TAU GLYCOSYLATION IN ALZHEIMERS DISEASE

这项建议的长期目标是了解阿尔茨海默病(AD)中神经纤维变性的机制。本项目的具体目的是揭示阿尔茨海默病神经纤维变性不同阶段微管相关蛋白tau糖基化的性质和程度，并了解tau糖基化在AD脑内tau转化为成对螺旋细丝缠结(PHF)中的作用。为此，我们建议：(1)鉴定代表神经原纤维变性不同阶段的不同种类tau的糖类和糖基化位点。来自正常人脑的正常tau，以及来自AD脑中的AD非过度磷酸化tau、可溶AD异常磷酸化tau、易洗涤剂可溶性phf-tau和少量洗涤剂可溶性phf-tau将被批量分离。结合这些蛋白质的糖的存在和数量将分别通过凝集素结合技术和气相色谱-质谱仪来确定。糖苷键的类型将通过它们对选择性糖苷酶的敏感性来确定。糖基化位点将通过糖肽的产生和分离，以及它们的氨基酸测序和基质辅助激光解吸/电离-质谱学来确定。(2)研究tau糖基化异常与tau异常过度磷酸化之间的相互作用，探讨tau糖基化在AD发病中的作用。两种tau翻译后修饰中的一种(糖基化和磷酸化)的相互影响将通过比较存在和不存在另一种修饰的修饰的动力学来研究。糖基化的tau对蛋白质分解的敏感性以及它聚合成细丝的趋势将在从蛋白质中去除多糖之前和之后进行研究。最后，将PHF缠结脱糖后用电子显微镜观察其结构。这些研究将阐明tau糖基化异常及其对PHF缠结形成的影响，从而为阿尔茨海默病神经原纤维变性的机制提供一个新的内观。

The long term objective of this proposal is to understand the mechanism of neurofibrillary degeneration in Alzheimer's disease (AD). The specific aims of this project are to reveal the nature and extent of glycosylation of microtubule associated protein tau during various stages of Alzheimer neurofibrillary degeneration, and to understand the role of tau glycosylation in the conversion of tau into tangles of paired helical filaments (PHF) in AD brain. Towards these aims it is proposed: (1) to identify the saccharides and the glycosylation sites of various species of tau representing different stages of neurofibrillary degeneration. Normal tau from normal human brains, and AD non- hyperphosphorylated tau, soluble AD abnormally phosphorylated tau, readily detergent soluble PHF-tau and sparingly detergent soluble PHF-tau from AD brains will be bulk isolated. The presence and the quantity of saccharides conjugated to these proteins will be determined by lectin binding techniques and gas chromatography-Mass spectrometry, respectively. The types of glycosidic linkages will be identified by their sensitivity to selective glycosidases. The glycoylation sites will be mapped by generation and isolation of glycopeptides, and their amino acid sequencing and matrix-assisted laser desorption/ionization-Mass spectrometry. (2) To study the interactions between abnormal glycosylation and abnormal hyperphosphorylation of tau and the role of tau glycosylation in the AD pathogenesis. The effects of one of the two tau posttranslational modifications (glycosylation and phosphorylation) on each other will be investigated by comparing the kinetics of the modification with and without the prior presence of the other modification. The sensitivity of glycosylated tau to proteolysis and its tendency to polymerize into filaments will be studied before and after removal of glycans from the proteins. Finally, the structure of PHF tangles will be examined by electron microscopy after the tangles are deglycosylated. These studies will elucidate the abnormal tau glycosylation and its impact on the formation of PHF tangles and, therefore, should provide a new inside view of the mechanism of Alzheimer neurofibrillary degeneration.