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

促进细胞中蛋白质特异性糖基化化学计量操作的工具

基本信息

批准号：
9814357
负责人：
Christina Woo
金额：
$ 49.47万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-05 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9814357
关键词：
Affinity Antibodies Binding Biological Biological Process Biological Sciences Biology Cell physiology Cells Cellular Assay Chimeric Proteins Collection Complement Data Detection Development Dissociation Drosophila genus Emerging Technologies Engineering Enzymes Evaluation Excision Fucosyltransferase Glucosamine Glycoproteins Goals In Vitro Individual Knock-out Laboratories Libraries Link Mass Spectrum Analysis Methods Mitochondria Modeling Modification Molecular Biology Molecular Biology Techniques Monosaccharides Mutagenesis Nuclear O-GlcNAc transferase Organ Peptides Performance Play Polysaccharides Post-Translational Protein Processing Procedures Protein Glycosylation Protein Isoforms Proteins Proteome Protocols documentation Publishing Reaction Reagent Reporting Reproducibility Role Sampling Signal Transduction Site Specificity Structure System Techniques Technology Tertiary Protein Structure Testing Time Transfection Transferase Up-Regulation Validation Western Blotting base biological systems cell type data integration data warehouse experience glycoproteomics glycosylation in vivo insight interest nanobodies nanomolar new technology peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase personalized approach reagent standardization stable cell line stoichiometry sugar tool vector web site

项目摘要

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.

项目概要/摘要 O-连接的 N-乙酰氨基葡萄糖 (O-GlcNAc) 是一种单糖，可修饰核细胞质蛋白和然而，如何从机制上理解它在细胞过程中的传感和信号传导中发挥着重要作用。 O-GlcNAc 在许多这些蛋白质上的功能仍然难以捉摸，部分原因是诱导蛋白质- 细胞中特异性 O-GlcNAc 酰化作用的两种酶，即 O-GlcNAc 转移酶 (OGT)。和 O-GlcNAcase (OGA)，通过不存在的机制从数千种蛋白质中添加和去除 O-GlcNAc 因此，蛋白质糖基化的操纵是通过针对个体量身定制的方法来实现的。在数量非常有限的专家实验室中对蛋白质进行研究，并对糖蛋白的功能进行深入了解，其中实现的，通常很难与更广泛的生物系统整合。最近，我们开发了一种。使用纳米抗体操纵活细胞中特定蛋白质的 O-GlcNAc 化学计量的机制纳米抗体是小的（12-15 kDa）单结构域蛋白质，具有具有纳摩尔结合亲和力的高变结构域，类似于抗体的融合。 OGT 或 OGA 的纳米抗体能够轻松地将酶引导至多种蛋白质靶标库纳米抗体融合蛋白揭示了诱导或减少 O-GlcNAc 对细胞中蛋白质的占据。新功能归因于O-GlcNAcylated蛋白质或OGT本身的实现。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现成的蛋白质印迹或 O-GlcNAc 化学计量检测目标蛋白质谱技术对操作特定的 O-GlcNAc 蛋白具有显着的障碍。减少，我们的目标是建立标准化试剂、协议和方法，供非专家实验室操作细胞中所需靶蛋白的聚糖化学计量学为了实现这一目标，我们将首先优化纳米抗体-OGT 系统用于体外和体内蛋白质特异性 O-GlcNAc 酰化，并开发基于这些优化的程序将得到扩展的补充。评估纳米抗体识别技术，以识别选择性的最佳结合亲和力将插入短肽标签或内源蛋白质靶标的糖基化。到标准载体，以便非专家实验室随时测试纳米抗体或其他聚糖编辑酶。此外，我们还将开发纳米抗体与其他聚糖编辑酶的融合，这些酶可以操纵其他聚糖最后，我们将提供试剂和方案给几个合作实验室进行试用，除了标准样品外，还可应用于感兴趣的生物系统。产生的结果将被整合并发布，目标是该方法可以在任何实验室轻松执行，无论之前是否具备糖蛋白组学方面的专业知识。