Chemical Tools for Studying Fucosylated Glycans

 描述（由申请方提供）：岩藻糖基化聚糖参与真核生物的多种生理和病理过程，包括血管生成、受精、胚胎发生、细胞粘附、炎症和肿瘤转移。我们对岩藻糖基化聚糖的认识存在一些主要空白，包括它们在胚胎发育和癌症进展过程中如何动态调节，以及它们的特定细胞功能。该项目的主要目标是开发化学工具，以促进我们对岩藻糖基化聚糖在这些过程中的生物学功能的理解。 在上一个授权期，我们设计了化学酶法合成5′-二磷酸胍-β-L-岩藻糖（通用岩藻糖基供体）和化学上确定的岩藻糖苷及其结构相关的衍生物。利用这些化学工具，我们开发了一些平台来研究岩藻糖基化聚糖在体外和体内的生物学功能，包括一个微阵列平台来进行岩藻糖苷与岩藻糖结合蛋白的构效关系分析。在与佛罗伦萨马洛（爱因斯坦）教授的合作中，我们开发了一种方法来检测和成像新合成的岩藻糖苷在斑马鱼胚胎使用GDP-岩藻糖功能化的生物正交标签作为代谢前体。我们发现N-连接岩藻糖基化的上调通过抑制Wnt信号通路破坏了斑马鱼胚胎发育早期的背腹图案。使用岩藻糖基转移酶和点击化学的组合，我们开发了一种方法来分析鼠和人患者样品中细胞表面外周N-乙酰基乳糖胺承载聚糖的动态变化。这些结果验证了我们的化学酶工具用于解开岩藻糖基化聚糖生物学的功能相关性。在这项工作的基础上，在下一个资助期内，我们将发明新的化学工具，与我们现有的工具结合使用，以研究岩藻糖基化聚糖在免疫系统和肿瘤之间相互作用中的作用。目的一：从肿瘤细胞的角度研究岩藻糖基化多糖的作用。具体来说，我们将研究肿瘤细胞利用岩藻糖基化聚糖抑制免疫细胞活化的机制（目的1）。在本项目的第二部分，我们将从免疫系统的角度研究岩藻糖基化糖的作用。我们将全面表征免疫细胞活化过程中细胞表面岩藻糖基化的变化（目的2）。最后，我们将设计一种在免疫细胞中工程化岩藻糖基化聚糖的方法，并评估其抗肿瘤活性（目标3）。 在这个项目中开发的化学生物学工具将提供给非专业人员，用于研究岩藻糖苷或相关聚糖在他们自己系统中的生物学功能。

 DESCRIPTION (provided by applicant): Fucosylated glycans are involved in a variety of physiological and pathological processes in eukaryotic organisms including angiogenesis, fertilization, embryogenesis, cell adhesion, inflammation, and tumor metastasis. Some major gaps in our knowledge of fucosylated glycans include how they are dynamically regulated during embryo development and cancer progression, and their specific cellular functions. The broad objective of this project is to develop chemical tools to advance our understanding of the biological functions of fucosylated glycans in these processes. In the last granting period, we designed chemoenzymatic methods to synthesize guanidine 5′- diphosphate-β-L-fucose, the universal fucosyl donor, and chemically defined fucosides, as well as their structurally related derivatives. Using these chemical tools, we developed a few platforms to study the biological functions of fucosylated glycans both in vitro and in vivo, including a microarray platform to perform structure-activity relationship analysis of fucosides with fucose-binding proteins. In collaboration with Prof. Florence Marlow (Einstein), we developed a method to detect and image newly synthesized fucosides in zebrafish embryos using GDP-fucose functionalized with a bioorthogonal tag as the metabolic precursor. We discovered that up-regulation of N-linked fucosylation disrupts zebrafish dorsal-ventral patterning in the early embryogenesis by inhibiting the Wnt signaling pathway. Using a combination of fucosyltransferase and click chemistry, we developed a method to analysis the dynamic changes of cell-surface peripheral N- acetyllactosamine-bearing glycans in murine and human patient samples. These results validated the functional relevance of our chemoenzymatic tools for unraveling the biology of fucosylated glycans. Building upon this work, in the next granting period, we will invent new chemical tools, which will be used in conjunction with our established tools, to study the role of fucosylated glycans in the interaction between the immune system and tumors. In Aim 1, we will study the role of fucosylated glycans from the aspect of tumor cells. Specifically, we will investigate mechanisms by which tumor cells use fucosylated glycans to suppress immune cell activation (Aim 1). In the second part of this project, we will study the role of fucosylated glycas from the aspect of the immune system. We will comprehensively characterize the changes in cell-surface fucosylation during immune cell activation (Aim 2). Finally, we will design a method to engineer fucosylated glycans in immune cells and evaluate their anti-tumor activities (Aim 3). The chemical biology tools developed in this project will be made accessible to non-specialists for studying the biological functions of fucosides or the related glycans in their own systems.