Mechanism of Prebeta-HDL Formation and Activation

Abstract

The mechanism of formation of functional high-density lipoprotein (HDL) from secreted lipid-free apolipoprotein A1 (apo A1) was determined using human liver-derived (HepG2) cells, human intestine-derived (CaCO2) cells, and CHO cells stably expressing full-length human apo A1 (CHO-A1 cells). In each cell line, a significant proportion of secreted apo A1 had a Stokes radius of 2.6 nm and was inactive in binding phospholipids (PL) or free cholesterol (FC). Extracellularly, in a reaction dependent on membrane transporter ABCA1, prealpha-migrating 2.6 nm apo A1 was converted to a prebeta-migrating product that was able to bind PL. Both forms were reactive with mAb55201, a monoclonal antibody specific for native plasma lipid-poor (prebeta1) HDL [Nakamura, Y., et al. (2004) Biochemistry 43, 14311-14318]. The physical properties of precursor and product apo A1 suggested that both are monomers, with Stokes radii of 2.6 and 3.6 nm, respectively, consistent with the absence of intermolecular cross-linking of apo A1 in lipid-poor HDL, reported previously. Product but not precursor apo A1 promoted reverse cholesterol transport (RCT) from human aortic smooth muscle cells. These studies suggest an important contribution of secreted lipid-free apo A1 to HDL formation.

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

“…3, lane 4). The finding that apoA-I in preb-HDL is monomeric is consistent with prior reports (11,12,15,21,31).…”

“…Direct analysis of the PL and FC contents of the preb-HDL confirms that there are three to four PL and one to two FC molecules associated with each apoA-I molecule ( Table 1). This PL/apoA-I ratio agrees with reports of preb-HDL composition using other cell types (11,21), reconstituted particles (26,31), and human plasma preb-HDL (12). However, the reported FC contents of preb-HDL particles vary widely.…”

“…Lipid-free apoA-I in the monomeric state exists in a relatively expanded form because the hydrodynamic diameter of ?6 nm is larger than that expected for a globular protein of the same molecular weight (8). The diameter of 7.5 nm for the preb-HDL particles created by fibroblasts is consistent with the sizes of equivalent particles formed by other cell types (11,21). The two-dimensional gel in Fig.…”

“…1B, D). Because larger, discoidal nascent HDL particles are not formed in this time frame, it seems that lipid-poor apoA-I molecules can be formed directly during the efflux of cellular PL via ABCA1 (21). These lipid-poor apoA-I molecules are apparently intermediates in the formation of larger discoidal HDL particles, because, upon longer incubation with the cells, they are converted into discs that contain 100-200 PL molecules (10).…”

“…The apoA-I molecules that form the preb1-HDL pool presumably acquire their lipid via the ABCA1 pathway. Indeed, there is evidence that monomeric preb1-HDL particles containing a little PL are formed when lipid-free apoA-I reacts with ABCA1 (11,21).…”