The C-terminal lipid-binding domain of apolipoprotein E is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high-density lipoproteins
The C-terminal lipid-binding domain of apolipoprotein E is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high-density lipoproteins
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DOI：
10.1021/bi602407r
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发表时间：
2007-03-13
期刊：
影响因子：
2.9
通讯作者：
Bielicki, John K.
中科院分区：
文献类型：
--
作者：
Vedhachalam, Charulatha;Narayanaswami, Vasanthy;Bielicki, John K.
本研究旨在鉴定人apoE的α-螺旋结构域，该结构域通过ATP结合盒转运蛋白A1（ABCA 1）介导细胞胆固醇流出和HDL组装。在使用J774巨噬细胞和HeLa细胞的研究中，发现apoE的C-末端（CT）结构域（残基222-299）以与完整apoE 2、-E3和-E4相似的方式刺激ABCA 1依赖性胆固醇流出。N-末端（NT）四螺旋束结构域（残基1-191）是胆固醇流出的相对较差的介质。在每个分子的基础上，CT结构域刺激胆固醇流出具有相同的效率（K-m类似于0.2 μ M）作为完整的apoA-I和apoE。凝胶过滤色谱条件培养基从ABCA 1表达J774细胞显示，像完整的apoE亚型，CT域促进大会的HDL颗粒直径为8和13 nm。去除CT域消除了HDL大小的颗粒的形成，并且仅形成在空隙体积中洗脱的较大颗粒。与CT截短突变体的apoE 3和肽的研究表明，疏水性螺旋段的细胞胆固醇流出的效率和连体A类和G两亲性α-螺旋需要最佳的流出活性。总的来说，这些数据表明，CT脂质结合域的apoE涵盖氨基酸222-299是必要的和足够的介导ABCA 1脂质流出和HDL颗粒组装。
This study was undertaken to identify the alpha-helical domains of human apoE that mediate cellular cholesterol efflux and HDL assembly via ATP-binding cassette transporter A1 (ABCA1). The C-terminal (CT) domain (residues 222-299) of apoE was found to stimulate ABCA1-dependent cholesterol efflux in a manner similar to that of intact apoE2, -E3, and -E4 in studies using J774 macrophages and HeLa cells. The N-terminal (NT) four-helix bundle domain (residues 1-191) was a relatively poor mediator of cholesterol efflux. On a per molecule basis, the CT domain stimulated cholesterol efflux with the same efficiency (K-m similar to 0.2 mu M) as intact apoA-I and apoE. Gel filtration chromatography of conditioned medium from ABCA1-expressing J774 cells revealed that, like the intact apoE isoforms, the CT domain promoted the assembly of HDL particles with diameters of 8 and 13 nm. Removal of the CT domain abolished the formation of HDL-sized particles, and only larger particles eluting in the void volume were formed. Studies with CT truncation mutants of apoE3 and peptides indicated that hydrophobic helical segments governed the efficiency of cellular cholesterol efflux and that conjoined class A and G amphipathic alpha-helices were required for optimal efflux activity. Collectively, the data suggest that the CT lipid-binding domain of apoE encompassing amino acids 222-299 is necessary and sufficient for mediating ABCA1 lipid efflux and HDL particle assembly.