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

• 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
• 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

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.

项目摘要/摘要 O连接的N-乙酰葡萄糖（O-GLCNAC）是一种单糖，可修饰核细胞质蛋白和 在细胞过程中的灵敏度和信号传导中起着至关重要的作用。但是，机械理解如何 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的难度 细胞中的特定O-Glcnacylation。操纵O-GLCNAC，O-GLCNAC转移酶（OGT）的两种酶 和O-GlcNACase（OGA），通过不属于的机制从数千种蛋白质中添加并去除O-GlCNAC 尚未理解。这是通过量身定制的方法来实现蛋白质糖基化的操纵 蛋白质数量非常有限的专家实验室，以及对糖蛋白的功能见解， 实现的，通常很难与更广泛的生物系统集成。最近，我们开发了一个 使用纳米结词在活细胞中特异性蛋白上操纵O-GLCNAC化学计量的机制 接近指导剂。纳米型很小（12-15 kDa），单域蛋白具有 具有纳摩尔结合亲和力的高变量结构域，类似于抗体中发现的域。融合 纳米机构或OGA或OGA启用了酶的便利方向， 细胞中蛋白质上O-GlCNAC占用率的诱导或降低。纳米型融合蛋白显示 归因于O-Glcnacylated蛋白或OGT本身的新功能。实施接近度 - 定向OGT或OGA融合蛋白仅需要通过标准分子生物学对细胞进行转化 使用随时可用的蛋白质印迹或 质谱技术。在操纵特定O-Glcnacylated蛋白的障碍中 减少，我们旨在建立非专家实验室的标准化试剂，协议和方法来操纵 细胞中所需靶蛋白的聚糖化学计量法。为此，我们将首先优化 用于蛋白质特异性O-Glcnacylation的纳米型OGT系统体外和体内以及基于方案的发展 在这些优化的过程中。这些接近指导的OGT试剂将通过扩展 评估纳米型识别技术，用于识别选择性的最佳结合亲和力 短肽标签或内源性蛋白靶标的糖基化。评估的纳米体将插入 通过非专家实验室的标准矢量进行纳米生物的现成测试或其他聚糖编辑酶。 除了，我们还将开发纳米融合，以操纵额外的额外的聚糖编辑酶 聚糖结构。最后，我们将向几个合作实验室提供试剂和协议，以供试用， 除标准样本外，还适用于其感兴趣的生物系统。数据和方法 生产将被整合并发布，目的是在任何实验室中易于执行的方法， 不论糖蛋白质组学的先前专业知识如何。