Nascent high density lipoproteins formed by ABCA1 resemble lipid rafts and are structurally organized by three apoA-I monomers

Abstract

This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ∼ 12, 10, 7.5, and <6 nm) and composition were purified by size-exclusion chromatography. Electron microscopy suggested that the nHDL were mostly spheroidal. The proportions of the principal nHDL lipids, free cholesterol, glycerophosphocholine, and sphingomyelin were similar to that of lipid rafts, suggesting that the lipid originated from a raft-like region of the cell. Smaller amounts of glucosylceramides, cholesteryl esters, and other glycerophospholipid classes were also present. The largest particles, ∼ 12 nm and 10 nm diameter, contained ∼ 43% free cholesterol, 2-3% cholesteryl ester, and three apoA-I molecules. Using chemical cross-linking chemistry combined with mass spectrometry, we found that three molecules of apoA-I in the ∼ 9-14 nm nHDL adopted a belt-like conformation. The smaller (7.5 nm diameter) spheroidal nHDL particles carried 30% free cholesterol and two molecules of apoA-I in a twisted, antiparallel, double-belt conformation. Overall, these new data offer fresh insights into the biogenesis and structural constraints involved in forming nascent HDL from ABCA1.

Fig. 1.

Nascent HDL particle diameter distribution. A: Distribution of nHDL particles from cells incubated for 18–24 h with 10 µg/ml of lipid-free human ¹²⁵I-apoA-I in serumfree media. After incubation, the medium was dialyzed and concentrated, and nHDL particles were purified based on size using FPLC as described in Materials and Methods. The radioactive content of each fraction was determined and plotted as the percentage of total ¹²⁵I-apoA-I (filled circles) per fraction. As a control, HEK cells not expressing ABCA1 (Flp-In™) were incubated in the same manner as ABCA1-expressing cells and plotted as the percentage of total ¹²⁵I-apoA-I (filled squares) per fraction. In other experiments, cells were preincubated with 1 µCi/ml of ³H-cholesterol overnight and incubated with 10 µg/ml unlabeled lipid-free apoA-I. The radioactive content of each fraction was determined and plotted as the percentage of total ³H-cholesterol from ABCA1-expressing cells (open circles) or nonABCA1-expressing cells (open squares) per fraction. Aliquots of peak fractions (Fx) were taken and separated using 4–30% NDGE and are shown in the inset. Radioactive protein bands were visualized on the gel using phosphorimaging. From these experiments, Peaks 1–4 were defined: Peak 1 = fractions 110–125, Peak 2 = fractions 126–139, Peak 3 = fractions 140–157, and Peak 4 = fractions 158–175. B: Analysis of nHDL from ABCA1-expressing cells. In these experiments, ABCA1-expressing cells were incubated with 10 µg/ml of unlabeled apoA-I for 24 h as outlined above and in Materials and Methods. Fractions were pooled as described in A, and aliquots containing 10 µg of apoA-I mass were analyzed by 4–30% NDGE using Coomassie Blue G for visualization: Lane 1, pooled Peak 1 nHDL; Lane 2, pooled Peak 2 nHDL; Lane 3, pooled Peak 3 nHDL; Lane 4, pooled Peak 4 nHDL. Lane MW shows the Stoke's diameter for high molecular weight standards. C: To determine the number of apoA-I molecules per particle, aliquots of pooled nHDL fractions were subjected to cross-linking using a 100:1 molar ratio of DSP to apoA-I. Cross-linked nHDL were separated using nonreducing 4–12% SDS-PAGE. The lanes are as follows: ApoA-I Ladder; Pk1 = cross-linked Peak 1 nHDL; Pk2 = cross-linked Peak 2 nHDL; Pk3 = cross-linked Peak 3 nHDL; Dimer Mix = unreduced mixture of Q109C apoA-I homodimer and A176C apoA-I homodimer (24). These figures represent typical analyses from four independent experiments.

Fig. 2.

Electron microscopy evaluation of nascent HDL. Aliquots of Peaks 1–4 prepared and purified as described in Materials and Methods were adjusted to 0.1 µg/ml of protein and evaluated by electron microscopy. A–D: Representative images of nHDL. A′–D′: The corresponding histogram distribution of particle diameters in nm. A and A′, Peak 1 nHDL; B and B′, Peak 2 nHDL; C and C′, Peak 3 nHDL; D and D′, Peak 4 nHDL. Results shown are representative of data obtained from the analysis of particles from three independent experiments.

Fig. 3.

Distribution of major lipid components in nHDL. A: Total lipid (mol%) for each of the three major classes of lipids. Compositional analysis for FPLC Peaks 1–4 showed that the major lipids comprising nHDL particles were cholesterol (hatched columns), GP (open columns), and SM (filled columns). B: The nmole content of free (open columns) and CE (filled columns) for Peaks 1–4. C: the GP class distribution for FPLC Peaks 1–4 are expressed as nmol total lipid per particle. D: GP class distribution for FPLC Peaks 1–4 expressed as mol%. GP accounts for 40–70% of the total nHDL lipids. Analyses were carried out as described in Materials and Methods. For each class, the GP was separated by HPLC, and individual fractions collected were analyzed for lipid phosphorus. Values represent total cell lipid extract. Total cholesterol and FC were measured using GC/MS methodology; GP, SM, and GC were analyzed using LC/MS/MS. Values are from three or more independent experiments. Statistical differences are indicated by different letters at P < 0.05.

Fig. 4.

Comparison of lipid raft composition and HEK293 cells and BMDM nHDL. A: The ratio of total cholesterol to PC for isolated plasma membrane, lipid rafts, and nHDL particles derived from HEK 293 cells overexpressing ABCA1 or the same fractions isolated from BMDMs. B: SM-PC ratio for the same fractions listed in A. Fractions from HEK cells or BMDM were obtained using the nondetergent procedure as described in Materials and Methods. C: Purity of the raft and nonraft fractions after Western blotting for Flotillin-1 and Transferrin receptor after 12% SDS-PAGE. Values are from two or three independent experiments for each cell type. FC and total cholesterol was measured using GC/MS methodology; PC, and SM were analyzed using LC/MS/MS.

Fig. 5.

Sphingomyelin fatty acyl chain length and saturation in nHDL. A: SM fatty acyl chain length shown as the total number of carbons for FPLC Peaks 1–4. B: Saturated and unsaturated fatty acyl chains (mol%) carried by PC for FPLC Peaks 1–4. All samples were analyzed by LC-MS/MS. Molar abundance for each SM chain length was calculated from the SM mass spectra after correction for isotope and the mass dependent instrument response. Results shown are the mean ± SD of four independent experiments. Within a specific class, statistically significant differences are indicated by different letters at P < 0.05.

Fig. 6.

Conformation of two ApoA-I molecules on 7.5 nm nascent HDL particles. Molecular models of 7–8 nm nHDL particles (Peak 3) containing 26 molecules of total lipid (Table 2). The lipids include representative PC, SM, GCer, CE, and FC in the same ratio reported in Table 1. A: Two apoA-I molecules arranged in an antiparallel orientation. The regions of the two strands are indicated by the residue number. Note that the configuration of the protein is helical. B: The two strands shown in A, including 26 lipids. C: The particle in B with the apoA-I displayed as a space-filling surface. Glycerophospholipid is indicated by chains having a green backbone and white hydrogen atoms, ceramides are in blue, cholesteryl ester is in green, and free cholesterol is in solid red.

Fig. 7.

Conformation of three ApoA-I molecules on ∼9–11 nm nascent HDL particles. Molecular models of 9–11 nm diameter nHDL particles (Peak 2) containing 266 molecules of total lipid. The lipids include representative PC, SM, GCer, CE, and FC in the same ratio reported in Table 2. A: Three apoA-I strands in an antiparallel orientation. The two outer strands are in the same orientation but are antiparallel to the center strand. Regions of the three strands are indicated by residue numbers. Note that apoA-I assumes a belt configuration. B: The three strands shown in A wrapped around 239 lipids. C: B with the apoA-I displayed as a space-filling surface. Glycerophospholipid is indicated by chains having a green backbone and white hydrogen atoms, ceramides are in blue, CE in green, and FC in solid red.

Fig. 8.

Formation of nHDL Particles by ABCA1. Lipidation of nascent HDL particles by ABCA1. A: The large jump in total lipid content per apoA-I between the lipid rich 9–11 nm diameter nHDL and the lipid-poor 7.5 nm diameter nHDL. These data suggest that accessory or chaperone protein(s) may aid in coordination and opening of lipid-poor apoA-I for assembly of the 3-apoA-I lipid-rich particles. B: Mechanism for the ABCA1-mediated lipidation of apoA-I. apoA-I binds at the membrane surface, possibly to accessory protein(s), and then moves to ABCA1. These accessory protein(s) are probably involved in how the apoA-I /ABCA1 complex adds a second apoA-I or how lipid-poor apoA-I is released. ABCA1 is not located in lipid rafts (regions of high cholesterol and sphingomyelin content), but these regions participate as the source of lipids transferred to nHDL. When a second molecule of apoA-I binds, then the two apoA-I/ABCA1 complex can package small amounts of cholesterol and then be released as the 7.5 nm nHDL particle. However, to package maximal amounts of cholesterol, a third molecule of apoA-I must be present to adapt to the proper conformation and generate the ∼9–11 nm diameter nHDL, thus allowing ABCA1 to coordinate the removal of excess membrane cholesterol and maintain lipid raft integrity.