Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners
Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners
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DOI：
10.1073/pnas.1114356109
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发表时间：
2012-03-27
影响因子：
11.1
通讯作者：
Kohler, Jennifer J.
中科院分区：
文献类型：
--
作者：
Yu, Seok-Ho;Boyce, Michael;Kohler, Jennifer J.
O-linked β - n -乙酰氨基葡萄糖（O-GlcNAc）是一种可逆的翻译后修饰，存在于高等真核生物的数百种核蛋白和细胞质蛋白上。尽管O-GlcNAc修饰在哺乳动物中普遍存在且至关重要，但其功能作用仍不明确。在这里，我们开发了一种结合遗传和化学的方法，可以将重氮嘧啶光交联剂引入到细胞中的O-GlcNAc修饰上。我们通过表达一种突变形式的UDP-GlcNAc焦磷酸化酶，设计哺乳动物细胞产生重氮嘧啶修饰的O-GlcNAc，随后用细胞渗透性、重氮嘧啶修饰的glcnac -1-磷酸培养这些细胞。紫外光照射细胞激活交联剂，导致o - glcnac修饰蛋白与邻近分子之间形成共价键，可以通过质谱法鉴定。我们用这种方法鉴定了o - glcnac修饰的FG-repeat核孔蛋白的相互作用伙伴。我们观察到fg重复核孔蛋白和核转运因子之间的交联，这表明O-GlcNAc残基与核转运中的基本识别事件密切相关。此外，我们认为本文报道的方法可以广泛应用于研究o - glcn酰化的功能后果。
O-linked beta-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic 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 nucleoporins. We observed crosslinking between FG-repeat nucleoporins 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.