A genetically encoded reporter platform to dissect the O-glycoproteome

用于剖析 O-糖蛋白质组的基因编码报告平台

• 批准号: BB/V008439/1
• 负责人: Benjamin Schumann
• 金额: $ 58.4万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV008439%2F1
• 关键词: genetically; encoded; reporter; platform; dissect

Every single living cell carries on its surface a protective layer of sugar molecules called the glycocalyx. These molecules are much more complex than dietary sugar and are an important component of life. As the outermost layer of cells, the glycocalyx is often the first part that interacts with other cells, pathogens such as viruses, and signalling molecules. The structure of the sugar molecules is highly variable, and small changes can have a profound impact, for example on metabolism and on mounting an effective immune response.Unlike other biomolecules, sugars are not directly encoded in the genome - there is no DNA template that codes for them. Instead, molecular machines called enzymes build complex sugars from simple building blocks. These enzymes form an assembly line that sequentially incorporates each building block into proteins, creating a huge complexity of sugar structures. The interplay between enzymes determines which structures are eventually made. In order to understand the roles of sugar molecules in health and disease, it is thus important to understand how enzymes function.Our focus is on a large class of enzymes called GalNAc-Ts that incorporate a certain sugar building block called GalNAc into proteins. These proteins eventually end up on the cell surface or in the bloodstream and impact lots of different processes. When GalNAc-Ts do not function properly, a range of severe effects are seen. For instance, cancer cells often have too many GalNAc-T enzymes. As there are many different GalNAc-Ts with slightly different roles in a cell, it is very difficult to understand on a molecular level how they work together. This understanding is important as it will shed light on some of the most fundamental processes in biology and give clues about the design of new drugs.To understand how GalNAc-Ts work together, we will develop reagents that act as so-called reporters. If these reagents are specific for a certain GalNAc-T enzyme, they should tell us which target protein the GalNAc-T worked on to transfer the sugar GalNAc to. In order to make the reagents specific, we will use a trick: the reagents are designed such that they are only used by a single GalNAc-T that has been slightly altered or engineered. Since none of the normal, unchanged GalNAc-Ts can bind the reagents, they won't give us a signal. We can then use these reporter reagents to tell us which GalNAc-T worked on which protein.We will use this technique to study the entire GalNAc-T family. We will set up a platform of specific reagents, and generate data that will be shared with the scientific community. Our approach will give us important insight into the way sugars are incorporated into living cells. These studies will pave the way to many different aspects of basic and applied research, from understanding molecular mechanisms of physiology to generating drugs.

每个活细胞的表面都带有一层糖分子保护层，称为糖萼。这些分子比膳食糖复杂得多，是生命的重要组成部分。作为细胞的最外层，糖萼通常是与其他细胞、病毒等病原体和信号分子相互作用的第一个部分。糖分子的结构高度可变，微小的变化可能会产生深远的影响，例如对新陈代谢和有效免疫反应的影响。与其他生物分子不同，糖不是直接在基因组中编码的 - 没有 DNA 模板他们的代码。相反，称为酶的分子机器从简单的构建块构建复杂的糖。这些酶形成一条装配线，依次将每个构建模块整合到蛋白质中，从而形成极其复杂的糖结构。酶之间的相互作用决定了最终形成的结构。为了了解糖分子在健康和疾病中的作用，了解酶的功能非常重要。我们的重点是一大类称为 GalNAc-T 的酶，它将某种称为 GalNAc 的糖构建块整合到蛋白质中。这些蛋白质最终出现在细胞表面或血液中，并影响许多不同的过程。当 GalNAc-T 不能正常发挥作用时，会出现一系列严重的影响。例如，癌细胞通常含有过多的 GalNAc-T 酶。由于细胞中有许多不同的 GalNAc-T，它们的作用略有不同，因此很难在分子水平上理解它们如何协同工作。这种理解很重要，因为它将揭示生物学中一些最基本的过程，并为新药的设计提供线索。为了了解 GalNAc-T 如何协同工作，我们将开发充当所谓记者的试剂。如果这些试剂对某种 GalNAc-T 酶具有特异性，它们应该告诉我们 GalNAc-T 将糖 GalNAc 转移到哪个靶蛋白上。为了使试剂具有特异性，我们将使用一个技巧：试剂的设计使其仅由经过轻微改变或设计的单个 GalNAc-T 使用。由于正常的、未改变的 GalNAc-T 都不能结合试剂，因此它们不会给我们信号。然后我们可以使用这些报告试剂来告诉我们哪种 GalNAc-T 对哪种蛋白质起作用。我们将使用这种技术来研究整个 GalNAc-T 家族。我们将建立一个特定试剂的平台，并生成与科学界共享的数据。我们的方法将使我们对糖融入活细胞的方式有重要的了解。这些研究将为基础和应用研究的许多不同方面铺平道路，从理解生理学的分子机制到生产药物。

项目成果

O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Likely Contribute to the Mutational Trajectory in Variants of Concern

O-连接唾液酸聚糖调节 SARS-CoV-2 刺突的蛋白水解，并可能有助于引起关注的变体的突变轨迹

• DOI: http://dx.10.1021/acscentsci.2c01349
• 发表时间: 2023
• 期刊: ACS Central Science
• 影响因子: 18.2
• 作者: Gonzalez

Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.

对人多肽 N-乙酰半乳糖胺转移酶进行凹凸工程，以剖析其蛋白质底物和细胞中的糖基化位点。

• DOI: http://dx.10.1016/j.xpro.2022.101974
• 发表时间: 2023
• 期刊: STAR protocols
• 作者: Calle B