Hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry determine the interface and dynamics of apolipoprotein E oligomerization.
Hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry determine the interface and dynamics of apolipoprotein E oligomerization.
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DOI：
10.1021/bi2010027
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发表时间：
2011-11-01
期刊：
影响因子：
2.9
通讯作者：
Gross, Michael L.
中科院分区：
文献类型：
--
作者：
Huang, Richard Y-C.;Garai, Kanchan;Frieden, Carl;Gross, Michael L.
载脂蛋白E是一种34 kDa的蛋白质，在甘油三酯和胆固醇代谢中起关键作用。在三种常见的同种型（ApoE 2、3和4）中，只有ApoE 4是阿尔茨海默病的危险因素。野生型ApoE的所有三种亚型自缔合形成寡聚体，这是一个可能具有功能后果的过程。虽然认为ApoE的C-末端结构域（残基216-299）介导自缔合，但参与该过程的特定残基尚不清楚。在这里，我们报告使用氢/氘交换（H/DX）加上酶消化，以确定这些地区的全长载脂蛋白E的序列参与寡聚化。对于这一决定，我们比较了野生型蛋白质的H/DX的结果和通过修饰C-末端结构域中的四个残基获得的单体形式的结果。三种野生型和突变体同种型基于其相似的H/DX动力学和交换程度显示出相似的结构。ApoE亚型的C-末端区域（残基230-270）显示寡聚体和单体形式之间氘摄取的显著差异，证实在这些区域发生寡聚化。为了实现单个氨基酸的分辨率，我们研究了H/DX的程度，通过使用电子转移解离（ETD）片段化的肽代表的单体和寡聚体形式的ApoE 4的选定区域。从这些实验中，我们可以确定参与ApoE寡聚化的特定残基。此外，我们的研究结果证实，ApoE 4是由一个紧凑的结构在其N-末端域。然而，C-末端结构域的区域似乎缺乏确定的结构。
Apolipoprotein E, a 34 kDa protein, plays a key role in triglyceride and cholesterol metabolism. Of the three common isoforms (ApoE2, 3 and 4), only ApoE4 is a risk factor for Alzheimer’s Disease. All three isoforms of wild-type ApoE self-associate to form oligomers, a process that may have functional consequences. Although the C-terminal domain, residues 216–299, of ApoE is believed to mediate self-association, the specific residues involved in this process are not known. Here we report the use of hydrogen/deuterium exchange (H/DX) coupled with enzymatic digestion to identify those regions in the sequence of full-length apoE involved in oligomerization. For this determination, we compared the results of H/DX of the wild-type proteins and those of monomeric forms obtained by modifying four residues in the C-terminal domain. The three wild type and mutant isoforms show similar structures based on their similar H/DX kinetics and extents of exchange. Regions of the C-terminus (residues 230–270) of the ApoE isoforms show significant differences of deuterium uptake between oligomeric and monomeric forms, confirming that oligomerization occurs at these regions. To achieve single amino acid resolution, we examined the extents of H/DX by using electron transfer dissociation (ETD) fragmentation of peptides representing selected regions of both the monomeric and the oligomeric forms of ApoE4. From these experiments, we could identify the specific residues involved in ApoE oligomerization. In addition, our results verify that ApoE4 is composed of a compact structure at its N-terminal domain. Regions of C-terminal domain, however, appear to lack defined structure.
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