Formation of two intramolecular disulfide bonds is necessary for apoA-I-dependent cholesterol efflux mediated by ABCA1

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 (Cys75 and Cys309) in ECD1 were found to be essential for apoA-I binding. In contrast, in ECD2, only Cys1477 was found to be essential for HDL formation, and no single cysteine replacement impaired apoA-I binding. The concurrent replacement of two cysteines, Cys1463 and Cys1465, impaired apoA-I binding and HDL formation, suggesting that four of five extracellular cysteines (Cys75, Cys309, Cys1463, Cys1465, and Cys1477) 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 Cys75 and Cys309 in ECD1 and either Cys1463 or Cys1465 and Cys1477 in ECD2) are required for ABCA1 to be fully functional. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.