Apolipoprotein A-I structural modification and the functionality of reconstituted high density lipoprotein particles in cellular cholesterol efflux

The role of HDL and its major protein constituent, apolipoprotein (ape) A-I, in promoting the removal of excess cholesterol from cultured cells has been well es tablished; however, the mechanisms by which this occurs are not completely understood, To address the effects of apoA-I modification on cellular unesterified (free) cholesterol (FC) efflux, three recombinant human apoA-I deletion mutants and plasma apoA-I were combined with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and FC to make reconstituted high density lipoprotein (rHDL) discoidal complexes, These particles were characterized structurally and for their efficiency as accepters of mouse L-cell fibroblast cholesterol, The deletion mutant proteins lacked NH2-terminal (apoA-I (Delta 44-126)), central (apoA-I (Delta 139-170)), or COOH-terminal (apoA-I (Delta 190-243)) domains of apoA-I, The three deletion mutants all displayed lipid-binding abilities and formed discoidal complexes that were similar in major diameter (13.2 +/- 1.5 nm) to those formed by human apoA-I when reconstituted at a 100:5:1 (POPC:FC: protein) mole ratio, Gel filtration profiles indicated unreacted protein in the preparation made with apoA-I (Delta 190-243), which is consistent with the COOH terminus portion of apoA-I being an important determinant of lipid binding, Measurements of the percent cu-helix content of the proteins, as well as the number of protein molecules per rHDL particle, gave an indication of the arrangement of the deletion mutant proteins in the discoidal complexes, The rHDL particles containing the deletion mutants had more molecules of protein present than particles containing intact apoA-I, to the extent that a similar number of helical segments was incorporated into each of the discoidal species, Comparison of the experimentally determined number of helical segments with an estimate of the available space indicated that the deletion mutant proteins are probably more loosely arranged than apoA-I around the edge of the rHDL, The abilities of the complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts, All four discoidal complexes displayed similar abilities to remove FC from the plasma membrane of L-cells when compared at an acceptor concentration of 50 mu g of phospholipid/ml. Thus, none of the deletions imposed in this study notably altered the ability of the rHDL particles to participate in cellular FC efflux, These results suggest that efficient apoA-I-mediated FC efflux requires the presence of amphipathic alpha-helical segments but is not dependent on specific helical segments.