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 ef flux from human skin fibroblasts and macrophage was examined. Four apoA-I m utants were designed, each by deletion of a pair of predicted adjacent heli ces; 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-t ype apoA-I and mutants with central domain deletions, Delta(187-243) exhibi ted a marked reduction in its ability to promote either cholesterol or phos pholipid efflux from THP-1 macrophages. This mutant also demonstrated a dec reased ability to bind lipids and to form lipoprotein complexes. In contras t, the four mutants and apoA-I equally supported cholesterol efflux from fi broblasts, albeit with a reduced capacity when compared to macrophages. Del ta(187-243) bound poorly to the macrophage cell surface when compared to ap oA-I, and competitive binding studies with the central domain and C-termina l deletions mutants showed that only Delta(187-243) did not compete effecti vely with [I-125]apoA-I. Omission of PMA during cholesterol loading enhance d 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 l oading and, in the absence of PMA, increased and equalized cholesterol effl ux to apoA-I and a(187-243). Surprisingly, omission of PMA during cholester ol loading had minimal effects on the binding of apoA-I or Delta(187-243) t o the THP-1 cell surface. Overall, these results show that cholesterol effl ux from cells such as fibroblasts does not require:any specific sequence be tween residues 100 and 243 of apoA-I. In contrast, optimal cholesterol effl ux 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.