O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.

中文翻译：

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对细胞中选择性蛋白质O-GlcNAcy的近邻定向O-GlcNAc转移酶进行工程设计。

O-连接的β-N-乙酰氨基葡萄糖（O-GlcNAc）是一种单糖，在真核细胞整个核质蛋白组中的细胞信号传导中起着至关重要的作用。选择性增加细胞中靶蛋白上O-GlcNAc水平的策略将加速这种基本修饰的研究。在这里，我们报告了一种通用的策略，用于将O-GlcNAc引入到细胞中的选定靶蛋白中，使用纳米抗体作为融合到O-GlcNAc转移酶（OGT）的邻近导向剂。识别GFP（nGFP）的纳米抗体或识别四氨基酸序列EPEA（nEPEA）的纳米抗体与OGT的融合产生了纳米抗体-OGT构建体，该构建体选择性地将O-GlcNAc递送至一系列标记的靶蛋白（例如，JunB， cJun和Nup62）。截断OTG（4）中的四肽重复结构域可以通过降低细胞中O-GlcNAc水平的整体升高来提高通过纳米抗体对靶蛋白的选择性。定量化学蛋白质组学证实了纳米抗体-OGT（4）对目标蛋白质的O-GlcNAc含量增加。糖皮质激素学表明，纳米抗体-OGT（4）或全长OGT在目标蛋白JunB和Nup62上产生相似的糖位分布。最后，我们证明了在HEK293T细胞中选择性靶向内源性α-突触核蛋白以进行O-GlcNAcylation的能力。这些首批邻近导向的OGT构建体为靶向其他蛋白质提供了灵活的策略，并为将来进一步改造OGT和O-GlcNAc蛋白质组提供了模板。