Spatiotemporal Proximity Labeling Tools to Track GlcNAc Sugar-Modified Functional Protein Hubs during Cellular Signaling

A fundamental mechanism that all eukaryotic cells use to adapt to their environment is dynamic protein modification with monosaccharide sugars. In humans, O-linked N-acetylglucosamine (O-GlcNAc) is rapidly added to and removed from diverse protein sites as a response to fluctuating nutrient levels, stressors, and signaling cues. Two aspects remain challenging for tracking functional O-GlcNAc events with chemical strategies: spatial control over subcellular locations and time control during labeling. The objective of this study was to create intracellular proximity labeling tools to identify functional changes in O-GlcNAc patterns with spatiotemporal control. We developed a labeling strategy based on the TurboID proximity labeling system for rapid protein biotin conjugation directed to O-GlcNAc protein modifications inside cells, a set of tools called “GlycoID.” Localized variants to the nucleus and cytosol, nuc-GlycoID and cyt-GlycoID, labeled O-GlcNAc proteins and their interactomes in subcellular space. Labeling during insulin and serum stimulation revealed functional changes in O-GlcNAc proteins as soon as 30 min following signal initiation. We demonstrated using proteomic analysis that the GlycoID strategy captured O-GlcNAcylated “activity hubs” consisting of O-GlcNAc proteins and their associated protein–protein interactions. The ability to follow changes in O-GlcNAc hubs during physiological events such as insulin signaling allows these tools to determine the mechanisms of glycobiological cell regulation. Our functional O-GlcNAc data sets in human cells will be a valuable resource for O-GlcNAc-driven mechanisms.

真核细胞适应外界环境的核心机制之一，是借助单糖（monosaccharide sugars）开展动态蛋白质修饰。在人体中，O-连接N-乙酰葡糖胺（O-linked N-acetylglucosamine, O-GlcNAc）会响应波动的营养水平、应激因素与信号提示，快速结合并脱离不同蛋白质位点。利用化学策略追踪功能性O-GlcNAc事件仍存在两大挑战：一是对亚细胞位置的空间调控，二是标记过程中的时间控制。本研究旨在开发胞内邻近标记工具，以实现时空可控的O-GlcNAc修饰模式功能变化鉴定。我们基于TurboID邻近标记系统开发了一种标记策略，可在细胞内靶向O-GlcNAc修饰的蛋白质实现快速蛋白质生物素共轭，该套工具被命名为‘GlycoID’。针对细胞核与细胞质的定位变体——nuc-GlycoID与cyt-GlycoID，可在亚细胞空间内标记O-GlcNAc修饰蛋白及其相互作用组。在胰岛素与血清刺激过程中开展标记，可在信号启动后仅30分钟即检测到O-GlcNAc修饰蛋白的功能变化。我们通过蛋白质组学分析证实，GlycoID策略可捕获由O-GlcNAc修饰蛋白及其相关蛋白质-蛋白质相互作用构成的O-GlcNAc化‘活性枢纽’。该工具能够追踪胰岛素信号通路等生理过程中O-GlcNAc活性枢纽的变化，从而助力解析糖生物学介导的细胞调控机制。本研究在人体细胞中获得的功能性O-GlcNAc数据集，将为O-GlcNAc介导的细胞机制研究提供宝贵的参考资源。