New tools for studying GlcNAc biology

项目摘要/摘要 该项目的目标是开发可获得和有效的方法来监测水平和相互作用 含GlcNAc的糖偶联物的伙伴。N-乙酰氨基葡萄糖(GlcNAc)是一种发现于 哺乳动物的许多种类的糖结合物。GlcNAc与丝氨酸和苏氨酸残基相加形成 胞内O-GlcNAc修饰，GlcNAc也被结合到许多胞外糖偶联物中 包括N-连接的多聚糖和GalNAc-型多聚糖。将GlcNAc掺入糖共轭化合物是 由N-乙酰氨基葡萄糖转移酶(GlcNAc-转移酶)的活性催化 供体UDP-GlcNAc为(糖)蛋白和(糖)脂受体。UDP-GlcNAc是通过 营养感应氨基己糖胺的生物合成途径，它整合了碳水化合物，蛋白质， 脂类和核苷酸的可用性。UDP-GlcNAc水平调节关键的糖链结构的产生，即O- GlcN酰化和N-连接的糖链分支，进而控制重要的信号转导通路。 因此，含有GlcNAc的糖链是新陈代谢状态和细胞信号之间的关键环节。 然而，基于细胞的方法来表征这些分子的水平和相互作用伙伴仍然存在 不够充分。AIM 1将提供非侵入性、非扰动的荧光和发光记者 细胞内O-GlcNAc水平。这一目的建立在发现O-GlcNAc转移酶的剪接 (OGT)转录本对O-GlcNAc水平的变化做出快速反应。AIMS 2-4在先前报道的基础上改进 光交联糖技术，其中二氮杂光活化交联基安装在 活细胞中的GlcNAc残基。AIM 2将通过简化试剂使这项技术更容易使用， 提高了交联率，有利于交联物的提纯。《目标3》会做到这一点 通过将光交联GlcNAc引入到更多的类别中来扩大范围的技术 糖共轭，包括N-连接的多聚糖。Aim 4将通过开发一种 质谱学策略，不仅识别结合伙伴的位点的身份，而且还 交联点。基于质谱学的交联分析方法将捕获分子 活细胞中发生的依赖O-GlcNAc的相互作用的细节。中开发的试剂和方法 这项建议将与其他研究小组分享，以实现对各种O-GlcN酰化的研究 和具有多种生物学功能的N-糖基化蛋白。拟议的工作确定了以下方法的优先顺序 易于实施和使用“现成”试剂和程序。制作这些方法 对广泛的生物医学界是重要的，因为含有GlcNAc的失调 糖共轭化合物与多种疾病状态有关，包括糖尿病、神经退行性疾病和 癌症。

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.