Metabolic cross-talk allows labeling of O-linked β-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway
Metabolic cross-talk allows labeling of O-linked β-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway
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DOI：
10.1073/pnas.1010045108
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发表时间：
2011-02-22
影响因子：
11.1
通讯作者：
Bertozzi, Carolyn R.
中科院分区：
文献类型：
--
作者：
Boyce, Michael;Carrico, Isaac S.;Bertozzi, Carolyn R.
数百种哺乳动物细胞核和细胞质蛋白被O-连接的β-N-乙酰葡萄糖胺（O-GlcNAc）可逆地糖基化，以调节其功能、定位和稳定性。尽管O-GlcNAc信号传导具有广泛的功能意义，但其动态和翻译后性质使得单独使用传统分子和细胞生物学技术进行研究具有挑战性。在这里，我们报告的N-乙酰半乳糖胺补救和O-GlcNAc酰化途径之间的代谢串扰可以利用标记和鉴定O-GlcNAc酰化蛋白。我们发现N-叠氮乙酰半乳糖胺（GalNAz）被内源性哺乳动物生物合成酶转化为UDP-GalNAz，然后差向异构化为UDP-N-叠氮乙酰葡萄糖胺（GlcNAz）。O-GlcNAc转移酶接受UDP-GlcNAz作为核苷酸-糖供体，将叠氮糖附加到其天然底物上，然后可以使用叠氮反应性化学探针通过共价标记进行检测。在原理性蛋白质组学实验中，我们使用人细胞的代谢GalNAz标记和生物正交化学探针来亲和纯化和鉴定许多O-GlcNAc酰化蛋白质。我们的工作为未来各种化学方法提供了蓝图，以识别，可视化和表征动态O-GlcNAc信号。
Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked beta-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.