Abstract
The role of HDL and its major protein constituent, apolipoprotein (apo) A-I, in promoting the removal of excess cholesterol from cultured cells has been well established; 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 acceptors of mouse L-cell fibroblast cholesterol. The deletion mutant proteins lacked NH2-terminal (apoA-I (Delta44-126)), central (apoA-I (Delta139-170)), or COOH-terminal (apoA-I (Delta190-243)) 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 (Delta190-243), which is consistent with the COOH terminus portion of apoA-I being an important determinant of lipid binding. Measurements of the percent alpha-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 microg 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.
Highlights
High density lipoproteins (HDL)1 are a heterogeneous class of particles thought to mediate the flux of unesterified cholesterol (FC) from peripheral cells to the liver in the process of reverse cholesterol transport [1]
In an attempt to define the domain of apoA-I that is crucial for cholesterol efflux from cells, the present study uses acceptor particles containing these engineered apoA-I molecules reconstituted with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) into homogeneous and highly defined discoidal complexes
Cholesterol efflux studies indicate that when reconstituted HDL (rHDL) particles are utilized as acceptors of FC, there are no significant differences in the rates of efflux to the discoidal HDL containing either intact apoA-I or the deletion mutant proteins
Summary
High density lipoproteins (HDL) are a heterogeneous class of particles thought to mediate the flux of unesterified (free) cholesterol (FC) from peripheral cells to the liver in the process of reverse cholesterol transport [1]. In an attempt to define the domain of apoA-I that is crucial for cholesterol efflux from cells, the present study uses acceptor particles containing these engineered apoA-I molecules reconstituted with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) into homogeneous and highly defined discoidal complexes. Cholesterol efflux studies indicate that when rHDL particles are utilized as acceptors of FC, there are no significant differences in the rates of efflux to the discoidal HDL containing either intact apoA-I or the deletion mutant proteins. These data indicate that the deletion of large segments from either the NH2-terminal, central or COOH-terminal regions of the apoA-I molecule does not significantly impair the functionality of the rHDL complexes as acceptors of cellular cholesterol
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