Purification and ATPase activity of human ABCA1
Purification and ATPase activity of human ABCA1
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DOI：
10.1074/jbc.m513783200
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发表时间：
2006-04-21
影响因子：
4.8
通讯作者：
Ueda, K
中科院分区：
文献类型：
--
作者：
Takahashi, K;Kimura, Y;Ueda, K
atp结合盒蛋白A1 （ABCA1）在胆固醇稳态和高密度脂蛋白代谢中起重要作用。载脂蛋白A-I与ABCA1结合，细胞胆固醇和磷脂（主要是磷脂酰胆碱）被装载到载脂蛋白A-I上，形成β前高密度脂蛋白（HDL）。ABCA1直接或间接地易位磷脂和胆固醇形成β - HDL。为了探究ABCA1介导前β - HDL形成的机制，我们在昆虫Sf9细胞中表达了人ABCA1并对其进行了纯化。胰蛋白酶有限消化纯化的洗涤剂可溶性形式的ABCA1表明，它保留了类似于人成纤维细胞WI-38细胞膜上表达的ABCA1的构象。纯化后的ABCA1在合成磷脂酰胆碱脂质体中重组后显示出强大的atp酶活性。当ABCA1在含有磷脂酰丝氨酸、磷脂酰乙醇胺或磷脂酰甘油的脂质体中重组时，其atp酶活性较低，并且在酰基链物种中表现出较弱的特异性。三磷酸腺苷酶活性随着胆固醇的增加而降低，在20%胆固醇的情况下降低了25%。-谷甾醇和油菜甾醇具有相似的抑制作用，而豆甾醇则没有，这表明ABCA1与甾醇之间存在结构特异性相互作用。格列本脲抑制ABCA1 atp酶，提示其通过抑制ABCA1 atp酶活性抑制apoa - i依赖性细胞胆固醇外排。这些结果表明，具有胆碱头基团的磷脂、磷脂酰胆碱和鞘磷脂优先刺激ABCA1的atp酶活性。利用纯化的人ABCA1进行的研究，为ABCA1介导HDL形成的机制提供了第一个生化基础。
ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. beta-Sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.