Abstract 1203: Complexation of an ApoA-I Mimetic Peptide with Phospholipid Increases ABCA1-specific Cholesterol Efflux
The ABCA1 transporter promotes the efflux of cholesterol to amphipathic helical containing peptides, but the optimum state of lipidation for mediating ABCA1-specific efflux and for reducing the hemolytic properties of these peptides is unknown. The synthetic peptide 37pA containing two class-A amphipathic helices linked by proline was complexed with various amounts of phosphatidylcholine (DPPC) to produce a small (peptide:lipid; 1:9), medium (1:15), and large (1:30) discoidal complexes (10–20 nm). The detergent solubilizing capacity, as assessed by DMPC vesicle solubiliztion, of the small complex was reduced by approximately 65% compared to the free peptide. The medium and large complexes were almost completely ineffective in solubilizing DMPC vesicles. Similarly, hemolysis was significantly reduced by addition of the smallest amount of DPPC when compared with the free peptide (7% RBC hemolysis/2h/80μg/mL vs. 27%, respectively). Finally, the free peptide and the three complexes were tested for cholesterol efflux from ABCA1 transfected and control HeLa cells. The addition of 9 mole of phospholipid per mole of 37pA to produce the small complex increased ABCA1-specific cholesterol efflux by an almost doubling of the Vmax when compared to the free peptide but with a reduced affinity (Vmax=9.7% cholesterol efflux/18h; Km=8.1μg/mL vs. Vmax=5.0%/18h; Km=1.2μg/mL, respectively). The addition of more phospholipid to produce the medium size complex increased Vmax to 13.0%/18h and Km to 21.9μg/mL. The addition of more phospholipid did not further increase ABCA1-specific cholesterol efflux but increased ABCA1-independent efflux by more than 5-fold from the control cell line. ABCA1-specific phospholipid efflux showed a similar pattern but ABCA1-nonspecific efflux of phospholipids from control HeLa cells was almost completely abolished with the addition of any phopsholipid to the peptide. In summary, addition of phospholipd to 37pA can enhance its ability to efflux cholesterol by the ABCA1 transporter and reduces its hemolytic potential, suggesting that partially lipidated apolipoproteins may be the endogenous lipid acceptors for ABCA1 and that partially lipidated apoA-I mimetic peptides may be the preferred therapeutic form of such peptides.