A pyrene based fluorescence approach to study conformation of apolipoprotein E3 in macrophage-generated nascent high density lipoprotein.

Apolipoprotein E3 (apoE3) is an anti-atherogenic apolipoprotein with the ability to exist in lipid-free and lipoprotein-associated states. During atherosclerosis, its function in promoting cholesterol efflux from macrophages via the ATP-binding cassette transporter A1 (ABCA1) takes a prominent role, leading to generation of nascent high density lipoprotein (nHDL) particles. The objective of this study is to understand the conformation adopted by apoE3 in macrophage-generated nHDL using a fluorescence spectroscopic approach involving pyrene. Pyrene-labeled recombinant human apoE3 displayed a robust ability to stimulate ABCA1-mediated cholesterol efflux from cholesterol-loaded J774 macrophages (which do not express apoE), comparable to that elicited by unlabeled apoE3. The nHDL recovered from the conditioned medium revealed the presence of apoE3 by immunoblot analysis. A heterogeneous population of nHDL bearing exogenously added apoE3 was generated with particle size varying from ∼12 to ∼19 nm in diameter, corresponding to molecular mass of ∼450 to ∼700 kDa. The lipid: apoE3 ratio varied from ∼60:1 to 10:1. A significant extent of pyrene excimer emission was noted in nHDL, indicative of spatial proximity between Cys112 on neighboring apoE3 molecules similar to that noted in reconstituted HDL. Cross-linking analysis using Cys-specific cross-linkers revealed the predominant presence of dimers. Taken together the data indicate a double belt arrangement of apoE molecules on nHDL. A similar organization of the C-terminal tail of apoE on nHDL was noted when pyrene-apoEA277C(201-299) was used as the cholesterol acceptor. These studies open up the possibility of using exogenously labeled apoE3 to generate nHDL for structural and conformational analysis.

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 preß1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preß1-HDL ELISA. Here, we purified preß1-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 preß1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preß1-HDL and original plasma preß1-HDL, or between the preß1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preß1-HDL MAbs, reactivity with intact plasma preß1-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 preß1-HDL is lipid-free monomolecular apoA-1.

The low-resolution structure of nHDL reconstituted with DMPC with and without cholesterol reveals a mechanism for particle expansion.

Small-angle neutron scattering (SANS) with contrast variation was used to obtain the low-resolution structure of nascent HDL (nHDL) reconstituted with dimyristoyl phosphatidylcholine (DMPC) in the absence and presence of cholesterol, [apoA1:DMPC (1:80, mol:mol) and apoA1:DMPC:cholesterol (1:86:9, mol:mol:mol)]. The overall shape of both particles is discoidal with the low-resolution structure of apoA1 visualized as an open, contorted, and out of plane conformation with three arms in nascent HDL/dimyristoyl phosphatidylcholine without cholesterol (nHDL(DMPC)) and two arms in nascent HDL/dimyristoyl phosphatidylcholine with cholesterol (nHDL(DMPC+Chol)). The low-resolution shape of the lipid phase in both nHDL(DMPC) and nHDL(DMPC+Chol) were oblate ellipsoids, and fit well within their respective protein shapes. Modeling studies indicate that apoA1 is folded onto itself in nHDL(DMPC), making a large hairpin, which was also confirmed independently by both cross-linking mass spectrometry and hydrogen-deuterium exchange (HDX) mass spectrometry analyses. In nHDL(DMPC+Chol), the lipid was expanded and no hairpin was visible. Importantly, despite the overall discoidal shape of the whole particle in both nHDL(DMPC) and nHDL(DMPC+Chol), an open conformation (i.e., not a closed belt) of apoA1 is observed. Collectively, these data show that full length apoA1 retains an open architecture that is dictated by its lipid cargo. The lipid is likely predominantly organized as a bilayer with a micelle domain between the open apoA1 arms. The apoA1 configuration observed suggests a mechanism for accommodating changing lipid cargo by quantized expansion of hairpin structures.

An apoA-I mimetic peptibody generates HDL-like particles and increases alpha-1 HDL subfraction in mice.

The aim of this study is to investigate the capability of an apoA-I mimetic with multiple amphipathic helices to form HDL-like particles in vitro and in vivo. To generate multivalent helices and to track the peptide mimetic, we have constructed a peptibody by fusing two tandem repeats of 4F peptide to the C terminus of a murine IgG Fc fragment. The resultant peptidbody, mFc-2X4F, dose-dependently promoted cholesterol efflux in vitro, and the efflux potency was superior to monomeric 4F peptide. Like apoA-I, mFc-2X4F stabilized ABCA1 in J774A.1 and THP1 cells. The peptibody formed larger HDL particles when incubated with cultured cells compared with those by apoA-I. Interestingly, when administered to mice, mFc-2X4F increased both pre-ß and α-1 HDL subfractions. The lipid-bound mFc-2X4F was mostly in the α-1 migrating subfraction. Most importantly, mFc-2X4F and apoA-I were found to coexist in the same HDL particles formed in vivo. These data suggest that the apoA-I mimetic peptibody is capable of mimicking apoA-I to generate HDL particles. The peptibody and apoA-I may work cooperatively to generate larger HDL particles in vivo, either at the cholesterol efflux stage and/or via fusion of HDL particles that were generated by the peptibody and apoA-I individually.

LDL-apheresis depletes apoE-HDL and pre-ß1-HDL in familial hypercholesterolemia: relevance to atheroprotection.

Subnormal HDL-cholesterol (HDL-C) and apolipoprotein (apo)AI levels are characteristic of familial hypercholesterolemia (FH), reflecting perturbed intravascular metabolism with compositional anomalies in HDL particles, including apoE enrichment. Does LDL-apheresis, which reduces HDL-cholesterol, apoAI, and apoE by adsorption, induce selective changes in HDL subpopulations, with relevance to atheroprotection? Five HDL subpopulations were fractionated from pre- and post-LDL-apheresis plasmas of normotriglyceridemic FH subjects (n = 11) on regular LDL-apheresis (>2 years). Apheresis lowered both plasma apoE (-62%) and apoAI (-16%) levels, with preferential, genotype-independent reduction in apoE. The mass ratio of HDL2:HDL3 was lowered from ~1:1 to 0.72:1 by apheresis, reflecting selective removal of HDL2 mass (80% of total HDL adsorbed). Pre-LDL-apheresis, HDL2 subpopulations were markedly enriched in apoE, consistent with ~1 copy of apoE per 4 HDL particles. Large amounts (50-66%) of apoE-HDL were removed by apheresis, preferentially in the HDL2b subfraction (-50%); minor absolute amounts of apoE-HDL were removed from HDL3 subfractions. Furthermore, pre-ß1-HDL particle levels were subnormal following removal (-53%) upon apheresis, suggesting that cellular cholesterol efflux may be defective in the immediate postapheresis period. In LDL-receptor (LDL-R) deficiency, LDL-apheresis may enhance flux through the reverse cholesterol transport pathway and equally attenuate potential biglycan-mediated deposition of apoE-HDL in the arterial matrix.

The origin and metabolism of a nascent pre-ß high density lipoprotein involved in cellular cholesterol efflux.

The pre-ß HDL fraction constitutes a heterogeneous population of discoid nascent HDL particles. They transport from 1 to 25 % of total human plasma apo A-I. Pre-ß HDL particles are generated de novo by interaction between ABCA1 transporters and monomolecular lipid-free apo A-I. Most probably, the binding of apo A-I to ABCA1 initiates the generation of the phospholipid-apo A-I complex which induces free cholesterol efflux. The lipid-poor nascent pre-ß HDL particle associates with more lipids through exposure to the ABCG1 transporter and apo M. The maturation of pre-ß HDL into the spherical α-HDL containing apo A-I is mediated by LCAT, which esterifies free cholesterol and thereby forms a hydrophobic core of the lipoprotein particle. LCAT is also a key factor in promoting the formation of the HDL particle containing apo A-I and apo A-II by fusion of the spherical α-HDL containing apo A-I and the nascent discoid HDL containing apo A-II. The plasma remodelling of mature HDL particles by lipid transfer proteins and hepatic lipase causes the dissociation of lipid-free/lipid-poor apo A-I, which can either interact with ABCA1 transporters and be incorporated back into pre-existing HDL particles, or eventually be catabolized in the kidney. The formation of pre-ß HDL and the cycling of apo A-I between the pre-ß and α-HDL particles are thought to be crucial mechanisms of reverse cholesterol transport and the expression of ABCA1 in macrophages may play a main role in the protection against atherosclerosis.

The impact of plasma triglyceride and apolipoproteins concentrations on high-density lipoprotein subclasses distribution.

OBJECTIVE: To investigate the effect of triglyceride (TG) integrates with plasma major components of apolipoproteins in HDL subclasses distribution and further elicited the TG-apolipoproteins (apos) interaction in the processes of high density lipoprotein (HDL) mature metabolic and atherosclerosis related diseases. METHODS: Contents of plasma HDL subclasses were quantities by two-dimensional gel electrophoresis associated with immunodetection in 500 Chinese subjects. RESULTS: Contents of preß1-HDL, HDL3a, and apoB-100 level along with apoB-100/A-I ratio were significantly increased, whereas there was a significant reduction in the contents of HDL2, apoA-I level as well as apoC-III/C-II ratio with increased TG concentration. Moreover, preß1-HDL contents is elevated about 9 mg/L and HDL2b contents can be reduced 21 mg/L for 0.5 mmol/L increment in TG concentration. Moreover, with increase of apoA-I levels, HDL2b contents were marginally elevated in any TG concentration group. Furthermore, despite of in the apoB-100/A-I < 0.9 group, the contents of preß1-HDL increased, and those of HDL2b decreased significantly for subjects in both high and very high TG levels compared to that in normal TG levels. Similarly, in the apoB-100/A-I ≥ 0.9 group, the distribution of HDL subclasses also showed abnormality for subjects with normal TG levels. CONCLUSIONS: The particle size of HDL subclasses tend to small with TG levels increased which indicated that HDL maturation might be impeded and efficiency of reverse cholesterol transport(RCT) might be weakened. These data suggest that TG levels were not only significantly associated with but liner with the contents of preß1-HDL and HDL2b. They also raise the possibility that the TG levels effect on HDL maturation metabolism are subjected to plasma apolipoproteins and apolipoproteins ratios.

Apolipoprotein M expression increases the size of nascent pre beta HDL formed by ATP binding cassette transporter A1.

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre beta HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent pre beta HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that approximately 50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with (125)I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Pre beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre beta HDL formation but does stimulate the formation of larger-sized pre beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre beta HDL during or after their formation by ABCA1.

Analysis of lipid transfer activity between model nascent HDL particles and plasma lipoproteins: implications for current concepts of nascent HDL maturation and genesis.

The specifics of nascent HDL remodeling within the plasma compartment remain poorly understood. We developed an in vitro assay to monitor the lipid transfer between model nascent HDL (LpA-I) and plasma lipoproteins. Incubation of alpha-(125)I-LpA-I with plasma resulted in association of LpA-I with existing plasma HDL, whereas incubation with TD plasma or LDL resulted in conversion of alpha-(125)I-LpA-I to prebeta-HDL. To further investigate the dynamics of lipid transfer, nascent LpA-I were labeled with cell-derived [(3 )H]cholesterol (UC) or [(3)H]phosphatidylcholine (PC) and incubated with plasma at 37 degrees C. The majority of UC and PC were rapidly transferred to apolipoprotein B (apoB). Subsequently, UC was redistributed to HDL for esterification before being returned to apoB. The presence of a phospholipid transfer protein (PLTP) stimulator or purified PLTP promoted PC transfer to apoB. Conversely, PC transfer was abolished in plasma from PLTP(-/-) mice. Injection of (125)I-LpA-I into rabbits resulted in a rapid size redistribution of (125)I-LpA-I. The majority of [(3)H]UC from labeled r(HDL) was esterified in vivo within HDL, whereas a minority was found in LDL. These data suggest that apoB plays a major role in nascent HDL remodeling by accepting their lipids and donating UC to the LCAT reaction. The finding that nascent particles were depleted of their lipids and remodeled in the presence of plasma lipoproteins raises questions about their stability and subsequent interaction with LCAT.

Small discoidal pre-beta1 HDL particles are efficient acceptors of cell cholesterol via ABCA1 and ABCG1.

The aim of this study was to correlate the lipid content and size of discoidal reconstituted HDL particles with their ability to promote cellular cholesterol efflux. Homogeneous discoidal rHDL particles containing apoA-I and POPC, with diameters of 7.8, 9.6, 10.8, 12.5, and 17.0 nm, were prepared by the cholate dialysis technique. Cholesterol efflux to rHDL was evaluated in pathway-specific cell models for ABCA1-, ABCG1-, and SR-BI-mediated efflux. ABCA1-mediated efflux was efficiently promoted by the 7.8 nm rHDL containing 82 POPC molecules per particle. This rHDL also promoted ABCG1, but not SR-BI, cholesterol efflux. All large and lipid-rich rHDLs, with a diameter of >or=9.6 nm and a phospholipid content of >/=202 molecules per particle, promoted both SR-BI- and ABCG1-mediated efflux. Our results indicated that the ABCA1-mediated cell cholesterol efflux can be efficiently driven not only by monomolecular lipid free/poor apoA-I but also by a small discoidal phospholipid-containing particle resembling plasma pre-beta1 HDL. This same particle also promotes ABCG1- but not SR-BI-mediated efflux. These results help to clarify the role of plasma pre-beta1 HDL in reverse cholesterol transport.

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

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

Structural basis of human high-density lipoprotein formation and assembly at sub nanometer resolution.

Human high-density lipoproteins (HDL) are protein/lipid particles of nanometer sizes. These nano particles are critical for transportation of the "bad cholesterol" from peripheral tissues back to the liver for clearance. An inverse correlation has been observed between the plasma HDL concentration and atherosclerosis. Furthermore, the HDL particle has also been utilized as a vehicle for drug delivery and for intracellular cell biology studies of membrane proteins. The structural basis of HDL formation and assembly, however, is poorly understood. Using high-resolution structural approaches, the formation and assembly of the HDL particle is being examined at atomic resolution, which is reviewed in this chapter. We will mainly focus on our own NMR studies of different apoAI conformations with a brief summary of previously published work by other laboratories.

A unique protease-sensitive high density lipoprotein particle containing the apolipoprotein A-I(Milano) dimer effectively promotes ATP-binding Cassette A1-mediated cell cholesterol efflux.

Carriers of the apolipoprotein A-I(Milano) (A-I(M)) variant present with severe reductions of plasma HDL levels, not associated with premature coronary heart disease (CHD). Sera from 14 A-I(M) carriers and matched controls were compared for their ability to promote ABCA1-driven cholesterol efflux from J774 macrophages and human fibroblasts. When both cell types are stimulated to express ABCA1, the efflux of cholesterol through this pathway is greater with A-I(M) than control sera (3.4 +/- 1.0% versus 2.3 +/- 1.0% in macrophages; 5.2 +/- 2.4% versus 1.9 +/- 0.1% in fibroblasts). A-I(M) and control sera are instead equally effective in removing cholesterol from unstimulated cells and from fibroblasts not expressing ABCA1. The A-I(M) sera contain normal amounts of apoA-I-containing prebeta-HDL and varying concentrations of a unique small HDL particle containing a single molecule of the A-I(M) dimer; chymase treatment of serum degrades both particles and abolishes ABCA1-mediated cholesterol efflux. The serum content of chymase-sensitive HDL correlates strongly and significantly with ABCA1-mediated cholesterol efflux (r = 0.542, p = 0.004). The enhanced capacity of A-I(M) serum for ABCA1 cholesterol efflux is thus explained by the combined occurrence in serum of normal amounts of apoA-I-containing prebeta-HDL, together with a unique protease-sensitive, small HDL particle containing the A-I(M) dimer, both effective in removing cell cholesterol via ABCA1.

A complete backbone spectral assignment of human apolipoprotein AI on a 38 kDa prebetaHDL (Lp1-AI) particle.

ApoAI is the major protein component of the high-density lipoprotein (HDL) that has been a hot subject of interests because of its anti-atherogenic properties. ApoAI/prebeta-HDL is the most effective acceptors specifically for free cholesterol in human plasma and serves as the precursor of HDL particles. Here we report a complete backbone assignment of human apoAI on a 38 kDa prebetaHDL (Lp1-AI) particle.