Abstract
The nascent HDL created by ABCA1-mediated efflux of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apolipoprotein A-I (apoA-I) has not been defined. To address this issue, we characterized the lipid particles released when J774 mouse macrophages and human skin fibroblasts in which ABCA1 is activated are incubated with human apoA-I. In both cases, three types of nascent HDL containing two, three, or four molecules of apoA-I per particle are formed. With J774 cells, the predominant species have hydrodynamic diameters of approximately 9 and 12 nm. These discoidal HDL particles have different FC contents and PL compositions, and the presence of acidic PL causes them to exhibit alpha-electrophoretic mobility. These results are consistent with ABCA1 located in more than one membrane microenvironment being responsible for the production of the heterogeneous HDL. Activation of ABCA1 also leads to the release of apoA-I-free plasma membrane vesicles (microparticles). These larger, spherical particles released from J774 cells have the same PL composition as the 12 nm HDL and contain CD14 and ganglioside, consistent with their origin being plasma membrane raft domains. The various HDL particles and microparticles are created concurrently, and there is no precursor-product relationship between them. Importantly, a large fraction of the cellular FC effluxed from these cells by ABCA1 is located in microparticles. Collectively, these results show that the products of the apoA-I/ABCA1 interaction include discoidal HDL particles containing different numbers of apoA-I molecules. The cellular PLs and cholesterol incorporated into these nascent HDL particles originate from different cell membrane domains.
Highlights
The goal of this study was to clarify the situation by focusing on the characterization of the nascent HDL particles formed by the interaction of apolipoprotein A-I (apoA-I) with ABCA1 [39, 40]
Because both lipidfree apoA-I and apoA-I/PC discoidal complexes exhibit preb-electrophoretic mobility, the extra negative surface charge leading to a-mobility arises from the presence of acidic PS and phosphatidylinositol molecules in the nascent HDL particles (Table 2) [42]
The ability of cellular acidic PL to induce a-electrophoretic mobility in apoA-I-containing lipoprotein particles has been demonstrated with fibroblasts [43] and other cell types [14, 15]
Summary
FC efflux (in the absence of apoA-I) was subtracted. For PL efflux, after the medium was filtered, the lipids in the medium were extracted by the procedure of Bligh and Dyer [29]; the aqueous phase containing any free [3H]choline was aspirated, and the chloroform phase was washed three times with 10:9 (v/v) methanol-water. Lipids were extracted from aliquots of the concentrated medium by the method of Bligh and Dyer [29]; cholesteryl methyl ether was added as an internal standard for the gas-liquid chromatographic assay of FC [30]. This method can detect as little as 1.5 mg of FC. To analyze the PL subclasses present in the particles, lipids from a 250 mg PL aliquot of apoA-I-containing particles were extracted by the method of Bligh and Dyer [29] and separated by TLC using silica gel H plates with a mobile phase of chloroform-methanol-acetic acid-water (50:25:8:1, v/v). Components of the nascent HDL particles present at >1% of total PL could be detected
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