Abstract
ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. ABCA1 contains disulfide bond(s) between its N- and C-terminal halves, but it remains unclear whether disulfide bond formation is important for the functions of ABCA1 and which cysteines are involved in disulfide bond formation. To answer these questions, we constructed >30 ABCA1 mutants in which 16 extracellular domain (ECD) cysteines were replaced with serines and examined disulfide bond formation, apoA-I binding, and HDL formation in these mutants. From the single cysteine replacements, two cysteines (Cys(75) and Cys(309)) in ECD1 were found to be essential for apoA-I binding. In contrast, in ECD2, only Cys(1477) was found to be essential for HDL formation, and no single cysteine replacement impaired apoA-I binding. The concurrent replacement of two cysteines, Cys(1463) and Cys(1465), impaired apoA-I binding and HDL formation, suggesting that four of five extracellular cysteines (Cys(75), Cys(309), Cys(1463), Cys(1465), and Cys(1477)) are involved in these functions of ABCA1. Trypsin digestion experiments suggested that one disulfide bond is not sufficient and that two intramolecular disulfide bonds (between Cys(75) and Cys(309) in ECD1 and either Cys(1463) or Cys(1465) and Cys(1477) in ECD2) are required for ABCA1 to be fully functional.
Highlights
Maintenance of cellular cholesterol homeostasis is important for normal human physiology; its disruption can lead to a variety of pathological conditions, including cardiovascular disease [1]
ABCA1(Cys5) showed apoA-Idependent cholesterol efflux as efficiently as the wild type, whereas C2/6/13/14/15S did not show any activity. These results suggest that four cysteine residues (C2, C6, C15, and either C13 or C14) in ECD1 and ECD2 of ABCA1 are required for apoA-I-dependent cholesterol efflux
ApoA-I-dependent cholesterol efflux from these cells was as high as that from cells expressing wild-type ABCA1 (Fig. 7B). These results suggest that four cysteine residues in ECD1 and ECD2 of ABCA1 are sufficient for apoA-I binding and apoA-I-dependent cholesterol efflux
Summary
Materials—N-Ethylmaleimide was purchased from Nacalai Tesque; dithiothreitol (DTT) was from Wako Pure Chemical Industries. ApoA-I Binding Assay—HEK293 cells were subcultured on collagen-coated glass coverslips in 12-well dishes at a density of 2 ϫ 104 cells in DMEM containing 10% fetal bovine serum. After 18 h of incubation, cells were transfected with green fluorescent protein (GFP)-tagged ABCA1 using calcium phosphate buffer. 28 h after transfection, cells were washed with ice-cold phosphate-buffered saline containing 0.1 g/liter CaCl2 and MgCl21⁄76H2O (PBSϩ) and incubated with 0.5 mg/ml sulfo-NHS-biotin solubilized in PBSϩ for 30 min on ice in the dark. Cellular Lipid Release Assay—HEK293 cells subcultured on 100-mm dishes were transfected with GFP-tagged ABCA1 using Lipofectamine 2000. 12 h after transfection, cells were trypsinized and subcultured in 6-well dishes at a density of 6 ϫ 105 cells in DMEM with 10% fetal bovine serum. After 12 h of incubation, the culture medium was removed, and cells were washed with DMEM containing 0.02% bovine serum albumin. Cells were viewed using a Zeiss confocal microscope (LSM 5 Pascal)
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