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

聚糖是生命所必需的，也是生物过程的重要介质。尽管从一个谜慢慢发展成为科学主流，但它们的生物合成是细胞生物学中最不了解的问题之一。在这个多学科的提案中，我将开发“精确的工具”，这些工具可以定制成活细胞中将聚糖添加到蛋白质中的复杂过程的图像和地图。当涉及酶促反应时，定量生物学的创新往往不足，而酶促反应不能单独从核酸序列预测。这对于在由250种糖基转移酶的组合相互作用介导的分泌途径中向蛋白质添加聚糖是一个特别的挑战。为了描述聚糖的生物合成是如何协调的，我们需要一些方法来了解糖基转移酶之间的功能相互作用。为了解决这个未满足的需求，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报告者。这些工具将允许直接洞察大型GalNAc转移酶家族，该家族引入O-连接聚糖作为最丰富和最复杂的蛋白质修饰之一。质谱、超分辨率显微镜和相关光学和电子显微镜的尖端方法将共同揭示哪种酶何时何地作用于通过分泌途径运输的糖蛋白。这种范式转变的工作只有通过我发现一种策略才有可能工程GalNAc转移酶选择性地容纳可追踪的糖类似物作为野生型转移酶不使用的底物。这里提出的糖配备了可以用荧光团追踪的化学标签。因此，我的工作将为分泌途径中蛋白质成熟的细节提供前所未有的见解，这是细胞生物学中的一个主要过程，有许多未解之谜。

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.