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.     

Apolipoprotein A-I adopts a belt-like orientation in reconstituted high density lipoproteins

Apolipoprotein A-I (apoA-I) is the major protein associated with high density lipoprotein (HDL), and its plasma levels have been correlated with protection against atherosclerosis. Unfortunately, the structural basis of this phenomenon is not fully understood. Over 25 years of study have produced two general models of apoA-I structure in discoidal HDL complexes. The belt model states that the amphipathic helices of apoA-I are aligned perpendicular to the acyl chains of the lipid bilayer, whereas the picket fence model argues that the helices are aligned parallel with the acyl chains. To distinguish between the two models, various single tryptophan mutants of apoA-I were analyzed in reconstituted, discoidal HDL particles composed of phospholipids containing nitroxide spin labels at various positions along the acyl chain. We have previously used this technique to show that the orientation of helix 4 of apoA-I is most consistent with the belt model. In this study, we performed additional control experiments on helix 4, and we extended the results by performing the same analysis on the remaining 22-mer helices (helices 1, 2, 5, 6, 7, 8, and 10) of human apoA-I. For each helix, two different mutants were produced that each contained a probe Trp occurring two helical turns apart. In the belt model, the two Trp residues in each helix should exhibit maximal quenching at the same nitroxide group position on the lipid acyl chains. For the picket fence model, maximal quenching should occur at two different levels in the bilayer. The results show that the majority of the helices are in an orientation that is consistent with a belt model, because most Trp residues localized to a position about 5 A from the center of the bilayer. This study corroborates a belt hypothesis for the majority of the helices of apoA-I in phospholipid discs.     

Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.     

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.     

Guinea pig low density lipoproteins: structural and metabolic heterogeneity

The structural and metabolic heterogeneity of low density lipoproteins (LDL, d 1.024-1.100 g/ml) has been investigated in the guinea pig. Two LDL subfractions, of d 1.024-1.050 and 1.050-1.100 g/ml, respectively, were isolated by sequential ultracentrifugation; while both were enriched in cholesteryl ester and apoB-100, the former was heterogeneous displaying three particle size species of diameters 26.9, 25.6, and 24.7 nm, whereas the denser subfraction was relatively homogeneous containing a single, smaller species (diam. 23.6 nm). The fractional catabolic rates (FCR) of the two LDL subfractions were alike (approximately 0.090 pools/hr) in the guinea pig in vivo. After modification of each subfraction by reductive methylation, the FCRs were reduced similarly and indicated that 70-80% of degradation occurred via the cellular LDL receptor pathway. However, the intravascular metabolism of these LDL subfractions, determined from the radioactive content of density gradient fractions as a function of time after injection of radiolabeled native or chemically modified LDL, tended to be distinct. Thus, while radiolabeled apoB-100 in the lighter subfraction maintained the initial density profile up to 48 hr, the radioactive profile of its methylated counterpart changed, the proportion of radioactivity in the lighter gradient fractions (d 1.027-1.032 g/ml) increasing while that in the denser (d 1.037-1.042 g/ml) fractions diminished. A more marked transformation occurred in LDL of d 1.050-1.100 g/ml, in which the radioactive profile shifted towards lighter particles of the d 1.024-1.050 g/ml species; this shift was partially dependent on the LDL receptor, since it was more pronounced in the methylated subfraction. Furthermore, a net increase in the radioactive content of gradient subfractions 7 to 9 (d 1.032-1.042 g/ml) was found 10 hr after injection of methylated LDL of d 1.050-1.100 g/ml, at which time the bulk of LDL radioactivity had been removed from plasma. Several mechanisms, acting alone or in combination, may account for these findings; among them, some degree of transformation of dense to lighter LDL species appears a prerequisite. In conclusion, our data attest to the structural heterogeneity of circulating LDL in the guinea pig, and suggest that the intravascular processing and metabolism of LDL particle subspecies is directly related to their structure and physicochemical properties.     

On the structural similarity of ApoA-I and ApoA-II of human high density lipoproteins.

The possible evolutionary origin of apolipoproteins was studied by comparing the primary structures of different plasma apolipoproteins and other phospholipid-binding proteins. Apolipoprotein A-I (ApoA-I) and apolipoprotein A-II (ApoA-II) of human high density lipoprotein (HDL) are related. The resemblance of these two HDL apolipoproteins are apparently restricted to the carboxyl terminal regions suggesting that these portions of the molecules are derived from the same ancestor. The homologous carboxyl terminal segments may be involved in the regulation of HDL metabolism or in the interaction with phospholipids.     

Crystallization effects in serum high density lipoproteins

Peculiarities of aggregation in the samples of high density serum lipoproteins LHD2 and LHD3 obtained from healthy donors and patients with ishaemic heart disease were studied under isothermal exposition. It was found that lipoprotein samples from defective serum were characterized by chemical and structural inhomogeneity. It is reflected in the formation of spherolites of various types and of well-grown dendrite forms. Growth of stepped lens-shaped crystals of cholesterol takes place in the normal serum LHD3 preparations after long exposition. Crystal growth is thought to begin according to dislocational mechanism on linear defects existing in the liquid crystalline phase; then transformation to kinetically rough surface and to normal growth of the face occurs with the concentration increase.     

Effect of low density lipoproteins, high density lipoproteins, and cholesterol on apolipoprotein A-I mRNA in Hep G2 cells

We have utilized the human hepatocellular carcinoma cell line, Hep G2, to study the effects of low density lipoproteins (LDL), high density lipoproteins (HDL), and free cholesterol on apolipoprotein (apo) A-I mRNA levels. Incubation of the Hep G2 cells with LDL and free cholesterol led to a significant increase in the cellular content of cholesterol without any effect on the yield of total RNA or in the cellular protein content. Our studies established that incubation with LDL or free cholesterol increased the relative levels of apoA-I mRNA in the Hep G2 cells. In contrast with cholesterol loading, HDL had the effect of lowering the levels of apoA-I mRNA. These results indicate the LDL and HDL pathways as well as intracellular cholesterol may be important in apoA-I gene expression and regulation.     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.     

Purification and characterization of bovine lipoproteins: resolution of high density and low density lipoproteins using heparin-Sepharose chromatography

The selective and reversible adsorption of bovine low density lipoproteins (LDL) by heparin-Sepharose has been exploited as the critical step in a procedure for the preparative isolation of very low density lipoproteins (VLDL)/chylomicrons, LDL, and high density lipoproteins (HDL) from bovine plasma. Molecular size exclusion chromatography and isopycnic density gradient separation steps are also involved in the method described. The resulting HDL and LDL fractions are free from contamination by one another as judged by electrophoretic mobility in agarose gels. The major lipid and apolipoprotein compositions of the three resolved lipoprotein classes have been determined.     

Transthyretin in high density lipoproteins: association with apolipoprotein A-I

Previous studies have revealed the presence of transthyretin (TTR) on lipoproteins. To further address this issue, we fractionated plasma lipoproteins from 9 normal individuals, 10 familial amyloidotic polyneuropathy (FAP) patients, and 19 hyperlipidemic subjects using gel filtration. In the majority of the subjects, as well as in 9 of the 10 FAP patients and 14 of the 19 patients with hyperlipidemia, TTR was detected by ELISA in the high density lipoprotein (HDL) fraction. The presence of TTR in HDL was confirmed by direct sequencing and by immunoblotting; using non-reducing conditions, TTR was found by immunoblotting in a high molecular weight complex, which reacted also for apolipoprotein A-I (apoA-I). The amount of TTR present in HDL (HDL-TTR), as quantified by ELISA corresponded to 1;-2% of total plasma TTR. However, no detectable TTR levels were found in HDL fraction from 6 of the hyperlipidemic subjects. No correlation was found between the lack of TTR in HDL and plasma levels of total, LDL-, or HDL-associated cholesterol as well as levels of apoA-I and total plasma TTR. Ligand binding experiments showed that radiolabeled TTR binds to the HDL fraction of individuals with HDL-TTR but not to the corresponding fractions of individuals devoid of HDL-TTR, suggesting that HDL composition may interfere with TTR binding. The component(s) to which TTR binds in the HDL fraction were investigated. Polyclonal antibody against apoA-I was able to block the interaction of TTR with HDL, suggesting that the interaction of TTR with the HDL particle occurs via apoA-I. This hypothesis was further demonstrated by showing the formation of a complex of TTR with HDL and apoA-I by crosslinking experiments. Furthermore, anti-apoA-I immunoblot under native conditions suggested the existence of differences in HDL particle properties and/or stability between individuals with and without HDL-TTR.     

Synthesis, transport, and processing of apolipoproteins of high density lipoproteins

Cell biology methods have greatly influenced the elucidation of the biosynthetic pathways of apolipoproteins. In vitro and tissue culture systems allow the study, to a large extent, of the process of synthesis, intracellular processing, secretion, and extracellular processing of the major high density lipoprotein apoproteins apoA-I and A-II and also of a minor component, apoA-IV. Whereas the latter apoprotein is equipped only with a signal sequence, the primary translation products of apoA-I and apoA-II carry N-terminal extensions of preprosequence of 24 amino acids for apoA-I and 23 amino acid residues for apoA-II. The pro-form of apoA-I characterized by a hexapeptide extension is completely stable intracellularly and is secreted as such. The pro-form is further processed by a serum protease specific for an unusual -Gln-Gln-Asp-Glu-sequence site. Pro-apoA-II, a pentapeptide sequence, is partially processed intracellularly to its mature form and secreted together with the residual pro-form. The cleavage site of pro-apoA-II is characterized by two basic amino acid residues Arg-Arg, present also in other known pro-proteins. The biological function of the N-terminal pro-sequences and details of their final processing by the serum protease(s) have yet to be established.     

Protective effect of high density lipoproteins, their subfractions and lecithin-cholesterol-acyltransferases on the peroxidation modification of low density lipoproteins

The role of high density lipoproteins (HDL), their subfractions (HDL2 and HDL3) and lecithin: cholesterol acyltransferase (LCAT) on peroxidative modification of low density lipoproteins (LDL) in vitro was studied. Peroxidative modification was estimated by the formation of malonic dialdehyde (MDA) and LDL aggregates during LDL incubation at 37 degrees C for several days without Fe2+ or for 2 hours in the presence of Fe2+ in EDTA-free media. It was shown that the addition of HDL3 (but not HDL2) markedly decreases the formation of both MDA and LDL aggregates. Since LCAT is bound to HDL3, its effect was examined. An addition of LCAT isolated from human plasma (650-fold purification) at a concentration of 450 micrograms/ml resulted in a complete inhibition of LDL peroxidation and LDL aggregate formation. Heat-inactivated LCAT had no effect. Possible mechanisms of the protective effect of LCAT on LDL peroxidative modification are discussed.     

Ethanol induced alterations in low and high density lipoproteins

Male squirrel monkeys fed ethanol (ETOH) at variable doses were used to determine whether alcohol modifies levels of plasma low density lipoproteins (LDL) in addition to increasing high density lipoproteins (HDL). Because we earlier showed that high alcohol consumption enhances lipoprotein cholesterol synthesis, experiments were also performed to further assess whether ETOH alters lipoprotein clearance and plasma transfer processes in vivo. Monkeys were divided into three groups: Controls fed isocaloric liquid diet; and Low and High ETOH animals fed liquid diet with vodka substituted isocalorically for carbohydrate at 12 and 24 of the calories, respectively. High ETOH primates had significantly more LDL lipid and protein while serum glutamate oxaloacetate transaminase was similar for the three groups. Although removal of 3H LDL cholesteryl ester (CE) from the plasma compartment was not affected by dietary ETOH, transfer of LDL CE to HDL was impaired in the High ETOH group suggesting a mechanism for the enlarged circulating pool of LDL. Transfer of 14C HDL CE to lower density lipoproteins was similar for the three groups. However, ETOH at both doses delayed clearance of radiolabeled HDL CE from circulation. Thus besides enhancing synthesis of lipoproteins, ETOH at a moderately high dose (24% of calories) influences lipoprotein levels in primates by modifying lipid transfer processes (LDL) as well as by altering clearance (HDL) without adversely affecting liver function.     

氧化修饰高密度脂蛋白的致动脉粥样硬化作用

高密度脂蛋白（ｈｉｇｈ ｄｅｎｓｉｔｙ ｌｉｐｏｐｒｏｔｅｉｎ ,ＨＤＬ）具有抗动脉粥样硬化（ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ,ＡＳ）作用,一旦发生氧化修饰后,即具致ＡＳ作用。氧化修饰ＨＤＬ（ｏｘｉｄｉｚｅｄ ＨＤＬ,ＯＸ－ＨＤＬ）通过减少胆固酥因管平滑肌细胞（ｓｍｏｏｔｈ ｍｕｓｃｌｅ ｃｅｌｌ,ＳＭＣ）,内皮细胞及巨噬细胞流出,抑制胆固醇逆向转运;     

Role of lipids in spheroidal high density lipoproteins

We study the structure and dynamics of spherical high density lipoprotein (HDL) particles through coarse-grained multi-microsecond molecular dynamics simulations. We simulate both a lipid droplet without the apolipoprotein A-I (apoA-I) and the full HDL particle including two apoA-I molecules surrounding the lipid compartment. The present models are the first ones among computational studies where the size and lipid composition of HDL are realistic, corresponding to human serum HDL. We focus on the role of lipids in HDL structure and dynamics. Particular attention is paid to the assembly of lipids and the influence of lipid-protein interactions on HDL properties. We find that the properties of lipids depend significantly on their location in the particle (core, intermediate region, surface). Unlike the hydrophobic core, the intermediate and surface regions are characterized by prominent conformational lipid order. Yet, not only the conformations but also the dynamics of lipids are found to be distinctly different in the different regions of HDL, highlighting the importance of dynamics in considering the functionalization of HDL. The structure of the lipid droplet close to the HDL-water interface is altered by the presence of apoA-Is, with most prominent changes being observed for cholesterol and polar lipids. For cholesterol, slow trafficking between the surface layer and the regimes underneath is observed. The lipid-protein interactions are strongest for cholesterol, in particular its interaction with hydrophobic residues of apoA-I. Our results reveal that not only hydrophobicity but also conformational entropy of the molecules are the driving forces in the formation of HDL structure. The results provide the first detailed structural model for HDL and its dynamics with and without apoA-I, and indicate how the interplay and competition between entropy and detailed interactions may be used in nanoparticle and drug design through self-assembly.     

Human apolipoprotein A-I kinetics within triglyceride-rich lipoproteins and high density lipoproteins.

Stable isotope methodology was used to determine the kinetic behavior of apolipoprotein (apo) A-I within the triglyceride-rich lipoprotein (TRL) fraction and to compare TRL apoA-I kinetics with that of apoA-I in high density lipoprotein (HDL) and TRL apoB-48. Eight subjects (5 males and 3 females) over the age of 40 were placed on a baseline average American diet and after 6 weeks received a primed-constant infusion of [5,5,5-(2)H(3)]-l-leucine for 15 h while consuming small hourly meals of identical composition. HDL and TRL apoA-I and TRL apoB-48 tracer/tracee enrichment curves were obtained by gas chromatography;-mass spectrometry. Data were fitted to a compartmental model to determine the fractional secretion rates of apoA-I and apoB-48 within each lipoprotein fraction. Mean plasma apoA-I levels in TRL and HDL fractions were 0. 204 +/- 0.057 and 134 +/- 15 mg/dl, respectively. The mean fractional catabolic rate (FCR) of TRL apoA-I was 0.250 +/- 0.069 and HDL apoA-I was 0.239 +/- 0.054 pools/day, with mean estimated residence times (RT) of 4.27 and 4.37 days, respectively. The mean TRL apoB-48 FCR was 5.2 +/- 2.0 pools/day and the estimated mean RT was 5.1 +/- 1.8 h. Our results indicate that apoA-I is catabolized at a slower rate than apoB-48 within TRL, and that apoA-I within TRL and HDL fractions are catabolized at similar rates.