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 ce lls 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 wer e combined with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and FC to make reconstituted high density lipoprotein (rHDL) discoidal compl exes, These particles were characterized structurally and for their ef ficiency as accepters of mouse L-cell fibroblast cholesterol, The dele tion mutant proteins lacked NH2-terminal (apoA-I (Delta 44-126)), cent ral (apoA-I (Delta 139-170)), or COOH-terminal (apoA-I (Delta 190-243) ) domains of apoA-I, The three deletion mutants all displayed lipid-bi nding abilities and formed discoidal complexes that were similar in ma jor diameter (13.2 +/- 1.5 nm) to those formed by human apoA-I when re constituted at a 100:5:1 (POPC:FC: protein) mole ratio, Gel filtration profiles indicated unreacted protein in the preparation made with apo A-I (Delta 190-243), which is consistent with the COOH terminus portio n of apoA-I being an important determinant of lipid binding, Measureme nts of the percent cu-helix content of the proteins, as well as the nu mber of protein molecules per rHDL particle, gave an indication of the arrangement of the deletion mutant proteins in the discoidal complexe s, The rHDL particles containing the deletion mutants had more molecul es 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 deter mined number of helical segments with an estimate of the available spa ce indicated that the deletion mutant proteins are probably more loose ly arranged than apoA-I around the edge of the rHDL, The abilities of the complexes to remove radiolabeled FC were compared in experiments u sing cultured mouse L-cell fibroblasts, All four discoidal complexes d isplayed similar abilities to remove FC from the plasma membrane of L- cells when compared at an acceptor concentration of 50 mu g of phospho lipid/ml. Thus, none of the deletions imposed in this study notably al tered 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 no t dependent on specific helical segments.