Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan.
Sci Rep. 2018 Apr 18;8(1):6170. doi: 10.1038/s41598-018-24428-6.
The quality and quantity of high-density lipoprotein (HDL) in blood plasma are important for preventing coronary artery disease. ATP-binding cassette protein A1 (ABCA1) and apolipoprotein A-I (apoA-I) play essential roles in nascent HDL formation, but controversy persists regarding the mechanism by which nascent HDL is generated. In the "direct loading model", apoA-I acquires lipids directly from ABCA1 while it is bound to the transporter. By contrast, in the "indirect model", apoA-I acquires lipids from the specific membrane domains created by ABCA1. In this study, we found that trypsin treatment causes rapid release of phosphatidylcholine (PC) and cholesterol from BHK/ABCA1 cells, and that the time course of lipid release coincides with those of trypsin digestion of extracellular domains (ECDs) of surface ABCA1 and of release of ECD fragments into the medium. This trypsin-dependent lipid release was dependent on ABCA1 ATPase activity, and did not occur in cells that express ABCG1, which exports lipids like ABCA1 but does not have large ECDs. These results suggest that the trypsin-sensitive sites on the cell surface are the large ECDs of ABCA1, and that lipids transported by ABCA1 are temporarily sequestered within the ECDs during nascent HDL formation.
血液血浆中高密度脂蛋白（HDL）的质量和数量对于预防冠状动脉疾病很重要。ATP 结合盒蛋白 A1（ABCA1）和载脂蛋白 A-I（apoA-I）在新生 HDL 形成中起着重要作用，但关于新生 HDL 生成的机制仍存在争议。在“直接加载模型”中，apoA-I 在与转运蛋白结合时直接从 ABCA1 获得脂质。相比之下，在“间接模型”中，apoA-I 从 ABCA1 形成的特定膜域中获得脂质。在这项研究中，我们发现胰蛋白酶处理会导致 BHK/ABCA1 细胞中磷脂酰胆碱（PC）和胆固醇的快速释放，并且脂质释放的时间过程与细胞表面 ABCA1 的细胞外结构域（ECD）的胰蛋白酶消化以及 ECD 片段释放到介质中的时间过程相吻合。这种依赖于胰蛋白酶的脂质释放依赖于 ABCA1 ATP 酶活性，并且不会发生在表达 ABCG1 的细胞中，ABCG1 像 ABCA1 一样转运脂质，但没有大的 ECD。这些结果表明，细胞表面上的胰蛋白酶敏感位点是 ABCA1 的大 ECD，并且在新生 HDL 形成过程中，由 ABCA1 转运的脂质暂时被隔离在 ECD 内。
