Hozoji Masako, Kimura Yasuhisa, Kioka Noriyuki, Ueda Kazumitsu
Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Kyoto University Graduate School of Agriculture, Kyoto, Japan.
J Biol Chem. 2009 Apr 24;284(17):11293-300. doi: 10.1074/jbc.M900580200. Epub 2009 Mar 3.
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.
ABCA1在胆固醇稳态和高密度脂蛋白（HDL）代谢中起主要作用。ABCA1在其N端和C端之间含有二硫键，但二硫键的形成对ABCA1的功能是否重要以及哪些半胱氨酸参与二硫键的形成仍不清楚。为了回答这些问题，我们构建了30多个ABCA1突变体，其中16个细胞外结构域（ECD）半胱氨酸被丝氨酸取代，并检测了这些突变体中二硫键的形成、载脂蛋白A-I结合和HDL形成。从单个半胱氨酸取代中发现，ECD1中的两个半胱氨酸（Cys(75)和Cys(309)）对载脂蛋白A-I结合至关重要。相比之下，在ECD2中，仅发现Cys(1477)对HDL形成至关重要，且单个半胱氨酸取代不会损害载脂蛋白A-I结合。同时取代两个半胱氨酸Cys(1463)和Cys(1465)会损害载脂蛋白A-I结合和HDL形成，这表明五个细胞外半胱氨酸中的四个（Cys(75)、Cys(309)、Cys(1463)、Cys(1465)和Cys(1477)）参与了ABCA1的这些功能。胰蛋白酶消化实验表明，一个二硫键是不够的，ABCA1要完全发挥功能需要两个分子内二硫键（ECD1中Cys(75)和Cys(309)之间以及ECD2中Cys(1463)或Cys(1465)与Cys(1477)之间）。
