New tools for studying GlcNAc biology

项目总结/摘要 该项目的目标是开发可访问和有效的方法来监测水平和互动 含有GlcNAc的糖缀合物的配偶体。N-乙酰葡糖胺（GlcNAc）是一种单糖， 许多种类的哺乳动物糖缀合物。将GlcNAc添加到丝氨酸和苏氨酸残基形成了GlcNAc。 细胞内的O-GlcNAc修饰，并且GlcNAc也掺入许多细胞外的糖缀合物中 包括N-连接聚糖和GalNAc型聚糖。将GlcNAc掺入糖缀合物中是 由N-乙酰葡糖胺基转移酶（GlcNAc-转移酶）的活性催化， 将供体UDP-GlcNAc转化为（糖）蛋白和（糖）脂质受体。UDP-GlcNAc是通过 营养传感己糖胺生物合成途径，它整合了碳水化合物，蛋白质， 脂质和核苷酸可用性。UDP-GlcNAc水平调节关键聚糖结构的产生，即O- GlcNAc酰化和N-连接聚糖分支，这反过来又控制着重要的信号转导途径。 因此，含GlcNAc的聚糖代表了代谢状态和细胞信号传导之间的关键联系。 然而，基于细胞的方法来表征这些分子的水平和相互作用伴侣仍然存在 不足Aim 1将提供非侵入性，非干扰性的荧光和发光报告基因， 细胞内O-GlcNAc水平。这一目标建立在O-GlcNAc转移酶的剪接 (OGT)转录物迅速响应O-GlcNAc水平的变化。目标2-4比以前报告的有所改进 光交联糖技术，其中二氮丙环可光活化的交联基团安装在 活细胞中的GlcNAc残基。目标2将通过简化试剂使这项技术更容易使用， 提高了交联产率，有利于交联复合物的纯化。目标3将使这个 通过将光交联GlcNAc引入到其他类别的 糖缀合物，包括N-连接的聚糖。Aim 4将通过开发一种 质谱分析策略，以确定不仅结合配偶体的位点的身份，而且还 交联位点。基于质谱的交联分析方法将捕获分子 活细胞中发生的O-GlcNAc依赖性相互作用的详细信息。开发的试剂和方法 这一建议将与其他研究小组分享，以便能够对各种O-GlcNAc酰化进行研究 和具有多种生物学功能的N-糖基化蛋白质。拟议的工作优先考虑的方法， 易于实施，并且使用“现成的”试剂和程序。使这些方法 广泛的生物医学界可用的是重要的，因为含有GlcNAc的 糖缀合物与多种疾病状态相关，包括糖尿病、神经退行性疾病和 癌

Project Summary/Abstract The goal of this project is to develop accessible and effective methods to monitor the levels and interaction partners of GlcNAc-containing glycoconjugates. N-acetylglucosamine (GlcNAc) is a monosaccharide found in many classes of mammalian glycoconjugates. Addition of GlcNAc to serine and threonine residues forms the intracellular O-GlcNAc modification, and GlcNAc is also incorporated into many extracellular glycoconjugates including N-linked glycans and GalNAc-type glycans. Incorporation of GlcNAc into glycoconjugates is catalyzed by the activity of N-acetylglucosaminyltransferases (GlcNAc-transferases) that transfer GlcNAc from the donor UDP-GlcNAc to (glyco)protein and (glyco)lipid acceptors. UDP-GlcNAc is produced through the nutrient-sensing hexosamine biosynthetic pathway, which integrates information about carbohydrate, protein, lipid, and nucleotide availability. UDP-GlcNAc levels regulate production of key glycan structures, namely O- GlcNAcylation and N-linked glycan branching, which in turn control essential signal transduction pathways. Thus, GlcNAc-containing glycans represent a crucial link between metabolic state and cellular signaling. However, cell-based methods to characterize the levels and interaction partners of these molecules remain inadequate. Aim 1 will deliver non-invasive, non-perturbing fluorescent and luminescent reporters of intracellular O-GlcNAc levels. This Aim builds on the discovery that splicing of the O-GlcNAc transferase (OGT) transcript responds rapidly to changes in O-GlcNAc levels. Aims 2-4 improve upon previously reported photocrosslinking sugar technology, in which the diazirine photoactivatable crosslinking group is installed on GlcNAc residues in living cells. Aim 2 will make this technology easier to use by simplifying the reagents, improving crosslinking yield, and facilitating purification of crosslinked complexes. Aim 3 will make this technology broader in scope by introducing photocrosslinking GlcNAc into additional classes of glycoconjugates, including N-linked glycans. Aim 4 will make the technology more powerful by developing a mass spectrometry strategy to identify not only the identity of the sites of the binding partners, but also the sites of crosslinking. The mass spectrometry-based approach to crosslinking analysis will capture molecular details of O-GlcNAc-dependent interactions that occur in living cells. The reagents and methods developed in this proposal will be shared with other research groups to enable study of a wide variety of O-GlcNAcylation and N-glycosylated proteins with diverse biological functions. The proposed work prioritizes approaches that are simple to implement and make use of “off-the-shelf” reagents and procedures. Making these methods available to the broad biomedical community is significant because dysregulation of GlcNAc-containing glycoconjugates is associated with multiple disease states including diabetes, neurodegenerative disease, and cancer.