Metabolic incorporation of photocrosslinking sugars to study sialoside function

描述（由申请人提供）：本提案描述了一项新技术，该技术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相互作用。该技术依赖于合成制备唾液酸或其前体n -乙酰甘露糖胺（ManNAc）的光交联类似物。将类似物添加到培养细胞中，使其代谢化合物，从而将光交联剂掺入细胞皂苷中。在紫外线照射下，光交联剂被激活并与邻近分子形成共价交联。Western blotting或质谱法用于鉴定交联配合物的组分。在Aim 1中，我们将通过合成一组类似物来开发最佳的光交联试剂，改变光交联剂的附着位置，光交联剂与吡喃环之间的间距以及光交联剂的特性。我们将测量这些分子对细胞涎苷的代谢，评估它们交联聚糖介导的相互作用的能力，并使用散弹枪蛋白质组学来鉴定交联复合物中存在的蛋白质。在目标2中，我们将通过确定单个唾液基转移酶的类似物耐受性来获得有关糖类似物被纳入唾液苷的信息。这些知识将有助于解释交联结果，并使我们能够为特定应用设计类似物。在Aim 3中，我们将扩大唾液基转移酶对非天然供体糖的耐受性，从而向唾液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、细胞分化和粘附、神经发育和癌症生物学中发挥重要作用。这种方法将立即适用于诸如识别唾液酸识别病毒（如流感病毒）的细胞受体或确定先天免疫系统组件（如siglece -10）的功能配体等问题。我的团队在非天然碳水化合物合成、哺乳动物细胞培养、试验开发和酵母工程方面具有必要的专业知识，可以进行拟议的实验。我们将依靠我的同事Steven Patrie博士的建议和仪器进行质谱实验。

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.