Glycosyltransferase Engineering to Dissect N-linked Protein Glycosylation

糖基转移酶工程剖析 N 连接蛋白糖基化

• 批准号: BB/V014862/1
• 负责人: Benjamin Schumann
• 金额: $ 58.56万
• 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: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV014862%2F1
• 关键词: Glycosyltransferase; Engineering; Dissect; linked; Protein

All living cells carry on their surfaces a layer of sugars called the glycocalyx. These sugars are made of approximately ten different building blocks called monosaccharides and protect cells from pathogens, desiccation and other types of stress. But the glycocalyx serves much more subtle functions, too - subtle changes to sugar molecules can facilitate or impair interactions between cells, modulate cell fate and survival. On human cells, sugars are usually attached to carrier molecules such as proteins and lipids, and fundamentally change the physical and biological properties of these carriers.Unlike other biomolecules, sugars are not directly encoded in the genome by DNA. Instead, they are synthesised by molecular machines called enzymes from simple monosaccharides. Enzymes form an assembly line to incorporate monosaccharides in a sequential manner and gradually "mature" sugars before they reach the cell surface. Unlike in most industrial manufacturing processes, sugar maturation underlies some flexibility: not all enzymes act on all sugars, and certain pathways are more often frequented than others. In order to understand the roles of cell surface sugars in processes of health and disease, it is important to understand how sugar-synthesising enzymes function.In this work, we focus on three enzymes called MGAT1, MGAT2 and MGAT5. Within the sugar assembly line, these enzymes are important bifurcation points yielding different sugars with different biological properties. Accordingly, a loss of these enzymes in patients due to gene mutation leads to severe symptoms including neurological and immunological disorders. If we can track how these enzymes function, we can thus begin to understand how sugar structures impact some of the most fundamental processes in physiology and possibly yield insights into new therapy options.To understand how MGAT1, MGAT2 and MGAT5 function, we will develop so-called reporter reagents. We will design these reporters to be specifically used by one of the three enzymes. That way, we can track the activities of the enzymes to see which sugars have incorporated the reporter reagents. We will develop such reagents using methods of biology and chemistry and establish them in living human cells. These studies will pave the way to understanding the biology of interactions happening on the cell surface and have many interesting applications in basic and applied research including the development of new treatment options against pathogenic viruses or cancer.

所有活细胞的表面都带有一层糖，称为糖萼。这些糖由大约十种不同的单糖组成，可以保护细胞免受病原体、干燥和其他类型的应激。但糖萼还具有更微妙的功能——糖分子的微妙变化可以促进或损害细胞之间的相互作用，调节细胞的命运和生存。在人体细胞上，糖通常附着在蛋白质和脂质等载体分子上，并从根本上改变这些载体的物理和生物学特性。与其他生物分子不同，糖不是由DNA直接编码在基因组中的。相反，它们是由称为酶的分子机器从简单的单糖合成的。酶形成一条装配线，以顺序方式掺入单糖，并在糖到达细胞表面之前逐渐“成熟”糖。与大多数工业制造过程不同，糖的成熟具有一定的灵活性：并非所有酶都作用于所有糖，并且某些途径比其他途径更常见。为了了解细胞表面糖在健康和疾病过程中的作用，了解糖合成酶的功能非常重要。在这项工作中，我们重点关注三种酶，即 MGAT1、MGAT2 和 MGAT5。在糖装配线中，这些酶是重要的分叉点，产生具有不同生物特性的不同糖。因此，由于基因突变而导致患者体内这些酶的损失会导致严重的症状，包括神经系统和免疫系统疾病。如果我们能够追踪这些酶的功能，我们就可以开始了解糖结构如何影响生理学中一些最基本的过程，并可能深入了解新的治疗方案。为了了解 MGAT1、MGAT2 和 MGAT5 的功能，我们将开发所谓的报告试剂。我们将设计这些报告基因以供三种酶中的一种专门使用。这样，我们就可以跟踪酶的活性，看看哪些糖结合了报告试剂。我们将利用生物学和化学方法开发此类试剂，并将其植入活的人体细胞中。这些研究将为理解细胞表面相互作用的生物学铺平道路，并在基础和应用研究中具有许多有趣的应用，包括开发针对病原病毒或癌症的新治疗方案。

项目成果

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

• DOI: 10.1016/j.xpro.2022.101974
• 发表时间: 2023-03-17
• 期刊: STAR PROTOCOLS
• 作者: Calle, Beatriz;Gonzalez-Rodriguez, Edgar;Mahoney, Keira E.;Cioce, Anna;Bineva-Todd, Ganka;Tastan, Omur Y.;Roustan, Chloe;Flynn, Helen;Malaker, Stacy A.;Schumann, Benjamin
• 通讯作者: Schumann, Benjamin