Apolipoprotein A-II inhibits high density lipoprotein remodeling and lipid-poor apolipoprotein A-I formation

The high density lipoproteins (HDL) in human plasma are classified on the basis of apolipoprotein composition into those containing apolipoprotein (apo) A-I but not apoA-II, (A-I)HDL, and those containing both apoA-I and apoA-II, (A-I/A-II)HDL. Cholesteryl ester transfer protein (CETP) transfers core lipids between HDL and other lipoproteins. It also remodels (A-I)HDL into large and small particles in a process that generates lipid-poor, pre-beta-migrating apoA-I. Lipid-poor apoA-I is the initial acceptor of cellular cholesterol and phospholipids in reverse cholesterol transport. The aim of this study is to determine whether lipid-poor apoA-I is also formed when (A-I/A-II)rHDL are remodeled by CETP. Spherical reconstituted HDL that were identical in size had comparable lipid/apolipoprotein ratios and either contained apoA-I only, (A-I)rHDL, or (A-I/A-II)rHDL were incubated for 0-24 h with CETP and Intralipid(R). At 6 h, the apoA-I content of the (A-I)rHDL had decreased by 25% and there was a concomitant formation of lipid-poor apoA-I. By 24 h, all of the (A-I)rHDL were remodeled into large and small particles. CETP remodeled approximately 32% (A-I/A-II)rHDL into small but not large particles. Lipid-poor apoA-I did not dissociate from the (A-I/A-II)rHDL. The reasons for these differences were investigated. The binding of monoclonal antibodies to three epitopes in the C-terminal domain of apoA-I was decreased in (A-I/A-II)rHDL compared with (A-I)rHDL. When the (A-I/A-II)rHDL were incubated with Gdn-HCl at pH 8.0, the apoA-I unfolded by 15% compared with 100% for the apoA-I in (A-I)rHDL. When these incubations were repeated at pH 4.0 and 2.0, the apoA-I in the (A-I)rHDL and the (A-I/A-II)rHDL unfolded completely. These results are consistent with salt bridges between apoA-II and the C-terminal domain of apoA-I, enhancing the stability of apoA-I in (A-I/A-II)rHDL and possibly contributing to the reduced remodeling and absence of lipid poor apoA-I in the (A-I/A-II)rHDL incubations.     

Oxidation of methionine residues affects the structure and stability of apolipoprotein A-I in reconstituted high density lipoprotein particles.

To determine the effect of oxidative damage to lipid-bound apolipoprotein A-I (apo A-I) on its structure and stability that might be related to previously observed functional disorders of oxidized apo A-I in high density lipoproteins (HDL), we prepared homogeneous reconstituted HDL (rHDL) particles containing unoxidized apo A-I and its commonly occurring oxidized form (Met-112, 148 bis-sulfoxide). The size of the obtained discoidal rHDL particles ranged from 9.0 to 10.0 nm and did not depend upon the content of the oxidized protein. Using circular dichroism methods, no change in the secondary structure of lipid-bound oxidized apo A-I was found. Isothermal and thermal denaturation experiments showed a significant destabilization of the oxidized protein to denaturation by guanidine hydrochloride or heat. This effect was observed with and without co-reconstituted apolipoprotein A-II. Limited tryptic digestion indicated that the central region of oxidatively damaged apo A-I becomes exposed to proteolysis in the rHDL particles. Implications of these data for apolipoprotein function are discussed.     

Evidence in vitro that hepatic lipase reduces the concentration of apolipoprotein A-I in rabbit high-density lipoproteins

Incubation of rabbit plasma in vitro with hepatic lipase resulted in the hydrolysis of triacylglycerol in high-density lipoproteins (HDL) and a reduction in HDL particle size. These changes were accompanied by a decrease in the concentration of apolipoprotein A-I (apo A-I) in the HDL. The loss of apo A-I was demonstrated independently by ultracentrifugation, size exclusion chromatography and gradient gel-immunoblot analysis. It was unrelated to hydrolysis of HDL phospholipids but did correlate with the reduction in HDL particle size. These studies suggest that the concentration of apo A-I in HDL may be influenced by factors which regulate the metabolism of HDL core lipid constituents.     

Apolipoprotein A-I localization and dipalmitoylphosphatidylcholine dynamics in reconstituted high density lipoproteins

The structure and molecular dynamics of recombinant high density lipoproteins (rHDL) were studied by non-radiative energy transfer (NRET), fluorescence anisotropy and intensity measurements. The rHDL particles contained human plasma apolipoprotein (apo) A-I and dipalmitoylphosphatidylcholine (DPPC). Fluorescent cis- and trans-parinaric acids were used both as probes of molecular motion in the particle lipid phase and as acceptors in the Forster's energy transfer from apo A-I tryptophan residues to determine particle dimensions, apolipoprotein localization and lipid dynamics. The probes are sensitive to thermal wobbling (macromobility) and conformational deformations (micromobility) of phospholipid acyl chains. The experimental data fitted to various models of the particle structure are compatible with the following: (a) at T < Tt the particles appeared as lens-like discs with a radius of the lipid phase of 5 nm and a mean thickness of 4 nm, the value being more by 20% in the particle centre, the alpha-helices of about 1 nm thickness were located around the edge of the lipid core. Compared to liposomes, both macro- and micromobility of DPPC molecules in rHDL were more rapid due to a significant disorder of the boundary lipid molecules close to the apo A-I molecule. This disorder led to the increase of the specific surface area per one lipid molecule, S(o). The lipid phase can be divided into three regions: (i) zone I of the most tightly packed lipid (0-1.7 nm from the disc axis) with a S(o) value small as 0.5 nm2; (ii) intermediate zone II (from 1.7 to 4.0 nm); and (iii) boundary lipid zone III (4-5 nm) of significantly disordered lipid with a S(o) value large as 0.65 nm2. (b) at T> Tt the S(o) heterogeneity disappeared, the radius of the lipid phase did not increase significantly, not exceeding 5.2-5.4 nm, but protein-induced immobilization of lipid molecules which affected about half or more of the total lipid, became remarkable. The overall effect was the suppression of the transition amplitude in rHDL compared to liposomes. The structural inhomogeneity might underlie the function of the native plasma HDL as the key component of the transport and metabolism of plasma lipids.     

Immunochemical characterization of two antigenic sites on human apolipoprotein A-I; localization and lipid modulation of these epitopes

Two monoclonal antibodies, A17 and A30, were raised against human apolipoprotein A-I (apo A-I). They were studied by competitive inhibition of 125I-labeled HDL3 with HDL subfractions, delipidated apo A-I, and complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I and apo A-II. Immunoblotting located the A17 antibody on CNBr fragment 4 of apo A-I and the A30 antibody on CNBr fragment 1. The A17 antigenic determinant was expressed identically in all HDL subclasses, on delipidated apo A-I as well as all on the DMPC-apo A-I and DMPC-apo A-I/apo A-II complexes. In contrast, the apparent affinity constant of the A30 antibody for delipidated apo A-I was about 30-times less than for HDL3 or for apo A-I/apo A-II-phospholipid complexes. These data suggest that the association of apo A-I with phospholipids improves the reactivity of the A30 monoclonal antibody towards apo A-I, and that this antigenic determinant has a different conformation in delipidated apo A-I compared to apo A-I complexed with phospholipids. Turbidimetric and fluorescence experiments monitoring the phospholipid-apo A-I association in the presence and in the absence of the A17 and A30 antibodies were consistent with the competition experiments carried out by solid phase radioimmunoassay (RIA). After reaction of apo A-I with the A30 antibody, we observed an enhancement of the degradation kinetics of large multilamellar vesicles (LMV), while the A17 antibody did not have a significant effect. Calcein leakage experiments carried out below the transition temperature of DPPC showed an enhancement of the degradation kinetics with both monoclonal antibodies, while the phase-transition release was independent of the reaction of apo A-I with the monoclonal antibodies. These data therefore suggest the existence of at least two different types of epitope on apo A-I, which might account for the differences in immunological reactivity of apo A-I that is either delipidated or present on HDL.     

Evidence that phospholipids play a key role in pre-beta apoA-I formation and high-density lipoprotein remodeling

The initial plasma acceptor of unesterified cholesterol and phospholipids from peripheral cells has been identified as pre-beta migrating, lipid-free, or lipid-poor apolipoprotein (apo) A-I (pre-beta apoA-I). Pre-beta apoA-I is formed when plasma factors, such as cholesteryl ester transfer protein (CETP), remodel high-density lipoproteins (HDL). The aim of this study is to determine how phospholipids influence pre-beta apoA-I formation during the CETP-mediated remodeling of HDL. Reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonyl phosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoyl phosphatidylcholine (PDPC) as the only phospholipid were prepared. The rHDL were comparable in size and core lipid/protein molar ratio and contained only cholesteryl esters in their core and apoA-I as the sole apolipoprotein. The (POPC)rHDL, (PLPC)rHDL, (PAPC)rHDL, and (PDPC)rHDL were respectively incubated for 0-24 h with CETP and microemulsions containing triolein and either POPC, PLPC, PAPC, or PDPC. The rate at which the rHDL were depleted of core lipids and remodeled to small particles varied widely with (POPC)rHDL < (PLPC)rHDL < (PDPC)rHDL approximately (PAPC)rHDL. Pre-beta apoA-I was not formed in the (POPC)rHDL incubations. Pre-beta apoA-I was apparent by 24 h in the (PLPC)rHDL incubations and by 12 h in the (PAPC)rHDL and (PDPC)rHDL incubations. The enhanced formation of pre-beta apoA-I in the (PAPC)rHDL and (PDPC)rHDL incubations reflected the increased core lipid depletion of the particles combined with the destabilization and progressive exclusion of apoA-I from the particle surface. In conclusion, these results show that phospholipids play a key role in the CETP-mediated remodeling of rHDL and pre-beta apoA-I formation.     

Molecular belt models for the apolipoprotein A-I Paris and Milano mutations

Models for the binding of the 200-residue carboxy-terminal domain of two mutants of apolipoprotein A-I (apo A-I), apo A-I(R173C)(Milano) and apo A-I(R151C)(Paris), to lipid in discoidal high-density lipoprotein (HDL) particles are presented. In both models, two monomers of the mutant apo A-I molecule bind to lipid in an antiparallel manner, with the long axes of their helical repeats running perpendicular to the normal of the lipid bilayer to form a single disulfide-linked homodimer. The overall structures of the models of these two mutants are very similar, differing only in helix-helix registration. Thus these models are consistent with experimental observations that reconstituted HDL particles containing apo A-I(Milano) and apo A-I(Paris) are very similar in diameter to reconstituted HDL particles containing wild-type apo A-I, and they support the belief that apo A-I binds to lipid in discoidal HDL particles via the belt conformation.     

Apolipoproteins regulate the kinetics of endothelial lipase-mediated hydrolysis of phospholipids in reconstituted high-density lipoproteins

Endothelial lipase (EL) is a newly identified member of the triglyceride lipase gene family that hydrolyzes high-density lipoprotein (HDL) phospholipids. This study investigates the ability of the major apolipoproteins of rHDL to regulate the kinetics of EL-mediated phospholipid hydrolysis in well-characterized, homogeneous preparations of spherical rHDL. The rHDL contained either apoA-I as the only apolipoprotein, (A-I)rHDL, apoA-II as the only apolipoprotein, (A-II)rHDL, or apoA-I as well as apoA-II, (A-I/A-II)rHDL. The rHDL were comparable in terms of size and lipid composition and contained cholesteryl esters (CE) as their sole core lipid. Phospholipid hydrolysis was quantitated as the mass of nonesterified fatty acids (NEFA) released from the rHDL during incubation with EL. The V(max) of phospholipid hydrolysis for (A-I/A-II)rHDL [391.9 +/- 12.9 nmol of NEFA formed (mL of EL)(-1) h(-1)] was greater than (A-I)rHDL [152.8 +/- 4.7 nmol of NEFA formed (mL of EL)(-1) h(-1)]. The energy of activation (E(a)) for the hydrolysis reactions was calculated to be 52.1 and 34.8 kJ mol(-1) for (A-I)rHDL and (A-I/A-II)rHDL, respectively. Minimal phospholipid hydrolysis was observed for the (A-II)rHDL. Kinetic analysis showed that EL has a higher affinity for the phospholipids in (A-I)rHDL [K(m)(app) = 0.10 +/- 0.01 mM] than in (A-I/A-II)rHDL [K(m)(app) = 0.27 +/- 0.03 mM]. Furthermore, (A-I)rHDL is a competitive inhibitor of the EL-mediated phospholipid hydrolysis of (A-I/A-II)rHDL. These results establish that apolipoproteins are major determinants of the kinetics of EL-mediated phospholipid hydrolysis in rHDL.     

Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase

Previous studies have provided detailed information on the formation of spherical high density lipoproteins (HDL) containing apolipoprotein (apo) A-I but no apoA-II (A-I HDL) by an lecithin:cholesterol acyltransferase (LCAT)-mediated process. In this study we have investigated the formation of spherical HDL containing both apoA-I and apoA-II (A-I/A-II HDL). Incubations were carried out containing discoidal A-I reconstituted HDL (rHDL), discoidal A-II rHDL, and low density lipoproteins in the absence or presence of LCAT. After the incubation, the rHDL were reisolated and subjected to immunoaffinity chromatography to determine whether A-I/A-II rHDL were formed. In the absence of LCAT, the majority of the rHDL remained as either A-I rHDL or A-II rHDL, with only a small amount of A-I/A-II rHDL present. By contrast, when LCAT was present, a substantial proportion of the reisolated rHDL were A-I/A-II rHDL. The identity of the particles was confirmed using apoA-I rocket electrophoresis. The formation of the A-I/A-II rHDL was influenced by the relative concentrations of the precursor discoidal A-I and A-II rHDL. The A-I/A-II rHDL included several populations of HDL-sized particles; the predominant population having a Stokes' diameter of 9.9 nm. The particles were spherical in shape and had an electrophoretic mobility slightly slower than that of the alpha-migrating HDL in human plasma. The apoA-I:apoA-II molar ratio of the A-I/A-II rHDL was 0.7:1. Their major lipid constituents were phospholipids, unesterified cholesterol, and cholesteryl esters. The results presented are consistent with LCAT promoting fusion of the A-I rHDL and A-II rHDL to form spherical A-I/A-II rHDL. We suggest that this process may be an important source of A-I/A-II HDL in human plasma.     

Effect in vitro of apolipoprotein A-V on the structure and functions of recombinant high density lipoprotein

The neighboring position of apolipoprotein A-I (apoA-I) and apolipoprotein A-V (apoA-V) gene and the modulation of apoA-V on the concentrations, size and maturation of high density lipoprotein (HDL) may indicate a special relationship between apoA-V and HDL. To assess the effects of apoA-V on HDL structure and related functions in vitro, a series of recombinant HDL (rHDL) were synthesized in vitro with various mass ratios of recombinant apoA-I: apoA-V. An increase in apoA-V in rHDL resulted in enhanced lipid-binding ability, increased phospholipid content and larger particle size. Furthermore, the lipid-free and lipid-bound apoA-V in rHDL showed antioxidant capacity against low density lipoprotein (LDL) in vitro. In THP-1 derived macrophages, apoA-V of rHDL was shown to have no influence on the uptake of oxidized LDL (oxLDL) and intracellular lipid accumulation. Thus, the addition of apoA-V to rHDL resulted in changes in several rHDL properties, including increased lipid-binding ability, phospholipid content, particle size and antioxidant capacity. These alterations may explain the modulation of apoA-V on HDL in vivo and the beneficial functions of apoA-V on atherosclerosis.     

The interaction of lipolysis products of very low density lipoprotein with plasma high density lipoprotein (HDL): perfusate HDL with plasma HDL subfractions

The nature of the interaction of high density lipoproteins (HDL), formed during lipolysis of human very low density lipoprotein (VLDL) by perfused rat heart, with subfractions of human plasma HDL was investigated. Perfusate HDL, containing apoliproproteins (apo) E, C-II, and C-III but no apo A-I or A-II, was incubated with a subfraction of HDL (HDL-A) containing apo A-I and A-II, but devoid of apo C-II, C-III, and E. The products of the incubation were resolved by heparin-Sepharose or hydroxylapatite chromatography under conditions which allowed the resolution of the initial HDL-A and perfusate HDL. The fractions were analyzed for apolipoprotein content and lipid composition and assessed for particle size by electron microscopy. Following the incubation, the apo-E-containing lipoproteins were distinct from perfusate HDL since they contained apo A-I as a major component and apo C-II and C-III in reduced proportions. However, the HDL-A fraction contained apo C-II and C-III as major constituents. Associated with these changes in apolipoprotein composition, the apo-E-rich lipoproteins acquired cholesteryl ester from the HDL-A fraction and lost phospholipid to the HDL-A fraction. The HDL-A fraction maintained a low unesterified cholesterol/phospholipid molar ratio (0.23), while the apo-E-containing lipoproteins possessed a high ratio (0.75) characteristic of the perfusate HDL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Limited proteolysis of a disulfide-linked apoA-I dimer in reconstituted HDL

The apolipoprotein A-I(Milano) (apoA-I(M)) is a molecular variant of apoA-I characterized by the Arg(173)-->Cys substitution, leading to the formation of homodimers A-I(M)/A-I(M). Upon interaction with palmitoyloleoylphosphatidylcholine, A-I(M)/A-I(M) forms only two species of reconstituted HDL (rHDL) particles, with diameters of 7.8 and 12.5 nm. We used limited proteolysis to analyze the conformation of A-I(M)/A-I(M) in the two rHDL particles, in comparison with that of apoA-I in rHDL of similar size. ApoA-I in the small, 7.8-nm rHDL is degraded to a greater extent (50% after 6 h) than in the large rHDL (<10% degraded after 6 h). The protease susceptibility of A-I(M)/A-I(M) in small and large rHDL is instead remarkably the same, with A-I(M)/A-I(M) being much more sensitive to proteolytic digestion (50% degraded after 10 min) than apoA-I. The identification of the proteolytic fragments by immunoblotting, N-terminal sequencing, and molecular mass determination, shows that the N-terminus of both proteins is resistant to proteolysis, with six cleavage sites located in the central and carboxy-terminal portions of the molecules. Cleavage in the middle of apoA-I occurs at distinct sites in 7.8-nm (Lys(118)) and 12.7-nm (Arg(123)) rHDL, indicating a different conformation in small and large rHDL particles. The A-I(M)/A-I(M) instead adopts a unique and identical conformation in small and large rHDL, with the carboxy-terminal portion of the molecule being remarkably more accessible to the proteases than in apoA-I. This suggests the presence of a novel carboxy-terminal domain in A-I(M)/A-I(M), not organized in a compact structure and not shared by wild-type apoA-I, which may account for the unique functional properties of A-I(M)/A-I(M).     

Speciation of human plasma high-density lipoprotein (HDL): HDL stability and apolipoprotein A-I partitioning

The distribution of apolipoprotein (apo) A-I between human high-density lipoproteins (HDL) and water is an important component of reverse cholesterol transport and the atheroprotective effects of HDL. Chaotropic perturbation (CP) with guanidinium chloride (Gdm-Cl) reveals HDL instability by inducing the unfolding and transfer of apo A-I but not apo A-II into the aqueous phase while forming larger apo A-I deficient HDL-like particles and small amounts of cholesteryl ester-rich microemulsions (CERMs). Our kinetic and hydrodynamic studies of the CP of HDL species separated according to size and density show that (1) CP mediated an increase in HDL size, which involves quasi-fusion of surface and core lipids, and release of lipid-free apo A-I (these processes correlate linearly), (2) >94% of the HDL lipids remain with an apo A-I deficient particle, (3) apo A-II remains associated with a very stable HDL-like particle even at high levels of Gdm-Cl, and (4) apo A-I unfolding and transfer from HDL to water vary among HDL subfractions with the larger and more buoyant species exhibiting greater stability. Our data indicate that apo A-I's on small HDL (HDL-S) are highly dynamic and, relative to apo A-I on the larger more mature HDL, partition more readily into the aqueous phase, where they initiate the formation of new HDL species. Our data suggest that the greater instability of HDL-S generates free apo A-I and an apo A-I deficient HDL-S that readily fuses with the more stable HDL-L. Thus, the presence of HDL-L drives the CP remodeling of HDL to an equilibrium with even larger HDL-L and more lipid-free apo A-I than with either HDL-L or HDL-S alone. Moreover, according to dilution studies of HDL in 3 M Gdm-Cl, CP of HDL fits a model of apo A-I partitioning between HDL phospholipids and water that is controlled by the principal of opposing forces. These findings suggest that the size and relative amount of HDL lipid determine the HDL stability and the fraction of apo A-I that partitions into the aqueous phase where it is destined for interaction with ABCA1 transporters, thereby initiating reverse cholesterol transport or, alternatively, renal clearance.     

Human apolipoprotein A-I forms small high density lipoprotein particles in rats in vivo.

The effects of injection of purified human or rat apolipoprotein (apo) A-I (1.7 mg/100 g body weight) on the size and composition of rat high density lipoprotein (HDL) particles have been investigated. The injection of human apo A-I results in the formation (over a period of 3 to 6 h) of a population of smaller HDL particles resembling human HDL3. This population of smaller particles contains human apo A-I and rat apo A-IV but lacks rat apo A-I and rat apo E. Small HDL3-like particles are not detected in rat plasma following the injection of rat apo A-I. Associated with the injection of either human or rat apo A-I is a gradual increase of plasma cholesterol levels of 20 to 50% (over 24 h) and the appearance of larger HDL particles. The results suggest that the smaller HDL particles in human plasma compared to rat plasma are not simply due to the action of lipid modifying enzymes or lipid transfer proteins but a specific property of human apo A-I.     

Influence of lipid environment on insulin binding in cultured hepatoma cells

Three mouse monoclonal antibodies (Mabs) to human apo A-I were produced using apolipoprotein A-I or HDL3 as immunogens. These monoclonal antibodies, 2G11, 4A12 and 4B11, were characterized for their reactivity with isolated apolipoprotein A-I and HDL in solution. The immunoblotting patterns of the HDL3 two-dimensional electrophoresis show that these three monoclonal antibodies reacted with all the polymorphic forms of apolipoprotein A-I. Cotitration experiments indicated that they correspond to three distinct epitopes. In order to locate these three antigenic determinants on the isolated apolipoprotein A-I, the reactivity of the three monoclonal antibodies has been studied on CNBr-cleaved apolipoprotein A-I. The monoclonal antibodies 2G11 and 4A12 addressed to the amino (CNBr 1) and carboxy (CNBr 4) terminal segments, respectively. In comparison with the monoclonal antibodies characterized by Weech et al. ((1985) Biochim. Biophys. Acta 835, 390-401), monoclonal antibody 4A12 is the only one described in the literature which is specific of the carboxy terminal segment of apolipoprotein A-I. Monoclonal antibody 4B11 does not react with any CNBr fragment, its binding is temperature dependent, it could be directed to a conformational epitope. Relative differences were demonstrated in the expression of the three epitopes in HDL subfractions isolated by density gradient ultracentrifugation. According to Curtiss and Edgington ((1985) J. Biol. Chem. 260, 2982-2993) our results indicate the existence of an immunochemical heterogeneity in the organization of apolipoprotein A-I at the surface of HDL particles as well as in the soluble form of apolipoprotein A-I.     

Role of apolipoprotein A-I in the structure of human serum high density lipoproteins. Reconstitution studies.

For a better definition of the role of human serum apolipoprotein A-I (apo A-I) in high density lipoprotein structure, a systematic investigation was carried out on factors influencing the in vitro association of this apoprotein with lipids obtained from the parent high density lipoprotein (HDL); these lipids include phospholipids, free cholesterol, cholesteryl esters, and triglycerides. Following equilibration, mixtures of apo A-I and lipids in varying stoichiometric amounts were fractionated by sequential flotation, CsCl density gradient ultracentrifugation, or gel-permeation chromatography, and the isolated complexes were characterized by physicochemical means. As defined by operational criteria (flotation at density 1,063 to 1.21 g/ml), only two types of HDL complexes were reassembled; one, reconstituted HDLS, small with a radius of 31 A, and the other, reconstituted HDLL, large with a radius of 39 A. The two types incorporated all of the lipid constituents of native HDL and contained 2 and 3 mol of apo A-I, respectively. A maximal yield of reconstituted HDL (R-HDL) was observed at an initial protein concentration of 0.1 muM, where apo A-I is predominantly monomeric. At increasing protein concentrations, the amount of apo A-I recovered in R-HDL was found to be proportional to the initial concentration of monomer and dimer in solution. The composition and yield of the complexes were independent of ionic strength and pH within the ranges studied. Both simple incubation and cosonication of apo A-I with HDL phospholipids produced complexes of identical composition, although the yeild of complexes was higher with co-sonication. When the comparison of the same methods was extended to mixtures of apo A-I and whole HDL lipids, the results confirmed previous observations that co-sonication is essential for the incorporation of the neutral lipid into the R-HDL complexes. The results indicate that (a) in vitro complexation of apo A-I with lipids is under kinetic control; (b) apo A-I can generate a lipid-protein complex with properties similar to those of the parent lipoprotein; (c) the process requires well defined experimental conditions and, most importantly, the presence in solution of monomers and dimers of apo A-I; (d) the number of apo A-I molecules incorporated into R-HDL determines the size and structure of the reassembled particle. All of these observations strongly support the essential role of apo A-I in the structure of human HDL.     

Apolipoprotein A-I structure and lipid properties in homogeneous, reconstituted spherical and discoidal high density lipoproteins

We prepared a spherical reconstituted high density lipoprotein (rHDL) particle in pure form and compared it with its homogeneous discoidal rHDL precursors, in terms of the structure and stability of the apolipoprotein A-I (apoA-I) component, the dynamics of the surface lipids, and the relative reactivity with lecithin-cholesterol acyltransferase. The apoA-I-structure was examined in the rHDL particles by circular dichroism and fluorescence spectroscopic methods, and the binding of monoclonal antibodies specific for apoA-I epitopes. The stability of apoA-I on the rHDL particles was assessed by the effects of guanidine hydrochloride on the wavelength of maximum intrinsic fluorescence of the apolipoprotein. Lipid dynamics in the acyl chain region and the polarity of the lipid-water interface were investigated by means of fluorescence probes. The conformation of apoA-I in the spherical 93-A rHDL particles was found to be very similar to that in the 96-A rHDL discs but distinct from the apoA-I structure in the 78-A rHDL discs. The stability of apoA-I to denaturation by guanidine hydrochloride was highest in the 93-A rHDL spheres. The experiments on the lipids indicate somewhat more ordered and motionally restricted acyl chains in the spheres, relative to the discs, but a similar surface polarity. These results suggest that the folding and organization of apoA-I on the three particles include protein domains consisting of interacting alpha-helical segments in the carboxyl-terminal region and a globular domain in the amino-terminal region of each apoA-I molecule. The reactivity with lecithin-cholesterol acyltransferase was highest for the 96-A rHDL disc, and 16- and 34-fold lower for the 78-A rHDL disc and the 93-A rHDL sphere, respectively, possibly as a result of differences in apoA-I structure and product inhibition in these particles.     

Evidence that hepatic lipase and endothelial lipase have different substrate specificities for high-density lipoprotein phospholipids

Hepatic lipase (HL) and endothelial lipase (EL) are both members of the triglyceride lipase gene family. HL hydrolyzes phospholipids and triglycerides in triglyceride-rich lipoproteins and high-density lipoproteins (HDL). EL hydrolyzes HDL phospholipids and has low triglyceride lipase activity. The aim of this study was to determine if HL and EL hydrolyze different HDL phospholipids and whether HDL phospholipid composition regulates the interaction of EL and HL with the particle surface. Spherical, reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonylphosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoylphosphatidylcholine (PDPC) as the only phospholipid, apolipoprotein A-I as the only apolipoprotein, and either cholesteryl esters (CE) only or mixtures of CE and triolein (TO) in their core were prepared. The rHDL were similar in size and had comparable core lipid/apoA-I molar ratios. The CE-containing rHDL were used to determine the kinetics of HL- and EL-mediated phospholipid hydrolysis. For HL the V(max) of phospholipid hydrolysis for (POPC)rHDL > (PLPC)rHDL approximately (PDPC)rHDL > (PAPC)rHDL, while the K(m)(app) for (POPC)rHDL > (PDPC)rHDL > (PLPC)rHDL > (PAPC)rHDL. For EL the V(max) for (PDPC)rHDL > (PAPC)rHDL > (PLPC)rHDL approximately (POPC)rHDL, while the K(m)(app) for (PAPC)rHDL approximately (PLPC)rHDL > (POPC)rHDL > (PDPC)rHDL. The kinetics of EL- and HL-mediated TO hydrolysis was determined using rHDL that contained TO in their core. For HL the V(max) of TO hydrolysis for (PLPC)rHDL > (POPC)rHDL > (PAPC)rHDL > (PDPC)rHDL, while the K(m)(app) for (PLPC)rHDL > (POPC)rHDL approximately (PAPC)rHDL > (PDPC)rHDL. For EL the V(max) and K(m)(app) for (PAPC)rHDL > (PDPC)rHDL > (PLPC)rHDL > (POPC)rHDL. These results establish that EL and HL have different substrate specificities for rHDL phospholipids and that their interactions with the rHDL surface are regulated by phospholipids.     

Oxidized phospholipid content destabilizes the structure of reconstituted high density lipoprotein particles and changes their function

High density lipoprotein (HDL) particles are made up of lipid and protein constituents and apolipoprotein A-I (apoA-I) is a principal protein component that facilitates various biological activities of HDL particles. Increase in Ox-PL content of HDL particles makes them 'dysfunctional' and such modified HDL particles not only lose their athero-protective properties but also acquire pro-atherogenic and pro-inflammatory functions. The details of Ox-PL-induced alteration in the molecular properties of HDL particles are not clear. Paraoxonase 1 (PON1) is an HDL-associated enzyme that possesses anti-inflammatory and anti-atherogenic properties; and many of the athero-protective functions of HDL are attributed to the associated PON1. In this study we have characterized the physicochemical properties of reconstituted HDL (rHDL) particles containing varying amounts of Ox-PL and have compared their PON1 stimulation capacity. Our results show that increased Ox-PL content (a) modifies the physicochemical properties of the lipid domain of the rHDL particles, (b) decreases the stability and alters the conformation as well as orientation of apoA-I molecules on the rHDL particles, and (c) decreases the PON1 stimulation capacity of the rHDL particles. Our data indicate that the presence of Ox-PLs destabilizes the structure of the HDL particles and modifies their function.     

Comparative models for human apolipoprotein A-I bound to lipid in discoidal high-density lipoprotein particles

We have constructed a series of models for apolipoprotein A-I (apo A-I) bound to discoidal high-density lipoprotein (HDL) particles, based upon the molecular belt model [Segrest, J. P., et al. (1999) J. Biol. Chem. 274, 31755-31758] and helical hairpin models [Rogers, D. P., et al. (1998) Biochemistry 37, 11714-11725], and compared these with picket fence models [Phillips, J. C., et al. (1997) Biophys. J. 73, 2337-2346]. Molecular belt models for discoidal HDL particles with differing diameters are presented, illustrating that the belt model can explain the discrete changes in HDL particle size observed experimentally. Hairpin models are discussed for the binding of apo A-I to discoidal HDL particles with diameters identical to those for the molecular belt model. Two models are presented for the binding of three monomers of apo A-I to a 150 A diameter discoidal HDL particle. In one model, two monomers of apo A-I bind to the exterior of the HDL particle in an antiparallel belt, with a third monomer of apo A-I bound to the disk in a hairpin conformation. In the second model, all three monomers of apo A-I are bound to the discoidal HDL particle in a hairpin conformation. Previously published experimental data for each model are reviewed, with FRET favoring either the belt or hairpin models over the picket fence models for HDL particles with diameters of 105 A. Naturally occurring mutations appear to favor the belt model for the 105 A particles, while the 150 A HDL particles favor the presence of at least one hairpin.