Univ. Lille, CNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France.
Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, US 41-UMS 2014-PLBS, F-59000 Lille, France.
Molecules. 2020 Oct 1;25(19):4501. doi: 10.3390/molecules25194501.
Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator β-catenin in its -GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of β-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the -GlcNAcylation status of β-catenin in HeLa cells. The changes in -GlcNAcylation of β-catenin were varied by perturbing global cellular -GlcNAc levels with the inhibitors of -GlcNAc transferase (OGT) and -GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.
由于这一家族庞大的翻译后修饰（PTM）的复杂性，监测细胞对刺激的糖基化变化仍然具有挑战性。我们开发了一种原始工具，该工具基于细胞内分子内Förster 共振能量转移（FRET）技术，可标记和可视化细胞周期关键调节因子β-连环蛋白的 -GlcNAc 形式。我们选择了基于生物正交化学报告策略，该策略通过用绿色荧光蛋白（GFP）对β-连环蛋白进行双标记来对蛋白质序列进行组合，并使用化学点击成像探针对 PTM 进行标记，从而实现了快速且易于监测的定性 FRET 测定。我们通过在 HeLa 细胞中成像β-连环蛋白的 -GlcNAc 化状态来验证该技术。通过用 -GlcNAc 转移酶（OGT）和 -GlcNAcase（OGA）抑制剂干扰细胞内整体 -GlcNAc 水平来改变β-连环蛋白的 -GlcNAc 化。最后，我们提供了一个流程图，展示了如何将该技术转移到任何类型的糖基化中。
