Deletion of the C-terminal domain of apolipoprotein A-I impairs cell surface binding and lipid efflux in macrophage.
The contribution of the amphipathic alpha-helices of apoA-I toward lipid efflux from human skin fibroblasts and macrophage was examined. Four apoA-I mutants were designed, each by deletion of a pair of predicted adjacent helices. Three mutants lacked two consecutive central alpha-helices [Delta(100-143), Delta(122-165), and Delta(144-186)], whereas the final mutant lacked the C-terminal domain [Delta(187-243)]. When compared to recombinant wild-type apoA-I and mutants with central domain deletions, Delta(187-243) exhibited a marked reduction in its ability to promote either cholesterol or phospholipid efflux from THP-1 macrophages. This mutant also demonstrated a decreased ability to bind lipids and to form lipoprotein complexes. In contrast, the four mutants and apoA-I equally supported cholesterol efflux from fibroblasts, albeit with a reduced capacity when compared to macrophages. Delta(187-243) bound poorly to the macrophage cell surface when compared to apoA-I, and competitive binding studies with the central domain and C-terminal deletions mutants showed that only Delta(187-243) did not compete effectively with [(125)I]apoA-I. Omission of PMA during cholesterol loading enhanced cholesterol efflux to both apoA-I (1.5-fold) and the C-terminal deletion mutant (2.5-fold). Inclusion of the Sandoz ACAT inhibitor (58-035) during loading and, in the absence of PMA, increased and equalized cholesterol efflux to apoA-I and Delta(187-243). Surprisingly, omission of PMA during cholesterol loading had minimal effects on the binding of apoA-I or Delta(187-243) to the THP-1 cell surface. Overall, these results show that cholesterol efflux from cells such as fibroblasts does not require any specific sequence between residues 100 and 243 of apoA-I. In contrast, optimal cholesterol efflux in macrophages requires binding of the C-terminal domain of apoA-I to a cell surface-binding site and the subsequent translocation of intracellular cholesterol to an efflux-competent pool.
Burgess
J W
JW
Lipoprotein & Atherosclerosis Group, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, Ontario, Canada.
Frank
P G
PG
Franklin
V
V
Liang
P
P
McManus
D C
DC
Desforges
M
M
Rassart
E
E
Marcel
Y L
YL
D000577:Amides / Q000494:pharmacology; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D017646:Organosilicon Compounds / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D002785:Sterol O-Acyltransferase / Q000037:antagonists & inhibitors; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000577:Amides; D016632:Apolipoprotein A-I; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D017646:Organosilicon Compounds; D010743:Phospholipids; D011994:Recombinant Proteins; C040256:SAN 58035; D002784:Cholesterol; D002785:Sterol O-Acyltransferase; D013755:Tetradecanoylphorbol Acetate