Spatiotemporal Activation of Protein O‑GlcNAcylation in Living Cells

O-linked N-acetylglucosamine
(O-GlcNAc) is a prevalent
protein modification that plays fundamental roles in both cell physiology
and pathology. O-GlcNAc is catalyzed solely by O-GlcNAc transferase
(OGT). The study of protein O-GlcNAc function is limited by the lack
of tools to control OGT activity with spatiotemporal resolution in
cells. Here, we report light control of OGT activity in cells by replacing
a catalytically essential lysine residue with a genetically encoded
photocaged lysine. This enables the expression of a transiently inactivated
form of OGT, which can be rapidly reactivated by photo-decaging. We
demonstrate the activation of OGT activity by monitoring the time-dependent
increase of cellular O-GlcNAc and profile glycoproteins using mass-spectrometry-based
quantitative proteomics. We further apply this activation strategy
to control the morphological contraction of fibroblasts. Furthermore,
we achieved spatial activation of OGT activity predominantly in the
cytosol. Thus, our approach provides a valuable chemical tool to control
cellular O-GlcNAc with much needed spatiotemporal precision, which
aids in a better understanding of O-GlcNAc function.

O-连接N-乙酰葡糖胺（O-GlcNAc）是一种广泛存在的蛋白质修饰，在细胞生理与病理过程中均发挥基础性作用。O-GlcNAc的糖基化修饰仅由O-GlcNAc转移酶（O-GlcNAc transferase, OGT）催化。当前蛋白质O-GlcNAc功能的研究受到限制，原因在于缺乏可在细胞内实现时空分辨率精准调控OGT活性的工具。本研究报道了一种细胞内OGT活性的光控策略：通过将催化必需的赖氨酸残基替换为遗传编码的光笼型赖氨酸，可获得瞬时失活的OGT变体，该变体可通过光脱笼作用快速恢复活性。我们通过监测细胞内O-GlcNAc水平的时间依赖性升高，并利用基于质谱的定量蛋白质组学技术对糖蛋白进行定性定量表征，验证了OGT活性的激活效果。我们进一步将该激活策略应用于调控成纤维细胞的形态收缩。此外，我们还实现了主要定位于细胞质的OGT活性空间精准激活。综上，本方法提供了一种极具价值的化学工具，可实现细胞内O-GlcNAc的时空精准调控，有助于更深入地解析O-GlcNAc的生物学功能。