously discriminate discoidal and spherical reconstituted HDL particles despite their great reactivities to lipid-free apoA-I and plasma HDL, suggesting the possibility of these mAbs to detect structural transition of apoA-I on HDL. Importantly, a helix-disrupting mutation of W50R into residues 44-65 restored the immunoreactivity of mAbs whose epitope being within residues 44-65 against reconstituted HDL particles, indicating that these mAbs specifically recognize the epitope region in a random coil state. These results encourage us to develop mAbs targeting epitopes in the N-terminal residues of apoA-I as useful probes for monitoring formation and remodeling of HDL particles.

Reverse cholesterol transport (RCT) is thought to be an atheroprotective function of HDL, and macrophage-specific RCT in mice is inversely associated with atherosclerosis. We developed a novel method using 3 H-cholesterol nanoparticles to selectively trace macrophage-specific RCT in vivo in humans. Use of 3 H-cholesterol nanoparticles was initially tested in mice to assess the distribution of tracer and response to interventions known to increase RCT. Thirty healthy subjects received 3 H-cholesterol nanoparticles intravenously, followed by blood and stool sample collection. Tracer counts were assessed in plasma, nonHDL, HDL, and fecal fractions. Data were analyzed by using multicompartmental modeling. Administration of 3 H-cholesterol nanoparticles preferentially labeled macrophages of the reticuloendothelial system in mice, and counts were increased in mice treated with a liver X receptor agonist or reconstituted HDL, as compared with controls. In humans, tracer disappeared from plasma rapidly after injection of nanoparticles, followed by reappearance in HDL and nonHDL fractions. Counts present as free cholesterol increased rapidly and linearly in the first 240 min after nadir; counts in cholesteryl ester increased steadily over time. Estimates of fractional transfer rates of key RCT steps were obtained. These results support the use of 3 H-cholesterol nanoparticles as a feasible approach for the measurement of macrophage RCT in vivo in humans., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter A1 (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190-243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223-243) is essential for the HDL formation, the function of low lipid affinity region (residues 191-220) remains unclear. To evaluate the role of residues 191-220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191-220 apoA-I, in comparison to wild-type and Δ223-243 apoA-I. Although deletion of residues 191-220 has a slight effect on the tertiary structure of apoA-I, the Δ191-220 variant showed intermediate behavior between wild-type and Δ223-243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δ191-220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABCA1-expressing cells of Δ191-220 apoA-I was also intermediate between wild-type and Δ223-243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191-220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation., (© 2013.)

Apolipoprotein (apo) A-I-containing nascent HDL particles produced by the ATP binding cassette transporter A1 have different sizes and compositions. To understand the molecular basis for this heterogeneity, the HDL particles produced by apoA-I-mediated solubilization of phospholipid (PL)/free (unesterified) cholesterol (FC) bilayer membranes in cell and cell-free systems are compared. Incubation of apoA-I with ATP binding cassette transporter A1-expressing baby hamster kidney cells leads to formation of two populations of FC-containing discoidal nascent HDL particles. The larger 11-nm diameter particles are highly FC-enriched (FC/PL = 1.2/1 mol/mol) relative to the smaller 8 nm particles and the cell plasma membrane (FC/PL = 0.4/1). ApoA-I-mediated spontaneous solubilization of either multilamellar or unilamellar vesicles made of a membrane-PL mixture and FC yields discoidal HDL particles with diameters in the range 9-17 nm and, as found with the cell system, the larger particles are relatively enriched in FC despite the fact that all particles are created by solubilization of a common FC/PL membrane domain. The size-dependent distribution of FC among HDL particles is due to varying amounts of PL being sequestered in a boundary layer by interaction with apoA-I at the disc edge. The presence of a relatively large boundary layer in smaller discoidal HDL promotes preferential distribution of phosphatidylserine to such particles. However, phosphatidylcholine and sphingomyelin which are the primary PL constituents of nascent HDL do not exhibit selective incorporation into HDL discs of different sizes. This understanding of the mechanisms responsible for the heterogeneity in lipid composition of nascent HDL particles may provide a basis for selecting subspecies with preferred cardio-protective properties.

Elucidation of apoA-I secondary structure in spherical plasma HDL particles is essential for understanding HDL structure and function at the molecular level. To provide this information, we have applied hydrogen exchange (HX) and mass spectrometry methods to compare apoA-I secondary structure in discoidal (two apoA-I molecules/particle) and spherical (five apoA-I molecules/particle) HDL particles. The HX kinetics indicate that the locations of helical segments within the apoA-I molecules are the same in both discoidal and spherical HDL particles (approximately 10 nm hydrodynamic diameter). Helix stabilities in both types of particles are 3-5 kcal/mol, consistent with the apoA-I molecules being in a highly dynamic state with helical segments unfolding and refolding in seconds. For the spherical HDL, apoA-I fragments corresponding to residues 115-158 exhibit bimodal HX kinetics consistent with this segment adopting an inter-converting (on the timescale of tens of minutes) helix-loop configuration. The segment adopting this configuration in the 10 nm disc is shorter because the surface area available to each apoA-I molecule is apparently larger. Loop formation in the central region of the apoA-I molecule contributes to the ability of the protein to adapt to changes in available space on the HDL particle surface. Overall, apoA-I secondary structure is largely unaffected by a change in HDL particle shape from disc to sphere.

Although the partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between high-density lipoprotein (HDL)-bound and -unbound states is an integral part of HDL metabolism, the factors that control binding of apoA-I to HDL particles are poorly understood. To address this gap in knowledge, we investigated how the properties of the apoA-I tertiary structure domains and surface characteristics of spherical HDL particles influence apoA-I binding. The abilities of (14)C-labeled human and mouse apoA-I variants to associate with human HDL and lipid emulsion particles were determined using ultracentrifugation to separate free and bound protein. The binding of human apoA-I (243 amino acids) to HDL is largely mediated by its relatively hydrophobic C-terminal domain; the isolated N-terminal helix bundle domain (residues 1-190) binds poorly. Mouse apoA-I, which has a relatively polar C-terminal domain, binds to human HDL to approximately half the level of human apoA-I. The HDL binding abilities of apoA-I variants correlate strongly with their abilities to associate with phospholipid (PL)-stabilized emulsion particles, consistent with apoA-I-PL interactions at the particle surface being important. When equal amounts of HDL2 and HDL3 are present, all of the apoA-I variants partition preferentially to HDL3. Fluorescence polarization measurements using Laurdan-labeled HDL2 and HDL3 indicate that PL molecular packing is looser on the more negatively charged HDL3 particle surface, which promotes apoA-I binding. Overall, it is clear that both apoA-I structural features, especially the hydrophobicity of the C-terminal domain, and HDL surface characteristics such as the availability of free space influence the ability of apoA-I to associate with HDL particles.

To understand high-density lipoprotein (HDL) structure at the molecular level, the location and stability of α-helical segments in human apolipoprotein (apo) A-I in large (9.6 nm) and small (7.8 nm) discoidal HDL particles were determined by hydrogen-deuterium exchange (HX) and mass spectrometry methods. The measured HX kinetics of some 100 apoA-I peptides specify, at close to amino acid resolution, the structural condition of segments throughout the protein sequence and changes in structure and stability that occur on incorporation into lipoprotein particles. When incorporated into the large HDL particle, the nonhelical regions in lipid-free apoA-I (residues 45-53, 66-69, 116-146, and 179-236) change conformation from random coil to α-helix so that nearly the entire apoA-I molecule adopts helical structure (except for the terminal residues 1-6 and 237-243). The amphipathic α-helices have relatively low stability, in the range 3-5 kcal/mol, indicating high flexibility and dynamic unfolding and refolding in seconds or less. A segment encompassed by residues 125-158 exhibits bimodal HX labeling indicating co-existing helical and disordered loop conformations that interchange on a time scale of minutes. When incorporated around the edge of the smaller HDL particle, the increase in packing density of the two apoA-I molecules forces about 20% more residues out of direct contact with the phospholipid molecules to form disordered loops, and these are the same segments that form loops in the lipid-free state. The region of disc-associated apoA-I that binds the lecithin-cholesterol acyltransferase enzyme is well structured and not a protruding unstructured loop as reported by others.

The apoA-I molecule adopts a two-domain tertiary structure and the properties of these domains modulate the ability to form HDL particles. Thus, human apoA-I differs from mouse apoA-I in that it can form smaller HDL particles; the C-terminal α-helix is important in this process and human apoA-I is unusual in containing aromatic amino acids in the non-polar face of this amphipathic α-helix. To understand the influence of these aromatic amino acids and the associated high hydrophobicity, apoA-I variants were engineered in which aliphatic amino acids were substituted with or without causing a decrease in overall hydrophobicity. The variants human apoA-I (F225L/F229A/Y236A) and apoA-I (F225L/F229L/A232L/Y236L) were compared to wild-type (WT) apoA-I for their abilities to (1) solubilize phospholipid vesicles and form HDL particles of different sizes, and (2) mediate cellular cholesterol efflux and create nascent HDL particles via ABCA1. The loss of aromatic residues and concomitant decrease in hydrophobicity in apoA-I (F225L/F229A/Y236A) has no effect on protein stability, but reduces by a factor of about three the catalytic efficiencies (V(max)/K(m)) of vesicle solubilization and cholesterol efflux; also, relatively large HDL particles are formed. With apoA-I (F225L/F229L/A232L/Y236L) where the hydrophobicity is restored by the presence of only leucine residues in the helix non-polar face, the catalytic efficiencies of vesicle solubilization and cholesterol efflux are similar to those of WT apoA-I; this variant forms smaller HDL particles. Overall, the results show that the hydrophobicity of the non-polar face of the C-terminal amphipathic α-helix plays a critical role in determining apoA-I functionality but aromatic amino acids are not required. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1-189 and 190-243) and mouse apoA-I (residues 1-186 and 187-240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.

Objective: Reconstituted high-density lipoprotein (rHDL) is of interest as a potential novel therapy for atherosclerosis because of its ability to promote free cholesterol (FC) mobilization after intravenous administration. We performed studies to identify the underlying molecular mechanisms by which rHDL promote FC mobilization from whole body in vivo and macrophages in vitro., Methods and Results: Wild-type (WT), SR-BI knockout (KO), ABCA1 KO, and ABCG1 KO mice received either rHDL or phosphate-buffered saline intravenously. Blood was drawn before and at several time points after injection for apolipoprotein A-I, phosphatidylcholine, and FC measurement. In WT mice, serum FC peaked at 20 minutes and rapidly returned toward baseline levels by 24 hours. Unexpectedly, ABCA1 KO and ABCG1 KO mice did not differ from WT mice regarding the kinetics of FC mobilization. In contrast, in SR-BI KO mice the increase in FC level at 20 minutes was only 10% of that in control mice (P<0.01). Bone marrow-derived macrophages from WT, SR-BI O, ABCA1 KO, and ABCG1 KO mice were incubated in vitro with rHDL and cholesterol efflux was determined. Efflux from SR-BI KO and ABCA1 KO macrophages was not different from WT macrophages. In contrast, efflux from ABCG1 KO macrophages was approximately 50% lower as compared with WT macrophages (P<0.001)., Conclusions: The bulk mobilization of FC observed in circulation after rHDL administration is primarily mediated by SR-BI. However, cholesterol mobilization from macrophages to rHDL is primarily mediated by ABCG1.

The partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between HDL-bound and -unbound states is an integral part of HDL metabolism. We used the surface plasmon resonance (SPR) technique to monitor in real time the reversible binding of apoA-I to HDL. Biotinylated human HDL(2) and HDL(3) were immobilized on a streptavidin-coated SPR sensor chip, and apoA-I solutions at different concentrations were flowed across the surface. The wild-type (WT) human and mouse apoA-I/HDL interaction involves a two-step process; apoA-I initially binds to HDL with fast association and dissociation rates, followed by a step exhibiting slower kinetics. The isolated N-terminal helix bundle domains of human and mouse apoA-I also exhibit a two-step binding process, consistent with the second slower step involving opening of the helix bundle domain. The results of fluorescence experiments with pyrene-labeled apoA-I are consistent with the N-terminal helix bundle domain interacting with proteins resident on the HDL particle surface. Dissociation constants (K(d)) measured for WT human apoA-I interactions with HDL(2) and HDL(3) are about 10 microM, indicating that the binding is low affinity. This K(d) value does not apply to all of the apoA-I molecules on the HDL particle but only to a relatively small, labile pool.

High density lipoprotein (HDL) possesses important anti-atherogenic properties and this review addresses the molecular mechanisms underlying these functions. The structures and cholesterol transport abilities of HDL particles are determined by the properties of their exchangeable apolipoprotein (apo) components. ApoA-I and apoE, which are the best characterized in structural terms, contain a series of amphipathic alpha-helical repeats. The helices located in the amino-terminal two-thirds of the molecule adopt a helix bundle structure while the carboxy-terminal segment forms a separately folded, relatively disorganized, domain. The latter domain initiates lipid binding and this interaction induces changes in conformation; the alpha-helix content increases and the amino-terminal helix bundle can open subsequently. These conformational changes alter the abilities of apoA-I and apoE to function as ligands for their receptors. The apoA-I and apoE molecules possess detergent-like properties and they can solubilize vesicular phospholipid to create discoidal HDL particles with hydrodynamic diameters of ~10 nm. In the case of apoA-I, such a particle is stabilized by two protein molecules arranged in an anti-parallel, double-belt, conformation around the edge of the disc. The abilities of apoA-I and apoE to solubilize phospholipid and stabilize HDL particles enable these proteins to be partners with ABCA1 in mediating efflux of cellular phospholipid and cholesterol, and the biogenesis of HDL particles. ApoA-I-containing nascent HDL particles play a critical role in cholesterol transport in the circulation whereas apoE-containing HDL particles mediate cholesterol transport in the brain. The mechanisms by which HDL particles are remodeled by lipases and lipid transfer proteins, and interact with SR-BI to deliver cholesterol to cells, are reviewed.

The exchangeability of apolipoprotein (apo) E between lipoprotein particles such as very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) is critical for lipoprotein metabolism, but despite its importance, the kinetics and mechanism of apoE-lipoprotein interaction are not known. We have used surface plasmon resonance (SPR) to monitor in real time the reversible binding of apoE to human VLDL and HDL(3); biotinylated lipoproteins were immobilized on a streptavidin-coated SPR sensor chip, and solutions containing various human apoE molecules at different concentrations were passed across the surface. Analysis of the resultant sensorgrams indicated that the apoE3-lipoprotein interaction is a two-step process. After an initial interaction, the second slower step involves opening of the N-terminal helix bundle domain of the apoE molecule. Destabilization of this domain leads to more rapid interfacial rearrangement which is seen when the lipoprotein binding of apoE4 is compared to that of apoE3. The resultant differences in interfacial packing seem to underlie the differing abilities of apoE4 and apoE3 to bind to VLDL and HDL(3). The measured dissociation constants for apoE binding to these lipoprotein particles are in the micromolar range. C-Terminal truncations of apoE to remove the lipid binding region spanning residues 250-299 reduce the level of binding to both types of lipoprotein, but the effect is weaker with HDL(3); this suggests that protein-protein interactions are important for apoE binding to this lipoprotein while apoE-lipid interactions are more significant for VLDL binding. The two-step mechanism of lipoprotein binding exhibited by apoE is likely to apply to other members of the exchangeable apolipoprotein family.

The ATP-binding cassette transporter A1 (ABCA1) plays a critical role in the biogenesis of high density lipoprotein (HDL) particles and in mediating cellular cholesterol efflux. The mechanism by which ABCA1 achieves these effects is not established, despite extensive investigation. Here, we present a model that explains the essential features, especially the effects of ABCA1 activity in inducing apolipoprotein (apo) A-I binding to cells and the compositions of the discoidal HDL particles that are produced. The apo A-I/ABCA1 reaction scheme involves three steps. First, there is binding of a small regulatory pool of apo A-I to ABCA1, thereby enhancing net phospholipid translocation to the plasma membrane exofacial leaflet; this leads to unequal lateral packing densities in the two leaflets of the phospholipid bilayer. Second, the resultant membrane strain is relieved by bending and by creation of exovesiculated lipid domains. The formation of highly curved membrane surface promotes high affinity binding of apo A-I to these domains. Third, this pool of bound apo A-I spontaneously solubilizes the exovesiculated domain to create discoidal nascent HDL particles. These particles contain two, three, or four molecules of apo A-I and a complement of membrane phospholipid classes together with some cholesterol. A key feature of this mechanism is that membrane bending induced by ABCA1 lipid translocase activity creates the conditions required for nascent HDL assembly by apo A-I. Overall, this mechanism is consistent with the known properties of ABCA1 and apo A-I and reconciles many of the apparently discrepant findings in the literature.

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.

The nascent HDL created by ABCA1-mediated efflux of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apolipoprotein A-I (apoA-I) has not been defined. To address this issue, we characterized the lipid particles released when J774 mouse macrophages and human skin fibroblasts in which ABCA1 is activated are incubated with human apoA-I. In both cases, three types of nascent HDL containing two, three, or four molecules of apoA-I per particle are formed. With J774 cells, the predominant species have hydrodynamic diameters of approximately 9 and 12 nm. These discoidal HDL particles have different FC contents and PL compositions, and the presence of acidic PL causes them to exhibit alpha-electrophoretic mobility. These results are consistent with ABCA1 located in more than one membrane microenvironment being responsible for the production of the heterogeneous HDL. Activation of ABCA1 also leads to the release of apoA-I-free plasma membrane vesicles (microparticles). These larger, spherical particles released from J774 cells have the same PL composition as the 12 nm HDL and contain CD14 and ganglioside, consistent with their origin being plasma membrane raft domains. The various HDL particles and microparticles are created concurrently, and there is no precursor-product relationship between them. Importantly, a large fraction of the cellular FC effluxed from these cells by ABCA1 is located in microparticles. Collectively, these results show that the products of the apoA-I/ABCA1 interaction include discoidal HDL particles containing different numbers of apoA-I molecules. The cellular PLs and cholesterol incorporated into these nascent HDL particles originate from different cell membrane domains.

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.

HDL and its associated apo, APOE, inhibit S-phase entry of murine aortic smooth muscle cells. We report here that the antimitogenic effect of APOE maps to the N-terminal receptor-binding domain, that APOE and its N-terminal domain inhibit activation of the cyclin A promoter, and that these effects involve both pocket protein-dependent and independent pathways. These antimitogenic effects closely resemble those seen in response to activation of the prostacyclin receptor IP. Indeed, we found that HDL and APOE suppress aortic smooth muscle cell cycle progression by stimulating Cox-2 expression, leading to prostacyclin synthesis and an IP-dependent inhibition of the cyclin A gene. Similar results were detected in human aortic smooth muscle cells and in vivo using mice overexpressing APOE. Our results identify the Cox-2 gene as a target of APOE signaling, link HDL and APOE to IP action, and describe a potential new basis for the cardioprotective effect of HDL and APOE.

Receptor-mediated trafficking of cholesterol between lipoproteins and cells is a fundamental biological process at the organismal and cellular levels. In contrast to the well-studied pathway of LDL receptor-mediated endocytosis, little is known about the trafficking of high-density lipoprotein (HDL) cholesterol by the HDL receptor, scavenger receptor BI (SR-BI). SR-BI mediates HDL cholesteryl ester uptake in a process in which HDL lipids are selectively transferred to the cell membrane without the uptake and degradation of the HDL particle. We report here the cell surface locale where the trafficking of HDL cholesterol occurs. Fluorescence confocal microscopy showed SR-BI in patches and small extensions of the cell surface that were distinct from sites of caveolin-1 expression. Electron microscopy showed SR-BI in patches or clusters primarily on microvillar extensions of the plasma membrane. The organization of SR-BI in this manner suggests that this microvillar domain is a way station for cholesterol trafficking between HDL and cells. The types of phospholipids in this domain are unknown, but SR-BI is not strongly associated with classical membrane rafts rich in detergent-resistant saturated phospholipids. We speculate that SR-BI is in a more fluid membrane domain that will favor rapid cholesterol flux between the membrane and HDL.

The mechanism of formation of high density lipoprotein (HDL) particles by the action of ATP-binding cassette transporter A1 (ABCA1) is not defined completely. To address this issue, we monitored efflux to apoA-I of phosphatidylcholine (PC), sphingomyelin (SM), and unesterified (free) cholesterol (FC) from J774 macrophages, in which ABCA1 is up-regulated, and investigated the nature of the particles formed. The various apoA-I/lipid particles appearing in the extracellular medium were separated by gel filtration chromatography. The presence of apoA-I in the extracellular medium led to the simultaneous formation of more than one type of poorly lipidated apoA-I-containing particle: there were 9- and 12-nm diameter particles containing approximately 3:1 and 1:1 phospholipid/FC (mol/mol), respectively, which were present together with 6-nm monomeric apoA-I. Removal of the C-terminal alpha-helix (residues 223-243) of apoA-I reduced phospholipid and FC efflux and prevented formation of the 9- and 12-nm HDL particles; the apoA-I variant formed larger particles that eluted in the void volume. FC loading of the J774 cells also led to the formation of larger apoA-I-containing particles that were highly enriched in FC. Besides creating HDL particles, ABCA1 mediated release of larger (20-450-nm diameter) FC-rich particles that were not involved in HDL formation and that are probably membrane vesicles. These particles contained 1:1 PC/SM in contrast to the HDL particles, which contained 2:1 PC/SM. This is consistent with lipid raft and non-raft plasma membrane domains being involved primarily in ABCA1-mediated vesicle release and nascent HDL formation, respectively.

Background: Factors that regulate the metabolism of HDL and apolipoprotein A-I (apoA-I) are incompletely understood. Overexpression of endothelial lipase (EL) markedly reduces plasma levels of HDL cholesterol and apoA-I in mice, but the mechanisms of this effect remain unknown., Methods and Results: We used different doses of a recombinant adenoviral vector to overexpress human EL in mice and studied the effects on plasma phospholipase activity, plasma lipids, HDL particle size, HDL turnover, and tissue sites of HDL degradation in mice. Overexpression of EL was associated with a significant dose-dependent increase in postheparin plasma phospholipase activity. Plasma phospholipid, HDL cholesterol, and apoA-I levels were markedly decreased, even at the lowest dose of vector. Kinetic studies demonstrated a significant dose-dependent increase in the fractional catabolic rate of HDL-apolipoprotein in EL-overexpressing mice. The postheparin plasma phospholipase activity was significantly positively correlated with HDL-apolipoprotein fractional catabolic rate. The uptake of apoA-I by the kidney and the liver was significantly increased by 2.5-fold and 3-fold, respectively, in mice overexpressing EL., Conclusions: Expression of EL in mice results in a dose-dependent increase in postheparin plasma phospholipase activity, catabolic rate of HDL-apolipoprotein, and uptake of apoA-I in both kidney and liver.

HDL particles possess important antiatherogenic functionalities and understanding of the molecular mechanisms underlying these effects requires detailed knowledge of HDL structure. This review summarizes current understanding of HDL structure. The various HDL subclasses are compared in terms of their lipid and protein compositions. The lipid-binding properties of the principal HDL apolipoprotein, apo A-I, permit plasticity in HDL structure. The amphipathic alpha-helical domains that are the major element of secondary structure mediate the interaction of apo A-I with phospholipid. Low resolution models of the structures of both discoidal and spherical HDL particles are evaluated. HDL particles are dynamic in that they are being remodeled constantly in vivo by interaction with lipases, lipid transfer proteins, and cell-surface HDL receptors. Current knowledge of the ways in which HDL particle structure is modulated by interactions with proteins such as LCAT, CETP, SR-BI and ABCA1 is reviewed.

Despite extensive studies and characterizations of the high density lipoprotein-cholesteryl ester (HDL-CE)-selective uptake pathway, the mechanisms by which the hydrophobic CE molecules are transferred from the HDL particle to the plasma membrane have remained elusive, until the discovery that scavenger receptor BI (SR-BI) plays an important role. To elucidate the molecular mechanism, we examined the quantitative relationships between the binding of HDL and the selective uptake of its CE in the murine adrenal Y1-BS1 cell line. A comparison of concentration dependences shows that half-maximal high affinity cell association of HDL occurs at 8.7 +/- 4.7 micrograms/ml and the Km of HDL-CE-selective uptake is 4.5 +/- 1.5 micrograms/ml. These values are similar, and there is a very high correlation between these two processes (r2 = 0.98), suggesting that they are linked. An examination of lipid uptake from reconstituted HDL particles of defined composition and size shows that there is a non-stoichiometric uptake of HDL lipid components, with CE being preferred over the major HDL phospholipids, phosphatidylcholine and sphingomyelin. Comparison of the rates of selective uptake of different classes of phospholipid in this system gives the ranking: phosphatidylserine > phosphatidylcholine approximately phosphatidylinositol > sphingomyelin. The rate of CE-selective uptake from donor particles is proportional to the amount of CE initially present in the particles, suggesting a mechanism in which CE moves down its concentration gradient from HDL particles docked on SR-BI into the cell plasma membrane. The activation energy for CE uptake from either HDL3 or reconstituted HDL is about 9 kcal/mol, indicating that HDL-CE uptake occurs via a non-aqueous pathway. HDL binding to SR-BI allows access of CE molecules to a "channel" formed by the receptor from which water is excluded and along which HDL-CE molecules move down their concentration gradient into the cell plasma membrane.

High density lipoprotein (HDL) is able to remove unesterified cholesterol from peripheral cells in the process of reverse cholesterol transport by an aqueous diffusion mechanism as well as by an apolipoprotein (apo)-mediated process. The aqueous diffusion mechanism is understood but the molecular mechanism of lipid-poor pre-beta-HDL-(apo-) mediated cholesterol removal is not known. Measurements of the initial rates of efflux of unesterified cholesterol and phospholipid from human fibroblasts to lipid-free, human apoA-I showed that both lipids are released from the cells during a 10-min incubation with apoA-I. The concentration-dependence of efflux of the lipids is the same (Km = 0.4 and 0.6 microg apoA-I/ml for cholesterol and phospholipid flux, respectively), suggesting a membrane microsolubilization process. A finite pool of about 1% of the plasma membrane cholesterol is accessible for release by solubilization; the limited size of this cholesterol pool is not due to a lack of availability of apoA-I, but rather to the restricted amount of phospholipid that is removed from the plasma membrane. Plasma membrane domains may be involved in membrane microsolubilization, but caveolar cholesterol seems not to be specifically accessed in this process. Membrane microsolubilization is the process by which pre-beta1-HDL removes cell cholesterol in the first step of reverse cholesterol transport. When apoA-I is present in the extracellular space, the relative contributions of cholesterol efflux by membrane microsolubilization and by aqueous diffusion are determined by the degree of lipidation of the apoA-I molecules.

African green monkeys fed fat-specific diets served as a model to investigate the effect of phospholipid acyl chain modification on high density lipoprotein (HDL)-mediated cellular cholesterol efflux. Diets enriched in saturated, monounsaturated, n-6 polyunsaturated, or n-3 polyunsaturated fats were provided during both low cholesterol and cholesterol-enriched stages; sera and HDL3 samples were obtained at specific points during the treatment period. Analysis of the HDL phospholipid composition revealed significant acyl chain modification, consistent with the respective fat-specific diet. Cholesterol efflux from mouse L-cell fibroblasts to HDL3 isolated from the specific diet groups was measured and revealed no differences in the abilities of the particles to accept cellular cholesterol; determination of the bidirectional flux of cholesterol between the cells and HDL3 species further demonstrated no effect of phospholipid acyl chain modification on this process. The effects of dietary modification of phospholipid acyl chains on cellular cholesterol efflux were directly examined by isolating the HDL phospholipid and combining it with human apolipoprotein A-I to form well-defined reconstituted HDL particles. These complexes did not display any differences with respect to their ability to stimulate cellular cholesterol efflux. Incubations with 5% sera further confirmed that the fat-specific diets do not influence cholesterol efflux. These results suggest that the established influences of specific dietary fats on the progression of atherosclerosis are due to effects on cholesterol metabolism other than the efflux of cellular cholesterol in the first step of reverse cholesterol transport.

The participation of HDL in the reverse cholesterol transport (RCT) from peripheral cells to the liver is critical for the antiatherogenic properties of this lipoprotein. Experimental results showing that efflux of cholesterol from cells growing in culture is mediated by HDL and lipoprotein particles containing apo A-I, in particular, support this conclusion. A bidirectional flux of unesterified cholesterol molecules between the plasma membrane of cells and HDL particles in the extracellular medium occurs. Net efflux of cholesterol mass from the cells involves passive diffusion of cholesterol molecules through the aqueous phase and down their concentration gradient between the membrane and HDL; the concentration gradient is maintained by LCAT-mediated esterification of cholesterol molecules in the HDL particles. Fully lipidated apo A-I is important in promoting this aqueous diffusion mechanism because it: (1) acts as a cofactor for LCAT; and (2) solubilizes phospholipid into small HDL-sized particles that are efficient at absorbing cholesterol molecules diffusing away from the cell surface. Apo A-I also exists in an incompletely lipidated state in plasma. Apo A-I molecules in this state are able to solubilize phospholipid and cholesterol from the plasma membrane of cells. This membrane-microsolubilization process is enhanced by enrichment of the plasma membrane with cholesterol and is the mechanism by which pre-beta-HDL particles in the extracellular medium remove cholesterol and phospholipid from cells. The relative contributions in vivo of the aqueous diffusion and membrane-microsolubilization mechanisms of apo A-I-mediated cell cholesterol efflux are not predicted readily from cell culture experiments. Confounding issues are the variations with cell type and the dependence on the degree of cholesterol loading of the cell plasma membrane.

High density lipoprotein (HDL) is thought to play a significant role in the process of reverse cholesterol transport. It has become clear that the apolipoprotein (apo) composition of HDL is important in determining the metabolic fate of this particle. The major proteins of human HDL are apoAI and APOAII; the latter protein is a disulfide-linked dimer in humans and higher primates but monomeric in the other species. The consequences of the apo Cys6-Cys6 disulfide bridge in apoAII for human HDL structure and function are not known. To address this issue, the influence of the Cys6-Cys6 disulfide bridge on the interaction of human apoAII with palmitoyl-oleoyl phosphatidylcholine has been studied. The size and valence of a series of homogeneous discoidal complexes containing either monomeric (reduced and carboxymethylated) or dimeric apoAII have been determined, and their ability to remove cholesterol from rat Fu5AH hepatoma cells grown in culture has been compared. The apoAII dimer and monomer form discoidal complexes of similar size, with twice as many of the latter molecule required per disc. Removal of the disulfide bond influences the stability of the helical segments around the edge of the disc as seen by a decrease in alpha-helix content of the monomeric protein. The discoidal particles containing the monomeric form of apoAII are somewhat more effective than particles containing either dimeric apoAII or apoAI in removing cellular cholesterol. Overall, reduction of the disulfide bridge of apoAII probably does not have a major effect in the determination of HDL particle size in vivo. It follows that the evolution of the Cys6-Cys6 disulfide bond in higher primates probably has not had a major effect on the function of the apoAII molecule.

The role of HDL and its major protein constituent, apolipoprotein (apo) A-I, in promoting the removal of excess cholesterol from cultured cells has been well established; however, the mechanisms by which this occurs are not completely understood. To address the effects of apoA-I modification on cellular unesterified (free) cholesterol (FC) efflux, three recombinant human apoA-I deletion mutants and plasma apoA-I were combined with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and FC to make reconstituted high density lipoprotein (rHDL) discoidal complexes. These particles were characterized structurally and for their efficiency as acceptors of mouse L-cell fibroblast cholesterol. The deletion mutant proteins lacked NH2-terminal (apoA-I (Delta44-126)), central (apoA-I (Delta139-170)), or COOH-terminal (apoA-I (Delta190-243)) domains of apoA-I. The three deletion mutants all displayed lipid-binding abilities and formed discoidal complexes that were similar in major diameter (13.2 +/- 1.5 nm) to those formed by human apoA-I when reconstituted at a 100:5:1 (POPC:FC:protein) mole ratio. Gel filtration profiles indicated unreacted protein in the preparation made with apoA-I (Delta190-243), which is consistent with the COOH terminus portion of apoA-I being an important determinant of lipid binding. Measurements of the percent alpha-helix content of the proteins, as well as the number of protein molecules per rHDL particle, gave an indication of the arrangement of the deletion mutant proteins in the discoidal complexes. The rHDL particles containing the deletion mutants had more molecules of protein present than particles containing intact apoA-I, to the extent that a similar number of helical segments was incorporated into each of the discoidal species. Comparison of the experimentally determined number of helical segments with an estimate of the available space indicated that the deletion mutant proteins are probably more loosely arranged than apoA-I around the edge of the rHDL. The abilities of the complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts. All four discoidal complexes displayed similar abilities to remove FC from the plasma membrane of L-cells when compared at an acceptor concentration of 50 microg of phospholipid/ml. Thus, none of the deletions imposed in this study notably altered the ability of the rHDL particles to participate in cellular FC efflux. These results suggest that efficient apoA-I-mediated FC efflux requires the presence of amphipathic alpha-helical segments but is not dependent on specific helical segments.

Alterations in high density lipoprotein (HDL) composition that occur in dyslipidemic states may modulate a number of events involved in cholesterol homeostasis. To elucidate the details of how HDL-core composition can affect the molecular structure of different kinds of HDL particles, the conformation and stability of apoA-I have been investigated in homogeneous recombinant HDL particles (LpA-I) containing palmitoyloleoyl phosphatidylcholine (POPC), triolein (TG), and/or cholesteryl linoleate (CE). In a discoidal particle containing two molecules of apoA-I and 85 molecules of POPC, apoA-I exhibits an alpha-helix content of 70% and a free energy of stability of its alpha-helical segments (delta G0D) of 2.2 kcal/mol. Inclusion of eight molecules of TG into the complex significantly reduces the alpha-helix content and stability of apoA-I, whereas inclusion of four molecules of CE into the complex has an opposite effect in that the alpha-helix content is significantly reduced and the stability of the remaining alpha-helical structure of apoA-I is increased. Neutral lipids have a different effect on apoA-I conformation in spherical LpA-I particles. In a sonicated-spherical LpA-I particle containing two molecules of apoA-I and 70 molecules of POPC, apoA-I exhibits an alpha-helix content of about 60% and a delta G0D of 1.2 kcal/mol apoA-I. Inclusion of either 10 molecules of TG or six molecules of CE into such a particle increases both the alpha-helix content and stability of apoA-I. Increasing the CE/TG ratio in LpA-I particles that contain both neutral lipids enhances the stability of the alpha-helical segments. ApoA-I molecules tend to dissociate and cause particle instability when delta G0D for the lipid-bound alpha-helices is less than that for helices in the lipid-free state. The stabilities of both discoidal and spherical LpA-I particles are relatively low when the only neutral lipid present is TG but the particle stability is enhanced by the presence of CE molecules. Such dissociation of apoA-I molecules from LpA-I particles that have a low CE/TG ratio would be promoted in the hypertriglyceridemic state in vivo.

Several subspecies of human high density lipoprotein (HDL) have been shown to exist, and particle size is one parameter that can be used to distinguish them. Recently, a small HDL subspecies has been described that may be a particularly efficient acceptor of peripheral cell unesterified (free) cholesterol (FC). To address the effects of particle size on the ability of HDL to remove FC from cells, homogeneous, well defined HDL particles were reconstituted (rHDL) that varied in particle diameter within the size range of human HDL particles (7-13 nm). The abilities of each of these particles to remove cellular FC from mouse L-cells and rat Fu5AH hepatoma cells were compared on the basis of their phospholipid (PL) content as well as on a per particle basis. The effect of particle size was also examined using small unilamellar vesicles (SUV) of 25 nm in diameter and large unilamellar vesicles (LUVs) of 70-180 nm in diameter. The SUV were prepared by sonication, and the LUVs were prepared by extrusion techniques. The FC efflux efficiency of these particles (in order of decreasing efficiency) was: rHDL > SUV > LUV when compared on the basis of acceptor PL content across a range of concentrations (i.e. at a given PL concentration for these three acceptor classes, smaller particles were more efficient). The FC efflux differences between the rHDL and the vesicles were not due to the absence of apolipoprotein in the vesicles. No difference was detected among the rHDL of varying size, nor was a difference detected among the LUVs of varying size when compared on the basis of PL content. When the FC efflux data for rHDL and LUVs were normalized on the basis of the number of acceptor particles present at a given PL concentration, a correlation was found between acceptor particle radius and the ability to accept cellular FC with larger particles being the most efficient. However, the dependence of the rate of FC efflux on acceptor particle size was not quantitatively the same within the rHDL and LUV classes of acceptor particles. The dependence of FC efflux on acceptor particle size may reflect differing abilities of the variously sized acceptor particles to access the region very close to the cell plasma membrane where most of the FC removal is expected to occur.

The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins and is associated with high density lipoproteins (HDL). To understand the mechanism of interaction of CETP with HDL, we studied the binding of pure recombinant CETP to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/apoA-I discoidal particles. Separating bound from free CETP using native gradient gel electrophoresis, complexes of CETP with 10-nm hydrodynamic diameter discoidal particles migrated with a diameter of 12-16 nm, compared with approximately 7.5 nm for CETP. At lower ratios of CETP to discs, CETP bound to discs without displacement of apoA-I. CETP alone was unable to generate discoidal complexes. Cross-linking and fluorescence resonance energy transfer experiments indicated that CETP bound to discs as monomers. Cross-linking of CETP to apoA-I in discs suggested proximity of apoA-I and CETP. By negative-stain electron microscopy, discoidal complexes containing CETP and CETP monoclonal antibody showed localization of antibody molecules to the disc edge, suggesting that CETP was bound to the disc edge. The binding of CETP to discs of different composition or size was studied. Discs (10-nm Stokes diameter) prepared with either apoA-I or apoA-II had a similar Kd (120 nM). Inclusion of 1 mol % cholesteryl oleate, 5 mol % cholesterol, or 6 mol % phosphatidylinositol increased the binding affinity of CETP 3-10 times (20-30 nM). In comparison, plasma HDL3 had a Kd of approximately 450 nM. For POPC/apoA-I discs, 10-nm discs bound CETP with much higher affinity than smaller 7.8-nm discs (Kd = 1-2 microM). 7.7-nm hydrodynamic diameter POPC/apoA-I spherical particles containing either triolein or cholesteryl oleate in their core bound CETP with higher affinity (Kd = 50-100 nM) than 7.8-nm POPC/apoA-I discs. Thus, CETP appears to bind to the perimeter of discoidal particles, possibly in a process in which flexible segments in apoA-I or apoA-II accommodate CETP at the disc edge. The binding of CETP to HDL is markedly influenced by overall particle size and shape and by lipid composition, and the increased binding affinity for cholesterol- and cholesteryl ester-containing discs suggests a higher affinity of CETP for nascent than mature HDL.

High density lipoprotein (HDL) phospholipid (PL) fatty acyl chain composition has been proposed to affect the ability of HDL to participate in the first step of reverse cholesterol transport. To examine the effects of PL fatty acid chain length and degree of unsaturation in this process, reconstituted HDL (rHDL) particles were made with human apolipoprotein (apo) A-I and PL containing fatty acid chains from 14 to 18 carbons in length, which were either fully saturated or unsaturated in one or both chains. These particles were characterized structurally and for their ability to promote free (unesterified) cholesterol (FC) efflux from cells growing in culture. The discoidal rHDL particles were homogeneous and exhibited similar hydrodynamic diameters (10.4 +/- 1.0 nm) indicating that apoA-I forms similarly sized discs with a variety of PL. Measurements of particle surface charge, apoA-I alpha-helix content, and conformational stability indicated that the conformation of apoA-I varies among the particles. These conformational effects on apoA-I are consistent with the PL fluidity influencing the interaction between the amphipathic alpha-helical segments and PL acyl chains. Differential scanning calorimetry demonstrated that the physical state of the rHDL PL at 37 degrees C varied according to acyl chain length and degree of unsaturation; the FC efflux efficiencies for particles with PL in either the gel or liquid crystal states were determined. The ability of the rHDL to accept cellular FC depended on the physical state of the PL in the rHDL. Liquid crystal PL formed the most efficient FC acceptor particles exhibiting a maximal efflux velocity (Vmax) of 12-14% release of total cellular FC per h. Gel-phase PL formed inefficient rHDL acceptors with a Vmax of about 3%/h. A similar hierarchy of FC efflux efficiency was noted when either mouse L-cells or rat Fu5AH hepatoma cells were used as the FC donors. Furthermore, this hierarchy was found to be due to the characteristics of the PL and not due to variable apoA-I conformation because protein-free, small unilamellar vesicles made with the same PL exhibited similar relative efflux capabilities. Generally, the ability of a given rHDL particle to accept cellular FC was related to rHDL PL acyl chain length and degree of unsaturation; decreases in PL acyl chain length and increases in chain unsaturation tended to result in more efficient FC acceptor particles.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of apolipoprotein conformation on the ability of high density lipoprotein (HDL) to remove cellular free cholesterol (FC) has not been studied in detail. To address the effects of amphipathic alpha-helix structure on cellular FC efflux, three class A helical peptides and apolipoprotein (apo) AI were complexed to dimyristoyl phosphatidylcholine (DMPC) to make discoidal complexes that were used as acceptors of cell cholesterol. The peptides consisted of an 18-amino acid, amphipathic, alpha-helical peptide with the sequence DWLKAFYDKVAEKLKEAF (18A), a dimer of 18A covalently linked by a proline residue (37pA), and acetyl-18A-amide (Ac-18A-NH2) that has a higher alpha-helix content than the unblocked 18A molecule. The three peptides strongly mimic the lipid-binding characteristics of the amphipathic segments of apolipoproteins and form discoidal complexes with DMPC that are similar in diameter (11-12 nm) to those formed by human apoAI when reconstituted at a 2.5:1 (w:w) phospholipid to protein ratio. The abilities of these complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts; efflux of FC from both the plasma membrane and the lysosomal pools was examined. For each of the acceptors, the removal of cholesterol from the plasma membrane and lysosomal pools was equally efficient. All four discoidal complexes were equally efficient cell membrane FC acceptors when compared at saturating acceptor concentrations of > 200 micrograms of DMPC/ml of medium. However, at the same lipid concentration, protein-free DMPC small unilamellar vesicles (SUV) were significantly less efficient. The initial rates of FC removal from cells at saturating concentrations of acceptor particles (Vmax) were 12, 10, 10, and 11% per h, respectively, for the complexes containing either 18A, Ac-18A-NH2, 37pA, or apoAI, but only 1% cellular FC per h for the DMPC SUV. The 10-fold higher Vmax for the apoprotein/peptide-containing acceptors was likely due to a reversible interaction of apoprotein or peptide with the plasma membrane that changed the lipid packing characteristics in such a way as to increase the rate of FC desorption from the cell surface. This interaction required amphipathic alpha-helical segments, but it was not affected by the length, number, or lipid-binding affinity of the helices. Furthermore, the efflux efficiency was not dependent on the amino acid sequence of the helical segments which suggests that this interaction is not mediated by a specific cell surface binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Several subspecies of human high density lipoprotein (HDL) exist, and particle surface charge is one parameter that can be used to distinguish them. For instance, pre-beta HDL has a lower negative surface charge than the bulk of plasma HDL resulting in slower migration when subjected to electrophoresis in an agarose gel at pH 8.6. The molecular basis for the variations in HDL particle surface charge have been established in this work. Quantitative analysis of electrophoretic mobilities in agarose gels indicates that the surface potentials of reconstituted pre-beta HDL and alpha-HDL3 are -7.6 and -11.4 mV, respectively. The difference of 3.8 mV corresponds to an apparent 1e more net negative charge on alpha-migrating HDL3. The contributions of the lipid and protein components to the charge of HDL3 were defined using reconstituted particles (rHDL) containing various combinations of HDL3 protein and lipid. The surface potentials of spherical and discoidal rHDL are not affected by the particle apoprotein composition. Thin layer chromatography of the HDL3 phospholipid fraction from five normolipidemic subjects revealed that it consists of approximately 84% phosphatidylcholine, 12% sphingomyelin, and 4% phosphatidylinositol (PI) by weight. PI significantly affects the charge of discoidal rHDL and is responsible for approximately 0.7 mV of the surface potential difference between pre-beta- and alpha-HDL. Reconstitution of spherical complexes containing apolipoprotein AI, palmitoyl-oleoyl phosphatidylcholine, and HDL3 neutral lipid esters showed that particles containing a neutral lipid core migrate to an alpha position in an agarose gel. The change in particle shape accounts for approximately 3 mV of the difference in surface potential between pre-beta and alpha-HDL. Overall, the charge of HDL particles is determined by 1) a direct contribution of negative charge from PI molecules in the surface of the lipoprotein and 2) the conformation of the resident apolipoprotein(s) which is influenced by the presence of neutral lipids and the particle shape. This quantitative understanding of surface charge provides a basis for understanding variations in function of HDL subspecies.

The effects of cholesterol on the conformation and net charge of apoA-I have been investigated in homogeneous recombinant high density lipoprotein (HDL) particles. ApoA-I charge and structure in discoidal recombinant HDL complexes containing palmitoyloleoylphosphatidylcholine and cholesterol have been quantitated by guanidine HCl denaturation, circular dichroism, electrokinetic analysis, and NMR spectroscopy of [13C]lysine-labeled apoA-I. In a discoidal particle containing 2 molecules of apoA-I and 160 molecules of palmitoyloleoylphosphatidylcholine, apoA-I exhibits an alpha-helix content of 75%, and the particle has a net negative surface charge of -5.2e/mol of apoA-I at pH 8.6. Addition of 2 molecules of cholesterol to this complex has no significant effect upon particle size, but slightly decreases the net charge (-5.0e) and alpha-helix content (68%) of apoA-I and enhances the stability of the helical segments, as reflected by an increase in the free energy of unfolding from 2.9 to 3.5 kcal/mol. In contrast, increasing the cholesterol content to 20 molecules/particle progressively increases particle size and apoA-I net negative charge (-6.1e), and there is a concomitant reduction in the free energy of stabilization of the alpha-helical structure in apoA-I to 2.2 kcal/mol. (13CH3)2-Lys resonances from apoA-I in discoidal recombinant HDL exhibit six chemical shifts at pH 10; these peaks originate from dimethyl-Lys residues that have pKa values ranging from 8.4 to 10.3. The titration behavior of apoA-I Lys residues is generally similar in the presence and absence of cholesterol, except that 4 Lys residues titrate at a significantly higher pH in the presence of cholesterol. These data are consistent with cholesterol having a direct effect on apoA-I conformation and charge in HDL. Structural changes of this magnitude can affect the interactions between HDL and various plasma proteins and cell surfaces. It is therefore likely that the cholesterol content of HDL plays an important role in regulating the metabolism of this lipoprotein.

The details of how high density lipoprotein (HDL) microstructure affects the conformation and net charge of apolipoprotein (apo) A-I in various classes of HDL particles have been investigated in homogeneous recombinant HDL (rHDL) particles containing apoA-I, palmitoyl-oleoyl phosphatidylcholine (POPC) and cholesteryl oleate. Isothermal denaturation with guanidine HCl was used to monitor alpha-helix structural stability, whereas electrokinetic analyses and circular dichroism were used to determine particle charge and apoA-I secondary structure, respectively. Electrokinetic analyses show that at pH 8.6 apoA-I has a net negative charge on discoidal (POPC.apoA-I) particles (-5.2 electronic units/mol of apoA-I) which is significantly greater than that of apoA-I either free in solution or on spherical (POPC.cholesteryl oleate.apoA-I) rHDL (approximately -3.5 electronic units). Raising the POPC content (32-128 mol/ml of apoA-I) of discoidal particles 1) increases the particle major diameter from 9.3 to 12.1 nm, 2) increases the alpha-helix content from 62 to 77%, and 3) stabilizes the helical segments by increasing the free energy of unfolding (delta GD degree) from 1.4 to 3.0 kcal/mol of apoA-I. Raising the POPC content (28-58 mol/mol of apoA-I) of spherical particles 1) increases the particle diameter from 7.4 to 12.6 nm, 2) increases the percent alpha-helix from 62 to 69%, and 3) has no significant effect on delta GD degree (2.2 kcal/mol of apoA-I). This study shows that different HDL subspecies maintain particular apoA-I conformations that confer unique charge and structural characteristics on the particles. It is likely that the charge and conformation of apoA-I are critical molecular properties that modulate the metabolism of HDL particles and influence their role in cholesterol transport.

To elucidate the molecular details of how high density lipoprotein (HDL) microstructure affects the conformation of apolipoprotein (apo) A-I in various classes of HDL particles, apoA-I structure in homogeneous recombinant HDL (rHDL) complexes containing palmitoyl-oleoyl phosphatidylcholine (POPC) and cholesteryl oleate has been investigated by NMR spectroscopy of [13C]lysine-labeled apoA-I. All Lys residues in rHDL apoA-I were labeled with 13C by reductive methylation, and then their ionization behavior was characterized by 13C NMR spectroscopy. Four discoidal particles were prepared to contain from 64 to 256 molecules of POPC and 2 molecules of apoA-I; their major diameters ranged from 9.3 to 12.1 nm. (13CH3)2-Lys resonances from apoA-I in discoidal complexes exhibit six distinct chemical shifts at pH 10. The various Lys have pKa values ranging from 8.3 to 10.5, indicating that they exist in different microenvironments. More than 80% of the Lys residues in small (9.3 nm) discoidal particles titrate at a significantly lower pH than in the large (12.1 nm) discoidal particles. This indicates that apoA-I has a different conformation on the differently size discs. Two spherical particles were prepared with POPC:cholesteryl oleate:apoA-I molar stoichiometries of 56:16:2 and 232:84:4 and diameters of 7.4 and 12.6 nm, respectively. On spherical rHDL, apoA-I (13CH3)2-Lys resonances exhibit five distinct chemical shifts at pH 10. The titration behavior of apoA-I Lys residues is the same in small and large spherical particles, indicating that apoA-I conformation is similar on the two particles. The Lys microenvironments indicate that the conformation of apoA-I in discoidal complexes is dependent on particle size and that these conformations are substantially different from that of apoA-I on spherical complexes. Lys microenvironments in discoidal complexes differ from that of spherical complexes by 4 to 5 ysines which titrate with relatively low pKa values on discs. This reflects apparent differences in conformation in the NH2-terminal one-third of apoA-I on discs and spheres.

The transfer of 14C-labeled, reduced and carboxymethylated human apolipoprotein A-II (RCM-AII) between small unilamellar vesicles (SUV) has been investigated. Ion-exchange chromatography was used for rapid separation of negatively charged egg phosphatidylcholine (PC)/dicetyl phosphate donor SUV containing bound 14C-labeled RCM-AII from neutral egg PC acceptor SUV present in 10-fold molar excess. The kinetics of 14C-labeled RCM-AII transfer in incubations of up to 12 h at 37 degrees C are consistent with the existence of fast, slow, and apparently "nontransferrable" pools of SUV-associated apolipoprotein; the transfers from these pools occur on the time scales of seconds or less, hours, and days/weeks, respectively. For donor SUV (0.15 mg of phospholipid/mL reaction mixture) containing about 15 RCM-AII molecules per vesicle, the sizes of the fast, slow, and nontransferrable pools are 13, 69, and 18%, respectively. The transfer of RCM-AII from the slow kinetic pool follows first-order kinetics, and the half-time (t 1/2) is about 3 h. The different kinetic pools of SUV-associated RCM-AII probably reflect apoprotein in different conformations of the SUV surface. Increasing the number of RCM-AII per donor SUV enlarges the size of the fast pool and increases the t 1/2 of transfer from the slow pool. In contrast, raising the incubation temperature reduces the t 1/2 of slow transfer. The t 1/2 of RCM-AII transfer from the slow kinetic pool is inversely proportional to the acceptor/donor SUV ratio which suggests that the transfer of apoprotein molecules in this kinetic pool is mediated by SUV collisions.(ABSTRACT TRUNCATED AT 250 WORDS)

The surface pressure (pi)-molecular area (A) isotherms for monolayers of human high-density lipoprotein (HDL3) and low-density lipoprotein (LDL) phospholipids and of mixed monolayers of these phospholipids with cholesterol spread at the air-water interface were used to deduce the likely molecular packing at the surfaces of HDL3 and LDL particles. LDL phospholipids form more condensed monolayers than HDL3 phospholipids; for example, the molecular areas of LDL and HDL3 phospholipids at pi = 10 dyn/cm are 88 and 75 A2/molecule, respectively. The closer packing in the LDL phospholipids monolayer can be attributed to the higher contents of saturated phosphatidylcholines and sphingomyelin relative to HDL3. Cholesterol condenses both HDL3 and LDL phospholipid monolayers but has a greater condensing effect on the LDL phospholipid monolayer. The pi-A isotherms for mixed monolayer of HDL3 phospholipid/cholesterol and LDL phospholipid/cholesterol at stoichiometries similar to those at the surfaces of lipoprotein particles suggest that the monolayer at the surface of the LDL particle is significantly more condensed than that at the surface of the HDL3 particle. The closer lateral packing in LDL is due to at least three factors: (1) the difference in phospholipid composition; (2) the higher unesterified cholesterol content in LDL; and (3) a stronger interaction between cholesterol and LDL phospholipids relative to HDL3 phospholipids. The influence of lipid molecular packing on the affinity of human apolipoprotein A-I (apo A-I) for HDL3 and LDL surface lipids was evaluated by monitoring the adsorption of 14C-methylated apo A-I to monolayers of these lipids spread at various initial surface pressures (pi i).(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of cholesterol and phosphatidylcholine (PC) exchange between human serum lipoproteins has been investigated by following the transfer of radiolabeled cholesterol and PC between high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Initially, [14C]cholesterol was present in the donor lipoprotein particle which was either HDL2, HDL3, or LDL. After incubation in saline solution for various times, the HDL and LDL were separated by precipitation of the LDL with Mn2+-heparin reagent. More than 90% of the [14C]cholesterol in donor HDL3 is transferred to LDL in a first-order process whose half-time is 2.9 min at 37 degrees C. This indicates that transfer of cholesterol molecules from the cholesterol ester/triglyceride core of HDL to the phospholipid/apoprotein monolayer at the surface of the particle is not rate limiting for exchange. The half-time for dipalmitoyl-PC exchange from HDL3 to LDL is 5 +/- h, indicating that the flux of PC is much lower than that of cholesterol. The half-times for [14C]cholesterol exchange from HDL2 and LDL at 37 degrees C are 4 and 45 min, respectively. The interfacial fluxes at 37 degrees C from the various lipoproteins are 4, 15, and 10 cholesterol molecules/(10 nm2 h), respectively, for LDL, HDL2, and HDL3. The rate of labeled cholesterol transfer from HDL3 is not affected when the concentration of LDL acceptor is increased 40-fold. The activation energies of cholesterol transfer between 4 and 37 degrees C for HDL3, HDL2, and LDL are 70 +/- 3, 75 +/- 3, and 78 +/- 3 KJ/mol, respectively. The general characteristics of the process of exchange of cholesterol between lipoproteins resemble those for exchange between small unilamellar vesicles. The results are only consistent with a mechanism of exchange in which cholesterol molecules diffuse through the aqueous phase; the experimental activation energy is associated with desorption of lipid from the donor lipoprotein into the aqueous phase.

Treatment of human high density lipoprotein (HDL) with tetranitromethane (TNM) inhibits its binding to HDL-specific binding sites of cells and isolated membranes. The mechanism of this inhibition, however, is not known; during treatment of HDL with TNM, in addition to the expected nitration of tyrosine residues, cross-linking of lipids to apoproteins and of apoproteins to one another occurs. In order to determine whether the cross-linking of lipids to apoproteins occurs through the carbon-carbon double bonds in the acyl chains, and to determine whether the cross-linking of phospholipids to apoproteins is a possible mechanism of inhibition of binding, we have prepared a reconstituted HDL3 in which the native phospholipids were replaced with dimyristoyl phosphatidylcholine (DMPC). As a control, a reconstituted HDL3 (C-r-HDL3) was also prepared using the total apoproteins and the total lipid constituents of native HDL3. The reconstituted DMPC-containing HDL3 (DMPC-r-HDL3) was similar to native HDL3 and to C-r-HDL3 in its agarose gel electrophoretic mobility, in its chemical composition, and in its binding to rat liver plasma membranes. When treated with TNM, DMPC-r-HDL3, like the native HDL3 and C-r-HDL3, lost its ability to bind to the HDL binding sites of rat liver plasma membranes, as determined by competitive binding assays with 125I-labeled human HDL3 as the tracer. Nitrated DMPC-r-HDL3 contained only traces of phospholipids covalently linked to apoproteins, whereas 21-26% of the total phospholipids were cross-linked to apoproteins of nitrated C-r-HDL3 and nitrated native HDL3.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution, proton-decoupled (13)C nuclear magnetic resonance spectra (90.55 MHz) of human high-density lipoproteins (HDL) have been employed to investigate the physical state of unesterified cholesterol molecules in such particles. The cholesterol molecules in HDL(2), and HDL(3) were replaced with [4-(13)C]cholesterol by either particle reconstitution or exchange from Celite. Two well-defined resonances from [4-(13)C]cholesterol molecules in HDL (2) and HDL(3) were observed at chemical shifts (delta) of 41.70 and 42.20 ppm, indicating that cholesterol molecules are present in two distinct environments. The signal at delta 41.70 arises from the C-4 atom of cholesterol molecules associated with the phospholipid monolayer at the surface of the particles. The resonance at delta 42.20 is due to the 4-(13)C atom of cholesterol molecules dissolved in the cholesterol ester/triglyceride core. Decomposition of the two [4-(13)C]cholesterol resonances shows that approximately 40% of the signal arises from molecules in the apolar core, with the remainder due to molecules in the surface. Spin-lattice relaxation time and line-width measurements indicate that the cholesterol molecules dissolved in the core are relatively disordered and mobile. The cholesterol molecules located among phospholipid molecules in the surface of the particle undergo relatively restricted, anisotropic motions. The chemical shifts and relaxation enhancements induced by the addition of paramagnetic ions to the aqueous phase indicate that the surface cholesterol molecules in HDL(2), and HDL(3) are exposed to the water and that the 4-(13)C atom of cholesterol is located in the region of the phospholipid acylcarboxyl groups.The NMR data indicate that the residence time for cholesterol molecules in either the surface or the core pools of HDL is greater than or equal to 10 ms. However, more than 90% of the unesterified cholesterol molecules in HDL are in a single kinetic pool for exchange with cholesterol molecules in other lipoprotein particles or cells. It follows that cholesterol molecules in the two microenvironments undergo fast exchange on the biological time scale and can equilibrate between the surface and core of HDL in the time scale 10 ms-ca. 300 s. Neither the surface nor the core microenvironments of human HDL particles are saturated with cholesterol.