在大肠杆菌中生产O-连接的N-乙酰葡糖胺修饰的重组tau蛋白并在体内检测Ser400 O-连接的N-乙酰葡糖胺修饰的tau蛋白

Production of O-GlcNAc Modified Recombinant Tau in E. coli and Detection of Ser400 O-GlcNAc Tau In Vivo.

作者信息

Yuzwa Scott A, Vocadlo David J

机构信息

Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Dr, Burnaby, BC, Canada, V5A 1S6.

Neurosciences and Mental Health program, The Hospital for Sick Children, Toronto, ON, Canada, M5G 0A4.

出版信息

Methods Mol Biol. 2017;1523:237-248. doi: 10.1007/978-1-4939-6598-4_13.

DOI:10.1007/978-1-4939-6598-4_13

PMID:27975253

Abstract

Assembly of the microtubule-associated protein tau (tau) into paired helical filaments that ultimately give rise to neurofibrillary tangles (NFTs) makes up one half of the two hallmark pathologies of Alzheimer's disease (AD). Tau has been shown to be modified with O-linked N-acetylglucosamine residues (O-GlcNAc), which is the modification of serine and threonine residues of nucleocytoplasmic proteins with N-acetyl-D-glucosamine (GlcNAc) moieties. Increasing O-GlcNAc in mouse models of tauopathy has been shown to hinder the progression of symptoms in these mice and impair the aggregation of tau into NFTs. In order to study how O-GlcNAc on tau may contribute to the protective effects observed in tauopathy mouse models, it is beneficial to study O-GlcNAc modified tau in vitro. Here we describe a method for producing, purifying and enriching recombinant tau that is O-GlcNAc modified. These methods have enabled the identification of O-GlcNAc modification sites on tau including Ser400. We further describe the detection of Ser400 O-GlcNAc on tau from brain lysates.

摘要

微管相关蛋白tau（tau）组装成最终导致神经原纤维缠结（NFTs）的双螺旋细丝，构成了阿尔茨海默病（AD）两种标志性病理的其中之一。已证明tau会被O-连接的N-乙酰葡糖胺残基（O-GlcNAc）修饰，这是用N-乙酰-D-葡糖胺（GlcNAc）部分对核质蛋白的丝氨酸和苏氨酸残基进行的修饰。在tau蛋白病小鼠模型中增加O-GlcNAc已被证明可阻碍这些小鼠症状的进展，并损害tau聚集成NFTs。为了研究tau上的O-GlcNAc如何有助于在tau蛋白病小鼠模型中观察到的保护作用，在体外研究O-GlcNAc修饰的tau是有益的。在这里，我们描述了一种生产、纯化和富集经O-GlcNAc修饰的重组tau的方法。这些方法已能够鉴定tau上包括Ser400在内的O-GlcNAc修饰位点。我们进一步描述了从脑裂解物中检测tau上的Ser400 O-GlcNAc。