GlycOTag - Precision Tools to unravel the fundamentals of O-glycan biosynthesis

多糖是生命所必需的，也是生物过程的重要介体。尽管它们的生物合成从一个谜慢慢地进入了科学主流，但它的生物合成是细胞生物学中最不被理解的问题之一。在这项多学科的提案中，我将开发定制的“精密工具”，以显示和绘制活细胞中蛋白质添加多聚糖的复杂过程。当涉及仅凭核酸序列无法预测的酶反应时，定量生物学的创新往往达不到要求。这对于在250个糖基转移酶的组合相互作用中介导的分泌途径中的蛋白质中添加糖是一个特别的挑战。为了描述糖的生物合成是如何协调的，需要一些方法来了解糖基转移酶之间的功能相互作用。为了解决这一未得到满足的需求，我将开发化学工具，作为活细胞中单个糖基转移酶活动的可追踪记者。这些工具将允许直接了解大型GalNAc转移酶家族，该家族引入O-连接的多糖作为最丰富和最复杂的蛋白质修饰之一。尖端的质谱仪、超分辨率显微镜和相关的光学和电子显微镜方法将共同揭示哪种酶在何时何地作用于通过分泌途径运输的糖蛋白。这种范式转换的工作只有通过我发现了一种策略来设计GalNAc转移酶，以选择性地适应可追踪的糖类似物作为底物而不被野生型转移酶使用，才有可能。这里提出的糖带有化学标签，可以通过荧光团进行追踪。因此，我的工作将前所未有地深入了解分泌途径中蛋白质成熟的细节，这是细胞生物学中的一个主要过程，有许多悬而未决的问题。

Glycans are essential for life and important mediators of biological processes. Despite slowly advancing from an enigma to scientific mainstream, their biosynthesis is one of the least understood issues in cell biology. In this multidisciplinary proposal, I will develop 'precision tools' that are customized to image and map the complex processes of adding glycans to proteins in the living cell.Innovations in quantitative biology often fall short when enzymatic reactions are involved that cannot be predicted from nucleic acid sequence alone. This is a particular challenge for the addition of glycans to proteins in the secretory pathway that is mediated by the combinatorial interplay of 250 glycosyltransferases. To delineate how glycan biosynthesis is coordinated, methods are needed to inform on the functional interplay between glycosyltransferases.To address this unmet need, I will develop chemical tools as traceable reporters for the activities of individual glycosyltransferases in the living cell. These tools will allow direct insight into the large GalNAc transferase enzyme family that introduces O-linked glycans as one of the most abundant and complex protein modifications. Cutting-edge methods of mass spectrometry, super-resolution microscopy and correlative light and electron microscopy will collectively reveal which enzyme acts when and where on glycoproteins trafficking through the secretory pathway. This paradigm-shifting work is only possible through my discovery of a tactic to engineer GalNAc transferases to selectively accommodate traceable sugar analogs as substrates that are not used by wildtype transferases. The sugars proposed here are armed with chemical tags that can be traced with fluorophores. My work will thus provide unprecedented insight into the details of protein maturation in the secretory pathway, a major process in cell biology with many unanswered questions.