Abstract：
The neuronal microtubule-associated tau protein is characterized in vivo by a large number of post-translational modifications along the entire primary sequence that modulates its function. The primary modification of tau is phosphorylation of serine/threonine or tyrosine residues that is involved in the regulation of microtubule binding and polymerization. In neurodegenerative disorders referred to as tauopathies including Alzheimer\'s disease, tau is abnormally hyperphosphorylated and forms fibrillar inclusions in neurons progressing throughout different brain area during the course of the disease. The O-β-linked N-acetylglucosamine (O-GlcNAc) is another reversible post-translational modification of serine/threonine residues that is installed and removed by the unique O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), respectively. This modification was described as a potential modulator of tau phosphorylation and functions in the physiopathology. Moreover, reducing protein O-GlcNAc levels in the brain upon treatment of tauopathy mouse models with an OGA inhibitor reveals a beneficial effect on tau pathology and neurodegeneration. However, whether the role of tau O-GlcNAcylation is responsible of the protective effect against tau toxicity remains to be determined. The production of O-GlcNAc modified recombinant tau protein is a valuable tool for the investigations of the impact of O-GlcNAcylation on tau functions, modulation of interactions with partners and crosstalk with other post-translational modifications, including but not restricted to phosphorylation. We describe here the in vitro O-GlcNAcylation of tau with recombinant OGT for which we provide an expression and purification protocol. The use of the O-GlcNAc tau protein in functional studies requires the analytical characterization of the O-GlcNAc pattern. Here, we describe a method for the O-GlcNAc modification of tau protein with recombinant OGT and the analytical characterization of the resulting O-GlcNAc pattern by a combination of methods for the overall characterization of tau O-GlcNAcylation by chemoenzymatic labeling and mass spectrometry, as well as the quantitative, site-specific pattern by NMR spectroscopy.
摘要：
神经元微管相关tau蛋白在体内的特征在于沿着调节其功能的整个一级序列的大量翻译后修饰。tau的主要修饰是参与微管结合和聚合调节的丝氨酸/苏氨酸或酪氨酸残基的磷酸化。在称为tau蛋白病的神经退行性疾病中，包括阿尔茨海默病，tau异常过度磷酸化，并在疾病过程中在整个不同大脑区域的神经元中形成纤维状内含物。O-β-连接的N-乙酰葡糖胺（O-GlcNAc）是丝氨酸/苏氨酸残基的另一种可逆翻译后修饰，通过独特的O-GlcNAc转移酶（OGT）和O-GlcNAc水解酶（OGA）安装和去除，分别。这种修饰被描述为tau磷酸化的潜在调节剂和在病理生理学中的功能。此外，在用OGA抑制剂治疗tau蛋白病小鼠模型后,降低脑中的蛋白质O-GlcNAc水平揭示了对tau病理学和神经变性的有益作用。然而,tauO-GlcNAcylation的作用是否负责对tau毒性的保护作用尚待确定。O-GlcNAc修饰的重组tau蛋白的生产是研究O-GlcNAc对tau功能的影响的有价值的工具，与伙伴的相互作用和与其他翻译后修饰的串扰的调制，包括但不限于磷酸化。我们在这里描述了用重组OGT对tau的体外O-GlcNAcylation，我们提供了表达和纯化方案。在功能研究中使用O-GlcNActau蛋白需要对O-GlcNAc模式进行分析表征。这里,我们描述了用重组OGT对tau蛋白进行O-GlcNAc修饰的方法，以及通过化学酶标记和质谱对tauO-GlcNAc酰化进行整体表征的方法的组合，对所得O-GlcNAc模式进行分析表征。以及定量，通过NMR光谱学确定的特定位点模式。
