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

Abstract

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.

Fig. 1.

Western blot analysis of two-dimensional native gel electrophoresis of apolipoprotein A-I (apoA-I)-containing nascent HDL particles generated by incubation of human skin fibroblasts and human lung fibroblasts with human apoA-I. After incubation of human skin fibroblasts and human lung fibroblasts (WI38V13) in which expression of ABCA1 was upregulated with apoA-I (15 μg/ml) for 2 h at 37°C, media were collected and prepared as described in Experimental Procedures. A total of 5 μg of apoA-I-containing particles from each medium was electrophoresed in the first dimension on a 0.7% agarose gel followed by electrophoresis in the second dimension on a 2–36% concave polyacrylamide gel. The nascent HDL bands from the two-dimensional gel were transferred onto a nitrocellulose membrane via a semidry blot system and probed with a polyclonal anti-apoA-I antibody. A: Lipid-free human apoA-I. B: Conditioned medium after 2 h of incubation with human skin fibroblasts. C: Twenty-four hour-conditioned medium from human skin fibroblasts. D: Two hour-conditioned medium from human lung fibroblasts. Molecular size markers (diameter in nanometers) are indicated. The HDL particle diameters are derived from the Rf values of the centers of the various bands as described (10).

Fig. 2.

Gel filtration elution profiles of medium collected after 2, 4, and 24 h incubations of [³H]choline-labeled and ABCA1-stimulated human skin fibroblasts with [¹⁴C]human apoA-I. Human skin fibroblasts were labeled with [³H]choline and treated with 9-cis-retinoic acid and 22-hydroxycholesterol as described in Experimental Procedures. [¹⁴C]human plasma apoA-I (15 μg/ml) was added to the cells. After 2 h (A), 4 h (B), and 24 h (C) incubations at 37°C, media were collected and subjected to Superdex 200 gel filtration chromatography. Fractions were collected and radioactivity was determined by liquid scintillation counting. Open circles, [³H]choline-phospholipid; closed circles, [¹⁴C]human plasma apoA-I. One representative profile is shown out of three independent experiments. PL, phospholipid.

Fig. 3.

Chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS³) of apoA-I in preβ-HDL particles created from ABCA1-expressing human skin fibroblasts. Fast-protein liquid chromatography fractions corresponding to elution volumes of 76–82 ml (see Fig. 2A) were pooled and dialyzed overnight against 0.1 M phosphate buffer, pH 7.4, concentrated to 1–2 mg apoA-I/ml using an Amicon Ultra-centrifugal filter, and then incubated with BS³ (10 mM) at room temperature. The cross-linked apoA-I samples were analyzed by 4–20% gradient SDS-PAGE. Lane 1, lipid-free apoA-I (applied 3 μg of apoA-I in 5 μl); lane 2, preβ-HDL particles (applied 5 μg of apoA-I in 5 μl); lane 3, lipid-free apoA-I plus BS³ (applied 15 μg of apoA-I in 15 μl); lane 4, preβ-HDL particles plus BS³ (applied 10 μg of apoA-I in 15 μl). Positions of monomer, dimer, trimer, and tetramer apoA-I were assigned according to their calculated molecular weights.

Fig. 4.

Protection of apoA-I in preβ-HDL particles from proteolysis. As described in Experimental Procedures, 50 μg of lipid-free apoA-I and 50 μg of preβ-HDL particles were incubated with enteropeptidase (0.13 U/μg apoA-I) for 4 h at 37°C and analyzed by 4–20% SDS-PAGE. Lane 1, 3 μg of lipid-free apoA-I; lane 2, 3 μg of enteropeptidase alone; lane 3, 5 μg of lipid-free apoA-I digested by enteropeptidase; lane 4, 5 μg of preβ-HDL particles alone; lane 5, 5 μg of preβ-HDL particles digested by enteropeptidase. Lanes 1–3 were visualized by Brilliant Colloidal G stain, whereas lanes 4, 5 were visualized by immunoblotting with anti-human apoA-I antibody.

Fig. 5.

Western blot analysis of two-dimensional native gel electrophoresis of apoA-I-containing particles generated by incubation of membrane lipid multilamellar vesicles (MLVs) with apoA-I. The MLVs were prepared with a mixture of natural phosphatidylcholine (PC), sphingomyelin, lyso-PC, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cholesterol and were incubated with human apoA-I for 6 h as described in Experimental Procedures. The particles were separated by two-dimensional gel electrophoresis as described for Fig. 1 and detected by immunoblotting using an antibody against apoA-I.

Fig. 6.

Western blot analysis of two-dimensional native gel electrophoresis of apoA-I-containing HDL particles generated by incubation of ABCA1-expressing human skin fibroblasts with human apoA-I (Δ223–243). Human skin fibroblasts were incubated with apoA-I (Δ223–243) (15 μg/ml) for 2 h at 37°C, and HDL particles in the media were separated by two-dimensional gel electrophoresis as described for Fig. 1. A: Lipid-free apoA-I (Δ223–243). B: Two hour-conditioned medium from human skin fibroblasts. C: Twenty-four hour-conditioned medium from human skin fibroblasts.

Fig. 7.

Cellular cholesterol efflux capabilities of preβ-HDL. A: Preβ-HDL particles were isolated from 2 h-conditioned medium of human fibroblasts by fast-protein liquid chromatography (see Fig. 2) and compared with lipid-free apoA-I in their abilities to mediate cholesterol efflux from ABCA1-expressing human skin fibroblasts. FC, free (unesterified) cholesterol. Error bars represent mean ± SD. B: Pure preβ-HDL particles (gel A) were incubated at 15 μg apoA-I/ml with ABCA1-expressing human skin fibroblasts for 24 h and analyzed by native two-dimensional gel electrophoresis as described for Fig. 1 (gel B).

Fig. 8.

Solubilization of dimyristoyl phosphatidylcholine (DMPC) MLVs by apoA-I and preβ-HDL. Representative time courses for the clearance of MLVs at 24°C are shown. The DMPC MLV concentration was 0.25 mg/ml, and the apoA-I concentration was 0.1 mg/ml.