Structural determination of lipid-bound ApoA-I using fluorescence resonance energy transfer

Based on the x-ray crystal structure of lipid-free Delta43 apoA-I, two monomers of apoA-I were suggested to bind to a phospholipid bilayer in an antiparallel paired dimer, or "belt orientation." This hypothesis challenges the currently held model in which each of the two apoA-I monomers fold as antiparallel alpha-helices or "picket fence orientation." When apoA-I is bound to a phospholipid disc, the first model predicts that the glutamine at position 132 on one apoA-I molecule lies within 16 A of glutamine 132 in the second monomer, whereas, the second model predicts glutamines at position 132 to be 104 A apart. To distinguish between these models, glutamine at position 132 was mutated to cysteine in wild-type apoA-I to produce Q132C apoA-I, which were labeled with thiol-reactive fluorescent probes. Q132C apoA-I was labeled with either fluorescein (donor probe) or tetramethylrhodamine (acceptor probe) and then used to make recombinant phospholipid discs (recombinant high density lipoprotein (rHDL)). The rHDL containing donor- and acceptor-labeled Q132C apoA-I were of similar size, composition, and lecithin:cholesterol acyltransferase reactivity when compared to rHDL-containing human plasma apoA-I. Analysis of donor probe fluorescence showed highly efficient quenching in rHDL containing one donor- and one acceptor-labeled Q132C apoA-I. rHDL containing only acceptor probe-labeled Q132C apoA-I showed rhodamine self-quenching. Both of these observations demonstrate that position 132 in two lipid-bound apoA-I monomers were in close proximity, supporting the "belt conformation" hypothesis for apoA-I on rHDL.     

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.     

Apolipophorin III from Hyphantria cunea shows different anti-oxidant ability against LDL oxidation in the lipid-free and lipid-bound state

Insect apolipophorin III (apoLp-III) and human apolipoprotein A-I (apoA-I) are major protein constituents of the lipoprotein system that share various properties. In order to compare the anti-oxidant ability of apoLp-III and apoA-I in the lipid-free and lipid-bound state, both proteins were purified and synthesized individually as a palmitoyloleoyl phosphatidylcholine (POPC)-reconstituted high-density lipoprotein (rHDL) using the same molar ratio. In the lipid-bound state, apoLp-III and apoA-I showed good anti-oxidant activities against copper-mediated LDL oxidation. Furthermore, apoLp-III and apoA-I, in the lipid-bound state, exhibited potent activities in ferric ion-reducing ability (FRA). However, lipid-free apoLp-III lost the anti-oxidant activity and FRA ability in contrast to lipid-free apoA-I. Lipid-free apoA-I treatment prevented the cellular uptake of oxLDL in macrophages, as visualized by oil-red O staining and detection assays for malondialdehyde (MDA) and lipid hydroperoxide (LPO) in the culture media. However, lipid-free apoLp-III did not prevent the uptake of oxLDL. These results indicate that the putative conformational change of apoLp-III during lipid association is critical for the maintenance of anti-oxidant activity and that the physiologic role of apoLp-III may differ when it is in the lipid-free state and the lipid-bound state.     

Structure and stability of apolipoprotein a-I in solution and in discoidal high-density lipoprotein probed by double charge ablation and deletion mutation

To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A/D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Delta(61-78) and Delta(121-142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid-cholesterol-apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4 and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation, suggesting involvement of R160/H162 in important interhelical interactions. The results on the Delta(121-142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by specific electrostatic interhelical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.     

Conformational adaptation of apolipoprotein A-I to discretely sized phospholipid complexes

The conformational constraints for apoA-I bound to recombinant phospholipid complexes (rHDL) were attained from a combination of chemical cross-linking and mass spectrometry. Molecular distances were then used to refine models of lipid-bound apoA-I on both 80 and 96 A diameter rHDL particles. To obtain molecular constraints on the protein bound to phospholipid complexes, three different lysine-selective homo-bifunctional cross-linkers with increasing spacer arm lengths (i.e., 7.7, 12.0, and 16.1 A) were reacted with purified, homogeneous recombinant 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) apoA-I rHDL complexes of each diameter. Cross-linked dimeric apoA-I products were separated from monomeric apoprotein using 12% SDS-PAGE, then subjected to in-gel trypsin digest, and identified by MS/MS sequencing. These studies aid in the refinement of our previously published molecular model of two apoA-I molecules bound to approximately 150 molecules of POPC and suggest that the protein hydrophobic interactions at the N- and C-terminal domains decrease as the number of phospholipid molecules or "lipidation state" of apoA-I increases. Thus, it appears that these incremental changes in the interaction between the N- and C-terminal ends of apoA-I stabilize its tertiary conformation in the lipid-free state as well as allowing it to unfold and sequester discrete amounts of phospholipid molecules.     

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.     

ApoA-I structure on discs and spheres. Variable helix registry and conformational states

Apolipoprotein A-I (apoA-I) readily forms discoidal high density lipoprotein (HDL) particles with phospholipids serving as an ideal transporter of plasma cholesterol. In the lipid-bound conformation, apoA-I activates the enzyme lecithin:cholesterol acyltransferase stimulating the formation of cholesterol esters from free cholesterol. As esterification proceeds cholesterol esters accumulate within the hydrophobic core of the discoidal phospholipid bilayer transforming it into a spherical HDL particle. To investigate the change in apoA-I conformation as it adapts to a spherical surface, fluorescence resonance energy transfer studies were performed. Discoidal rHDL particles containing two lipid-bound apoA-I molecules were prepared with acceptor and donor fluorescent probes attached to cysteine residues located at specific positions. Fluorescence quenching was measured for probe combinations located within repeats 5 and 5 (residue 132), repeats 5 and 6 (residues 132 and 154), and repeats 6 and 6 (residue 154). Results from these experiments indicated that each of the 2 molecules of discoidal bound apoA-I exists in multiple conformations and support the concept of a "variable registry" rather than a "fixed helix-helix registry." Additionally, discoidal rHDL were transformed in vitro to core-containing particles by incubation with lecithin:cholesterol acyltransferase. Compositional analysis showed that core-containing particles contained 11% less phospholipid and 633% more cholesterol ester and a total of 3 apoA-I molecules per particle. Spherical particles showed a lowering of acceptor to donor probe quenching when compared with starting rHDL. Therefore, we conclude that as lipid-bound apoA-I adjusts from a discoidal to a spherical surface its intermolecular interactions are significantly reduced presumably to cover the increased surface area of the particle.     

Fructated apolipoprotein A-I showed severe structural modification and loss of beneficial functions in lipid-free and lipid-bound state with acceleration of atherosclerosis and senescence

Non-enzymatic glycation of serum apolipoproteins is a main feature of diabetes mellitus under hyperglycemia. Advanced glycation end products are implicated in the development of aging and metabolic syndrome, including premature atherosclerosis in diabetic subjects. ApoA-I is the principal protein constituent of HDL. In this study, glycated human apoA-I (gA-I) by fructation was characterized on functional and structural correlations in lipid-free and lipid-bound states.The gA-I showed more spontaneous multimeric band formation up to pentamer and exhibited slower elution profile with more degraded fragments from fast protein liquid chromatography. The gA-I showed modified secondary structure from fluorescence and circular dichroism analysis. Reconstituted high-density lipoprotein (rHDL) containing the gA-I had less content of phospholipid with a much smaller particle size than those of rHDL-containing nA-I (nA-I-rHDL). The rHDL containing gA-I (gA-I-rHDL) consisted of less molecular number of apoA-I than nA-I-rHDL with decreased α-helical content. Treatment of the gA-I-rHDL induced more atherogenic process in macrophage cell and premature senescence in human dermal fibroblast cell.Conclusively, fructose-mediated apoA-I glycation resulted in severe loss of several beneficial functions of apoA-I and HDL regarding anti-senescence and anti-atherosclerosis activities due to a lack of anti-oxidant activity with increased susceptibility of protein degradation and structural modification.     

Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements

The central region of apolipoprotein A-I (apoA-I), spanning residues 143--165, has been implicated in lecithin:cholesterol acyltransferase (LCAT) activation and also in high density lipoprotein (HDL) structural rearrangements. To examine the role of individual amino acids in these functions, we constructed, overexpressed, and purified two additional point mutants of apoA-I (P143R and R160L) and compared them with the previously studied V156E mutant. These mutants have been reported to occur naturally and to affect HDL cholesterol levels and cholesterol esterification in plasma. The P143R and R160L mutants were effectively expressed in Escherichia coli as fusion proteins and were isolated in at least 95% purity. In the lipid-free state, the mutants self-associated similarly to wild-type protein. All the mutants, including V156E, were able to lyse dimyristoylphosphatidylcholine liposomes. In the lipid-bound state, the major reconstituted HDL (rHDL) of the mutants had diameters similar to wild type (96--98 A). Circular dichroism and fluorescence methods revealed no major differences among the structures of the lipid-free or lipid-bound mutants and wild type. In contrast, the V156E mutant had exhibited significant structural, stability, and self-association differences compared with wild-type apoA-I in the lipid-free state, and formed rHDL particles with larger diameters. In this study, limited proteolytic digestion with chymotrypsin showed that the V156E mutant, in lipid-free form, has a distinct digestion pattern and surface exposure of the central region, compared with wild type and the other mutants. Reactivity of rHDL with LCAT was highest for wild type (100%), followed by P143R (39%) and R160L (0.6%). Tested for their ability to rearrange into 78-A particles, the rHDL of the two mutants (P143R and R160L) behaved normally, compared with the rHDL of V156E, which showed no rearrangement after the 24-h incubation with low density lipoprotein (LDL). Similarly, the rHDL of V156E was resistant to rearrangement in the presence of apoA-I or apoA-II. These results indicate that structural changes are absent or modest for the P143R and R160L mutants, especially in rHDL form; that these mutants have normal conformational adaptability; and that LCAT activation is obliterated for R160L.Thus, individual amino acid changes may have markedly different structural and functional consequences in the 143--165 region of apoA-I. The R160L mutation appears to have a direct effect in LCAT activation, while the P143R mutation results in only minor structural and functional effects. Also, the processes for LCAT activation and hinge mobility appear to be distinct even if the same region of apoA-I is involved. -- Cho, K-H., D. M. Durbin, and A. Jonas. Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements. J. Lipid Res. 2001. 42: 379--389.     

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1.     

Lipid-free apolipoproteins A-I and A-II promote remodeling of reconstituted high density lipoproteins and alter their reactivity with lecithin:cholesterol acyltransferase

We examined the effect of lipid-free apolipoprotein A-I (apoA-I) and apoA-II on the structure of reconstituted high density lipoproteins (rHDL) and on their reactivity as substrates for lecithin:cholesterol acyltransferase (LCAT). First, homogeneous rHDL were prepared with either apoA-I or apoA-II using palmitoyloleoylphosphatidylcholine (POPC) and cholesterol. Lipid-free apoA-I and apoA-II were labeled with the fluorescent probe dansyl chloride (DNS). The binding kinetics of apoA-I-DNS to A-II-POPCrHDL and of apoA-II-DNS to A-I-POPCrHDL were monitored by fluorescence polarization, adding the lipid-free apolipoproteins to the rHDL particles in a 1:1 molar ratio. For both apolipoproteins, the binding to rHDL was rapid, occurring within 5 min. Next, the effect on rHDL structure and particle size was determined after incubations of lipid-free apolipoproteins with homogeneous rHDL at 37 degrees C from 0.5 to 24 h. The products were analyzed by non-denaturing gradient gel electrophoresis followed by Western blotting. The effect of apoA-I or apoA-II on 103 A A-II-POPCrHDL was a rearrangement into 78 A particles containing apoA-I and/or apoA-II, and 90 A particles containing only apoA-II. The effect of apoA-I or apoA-II on 98 A A-I-POPCrHDL was a rearrangement into complexes ranging in size from 78 A to 105 A containing apoA-I and/or apoA-II, with main particles of 78 A, 88 A, and 98 A. Finally, the effect of lipid-free apoA-I and apoA-II on rHDL as substrates for LCAT was determined. The addition of apoA-I to A-II-POPCrHDL increased its reactivity with LCAT 24-fold, reflected by a 4-fold increase in apparent V(m)ax and a 6-fold decrease in apparent K(m), while the addition of apoA-II to A-II-POPCrHDL had no effect on its minimal reactivity with LCAT. In contrast, the addition of apoA-II to A-I-POPCrHDL decreased the reaction with LCAT by about one-half. The inhibition was due to a 2-fold increase in apparent K(m); there was no significant change in apparent V(m)ax. Likewise, the addition of apoA-I to A-I-POPCrHDL inhibited the reaction with LCAT to about two-thirds that of A-I-POPCrHDL without added apoA-I. In summary, both lipid-free apoA-I and apoA-II can promote the remodeling of rHDL into hybrid particles of primarily smaller size. Both apoA-I and apoA-II affect the reactivity of rHDL with LCAT, when added to the reaction in lipid-free form. These results have important implications for the roles of lipid-free apoA-I and apoA-II in HDL maturation and metabolism.     

Inhibition of collar-induced carotid atherosclerosis by recombinant apoA-I cysteine mutants in apoE-deficient mice

The previous studies in our laboratory revealed that seven cysteine mutants of apolipoprotein A-I (apoA-I) have different structural features and biological activities in vitro and in vivo. To investigate the potential cardioprotective effects of apolipoprotein A-I(N74C) [apoA-I(N74C)], we examined the anti-inflammatory, antioxidant, and antiatherosclerotic effects of this cysteine mutant in a rapid atherosclerosis model induced by perivascular carotid collar placement in apoE^−/− mice. Lipid-free apoA-I(N74C) showed a significant increased antioxidant potency in low density lipoprotein (LDL) oxidation in vitro and reduced intracellular lipid accumulation in THP-1-derived macrophages, relative to wild-type apoA-I (apoA-Iwt). Mice injected with recombinant HDL (rHDL) reconstituted with apoA-I(N74C) (named rHDL74) through tail veins (40 mg/kg of body weight, three injections) had a significant lower level of serum interleukin-6 (IL-6) and enhanced serum antioxidation compared with mice receiving rHDL reconstituted with apoA-Iwt (named rHDLwt). Moreover, compared with rHDLwt, the rHDL74 in vivo injection resulted in a significant decrease in plaque size, ratio of aorta intima to media, arterial remodeling, and macrophage content in lesions. In summary, intravenous injection with rHDL74 reconstituted with apoA-I cysteine mutant apoA-I (N74C) dramatically delays the development of atherosclerosis induced by perivascular carotid collar placement and reduces vascular remodeling in the carotid artery in apoE^−/− mice.     

Volumetric determination of apolipoprotein stoichiometry of circulating HDL subspecies

Although HDL is inversely correlated with coronary heart disease, elevated HDL-cholesterol is not always protective. Additionally, HDL has biological functions that transcend any antiatherogenic role: shotgun proteomics show that HDL particles contain 84 proteins (latest count), many correlating with antioxidant and anti-inflammatory properties of HDL. ApoA-I has been suggested to serve as a platform for the assembly of these protein components on HDL with specific functions - the HDL proteome. However, the stoichiometry of apoA-I in HDL subspecies is poorly understood. Here we use a combination of immunoaffinity chromatography data and volumetric analysis to evaluate the size and stoichiometry of LpA-I and LpA-I,A-II particles. We conclude that there are three major LpA-I subspecies: two major particles, HDL[4] in the HDL₃ size range (d = 85.0 ± 1.2 Å) and HDL[7] in the HDL₂ size range (d = 108.5 ± 3.8 Å) with apoA-I stoichiometries of 3 and 4, respectively, and a small minor particle, HDL[1] (d = 73.8 ± 2.1Å) with an apoA-I stoichiometry of 2. Additionally, we conclude that the molar ratio of apolipoprotein to surface lipid is significantly higher in circulating HDL subspecies than in reconstituted spherical HDL particles, presumably reflecting a lack of phospholipid transfer protein in reconstitution protocols.     

Cholesterol mobilization by free and lipid-bound apoAI(Milano) and apoAI(Milano)-apoAII heterodimers

Despite very low plasma levels of HDL, carriers of the apolipoprotein AI Arg173 --> Cys mutation apoAI(Milano) (AIM) have no apparent increase in risk for atherosclerotic vascular disease. HDL apolipoprotein species in AIM carriers include apoAI-AII heterodimers, previously found to confer the enhanced ability of tyrosyl radical-oxidized HDL to mobilize cholesterol for removal from cultured cells. To determine whether enhanced mobilization of cholesterol by apoprotein species in AIM explains a cardioprotective action of this mutation, we examined the ability of lipid-free and lipid-bound AIM and AIM-AII heterodimers to deplete cholesterol from cultured cells. Free AIM and AIM-AII heterodimers showed a decreased capacity to act as acceptors of cholesterol from cholesterol-loaded human fibroblasts compared with native apoAI but similar capacities to deplete fibroblasts of the pool of cholesterol available for esterification by acyl-CoA:cholesterol acyltransferase (ACAT). Discoidal reconstituted HDL (rHDL) containing apoAI depleted both of these cholesterol pools more readily than AIM-containing rHDL when compared at equivalent rHDL protein levels, but similar abilities of these rHDL to deplete cell cholesterol were seen when compared at equivalent phospholipid levels. Spherical rHDL generated using the whole lipid fraction of HDL and apoAI or AIM showed similar capacities to deplete total and ACAT-accessible cell cholesterol when compared at similar protein levels, but an increased capacity of AIM-containing particles was seen when compared at equivalent phospholipid levels. Unlike the apoAI-AII heterodimer in tyrosylated HDL, AIM-AII heterodimer-containing spherical rHDL showed no increased capacity to deplete either of these pools of cholesterol. These results suggest a similar or better capacity of native apoAI in lipid-free or lipid-bound form in discoidal rHDL to enhance the mobilization of cellular cholesterol when compared to AIM in its free or lipid-bound forms. Any increase in depletion of cellular cholesterol by lipid-bound AIM in spherical rHDL appears related to altered phospholipid-binding rather than intrinsic cholesterol-mobilizing characteristics of this protein compared to native apoAI. The lack of major differences in these studies in cholesterol mobilization by native apoAI and AIM, or by apoAIM-AII heterodimers, suggests that any protection against atherosclerosis conferred by this mutation is likely related to other beneficial vascular effects of AIM.     

Synthesis of reconstituted high density lipoprotein (rHDL) containing apoA-I and apoC-III: the functional role of apoC-III in rHDL

Apolipoprotein (apo) C-III is a marker protein of triacylglycerol (TG)-rich lipoproteins and high-density lipoproteins (HDL), and has been proposed as a risk factor of coronary heart disease. To compare the physiologic role of reconstituted HDL (rHDL) with or without apoC-III, we synthesized rHDL with molar ratios of apoA-I:apoC-III of 1:0, 1:0.5, 1:1, and 1:2. Increasing the apoC-III content in rHDL produced smaller rHDL particles with a lower number of apoA-I molecules. Furthermore, increasing the molar ratio of apoC-III in rHDL enhanced the surfactant-like properties and the ability to lyse dimyristoyl phosphatidylcholine. Furthermore, rHDL containing apoC-III was found to be more resistant to particle rearrangement in the presence of low-density lipoprotein (LDL) than rHDL that contained apoA-I alone. In addition, the lecithin:cholesterol acyltransferase (LCAT) activation ability was reduced as the apoC-III content of the rHDL increased; however, the CE transfer ability was not decreased by the increase of apoC-III. Finally, rHDL containing apoC-III aggravated the production of MDA in cell culture media, which led to increased cellular uptake of LDL. Thus, the addition of apoC-III to rHDL induced changes in the structural and functional properties of the rHDL, especially in particle size and rearrangement and LCAT activation. These alterations may lead to beneficial functions of HDL, which is involved in anti-atherogenic properties in the circulation.     

Remodeling of apolipoprotein E-containing spherical reconstituted high density lipoproteins by phospholipid transfer protein

Phospholipid transfer protein (PLTP) transfers phospholipids between HDL and other lipoproteins in plasma. It also remodels spherical, apolipoprotein A-I (apoA-I)-containing HDL into large and small particles in a process involving the dissociation of lipid-free/lipid-poor apoA-I. ApoE is another apolipoprotein that is mostly associated with large, spherical HDL that do not contain apoA-I. Three isoforms of apoE have been identified in human plasma: apoE2, apoE3, and apoE4. This study investigates the remodeling of spherical apoE-containing HDL by PLTP and the ability of PLTP to transfer phospholipids between apoE-containing HDL and phospholipid vesicles. Spherical reconstituted high density lipoproteins (rHDL) containing apoA-I [(A-I)rHDL], apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein were prepared by incubating discoidal rHDL with low density lipoproteins and lecithin:cholesterol acyltransferase. PLTP remodeled the spherical, apoE-containing rHDL into large and small particles without the dissociation of apoE. The PLTP-mediated remodeling of apoE-containing rHDL was more extensive than that of (A-I)rHDL. PLTP transferred phospholipids from small unilamellar vesicles to apoE-containing rHDL in an isoform-dependent manner, but at a rate slower than that for spherical (A-I)rHDL. It is concluded that apoE enhances the capacity of PLTP to remodel HDL but reduces the ability of HDL to participate in PLTP-mediated phospholipid transfers.     

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

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

Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states

Val156 of apolipoprotein A-I (apoA-I) was found to be a key amino acid in the structure and function of high density lipoprotein (HDL) (J. Biol. Chem., 275: 26821-26827, 2000). To determine more precisely the functions of the individual amino acids proximal to Val156, serial point mutants of proapoA-I, including V156K, D157K, and A158E, were overexpressed and purified to at least 95% purity. In the lipid-free state, A158E exhibited the most profound self-associative patterns and the least pronounced dimyristoyl phosphatidylcholine (DMPC) clearance activities. In the lipid-bound state, A158E formed a larger reconstituted HDL (rHDL) with palmitoyloleoyl phosphatidylcholine (POPC), approximately 120 A, whereas other mutants and the wild type (WT) formed 97 A of POPC-rHDL. Cross-linking analysis revealed that A158E-rHDL harbored at least four protein molecules in the particle, while other rHDL conformations contained only two protein molecules. All of the POPC-rHDL produced smaller HDL, around 78 A, after 24 h of incubation in the presence of low density lipoprotein at 37 degrees C. V156K and A158E exhibited decreased lecithin:cholesterol acyltransferase activation activity in the POPC-rHDL state, showing <2% of WT reactivity (apparent Vmax/Km). A158E also displayed markedly different properties in secondary structure, and its accessibility to proteolytic enzymes is different. These results suggest that the two amino acids in helix 6, Val156 and Ala158, are critical to both the structure and function of rHDL.     

Regulation of reconstituted high density lipoprotein structure and remodeling by apolipoprotein E

Apolipoprotein E (apoE) enters the plasma as a component of discoidal HDL and is subsequently incorporated into spherical HDL, most of which contain apoE as the sole apolipoprotein. This study investigates the regulation, origins, and structure of spherical, apoE-containing HDLs and their remodeling by cholesteryl ester transfer protein (CETP). When the ability of discoidal reconstituted high density lipoprotein (rHDL) containing apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein to act as substrates for LCAT were compared with that of discoidal rHDL containing apoA-I [(A-I)rHDL], the rate of cholesterol esterification was (A-I)rHDL > (E2)rHDL approximately (E3)rHDL > (E4)rHDL. LCAT also had a higher affinity for discoidal (A-I)rHDL than for the apoE-containing rHDL. When the discoidal rHDLs were incubated with LCAT and LDL, the resulting spherical (E2)rHDL, (E3)rHDL, and (E4)rHDL were larger than, and structurally distinct from, spherical (A-I)rHDL. Incubation of the apoE-containing spherical rHDL with CETP and Intralipid(R) generated large fusion products without the dissociation of apoE, whereas the spherical (A-I)rHDLs were remodeled into small particles with the formation of lipid-poor apoA-I. In conclusion, i) apoE activates LCAT less efficiently than apoA-I; ii) apoE-containing spherical rHDLs are structurally distinct from spherical (A-I)rHDL; and iii) the CETP-mediated remodeling of apoE-containing spherical rHDL differs from that of spherical (A-I)rHDL.     

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.