Fleming L M, Weisgraber K H, Strittmatter W J, Troncoso J C, Johnson G V
Department of Psychiatry, University of Alabama, Birmingham, 35294-0017, USA.
Exp Neurol. 1996 Apr;138(2):252-60. doi: 10.1006/exnr.1996.0064.
The apolipoprotein E4 (apoE4) gene dose is a major risk factor for late-onset and sporadic Alzheimer's disease with 50% of homozygous patients developing the disease by age 70. Previous studies have shown localization of apoE to the cytoplasm of certain neurons within the brain. In addition, apoE3, but not apoE4, forms SDS-stable complexes with the microtubule-associated proteins tau and MAP-2. To extend these studies and quantitate the association of apoE with other proteins, the association of apoE3 and apoE4 with several cytoskeletal proteins was examined using both gel shift and overlay assays. In the gel shift assay, apoE3 formed SDS-stable complexes with the longest isoform of human recombinant tau (T4L), the shortest isoform of human recombinant tau (T3), and the 160-kDa neurofilament protein (NFM). ApoE4 did not bind T3, T4L, or NFM in this assay. The association of apoE3 and apoE4 with T4L, actin, or tubulin was further examined in an overlay assay with known amounts of the cytoskeletal proteins slot-blotted onto nitrocellulose and incubated in 0.15 microM (5 microg/ml) apoE3 or apoE4. In this assay, apoE3 and apoE4 bound T4L and tubulin equally well. In contrast, apoE3 bound actin with a significantly greater affinity than did apoE4. These results indicate that apoE isoforms interact with cytoskeletal proteins with at least two different binding affinities. The more avid interaction results in the formation of complexes which are SDS stable and occurs almost exclusively with apoE3, while the other interactions between apoE and cytoskeletal proteins are specific for apoE3.
载脂蛋白E4（apoE4）基因剂量是晚发型和散发性阿尔茨海默病的主要危险因素，50%的纯合子患者在70岁前会患上该病。先前的研究表明，apoE定位于大脑内某些神经元的细胞质中。此外，apoE3而非apoE4能与微管相关蛋白tau和MAP-2形成SDS稳定复合物。为扩展这些研究并定量apoE与其他蛋白质的关联，使用凝胶迁移和覆盖分析检测了apoE3和apoE4与几种细胞骨架蛋白的关联。在凝胶迁移分析中，apoE3与人类重组tau的最长异构体（T4L）、人类重组tau的最短异构体（T3）以及160 kDa神经丝蛋白（NFM）形成了SDS稳定复合物。在该分析中，apoE4不与T3、T4L或NFM结合。用已知量的细胞骨架蛋白点样到硝酸纤维素膜上并在0.15 microM（5 microg/ml）的apoE3或apoE4中孵育，通过覆盖分析进一步检测apoE3和apoE4与T4L、肌动蛋白或微管蛋白的关联。在该分析中，apoE3和apoE4与T4L和微管蛋白的结合效果相当。相比之下，apoE3与肌动蛋白的结合亲和力明显高于apoE4。这些结果表明，apoE异构体与细胞骨架蛋白以至少两种不同的结合亲和力相互作用。更强烈的相互作用导致形成SDS稳定的复合物，且几乎仅与apoE3发生，而apoE与细胞骨架蛋白之间的其他相互作用则对apoE3具有特异性。
