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Deletion of the C-Terminal Domain of Apolipoprotein A-I Impairs Cell Surface Binding and Lipid Efflux in Macrophage

The contribution of the amphipathic α-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 α-helices [Δ(100−143), Δ(122−...

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Published in:Biochemistry (Easton) 1999-11, Vol.38 (44), p.14524-14533
Main Authors: Burgess, Jim W, Frank, Philippe G, Franklin, Vivian, Liang, Ping, McManus, Dan C, Desforges, Marc, Rassart, Eric, Marcel, Yves L
Format: Article
Language:English
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Summary:The contribution of the amphipathic α-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 α-helices [Δ(100−143), Δ(122−165), and Δ(144−186)], whereas the final mutant lacked the C-terminal domain [Δ(187−243)]. When compared to recombinant wild-type apoA-I and mutants with central domain deletions, Δ(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. Δ(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 Δ(187−243) did not compete effectively with [125I]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 Δ(187−243). Surprisingly, omission of PMA during cholesterol loading had minimal effects on the binding of apoA-I or Δ(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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi990930z