Design and synthesis of a next generation glycobiology toolbox for cell surface labeling

细胞膜是细胞与其周围环境相互作用和通信的界面， 包括附近的细胞、蛋白质和抗体。健康细胞的膜通常在患病状态下被修饰， 提供了一种外部手段来区分形态相似的细胞。通常，病变细胞会显示 它们细胞膜上的修饰碳水化合物和多糖。酶介导细胞的研究进展 表面工程为在不同细胞类型中标记这些修饰提供了有希望的途径。该方法 糖链标记结合了糖基转移酶和非天然的核苷酸糖供体，称为化学酶标记 配有化学标签，可直接标记生物样品中的特定表面多糖。最近，这个 一步法标记岩藻糖基转移酶和GDP-岩藻糖 生物素标签。与其他遗传和代谢技术相比，这种标记活细胞的方法是一种更 直接、简单的程序和更快的周转周期。它也被证明普遍适用于小鼠 肿瘤模型，因此有很高的转移到临床环境的潜力。虽然已发布的报告集中在 在少量的GDP-岩藻糖标记上，这种方法的高潜力仅限于 核苷酸糖标记物与可接受的糖基转移酶配对。为了加快医学进步的基础 在细胞相互作用方面，迫切需要扩大核苷酸糖标记和转移酶的可获得性。 可以由此方法使用的。 我们第一阶段项目的目标是满足这一需求，并为非专家用户提供一个 在它们选择的细胞膜上标记葡聚糖。实现这一目标将扩大可用 已发表研究以外的糖工程试剂和方法。在目标1中，我们将创建12个新的核苷酸糖 带有标签的衍生品。衍生品将基于对GDP-岩藻糖的成功研究，并扩展到CMP-唾液酸 酸。核苷酸将通过荧光染料、生物素或光探针的点击化学修饰 标签。在目标2中，我们将筛选和优化能够将标记的核苷酸糖连接到 多糖底物和细胞表面。我们将推出10个套件，它们结合了最有效的标签组合 和转移酶，以及最终用户可访问的标准操作程序和条件。用户 然后可以用用于细胞表面成像的荧光标记、用于细胞选择的生物素或光探针来标记膜 用于交联剂。数据将支持第二阶段的工作，将这种方法与现有技术进行比较，并探索 特定的细胞类型。

The cell membrane is the interface by which a cell interacts and communicates with its surroundings, including nearby cells, proteins, and antibodies. Membranes of healthy cells are often modified in diseased states, providing an external means to distinguish between morphologically similar cells. Often, diseased cells display modified carbohydrates and glycans on their cell membranes. Recent developments in enzyme-mediated cell surface engineering provide promising routes for labeling these modifications in different cell types. The approach known as chemoenzymatic glycan labeling combines glycosyltransferases and unnatural nucleotide sugar donors equipped with chemical tags to directly label specific surface glycans within biological samples. Recently, this single step labelling approach has been successfully used with fucosyltransferase and GDP-fucose equipped with biotin tags. Compared with other genetic and metabolic techniques, this method of labeling living cells is a more direct, simpler procedure with a faster turnover cycle. It has also been shown to be generally applicable to murine tumor models, and thus has a high potential for transfer to a clinical setting. While published reports have focused on a low number of GDP-fucose labels, the high potential of this method is limited only by the availability of nucleotide sugar labels paired with acceptable glycosyltransferases. In order to accelerate medical advances based on cellular interactions, there is an urgent need to expand the availability of nucleotide sugar labels and transferases that can be used by this method. The goal of our Phase I project is to meet this need and provide non-expert users with a platform for labeling glycans on the membranes of their choice cells. Meeting this goal will expand the availability of glycoengineering reagents and methods beyond published studies. In Aim 1, we will create 12 new nucleotide sugar derivatives with labels. Derivatives will be based on successful studies of GDP-fucose, and expanded to CMP-sialic acid. Nucleotide sugars will be modified through click-chemistry with a fluorescent dye, biotin, or photoprobe label. In Aim 2, we will screen and optimize glycosyltransferases capable of attaching labeled nucleotide sugars to glycan substrates and cell surfaces. We will advance 10 kits that combine the most effective combinations of label and transferase, along with standard operating procedures and conditions that are accessible to end users. Users may then label membranes with fluorescent tags for cell surface imaging, biotin for cell selection, or photoprobes for crosslinking. Data will support Phase II efforts to compare this method to existing technologies and probe specific cell types.