"Sticky" and "promiscuous", the yin and yang of apolipoprotein A-I termini in discoidal high-density lipoproteins: a combined computational-experimental approach

Apolipoprotein (apo) A-I-containing lipoproteins in the form of high-density lipoproteins (HDL) are inversely correlated with atherosclerosis. Because HDL is a soft form of condensed matter easily deformable by thermal fluctuations, the molecular mechanisms for HDL remodeling are not well understood. A promising approach to understanding HDL structure and dynamics is molecular dynamics (MD). In the present study, two computational strategies, MD simulated annealing (MDSA) and MD temperature jump, were combined with experimental particle reconstitution to explore molecular mechanisms for phospholipid- (PL-) rich HDL particle remodeling. The N-terminal domains of full-length apoA-I were shown to be "sticky", acting as a molecular latch largely driven by salt bridges, until, at a critical threshold of particle size, the associated domains released to expose extensive hydrocarbon regions of the PL to solvent. The "sticky" N-termini also associate with other apoA-I domains, perhaps being involved in N-terminal loops suggested by other laboratories. Alternatively, the overlapping helix 10 C-terminal domains of apoA-I were observed to be extremely mobile or "promiscuous", transiently exposing limited hydrocarbon regions of PL. Based upon these models and reconstitution studies, we propose that separation of the N-terminal domains, as particles exceed a critical size, triggers fusion between particles or between particles and membranes, while the C-terminal domains of apoA-I drive the exchange of polar lipids down concentration gradients between particles. This hypothesis has significant biological relevance since lipid exchange and particle remodeling are critically important processes during metabolism of HDL particles at every step in the antiatherogenic process of reverse cholesterol transport.     

MD simulations suggest important surface differences between reconstituted and circulating spherical HDL

Since spheroidal HDL particles (sHDL) are highly dynamic, molecular dynamics (MD) simulations are useful for obtaining structural models. Here we use MD to simulate sHDL with stoichiometries of reconstituted and circulating particles. The hydrophobic effect during simulations rapidly remodels discoidal HDL containing mixed lipids to sHDL containing a cholesteryl ester/triglyceride (CE/TG) core. We compare the results of simulations of previously characterized reconstituted sHDL particles containing two or three apoA-I created in the absence of phospholipid transfer protein (PLTP) with simulations of circulating human HDL containing two or three apoA-I without apoA-II. We find that circulating sHDL compared with reconstituted sHDL with the same number of apoA-I per particle contain approximately equal volumes of core lipid but significantly less surface lipid monolayers. We conclude that in vitro reconstituted sHDL particles contain kinetically trapped excess phospholipid and are less than ideal models for circulating sHDL particles. In the circulation, phospholipid transfer via PLTP decreases the ratio of phospholipid to apolipoprotein for all sHDL particles. Further, sHDL containing two or three apoA-I adapt to changes in surface area by condensation of common conformational motifs. These results represent an important step toward resolving the complicated issue of the protein and lipid stoichiometry of circulating HDL.     

Influence of Apolipoprotein (Apo) A-I Structure on Nascent High Density Lipoprotein (HDL) Particle Size Distribution

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.     

Structure of spheroidal HDL particles revealed by combined atomistic and coarse-grained simulations

Spheroidal high-density lipoprotein (HDL) particles circulating in the blood are formed through an enzymatic process activated by apoA-I, leading to the esterification of cholesterol, which creates a hydrophobic core of cholesteryl ester molecules in the middle of the discoidal phospholipid bilayer. In this study, we investigated the conformation of apoA-I in model spheroidal HDL (ms-HDL) particles using both atomistic and coarse-grained molecular dynamics simulations, which are found to provide consistent results for all HDL properties we studied. The observed small contribution of cholesteryl oleate molecules to the solvent-accessible surface area of the entire ms-HDL particle indicates that palmitoyloleoylphosphatidylcholines and apoA-I molecules cover the hydrophobic core comprised of cholesteryl esters particularly well. The ms-HDL particles are found to form a prolate ellipsoidal shape, with sizes consistent with experimental results. Large rigid domains and low mobility of the protein are seen in all the simulations. Additionally, the average number of contacts of cholesteryl ester molecules with apoA-I residues indicates that cholesteryl esters interact with protein residues mainly through their cholesterol moiety. We propose that the interaction of annular cholesteryl oleate molecules contributes to apoA-I rigidity stabilizing and regulating the structure and function of the ms-HDL particle.     

Structures of Discoidal High Density Lipoproteins: A COMBINED COMPUTATIONAL-EXPERIMENTAL APPROACH*

Conversion of discoidal phospholipid (PL)-rich high density lipoprotein (HDL) to spheroidal cholesteryl ester-rich HDL is a central step in reverse cholesterol transport. A detailed understanding of this process and the atheroprotective role of apolipoprotein A-I (apoA-I) requires knowledge of the structure and dynamics of these various particles. This study, combining computation with experimentation, illuminates structural features of apoA-I allowing it to incorporate varying amounts of PL. Molecular dynamics simulated annealing of PL-rich HDL models containing unesterified cholesterol results in double belt structures with the same general saddle-shaped conformation of both our previous molecular dynamics simulations at 310 K and the x-ray structure of lipid-free apoA-I. Conversion from a discoidal to a saddle-shaped particle involves loss of helicity and formation of loops in opposing antiparallel parts of the double belt. During surface expansion caused by the temperature-jump step, the curved palmitoyloleoylphosphatidylcholine bilayer surfaces approach planarity. Relaxation back into saddle-shaped structures after cool down and equilibration further supports the saddle-shaped particle model. Our kinetic analyses of reconstituted particles demonstrate that PL-rich particles exist in discrete sizes corresponding to local energetic minima. Agreement of experimental and computational determinations of particle size/shape and apoA-I helicity provide additional support for the saddle-shaped particle model. Truncation experiments combined with simulations suggest that the N-terminal proline-rich domain of apoA-I influences the stability of PL-rich HDL particles. We propose that apoA-I incorporates increasing PL in the form of minimal surface bilayers through the incremental unwinding of an initially twisted saddle-shaped apoA-I double belt structure.     

Validation of previous computer models and MD simulations of discoidal HDL by a recent crystal structure of apoA-I

HDL is a population of apoA-I-containing particles inversely correlated with heart disease. Because HDL is a soft form of matter deformable by thermal fluctuations, structure determination has been difficult. Here, we compare the recently published crystal structure of lipid-free (Δ185-243)apoA-I with apoA-I structure from models and molecular dynamics (MD) simulations of discoidal HDL. These analyses validate four of our previous structural findings for apoA-I: i) a baseline double belt diameter of 105 Å ii) central α helixes with an 11/3 pitch; iii) a “presentation tunnel” gap between pairwise helix 5 repeats hypothesized to move acyl chains and unesterified cholesterol from the lipid bilayer to the active sites of LCAT; and iv) interchain salt bridges hypothesized to stabilize the LL5/5 chain registry. These analyses are also consistent with our finding that multiple salt bridge-forming residues in the N-terminus of apoA-I render that conserved domain “sticky.” Additionally, our crystal MD comparisons led to two new hypotheses: i) the interchain leucine-zippers previously reported between the pair-wise helix 5 repeats drive lipid-free apoA-I registration; ii) lipidation induces rotations of helix 5 to allow formation of interchain salt bridges, creating the LCAT presentation tunnel and “zip-locking” apoA-I into its full LL5/5 registration.     

Assessment of the validity of the double superhelix model for reconstituted high density lipoproteins: a combined computational-experimental approach

For several decades, the standard model for high density lipoprotein (HDL) particles reconstituted from apolipoprotein A-I (apoA-I) and phospholipid (apoA-I/HDL) has been a discoidal particle ∼100 Å in diameter and the thickness of a phospholipid bilayer. Recently, Wu et al. (Wu, Z., Gogonea, V., Lee, X., Wagner, M. A., Li, X. M., Huang, Y., Undurti, A., May, R. P., Haertlein, M., Moulin, M., Gutsche, I., Zaccai, G., Didonato, J. A., and Hazen, S. L. (2009) J. Biol. Chem. 284, 36605–36619) used small angle neutron scattering to develop a new model they termed double superhelix (DSH) apoA-I that is dramatically different from the standard model. Their model possesses an open helical shape that wraps around a prolate ellipsoidal type I hexagonal lyotropic liquid crystalline phase. Here, we used three independent approaches, molecular dynamics, EM tomography, and fluorescence resonance energy transfer spectroscopy (FRET) to assess the validity of the DSH model. (i) By using molecular dynamics, two different approaches, all-atom simulated annealing and coarse-grained simulation, show that initial ellipsoidal DSH particles rapidly collapse to discoidal bilayer structures. These results suggest that, compatible with current knowledge of lipid phase diagrams, apoA-I cannot stabilize hexagonal I phase particles of phospholipid. (ii) By using EM, two different approaches, negative stain and cryo-EM tomography, show that reconstituted apoA-I/HDL particles are discoidal in shape. (iii) By using FRET, reconstituted apoA-I/HDL particles show a 28–34-Å intermolecular separation between terminal domain residues 40 and 240, a distance that is incompatible with the dimensions of the DSH model. Therefore, we suggest that, although novel, the DSH model is energetically unfavorable and not likely to be correct. Rather, we conclude that all evidence supports the likelihood that reconstituted apoA-I/HDL particles, in general, are discoidal in shape.     

Influence of C-terminal α-helix hydrophobicity and aromatic amino acid content on apolipoprotein A-I functionality

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).     

Serum amyloid A protein generates pre beta 1 high-density lipoprotein from alpha-migrating high-density lipoprotein

Serum amyloid A protein (SAA), an acute-phase reactant in reactive amyloidosis, has high affinity for high-density lipoprotein (HDL). When SAA is added to HDL, SAA displaces apolipoprotein A-I (apoA-I) and phospholipid from the HDL particles. These dissociated components may form prebeta1-HDL because free apoA-I can associate with phospholipid to become a lipoprotein having prebeta mobility. To determine whether SAA generates prebeta1-HDL from alpha-migrating HDL, we investigated the effects of recombinant SAA on HDL subfraction concentration using nondenaturing two-dimensional gradient gel electrophoresis. When we added SAA (0.5 mg/mL) to plasma, the prebeta1-HDL concentration increased by 164% (from 4.7% +/- 1.3% to 12.4% +/- 3.2% of apoA-I, p < 0.005). The increase in prebeta1-HDL was proportional to the dose of SAA. When we added SAA to a column of Sepharose beads coupled to the isolated HDL (alpha-migrating HDL), prebeta1-HDL was dissociated from the column together with the SAA-associated HDL. In summary, we demonstrate that SAA generates prebeta1-HDL from alpha-migrating HDL. We speculate that SAA-mediated HDL remodeling may take place in inflammation.     

High affinity, stability, and lactonase activity of serum paraoxonase PON1 anchored on HDL with ApoA-I

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme exhibiting antiatherogenic properties. This study examined the interaction of recombinant PON1 with reconstituted HDL comprised of PC, cholesterol, and various apolipoproteins (apoA-I, -II, and -IV). The affinity, stability, and lactonase activity were strongly correlated, with apoA-I exhibiting the strongest effects, apoA-IV exhibiting weaker yet significant effects, and apoA-II having a negative effect relative to protein-free particles. We found that PON1 binds apoA-I HDL with sub-nanomolar affinities (K(d) < 10(-)(9) M) and slow dissociation rates (t(1/2) > 80 min), while binding affinity for other particles was dramatically lower. A truncated form of PON1 lacking the N-terminal helix maintains considerable binding to apoA-I HDL (K(d) = 1.2 x 10(-)(7) M), validating the structural model which indicates additional parts of the enzyme involved in HDL binding. Kinetic inactivation assays revealed the existence of an equilibrium between two forms of PON1 differing in their stability by a factor of 100. Various lipoproteins and detergent preparations shift this equilibrium toward the more stable conformation. Consistent with its highest affinity, only apoA-I HDL is capable of totally shifting the equilibrium toward the stable form. The paraoxonase and arylesterase activities were stimulated by HDL by 2-5-fold as previously reported, almost independently of the apoliporotein content. In contrast, only apoA-I is capable of stimulating the lactonase activity by 相似文献   

A key point mutation (V156E) affects the structure and functions of human apolipoprotein A-I

A naturally occurring point mutant of human apolipoprotein A-I (apoA-I), V156E, which is associated with extremely low plasma apoA-I and high density lipoprotein (HDL) levels, and coronary artery disease (Huang, W., Sasaki, J., Matsunaga, A., Nanimatsu, H., Moriyama, K., Han, H. Kugi, M., Koga, T., Yamaguchi, K., and Arakawa, K. (1998) Arterioscler. Throm. Vasc. Biol. 18, 389-396), was produced in an Escherichia coli expression system. The purified recombinant proapoA-I V156E mutant was examined in its structural and functional properties, both, in the lipid-free and lipid-bound states. In the lipid-free form the mutant protein exhibited small changes in conformation, but was more stable, and quite resistant to self-association, compared with control apoA-I. The V156E mutant was able to interact with phospholipid (PL) at high PL:protein ratios (95:1, mol/mol), but was inefficient in forming reconstituted HDL (rHDL) complexes at lower PL:protein ratios (40:1). In the lipid-bound, rHDL state, the mutant protein was somewhat more alpha-helical and formed a larger complex (110 A) than control apoA-I (97 A). Furthermore, the rHDL particles containing the V156E mutant did not rearrange to smaller particles in the presence of low density lipoproteins, and had minimal reactivity with lecithin-cholesterol acyltransferase (LCAT), compared with rHDL particles made with control apoA-I. These results suggest a key role for Val-156, or the adjacent central region of apoA-I in the modulation of apoA-I conformation, stability, and self-association in solution, and in the formation of small HDL, the conformational adaptability of apoA-I leading to structural rearrangements of HDL, and the activation of LCAT.     

SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I-/- mice.

Serum amyloid A (SAA) is an acute phase protein of unknown function that is involved in systemic amyloidosis and may also be involved in atherogenesis. The precise role of SAA in these processes has not been established. SAA circulates in plasma bound to high density lipoprotein-3 (HDL3). The pathway for the production of SAA-containing HDL is not known. To test whether apolipoprotein (apo)A-I-HDL is required in the production of SAA-HDL, we analyzed the lipopolysaccharide (LPS)-induced changes in apoA-I+/+ and apoA-I-/- mice. In apoA-I+/+ mice, after injection of LPS, remodeling of HDL occurred: total cholesterol increased and apoA-I decreased slightly and shifted to lighter density. Dense (density of HDL3) but large (size of HDL2 ) SAA-containing particles were formed. Upon fast phase liquid chromatography fractionation of plasma, >90% of SAA eluted with HDL that was enriched in cholesterol and phospholipid and shifted "leftward" to larger particles. Non-denaturing immunoprecipitation with anti-mouse apoA-I precipitated all of the apoA-I but not all of the SAA, confirming the presence of SAA-HDL devoid of apoA-I. In the apoA-I-/- mice, which normally have very low plasma lipid levels, LPS injection resulted in significantly increased total and HDL cholesterol. Greater than 90% of the SAA was lipid associated and was found on dense but large, spherical HDL particles essentially devoid of other apolipoproteins.We conclude that serum amyloid A (SAA) is able to sequester lipid, forming dense but large HDL particles with or without apoA-I or other apolipoproteins. The capacity to isolate lipoprotein particles containing SAA as the predominant or only apolipoprotein provides an important system to further explore the biological function of SAA.     

Effects of apolipoprotein A-I on ATP-binding cassette transporter A1-mediated efflux of macrophage phospholipid and cholesterol: formation of nascent high density lipoprotein particles

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.     

Characterization of nascent HDL particles and microparticles formed by ABCA1-mediated efflux of cellular lipids to apoA-I

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.     

The central helices of ApoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type ApoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo

We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by approximately 80-92% by apoA-I[delta(185-243)], only by 15% by apoA-I[delta(1-41)] or apoA-I[delta(1-59)], and was restored to 75-80% of the wild-type apoA-I control value by double deletion mutants apoA-I[delta(1-41)delta(185-243)] and apoA-I[delta(1-59)delta(185-243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[delta(232-243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30-40% and 50-65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[delta(1-41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[delta(1-41)delta(185-243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220-231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.     

Identification and structural ramifications of a hinge domain in apolipoprotein A-I discoidal high-density lipoproteins of different size

Apolipoprotein (apo) A-I is the major protein constituent of human high-density lipoprotein (HDL) and is likely responsible for many of its anti-atherogenic properties. Since distinct HDL size subspecies may play different roles in interactions critical for these properties, a key question concerns how apoA-I can adjust its conformation in response to changes in HDL particle size. A prominent hypothesis states that apoA-I contains a flexible "hinge domain" that can associate/dissociate from the lipoprotein as its diameter fluctuates. Although flexible domains clearly exist within HDL-bound apoA-I, this hypothesis has not been directly tested by assessing the ability of such domains to modulate their contacts with the lipid surface. In this work, discoidal HDL particles of different size were reconstituted with a series of human apoA-I mutants containing a single reporter tryptophan residue within each of its 22 amino acid amphipathic helical repeats. The particles also contained nitroxide spin labels, potent quenchers of tryptophan fluorescence, attached to the phospholipid acyl chains. We then measured the relative exposure of each tryptophan probe with increasing quencher concentrations. We found that, although there were modest structural changes across much of apoA-I, only helices 5, 6, and 7 exhibited significant differences in terms of exposure to lipid between large (96 A) and small (78 A) HDL particles. From these results, we present a model for a putative hinge domain in the context of recent "belt" and "hairpin" models of apoA-I structure in discoidal HDL particles.     

Characterization and properties of pre beta-HDL particles formed by ABCA1-mediated cellular lipid efflux to apoA-I

The contribution of ABCA1-mediated efflux of cellular phospholipid (PL) and cholesterol to human apolipoprotein A-I (apoA-I) to the formation of pre beta 1-HDL (or lipid-poor apoA-I) is not well defined. To explore this issue, we characterized the nascent HDL particles formed when lipid-free apoA-I was incubated with fibroblasts in which expression of the ABCA1 was upregulated. After a 2 h incubation, the extracellular medium contained small apoA-I/PL particles (pre beta 1-HDL; diameter = 7.5 +/- 0.4 nm). The pre beta 1-HDL (or lipid-poor apoA-I) particles contained a single apoA-I molecule and three to four PL molecules and one to two cholesterol molecules. An apoA-I variant lacking the C-terminal alpha-helix did not form such particles when incubated with the cell, indicating that this helix is critical for the formation of lipid-poor apoA-I particles. These pre beta 1-HDL particles were as effective as lipid-free apoA-I molecules in mediating both the efflux of cellular lipids via ABCA1 and the formation of larger, discoidal HDL particles. In conclusion, pre beta 1-HDL is both a product and a substrate in the ABCA1-mediated reaction to efflux cellular PL and cholesterol to apoA-I. A monomeric apoA-I molecule associated with three to four PL molecules (i.e., lipid-poor apoA-I) has similar properties to the lipid-free apoA-I molecule.     

Point mutations in apolipoprotein A-I mimic the phenotype observed in patients with classical lecithin:cholesterol acyltransferase deficiency

We have analyzed the effect of charged to neutral amino acid substitutions around the kinks flanking helices 4 and 6 of apoA-I and of the deletion of helix 6 on the in vivo activity of LCAT and the biogenesis of HDL. The LCAT activation capacity of apoA-I in vitro was nearly abolished by the helix 6 point (helix 6P-apoA-I[R160V/H162A]) and deletion {helix 6Delta-apoA-I[Delta(144-165)]} mutants, but was reduced to 50% in the helix 4 point mutant (helix 4P-apoA-I[D102A/D103A]). Following adenovirus-mediated gene transfer in apoA-I deficient mice, the level of plasma HDL cholesterol was greatly reduced in helix 6P and helix 6Delta mutants. Electron microscopy and two-dimensional gel electrophoresis showed that the helix 6P mutant formed predominantly high levels of apoA-I containing discoidal particles and had an increased prebeta1-HDL/alpha-HDL ratio. The helix 6Delta mutant formed few spherical particles and had an increased prebeta1-HDL/alpha-HDL ratio. Mice infected with adenovirus expressing the helix 4P mutant or wild-type apoA-I had normal HDL cholesterol and formed spherical alpha-HDL particles. Coinfection of mice with adenoviruses expressing human LCAT and the helix 6P mutant dramatically increased plasma HDL and apoA-I levels and converted the discoidal into spherical HDL, indicating that the LCAT activity was rate-limiting for the biogenesis of HDL. The LCAT treatment caused only a small increase in HDL cholesterol and apoA-I levels and in alpha-HDL particle numbers in the helix 6Delta mutant. The findings indicate a critical contribution of residue 160 of apoA-I to the in vivo activity of LCAT and the subsequent maturation of HDL and explain the low HDL levels in heterozygous subjects carrying this mutation.     

Binding of high density lipoprotein (HDL) and discoidal reconstituted HDL to the HDL receptor scavenger receptor class B type I. Effect of lipid association and APOA-I mutations on receptor binding

The binding of apoA-I-containing ligands to the HDL receptor scavenger receptor class B type I (SR-BI) was characterized using two different assays. The first employed conventional binding or competition assays with (125)I-labeled ligands. The second is a new nonradioactive ligand binding assay, in which the receptor-associated ligand is detected by quantitative immunoblotting ("immunoreceptor assay"). Using both methods, we observed that the K(d) value for spherical HDL (density = 1.1-1.13 g/ml) was approximately 16 microgram of protein/ml, while the values for discoidal reconstituted HDL (rHDL) containing proapoA-I or plasma apoA-I were substantially lower (approximately 0.4-5 microgram of protein/ml). We also observed reduced affinity and/or competition for spherical (125)I-HDL cell association by higher relative to lower density HDL and very poor competition by lipid-free apoA-I and pre-beta-1 HDL. Deletion of either 58 carboxyl-terminal or 59 amino-terminal residues from apoA-I, relative to full-length control apoA-I, resulted in little or no change in the affinity of corresponding rHDL particles. However, rHDL particles containing a double mutant lacking both terminal domains competed poorly with spherical (125)I-HDL for binding to SR-BI. These findings suggest an important role for apoA-I and its conformation/organization within particles in mediating HDL binding to SR-BI and indicate that the NH(2) and COOH termini of apoA-I directly or indirectly contribute independently to binding to SR-BI.