Kl. Gillotte et al., APOLIPOPROTEIN-A-I STRUCTURAL MODIFICATION AND THE FUNCTIONALITY OF RECONSTITUTED HIGH-DENSITY-LIPOPROTEIN PARTICLES IN CELLULAR CHOLESTEROL EFFLUX, The Journal of biological chemistry, 271(39), 1996, pp. 23792-23798
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.