THE INFLUENCE OF APOLIPOPROTEIN STRUCTURE ON THE EFFLUX OF CELLULAR FREE-CHOLESTEROL TO HIGH-DENSITY-LIPOPROTEIN

The influence of apolipoprotein conformation on the ability of high de nsity lipoprotein (HDL) to remove cellular free cholesterol (FC) has n ot been studied in detail. To address the effects of amphipathic cu-he lix structure on cellular FC efflux, three class A helical peptides an d apolipoprotein (ape) All were complexed to dimyristoyl phosphatidylc holine (DMPC) to make discoidal complexes that were used as accepters of cell cholesterol. The peptides consisted of an 18-amino acid, amphi pathic, alpha-helical peptide with the sequence DWL-KAFYDKVAEKLKEAF (1 8A), a dimer of 18A covalently linked by a proline residue (37pA), and acetyl-18A-amide (Ac-18A-NH2) that has a higher alpha-helix content t han the unblocked 18A molecule. The three peptides strongly mimic the lipid-binding characteristics of the amphipathic segments of apolipopr oteins and form discoidal complexes with DMPC that are similar in diam eter (11-12 nm) to those formed by human apoAI when reconstituted at a 2.5:1 (w:w) phospholipid to protein ratio. The abilities of these com plexes to remove radiolabeled FC were compared in experiments using cu ltured mouse L-cell fibroblasts; efflux of FC from both the plasma mem brane and the lysosomal pools was examined. For each of the accepters, the removal of cholesterol from the plasma membrane and lysosomal poo ls was equally efficient. All four discoidal complexes were equally ef ficient cell membrane FC accepters when compared at saturating accepto r concentrations of >200 mu g of DMPC/ml of medium. However, at the sa me lipid concentration, protein-free DMPC small unilamellar vesicles ( SUV) were significantly less efficient. The initial rates of FC remova l from cells at saturating concentrations of acceptor particles (V-max ) were 12, 10, 10, and 11% per h, respectively, for the complexes cont aining either 18A, Ac-18A-NH2, 37pA, or apoAI, but only 1% cellular FC per h for the DMPC SUV. The 10-fold higher V-max for the apoprotein/p eptide containing accepters was likely due to a reversible interaction of apoprotein or peptide with the plasma membrane that changed the li pid packing characteristics in such a way as to increase the rate of F C desorption from the cell surface. This interaction required amphipat hic alpha-helical segments, but it was not affected by the length, num ber, or lipid-binding affinity of the helices. Furthermore, the efflux efficiency was not dependent on the amino acid sequence of the helica l segments which suggests that this interaction is not mediated by a s pecific cell surface binding site. At lower acceptor concentrations of <100 mu g of DMPC/ml of medium, differences in efflux efficiency were seen between the peptide-containing particles as a group and the apoA I-containing particle demonstrating that cu-helix structure can affect the ability of acceptor particles to either sequester or retain chole sterol molecules.