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

Abstract

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.

Influence of ApoA-I Structure on the ABCA1-mediated Efflux of Cellular Lipids

“…3B). These results are generally consistent with those of an earlier study in which it was shown that neither C-terminal nor central domain mutations of apoA-I alter lipid efflux from fibroblasts (27). However, as was seen with J774 cells (Fig.…”

“…It is apparent that deletion of the central domains (residues 44 -126 and 123-166) did not influence FC or PL efflux. These results agree with an earlier investigation in which it was shown that the elimination of the central domain of apoA-I had a minimal effect on ABCA1-dependent lipid efflux from THP-1 macrophages (27). Deletion of the N-terminal hydrophobic ␣-helix (residues 44 -65) and the N-terminal region (residues 1-43) led to a slight reduction in FC efflux relative to WT apoA-I (Fig.…”

“…In response to FC loading, a 2-2.5-fold enhancement in fractional PL and FC efflux to WT apoA-I was observed for fibroblasts and macrophages (16,17,27). It is clear that removal of segments in either the N-terminal or the central domains of apoA-I did not significantly affect the lipid efflux from FC-loaded fibroblasts and macrophages when they were exposed to 20 g/ml apoA-I (Fig.…”

“…Several laboratories have used engineered apoA-I variants to examine the contributions of different regions of the apoA-I molecule to membrane microsolubilization (17,26,27). Thus, the C-terminal region of apoA-I is essential for optimal ABCA1-mediated lipid efflux although there may be cell-specific effects because C-terminal truncation of apoA-I has been reported to significantly reduce lipid efflux from macrophages, but not from fibroblasts (27).…”

“…Thus, the C-terminal region of apoA-I is essential for optimal ABCA1-mediated lipid efflux although there may be cell-specific effects because C-terminal truncation of apoA-I has been reported to significantly reduce lipid efflux from macrophages, but not from fibroblasts (27). In addition to this uncertainty about the quantitative contribution of the apoA-I C-terminal domain, there is disagreement about the role of the helix bundle domain.…”