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

项目总结/摘要 O-连接的N-乙酰葡糖胺（O-GlcNAc）是一种修饰核质蛋白的单糖， 在细胞过程中的传感和信号传导中起着重要作用。然而，机械地理解 O-GlcNAc在这些蛋白质中的许多蛋白质上的功能仍然是难以捉摸的，部分原因是难以诱导蛋白质- 细胞中特异性O-GlcNAc化。操纵O-GlcNAc的两种酶，O-GlcNAc转移酶（OGT） 和O-GlcNAc酶（OGA），通过不适合的机制从数千种蛋白质中添加和去除O-GlcNAc。 但却被理解因此，蛋白质糖基化的操纵通过针对个体的定制方法来实现。 蛋白质在数量非常有限的专家实验室，和功能的见解，糖蛋白，其中 实现，通常难以与更广泛的生物系统整合。最近，我们开发了一个 在活细胞中使用纳米抗体对特定蛋白质操纵O-GlcNAc化学计量的机制 邻近导向剂。纳米抗体是小的（12-15 kDa）、单结构域蛋白，其具有 具有纳摩尔结合亲和力的高变结构域，类似于在抗体中发现的。融合 纳米抗体与OGT或OGA的结合使酶能够容易地定向到不同蛋白质靶点的文库， 诱导或减少细胞中蛋白质上的O-GlcNAc占据。纳米抗体融合蛋白揭示了 O-GlcNAc酰化蛋白或OGT本身的新功能。实施近距离- 定向OGT或OGA融合蛋白仅需要通过标准分子生物学转染细胞 方案和使用容易获得的蛋白质印迹法检测靶蛋白上的O-GlcNAc化学计量，或 质谱技术。由于操作特定O-GlcNAc酰化蛋白的障碍， 减少，我们的目标是建立标准化的试剂、方案和方法，供非专家实验室操作 在细胞中的所需靶蛋白上的聚糖化学计量。为此，我们将首先优化 用于体外和体内蛋白质特异性O-GlcNAc化的纳米抗体-OGT系统，并开发基于 这些优化的程序。这些邻近定向的OGT试剂将通过扩增来补充。 评价纳米抗体识别技术用于鉴定选择性抗体的最佳结合亲和力 短肽标签或内源性蛋白质靶标的糖基化。评估的纳米抗体将被插入 到标准载体，用于由非专家实验室准备测试纳米抗体或另外的聚糖编辑酶。 此外，我们将开发纳米抗体融合到额外的聚糖编辑酶， 聚糖结构。最后，我们将提供试剂和方案给几个合作实验室试用， 除了标准样品之外，还可以将其应用于感兴趣的生物系统。数据和方法 所产生的将被整合和出版，目的是使该方法在任何实验室都容易进行， 而不考虑糖蛋白组学的先前专业知识。

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.