Sakamoto Takaaki, Tanaka Masafumi, Vedhachalam Charulatha, Nickel Margaret, Nguyen David, Dhanasekaran Padmaja, Phillips Michael C, Lund-Katz Sissel, Saito Hiroyuki
Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan.
Biochemistry. 2008 Mar 4;47(9):2968-77. doi: 10.1021/bi701923h. Epub 2008 Jan 18.
To understand the molecular basis for the different self-association and lipoprotein preferences of apolipoprotein (apo) E isoforms, we compared the effects of progressive truncation of the C-terminal domain in human apoE3 and apoE4 on their lipid-free structure and lipid binding properties. A VLDL/HDL distribution assay demonstrated that apoE3 binds much better than apoE4 to HDL 3, whereas both isoforms bind similarly to VLDL. Removal of the C-terminal helical regions spanning residues 273-299 weakened the ability of both isoforms to bind to lipoproteins; this led to the elimination of the isoform lipoprotein preference, indicating that the C-terminal helices mediate the lipoprotein selectivity of apoE3 and apoE4 isoforms. Gel filtration chromatography experiments demonstrated that the monomer-tetramer distribution is different for the two isoforms with apoE4 being more monomeric than apoE3 and that removal of the C-terminal helices favors the monomeric state in both isoforms. Consistent with this, fluorescence measurements of Trp-264 in single-Trp mutants revealed that the C-terminal domain in apoE4 is less organized and more exposed to the aqueous environment than in apoE3. In addition, the solubilization of dimyristoylphosphatidylcholine multilamellar vesicles is more rapid with apoE4 than with apoE3; removal of the C-terminal helices significantly affected solubilization rates with both isoforms. Taken together, these results indicate that the C-terminal domain is organized differently in apoE3 and apoE4 so that apoE4 self-associates less and binds less than apoE3 to HDL surfaces; these alterations may lead to the pathological sequelae for cardiovascular and neurodegenerative diseases.
为了解载脂蛋白（apo）E 异构体不同的自我缔合和脂蛋白偏好的分子基础，我们比较了人 apoE3 和 apoE4 中 C 末端结构域逐步截短对其无脂结构和脂质结合特性的影响。一项极低密度脂蛋白/高密度脂蛋白分布测定表明，apoE3 与高密度脂蛋白 3 的结合能力比 apoE4 强得多，而两种异构体与极低密度脂蛋白的结合相似。去除跨越 273 - 299 位残基的 C 末端螺旋区域削弱了两种异构体与脂蛋白结合的能力；这导致异构体脂蛋白偏好的消除，表明 C 末端螺旋介导了 apoE3 和 apoE4 异构体的脂蛋白选择性。凝胶过滤色谱实验表明，两种异构体的单体 - 四聚体分布不同，apoE4 比 apoE3 更倾向于单体状态，并且去除 C 末端螺旋有利于两种异构体的单体状态。与此一致的是，单 Trp 突变体中 Trp - 264 的荧光测量显示，apoE4 中的 C 末端结构域比 apoE3 中的结构更松散，更暴露于水环境中。此外，apoE4 使二肉豆蔻酰磷脂酰胆碱多层囊泡的溶解比 apoE3 更快；去除 C 末端螺旋显著影响了两种异构体的溶解速率。综上所述，这些结果表明 apoE3 和 apoE4 中 C 末端结构域的组织方式不同，因此 apoE4 自我缔合较少，与高密度脂蛋白表面的结合也比 apoE3 少；这些改变可能导致心血管和神经退行性疾病的病理后果。
