O-GlcNAc Engineering on a Target Protein in Cells with Nanobody-OGT and Nanobody-splitOGA.
O-GlcNAc Engineering on a Target Protein in Cells with Nanobody-OGT and Nanobody-splitOGA.
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DOI：
10.1002/cpz1.117
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发表时间：
2021-05
期刊：
影响因子：
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通讯作者：
Woo CM
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文献类型：
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作者：
Ramirez DH;Ge Y;Woo CM
单糖O-GlcNAc是一种重要的动态翻译后修饰（PTM），可修饰数千种核质蛋白。询问O-GlcNAc对靶蛋白的作用是至关重要的，但在细胞中进行具有挑战性。我们最近报道了一对使用纳米抗体融合物与工程化的O-GlcNAc转移酶（OGT）或裂解的O-GlcNAc酶（OGA）在细胞中的靶蛋白上选择性地安装或去除O-GlcNAc的方法。靶蛋白O-GlcNAc化和de-O-GlcNAc化补充了在全球范围内或在单个糖位点上询问O-GlcNAc作用的方法。在本文中，我们描述了利用纳米抗体-OGT和-splitOGA系统来筛选靶蛋白上的O-GlcNAc功能的方案。我们还包括用于检测O-GlcNAc和克隆程序的相关协议，以适应用户感兴趣的靶蛋白的方法。
The monosaccharide O-GlcNAc is an essential and dynamic post-translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently reported a pair of methods to selectively install or remove O-GlcNAc on a target protein in cells using nanobody-fusions to an engineered O-GlcNAc transferase (OGT) or split O-GlcNAcase (OGA). Target protein O-GlcNAcylation and de-O-GlcNAcylation complements methods to interrogate the role of O-GlcNAc on a global scale or at individual glycosites. Herein, we describe a protocol for utilizing the nanobody-OGT and -splitOGA systems to screen for O-GlcNAc functionality on a target protein. We additionally include associated protocols for the detection of O-GlcNAc and cloning procedures to adapt the method for the user’s target protein of interest.