TAU GLYCOSYLATION IN ALZHEIMERS DISEASE

本研究的长期目标是了解阿尔茨海默病（AD）神经元退行性变的机制。 本项目的具体目的是揭示阿尔茨海默病神经退行性变不同阶段中微管相关蛋白tau糖基化的性质和程度，并了解tau糖基化在AD脑中tau转化为成对螺旋丝缠结（PHF）中的作用。 为了实现这些目标，提出：（1）鉴定代表神经元变性不同阶段的各种tau蛋白的糖基化位点和糖基化位点。将大量分离来自正常人脑的正常tau和来自AD脑的AD非过度磷酸化tau、可溶性AD异常磷酸化tau、易溶于去污剂的PHF-tau和微溶于去污剂的PHF-tau。 将分别通过凝集素结合技术和气相色谱-质谱法测定与这些蛋白质缀合的β-葡聚糖的存在和量。 糖苷键的类型将通过它们对选择性糖苷酶的敏感性来鉴定。 糖基化位点将通过糖肽的生成和分离、其氨基酸测序和基质辅助激光解吸/电离-质谱法进行作图。 (2)目的研究tau蛋白异常糖基化与异常过度磷酸化之间的相互作用及其在AD发病机制中的作用。 通过比较先前存在和不存在另一种修饰的修饰动力学，研究两种tau蛋白翻译后修饰（糖基化和磷酸化）之一对彼此的影响。 在从蛋白质中去除聚糖之前和之后，将研究糖基化tau蛋白对蛋白水解的敏感性及其降解成细丝的趋势。 最后，PHF缠结的结构将在缠结去糖基化后通过电子显微镜检查。 这些研究将阐明异常tau蛋白糖基化及其对PHF缠结形成的影响，因此，应该提供一个新的内部视图的阿尔茨海默病神经退行性变的机制。

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.