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.     

The mechanism of the remodeling of high density lipoproteins by phospholipid transfer protein

Phospholipid transfer protein (PLTP) remodels high density lipoproteins (HDL) into large and small particles. It also mediates the dissociation of lipid-poor or lipid-free apolipoprotein A-I (apoA-I) from HDL. Remodeling is enhanced markedly in triglyceride (TG)-enriched HDL (Rye, K.-A., Jauhiainen, M., Barter, P. J., and Ehnholm. C. (1998) J. Lipid. Res. 39, 613-622). This study defines the mechanism of the remodeling of HDL by PLTP and determines why it is enhanced in TG-enriched HDL. Homogeneous populations of spherical reconstituted HDL (rHDL) containing apoA-I and either cholesteryl esters only (CE-rHDL; diameter 9.3 nm) or CE and TG in their core (TG-rHDL; diameter 9.5 nm) were used. After 24 h of incubation with PLTP, all of the TG-rHDL, but only a proportion of the CE-rHDL, were converted into large (11.3-nm diameter) and small (7.7-nm diameter) particles. Only small particles were formed during the first 6 h of incubation of CE-rHDL with PLTP. The large particles and dissociated apoA-I were apparent after 12 h. In the case of TG-rHDL, small particles appeared after 1 h of incubation, while dissociated apoA-I and large particles were apparent at 3 h. The composition of the large particles indicated that they were derived from a fusion product. Spectroscopic studies indicated that the apoA-I in TG-rHDL was less stable than the apoA-I in CE-rHDL. In conclusion, these results show that (i) PLTP mediates rHDL fusion, (ii) the fusion product rearranges by two independent processes into small and large particles, and (iii) the more rapid remodeling of TG-rHDL by PLTP may be due to the destabilization of apoA-I.     

Phospholipid transfer is a prerequisite for PLTP-mediated HDL conversion

Phospholipid transfer protein (PLTP) is an important regulator of high-density lipoprotein (HDL) metabolism. The two main functions of PLTP are transfer of phospholipids between lipoprotein particles and modulation of HDL size and composition in a process called HDL conversion. These PLTP-mediated processes are physiologically important in the transfer of surface remnants from lipolyzed triglyceride-rich lipoproteins to nascent HDL particles and in the generation of prebeta-HDL, the initial acceptor of excess peripheral cell cholesterol. The aim of the study presented here was to investigate the interrelationship between the two functions of PLTP. Plasma PLTP was chemically modified using diethylpyrocarbonate or ethylmercurithiosalicylate. The modified proteins displayed a dose-dependent decrease in phospholipid transfer activity and a parallel decrease in the ability to cause HDL conversion. Two recombinant PLTP mutant proteins, defective in phospholipid transfer activity due to a mutation in the N-terminal lipid-binding pocket, were produced, isolated, and incubated together with radioactively labeled HDL(3). HDL conversion was analyzed using three methods: native gradient gel electrophoresis, ultracentrifugation, and crossed immunoelectrophoresis. The results demonstrate that the mutant proteins (i) are able to induce only a modest increase in HDL particle size compared to the wild-type protein, (ii) are unable to release apoA-I from HDL(3), and (iii) do not generate prebeta-mobile particles following incubation with HDL(3). These data suggest that phospholipid transfer is a prerequisite for HDL conversion and demonstrate the close interrelationship between the two main activities of PLTP.     

ApoE-containing high density lipoproteins and phospholipid transfer protein activity increase in patients with a systemic inflammatory response

High density lipoproteins (HDL) mediate reverse cholesterol transport as well as the clearance of oxidation products or inflammatory mediators, thereby contributing to tissue integrity. The decrease in HDL in inflammation has been attributed to decreased lecithin:cholesterol acyltransferase activity, whereas the role of phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein has not been analyzed in detail. We have studied the activities of HDL-modifying proteins and the heterogeneity of HDL in healthy control subjects and three groups of postsurgery patients: no bacterial infection (group 1), bacterial focus and systemic inflammatory response (group 2), and severe sepsis (group 3). For all patients, a decrease in total HDL could be demonstrated, with a loss of mainly large, apolipoprotein A-I (apoA-I) HDL particles, an almost total loss of apoC-I, and an increase in apoE HDL (200-500 kDa), which did not contain significant amounts of apoA-I, apoA-II, or apoC-I. PLTP activity was increased in patients of groups 2 and 3, paralleled by a redistribution of PLTP into a population of small (120- to 200-kDa) particles, probably representing PLTP homodimers or lipid-complexed PLTP.In summary, the increase in apoE HDL and PLTP activity may improve the delivery of energy substrates and phospholipids to tissues that must maintain cellular membrane homeostasis under conditions of inflammatory stress.     

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.     

Oxidation of methionine residues to methionine sulfoxides does not decrease potential antiatherogenic properties of apolipoprotein A-I

The initial stage of oxidation of high density lipoproteins (HDL) is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three Met residues of apolipoprotein A-I (apoA-I) to Met sulfoxides (Met(O)). Formation of such selectively oxidized apoA-I (i.e. apoA-I(+32)) may affect the antiatherogenic properties of HDL, because it has been suggested that Met(86) and Met(112) are important for cholesterol efflux and Met(148) is involved in the activation of lecithin:cholesterol acyl transferase (LCAT). We therefore determined which Met residues were oxidized in apoA-I(+32) and how such oxidation of apoA-I affects its secondary structure, the affinity for lipids, and its ability to remove lipids from human macrophages. We also assessed the capacity of discoidal reconstituted HDL containing apoA-I(+32) to act as substrate for LCAT, and the dissociation of apoA-I and apoA-I(+32) from reconstituted HDL. Met(86) and Met(112) were present as Met(O), as determined by amino acid sequencing and mass spectrometry of isolated peptides derived from apoA-I(+32). Selective oxidation did not alter the alpha-helicity of lipid-free and lipid-associated apoA-I as assessed by circular dichroism, and the affinity for LCAT was comparable for reconstituted HDL containing apoA-I or apoA-I(+32). Cholesteryl ester transfer protein mediated the dissociation of apoA-I more readily from reconstituted HDL containing apoA-I(+32) than unoxidized apoA-I. Also, compared with native apoA-I, apoA-I(+32) had a 2- to 3-fold greater affinity for lipid (as determined by the rate of clearance of multilamellar phospholipid vesicles) and its ability to cause efflux of [(3)H]cholesterol, [(3)H]phospholipid, and [(14)C]alpha-tocopherol from lipid-laden human monocyte-derived macrophages was significantly enhanced. By contrast, no difference was observed for cholesterol and alpha-tocopherol efflux to lipid-associated apolipoproteins. Together, these results suggest that selective oxidation of Met residues enhances rather than diminishes known antiatherogenic activities of apoA-I, consistent with the overall hypothesis that detoxification of lipid hydroperoxides by HDL is potentially antiatherogenic.     

Phospholipid transfer protein interacts with and stabilizes ATP-binding cassette transporter A1 and enhances cholesterol efflux from cells

Phospholipid lipid transfer protein (PLTP) is ubiquitously expressed in animal tissues and plays multiple roles in lipoprotein metabolism, but the function of peripheral PLTP is still poorly understood. Here we show that one of its possible functions is to transport cholesterol and phospholipids from cells to lipoprotein particles by a process involving PLTP interactions with cellular ATP-binding cassette transporter A1 (ABCA1). When ABCA1 was induced in murine macrophages or ABCA1-transfected baby hamster kidney cells, PLTP gained the ability to promote cholesterol and phospholipid efflux from cells. Although PLTP alone had lipid efflux activity, its maximum activity was observed in the presence of high density lipoprotein particles. Pulsechase studies showed that the interaction of PLTP with ABCA1-expressing cells played a role in promoting lipid efflux. Overexpression of ABCA1 dramatically increased binding of both PLTP and apoA-I to common sites on the cell surface. Both PLTP and apoA-I were covalently cross-linked to ABCA1, each protein blocked cross-linking of the other, and both PLTP and apoA-I stabilized ABCA1 protein. These results are consistent with PLTP and apoA-I binding to ABCA1 at the same or closely related sites. Thus, PLTP mimics apolipoproteins in removing cellular lipids by the ABCA1 pathway, except that PLTP acts more as an intermediary in the transfer of cellular lipids to lipoprotein particles.     

Thermal Stability of Apolipoprotein A-I in High-Density Lipoproteins by Molecular Dynamics

Apolipoprotein (apo) A-I is an unusually flexible protein whose lipid-associated structure is poorly understood. Thermal denaturation, which is used to measure the global helix stability of high-density lipoprotein (HDL)-associated apoA-I, provides no information about local helix stability. Here we report the use of temperature jump molecular dynamics (MD) simulations to scan the per-residue helix stability of apoA-I in phospholipid-rich HDL. When three 20 ns MD simulations were performed at 500 K on each of two particles created by MD simulations at 310 K, bilayers remained intact but expanded by 40%, and total apoA-I helicity decreased from 95% to 72%. Of significance, the conformations of the overlapping N- and C-terminal domains of apoA-I in the particles were unusually mobile, exposing hydrocarbon regions of the phospholipid to solvent; a lack of buried interhelical salt bridges in the terminal domains correlated with increased mobility. Nondenaturing gradient gels show that 40% expansion of the phospholipid surface of 100:2 particles by addition of palmitoyloleoylphosphatidylcholine exceeds the threshold of particle stability. As a unifying hypothesis, we propose that the terminal domains of apoA-I are phospholipid concentration-sensitive molecular triggers for fusion/remodeling of HDL particles. Since HDL remodeling is necessary for cholesterol transport, our model for remodeling has substantial biomedical implications.     

Avian phospholipid transfer protein causes HDL conversion without affecting cholesterol efflux from macrophages

Circulatory phospholipid transfer protein (PLTP) has two major functions: 1) transfer of phospholipids towards HDL particles; and 2) modulation of HDL size and composition via the HDL conversion process. In the laying hen (Gallus gallus), the massive oocyte-targeted lipid flow is achieved through the concerted actions of lipases, lipid transfer proteins, and relatives of the LDL receptor family. The aim of the study was to gain insights into the structure and functions of chicken PLTP. The results demonstrate that PLTP is highly conserved from chicken to mammals, as (i) chicken PLTP is associated with plasma HDL; (ii) it clearly possesses phospholipid transfer activity; (iii) it is inactivated at + 58 °C; and (iv) it mediates conversion of avian and human HDL into small preβ-mobile HDL and large fused α-mobile HDL particles. Our data show that HDL from different chicken models is similar in chemical and physical properties to that of man based on PLTP activity, cholesterol efflux, and HDL conversion assays. In contrast to mammals, PLTP-facilitated HDL remodeling did not enhance cholesterol efflux efficiency of chicken HDL particles.     

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

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

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

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

PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma

Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by Heparin-Sepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity. These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma.     

Unravelling the influence of surface lipids on the structure,dynamics and interactome of high-density lipoproteins

Surface lipids influence the biological activities of high-density lipoproteins (HDLs) but their species-specific effects on HDL structure, dynamics, and surface interactome has remained unclear. Building upon the five-lipid species HDL models developed and characterised in previous work, representative models of the major HDL subpopulations found in human plasma containing apolipoprotein A-I (apoA-I) have been studied using molecular dynamics simulation to describe their varying degrees of surface lipidome complexity. Specifically, two additional sets of representative HDL subpopulation particles were developed, one with sphingomyelin (SM) and the other with SM, phosphatidylethanolamine, phosphatidylinositol, and ceramide in quantities reflecting average levels characterised for HDL subpopulations derived from normolipidemic patients. These lipid species were assessed in terms of HDL size, morphology, dynamics, and overall interactome. The findings reveal that the presence of a representative SM fraction marginally enhanced HDL interfacial curvature and surface monolayer rigidity, manifesting in tighter phospholipid packing and slower surface lipid dynamics relative to SM-deficient HDL models. Furthermore, the presence of SM resulted in a reduction in the solvent exposure of core lipids and cholesterol molecules, whilst also enhancing apolipoprotein conformational flexibility and its overall twisting across the HDL surface. The hydrophobicity of apoA-I-bound lipid patches and the proportion of apoA-I hydrophobic surface area is enhanced by the overall lipidation of apoA-I irrespective of lipid composition. These findings offer new insights into how the surface lipid composition of different HDL subpopulations can significantly impact the overall interactome of HDL particles, potentially influencing subpopulation-specific biological functions like lipid scavenging and receptor interactions.     

Active plasma phospholipid transfer protein is associated with apoA-I- but not apoE-containing lipoproteins

Plasma phospholipid transfer protein (PLTP) is a multifaceted protein with diverse biological functions. It has been shown to exist in both active and inactive forms. To determine the nature of lipoproteins associated with active PLTP, plasma samples were adsorbed with anti-A-I, anti-A-II, or anti-E immunoadsorbent, and PLTP activity was measured in the resulting plasma devoid of apolipoprotein A-I (apoA-I), apoA-II, or apoE. Anti-A-I and anti-A-II immunoadsorbents removed 98 +/- 1% (n = 8) and 38 +/- 25% (n = 7) of plasma PLTP activity, respectively. In contrast, only 1 +/- 5% of plasma PLTP activity was removed by anti-E immunoadsorbent (n = 7). Dextran sulfate (DS) cellulose did not bind apoA-I, but it removed 83 +/- 5% (n = 4) of the PLTP activity in plasma. In size-exclusion chromatography, PLTP activity removed by anti-A-I or anti-A-II immunoadsorbent was associated primarily with particles of a size corresponding to HDL, whereas a substantial portion of the PLTP activity dissociated from DS cellulose was found in particles larger or smaller than HDL. These data show the following: 1) active plasma PLTP is associated primarily with apoA-I- but not apoE-containing lipoproteins; 2) active PLTP is present in HDL particles with and without apoA-II, and its distribution between these two HDL subpopulations varies widely among individuals; and 3) DS cellulose can remove active PLTP from apoA-I-containing lipoproteins, and this process creates new active PLTP-containing particles in vitro.     

Interfacial and lipid transfer properties of human phospholipid transfer protein: implications for the transfer mechanism of phospholipids

In circulation the phospholipid transfer protein (PLTP) facilitates the transfer of phospholipid-rich surface components from postlipolytic chylomicrons and very low density lipoproteins (VLDL) to HDL and thereby regulates plasma HDL levels. To study the molecular mechanisms involved in PLTP-mediated lipid transfer, we studied the interfacial properties of PLTP using Langmuir phospholipid monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) to follow the transfer of 14C-labeled phospholipids and [35S]PLTP between lipid vesicles and HDL particles. The AsFlFFF method was also used to determine the sizes of spherical and discoidal HDL particles and small unilamellar lipid vesicles. In Langmuir monolayer studies high-activity (HA) and low-activity (LA) forms of PLTP associated with fluid phosphatidylcholine monolayers spread at the air/buffer interphase. Both forms also mediated desorption of [14C]dipalmitoylphosphatidylcholine (DPPC) from the phospholipid monolayer into the buffer phase, even when it contained no physiological acceptor such as HDL. After the addition of HDL3 to the buffer, HA-PLTP caused enhanced lipid transfer to them. The particle diameter of HA-PLTP was approximately 6 nm and that of HDL3 approximately 8 nm as determined by AsFlFFF analysis. Using this method, it could be demonstrated that in the presence of HA-PLTP, but not LA-PLTP, [14C]DPPC was transferred from small unilamellar vesicles (SUV) to acceptor HDL3 molecules. Concomitantly, [35S]-HA-PLTP was transferred from the donor to acceptor, and this transfer was not observed for its low-activity counterpart. These observations suggest that HA-PLTP is capable of transferring lipids by a shuttle mechanism and that formation of a ternary complex between PLTP, acceptor, and donor particles is not necessary for phospholipid transfer.     

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

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

Folded functional lipid-poor apolipoprotein A-I obtained by heating of high-density lipoproteins: relevance to high-density lipoprotein biogenesis

HDL (high-density lipoproteins) remove cell cholesterol and protect from atherosclerosis. The major HDL protein is apoA-I (apolipoprotein A-I). Most plasma apoA-I circulates in lipoproteins, yet ~5% forms monomeric lipid-poor/free species. This metabolically active species is a primary cholesterol acceptor and is central to HDL biogenesis. Structural properties of lipid-poor apoA-I are unclear due to difficulties in isolating this transient species. We used thermal denaturation of human HDL to produce lipid-poor apoA-I. Analysis of the isolated lipid-poor fraction showed a protein/lipid weight ratio of 3:1, with apoA-I, PC (phosphatidylcholine) and CE (cholesterol ester) at approximate molar ratios of 1:8:1. Compared with lipid-free apoA-I, lipid-poor apoA-I showed slightly altered secondary structure and aromatic packing, reduced thermodynamic stability, lower self-associating propensity, increased adsorption to phospholipid surface and comparable ability to remodel phospholipids and form reconstituted HDL. Lipid-poor apoA-I can be formed by heating of either plasma or reconstituted HDL. We propose the first structural model of lipid-poor apoA-I which corroborates its distinct biophysical properties and postulates the lipid-induced ordering of the labile C-terminal region. In summary, HDL heating produces folded functional monomolecular lipid-poor apoA-I that is distinct from lipid-free apoA-I. Increased adsorption to phospholipid surface and reduced C-terminal disorder may help direct lipid-poor apoA-I towards HDL biogenesis.     

Lipid transfers between reconstituted high density lipoprotein complexes and low density lipoproteins: effects of plasma protein factors

In this study we examined the transfer of lipids between reconstituted high density lipoprotein discs (r-HDL) and human low density lipoproteins (LDL) in the presence and absence of lecithin:cholesterol acyltransferase (LCAT) or of plasma phospholipid transfer protein (PLTP). We found that spontaneous transfer of phospholipids from r-HDL to LDL occurred by an apparent first order reaction with a half-time of 5.8 to 6.9 hr depending on the phospholipid. During the time of incubation of r-HDL with LDL (from 0 to 25 hr), the phospholipid content of r-HDL decreased more than 30%, the free cholesterol content increased 2.5-fold, and low levels of cholesteryl esters appeared in r-HDL. These compositional changes gave rise to small discoidal particles with a limiting diameter of 77 A and two molecules of apoA-I per particle. When LCAT was included in the reaction mixture, the r-HDL lost even more phospholipid, lost some free cholesterol, and gained cholesteryl esters relative to the apolipoprotein content, due to the enzymatic reaction. The products of the LCAT reaction had a diameter of 93 A and three, rather than two, apoA-I molecules per particle. Inclusion of PLTP into the reaction mixture accelerated the transfer of phospholipids (half-time of 1.7 hr) and the formation of the 77 A product. In addition to these compositional and morphological changes, which may be important in the interconversions of native HDL subspecies, the prolonged incubations revealed some slow reactions, such as the esterification of LDL cholesterol by LCAT, a background formation of cholesteryl esters in r-HDL, and an apparent hydrolysis of cholesteryl esters in LDL in the presence of r-HDL.     

Isolation and partial characterization of the inactive and active forms of human plasma phospholipid transfer protein (PLTP)

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. Two forms of PLTP exist in human plasma, one catalytically active (high activity form, HA-PLTP) and the other inactive (low activity form, LA-PLTP) (Oka, T., Kujiraoka, T., Ito, M., Egashira, T., Takahashi, S., Nanjee, N. M., Miller, N. E., Metso, J., Olkkonen, V. M., Ehnholm, C., Jauhiainen, M., and Hattori, H. (2000) J. Lipid Res. 41, 1651-1657). The two forms are associated with macromolecular complexes of different size. The apparent size of LA-PLTP is 520 kDa and that of HA-PLTP is 160 kDa. Of the circulating PLTP mass only a minor portion is in the HA-PLTP form in normolipidemic subjects. In the present study we have isolated and partially characterized the LA and HA forms of PLTP. Both LA- and HA-PLTP bind to heparin-Sepharose and can be separated by elution with 0-0.5 m NaCl gradient, with HA-PLTP displaying higher affinity for the matrix. LA-PLTP was further purified using hydrophobic butyl-Sepharose and anti-PLTP immunoaffinity chromatography steps. HA-PLTP was subjected to a second heparin-Sepharose step and hydroxylapatite chromatography. Analysis of the two forms of PLTP by SDS-PAGE, Western blotting, immunoprecipitation, and gel filtration demonstrates that LA-PLTP is complexed with apoA-I whereas HA-PLTP is not. Instead, HA-PLTP copurified with apoE. Based on these findings we suggest a model in which nascent PLTP enters the circulation as a high specific activity form not associated with apoA-I. During or after the transfer of lipolytic surface remnants to HDL, PLTP is transferred to apoA-I-containing HDL particles and thereby becomes part of the low activity complex.     

Deletions of helices 2 and 3 of human apoA-I are associated with severe dyslipidemia following adenovirus-mediated gene transfer in apoA-I-deficient mice

The objective of this study was to determine the effect of two amino-terminal apolipoprotein A-I (apoA-I) deletions on high-density lipoprotein (HDL) biosynthesis and lipid homeostasis. Adenovirus-mediated gene transfer showed that the apoA-I[Delta(89-99)] deletion mutant caused hypercholesterolemia, characterized by increased plasma cholesterol and phospholipids, that were distributed in the very low density/intermediate density/low-density lipoprotein (VLDL/IDL/LDL) region, and normal triglycerides. The capacity of the mutant protein to promote ATP-binding cassette transporter A1- (ABCA1-) mediated cholesterol efflux and to activate lecithin:cholesterol acyltranserase (LCAT) was approximately 70-80% of the wild-type (WT) control. The phospholipid transfer protein (PLTP) activity of plasma containing the apoA-I[Delta(89-99)] mutant was decreased to 32% of the WT control. Similar analysis showed that the apoA-I[Delta(62-78)] deletion mutant in apoA-I-deficient mice caused combined hyperlipidemia characterized by increased triglycerides, cholesterol, and phospholipids in the VLDL/IDL region. There was enrichment of the VLDL/IDL with mutant apoA-I that resulted in reduction of in vitro lipolysis. The capacity of this mutant to promote ABCA1-mediated cholesterol efflux was normal, and the capacity to activate LCAT in vitro was reduced by 53%. The WT apoA-I and the apoA-I[Delta(62-78)] mutant formed spherical HDL particles, whereas the apoA-I[Delta(89-99)] mutant formed discoidal HDL particles. We conclude that alterations in apoA-I not only may have adverse effects on HDL biosynthesis but also may promote dyslipidemia due to interference of the apoA-I mutants on the overall cholesterol and triglycerides homeostasis.