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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners
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Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

机译：代谢标记能够使O-GlcNAc修饰的蛋白质与其结合伴侣进行选择性的光交联

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摘要

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification found on hundreds of nuclear and cyto-plasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucieoporins. We observed cross-linking between FG-repeat nucieoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.
机译：O-连接的β-N-乙酰氨基葡萄糖（O-GlcNAc）是一种可逆的翻译后修饰，存在于高级真核生物中的数百种核和细胞质蛋白上。尽管其在哺乳动物中无处不在且必不可少，但O-GlcNAc修饰的功能性作用仍不清楚。在这里，我们开发了一种遗传和化学相结合的方法，可以将重氮嗪光交联剂引入细胞中的O-GlcNAc修饰。我们通过表达UDP-GlcNAc焦磷酸化酶的突变体形式，然后通过细胞渗透性，重氮嗪修饰的GlcNAc-1-磷酸形式培养这些细胞，从而工程化哺乳动物细胞以产生重氮嗪修饰的O-GlcNAc。用紫外线照射细胞会激活交联剂，从而导致O-GlcNAc修饰的蛋白质与邻近分子之间形成共价键，这可以通过质谱鉴定。我们使用这种方法来确定O-GlcNAc修饰的FG-重复核素的相互作用伙伴。我们观察到FG重复的nuocioporins与核转运因子之间的交联，表明O-GlcNAc残基与核转运中的基本识别事件密切相关。此外，我们建议本文报道的方法可广泛用于研究O-GlcNAcylation的功能后果。

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