Metabolic incorporation of photocrosslinking sugars to study sialoside function

描述(由申请人提供)：这项提案描述了一种新技术，它使用代谢结合的光交联糖来发现和表征唾液酸修饰的糖蛋白和糖脂的结合作用。这项技术依赖于合成唾液酸光交联类似物或其前体N-乙酰甘露糖胺(ManNAc)。类似物被添加到培养的细胞中，这些细胞代谢化合物，导致光交联剂进入细胞唾液酸苷。在紫外光照射下，光交联剂被激活，并与邻近分子形成共价交联。Western blotting或质谱仪用于鉴定交联物的组成。在目标1中，我们将通过合成一组类似物，改变光交联剂的连接位置，光交联剂与吡喃糖环之间的间距，以及光交联剂的同一性来开发最好的光交联剂。我们将测量这些分子对细胞唾液酸苷的代谢，评估它们交联糖介导的相互作用的能力，并使用鸟枪式蛋白质组学来鉴定存在于交联物中的蛋白质。在目标2中，我们将通过确定单个唾液酸基转移酶的类似耐受性来获得糖类似物所结合的唾液酸苷的信息。这些知识将有助于解释交联结果，并使我们能够为特定的应用设计类似物。在目标3中，我们将扩大唾液酸基转移酶对非天然供体糖的耐受性，从而为唾液酸体的特定子集的特定功能化迈出第一步。唾液酸介导的相互作用在病原体识别、细胞分化和黏附、神经发育和癌症生物学中发挥着重要作用。这种方法将立即适用于识别唾液酸的细胞受体等问题-识别流感等病毒，或定义先天免疫系统组件的功能配体，如Siglec-10。我的团队拥有非天然碳水化合物合成、哺乳动物细胞培养、化验开发和酵母工程方面的基本专业知识，可以进行拟议的实验。我们将依靠我的同事史蒂文·帕特里博士的建议和仪器进行质谱学实验。 与公共健康相关：碳水化合物覆盖在所有真核细胞的表面，并决定它们如何与其他细胞、外来病原体和环境中的分子相互作用。尽管它们起着关键作用，但碳水化合物的相互作用是短暂的，因此很难研究。我建议使用非天然碳水化合物，当被光激活时，它们永久地与邻近的碳水化合物结合，提供碳水化合物相互作用的“快照”。

DESCRIPTION (provided by applicant): This proposal describes a new technology that uses metabolically incorporated photocrosslinking sugars to discover and characterize binding interactions of sialic acid- modified glycoproteins and glycolipids. This technology relies on the synthetic preparation of photocrosslinking analogs of sialic acid or of its precursor, N-acetylmannosamine (ManNAc). Analogs are added to cultured cells, which metabolize the compounds, resulting in incorporation of photocrosslinkers into cellular sialosides. Upon UV irradiation, photocrosslinkers are activated and form a covalent crosslink with neighboring molecules. Western blotting or mass spectrometry is used to identify components of crosslinked complexes. In Aim 1, we will develop the best photocrosslinking reagents by synthesizing a panel of analogs, varying the position of attachment of the photocrosslinker, the spacing between the photocrosslinker and the pyranose ring, and the identity of the photocrosslinker. We will measure the metabolism of these molecules to cellular sialosides, evaluate their ability to crosslink glycan-mediated interactions, and use shotgun proteomics to identify the proteins present in a crosslinked complex. In Aim 2, we will gain information about the sialosides into which sugar analogs are incorporated by determine the analog tolerance of individual sialyltransferases. This knowledge will aid in the interpretation of crosslinking results and enable us to design analogs for specific applications. In Aim 3, we will expand a sialyltransferase's tolerance for unnatural donor sugars, thereby making the first step toward specific functionalization of a defined subset of the sialome. Sialic acid-mediated interactions play essential roles in pathogen recognition, cell differentiation and adhesion, neural development, and cancer biology. This method will be immediately applicable to problems such as identifying cellular receptors for sialic acid-recognizing viruses like influenza or defining functional ligands for components of the innate immune system, such as Siglec-10. My group has the essential expertise in unnatural carbohydrate synthesis, mammalian cell culture, assay development, and yeast engineering to carry out the proposed experiments. We will rely on advice and instrumentation of my colleague, Dr. Steven Patrie, for mass spectrometry experiments. PUBLIC HEALTH RELEVANCE: Carbohydrates cover the surfaces of all eukaryotic cells and determine how they interact with other cells, with foreign pathogens, and with molecules in the environment. Despite their critical roles, carbohydrate interactions are fleeting and therefore difficult to study. I propose the use of unnatural carbohydrates that, when activated by light, permanently bind to their neighbors, providing a "snapshot" of carbohydrate interactions.