Tools to facilitate manipulation of protein-specific glycosylation stoichiometry in cells

项目摘要/摘要 O-连接N-乙酰氨基葡萄糖(O-GlcNAc)是一种单糖，可修饰核质蛋白和 在细胞过程中起着感知和信号传递的重要作用。然而，机械地理解如何 O-GlcNAc在其中许多蛋白质上的功能仍然难以捉摸，部分原因是诱导蛋白质- 细胞内特异的O-GlcN酰化。操纵O-GlcNAc，O-GlcNAc转移酶(OGT)的两种酶 和O-GlcNAcase(OGA)，在成千上万的蛋白质中添加和移除O-GlcNAc的机制不是 但还不能理解。因此，蛋白质糖基化的操作是通过个体定制的方法来实现的 在非常有限的几个专家实验室中的蛋白质，以及对糖蛋白的功能洞察，其中 通常很难与更广泛的生物系统相结合。最近，我们开发了一种 以纳米体为载体操纵活细胞中特定蛋白质的O-GlcNAc化学计量的机制 近距离引导剂。纳米体是小的(12-15 kDa)单域蛋白，具有 具有纳摩尔结合亲和力的高变区，与抗体中发现的类似。融合式的 纳米体到OGT或OGA使酶能够轻松地定向到不同蛋白质靶标的文库 诱导或减少O-GlcNAc在细胞内蛋白质上的占有率。纳米体融合蛋白揭示了 新的功能归因于O-GlcNacylated蛋白或OGT本身。实施邻近性- 定向的OGT或OGA融合蛋白只需要用标准分子生物学方法导入细胞 目的蛋白的O-GlcNAc化学计量比的检测方法和方法 质谱学技术。与操纵特定O-GlcNacylated蛋白的障碍显著 简而言之，我们的目标是建立标准化的试剂、规程和方法，供非专家实验室操作 细胞中目标蛋白的糖链化学计量学。要实现这一点，我们将首先优化 蛋白质特异性O-GlcN酰化纳米体-OGT系统的体内外研究及相关方案的建立 在这些优化的程序上。这些邻近导向的OGT试剂将得到扩展的补充 纳米体识别技术用于确定选择性结合的最佳亲和力的评价 短肽标签或内源性蛋白质靶标的糖基化。评估的纳米体将被插入 到标准载体，以便由非专家实验室随时测试纳米体或额外的多糖编辑酶。 此外，我们还将开发与额外的多糖编辑酶融合的纳米体，这些酶可以操纵额外的 糖链结构。最后，我们将把试剂和方案提供给几个合作的实验室进行试用， 除标准样品外，还可用于其感兴趣的生物系统。数据和方法 产生的产品将被集成并发布，其目标是该方法易于在任何实验室中执行， 不管先前在糖蛋白组学方面的专业知识如何。

PROJECT SUMMARY/ABSTRACT O-Linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide that modifies nucleocytoplasmic proteins and plays an essential role in sensing and signaling in cellular processes. Yet, a mechanistic understanding of how O-GlcNAc functions on many of these proteins remains elusive, partly due to difficulties in inducing protein- specific O-GlcNAcylation in cells. The two enzymes that manipulate O-GlcNAc, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), add and remove O-GlcNAc from thousands of proteins by mechanisms that are not yet understood. Thus, manipulation of protein glycosylation is achieved by tailored approaches to individual proteins in a very limited number of expert laboratories, and functional insights to the glycoprotein, where achieved, are typically difficult to integrate with the broader biological system. Recently, we developed a mechanism to manipulate O-GlcNAc stoichiometry on specific proteins in live cells using nanobodies as proximity-directing agents. Nanobodies are small (12–15 kDa), single domain proteins that possess a hypervariable domain with nanomolar binding affinities, similar to that found in antibodies. Fusion of the nanobody to OGT or OGA enabled facile direction of the enzyme to a library of diverse protein targets for induction or reduction of O-GlcNAc occupancy on the protein in cells. The nanobody fusion proteins revealed new functions attributable to the O-GlcNAcylated protein or OGT itself. Implementation of the proximity- directed OGT or OGA fusion proteins only requires transfection of cells by standard molecular biology protocols and detection of O-GlcNAc stoichiometry on the target protein using readily available Western blot or mass spectrometry techniques. With barriers to manipulation of specific O-GlcNAcylated proteins significantly reduced, we aim to build standardized reagents, protocols, and methods for non-expert labs to manipulate glycan stoichiometry on a desired target protein in cells. To accomplish this, we will first optimize the nanobody-OGT system for protein-specific O-GlcNAcylation in vitro and in vivo and develop protocols based on these optimized procedures. These proximity-directed OGT reagents will be complemented by expanded evaluation of the nanobody recognition technology for identification of optimal binding affinity for selective glycosylation of short peptide tags or endogenous protein targets. The evaluated nanobodies will be inserted to standard vectors for ready testing of nanobodies or additional glycan editing enzymes by non-expert labs. Alongside, we will develop nanobody fusions to additional glycan editing enzymes that manipulate additional glycan structures. Finally, we will provide the reagents and protocols to several collaborating labs for trial use, in addition to standard samples, for application to their biological systems of interest. The data and methods produced will be integrated and published with the goal of the method being readily performed in any lab, irrespective of prior expertise in glycoproteomics.