Chemical tools for mechanistic insights into O-mannosyl glycan biosynthesis

细胞表面的蛋白质经常被不同的、复杂的碳水化合物结构(多糖)修饰，这些结构在一系列细胞过程中发挥关键作用，包括细胞-细胞和宿主-病原体的相互作用。这个项目的重点是参与O-甘露糖基多糖生物合成的酶，O-甘露糖基是一种与苏氨酸或丝氨酸残基O-连接的糖链，由Aplha连接的甘露糖残基启动。这种类型的多糖的特性相对较差，但已知它在细胞与细胞外基质和病原体的相互作用中发挥基础作用。在过去的十年里，人们对蛋白质α-DG的O-甘露糖化越来越感兴趣，它通过连接细胞骨架和细胞外基质在肌肉组织和神经系统中发挥重要作用。由于参与其生物合成的一种酶的缺陷，未能在α-DG上组装正确的O-甘露糖聚糖，会导致一系列先天性肌肉营养不良，还可能促进癌细胞的转移特性。然而，O-甘露糖在α-DG上的生物合成途径直到最近才被阐明(2016/2017)，尽管大多数酶的细胞作用已经被分配，但关于底物的特异性、作用机制以及调节它们的定位、稳定性和活性的因素仍不清楚。不幸的是，很少有工具可用于研究所涉及的酶的活性和机制。利用化学生物学的方法，本项目旨在通过重点研究在糖链结构的生物合成中起关键作用的两种酶，来加深我们对α-DG O-甘露糖化机理的理解。这些蛋白质的缺陷会损害α-DG的正常功能，从而导致特定类型的先天性肌营养不良。我们的目标是将结构研究与新型化学工具的使用相结合，这些工具将被设计为一种或两种感兴趣的酶的底物类似物，以帮助我们更好地了解底物结合和目标酶的机制。这些结果还将指导新型抑制剂和探针的设计，使目标酶能够进行功能分析。为了达到这些目标，我们将使用将有机合成与生物化学和结构生物学相结合的跨学科方法。学生最初将专注于产生足够数量的可溶性蛋白质，以便进行动力学和结构研究。为了促进结晶学研究的进展，学生将由戴维斯教授指导，并将受益于他的实验室提供的资源和专业知识。通过使用由同时开始的第二个博士生合成的底物类似物，这些实验将进一步便利。从这些初步研究中获得的机理见解将被用来设计一套新的抑制剂和探针，专门结合在酶活性部位内。学生将合成这些化合物，并用它们来更详细地研究酶的活性和机制。因此，学生将接受高度跨学科的培训，包括化学合成、体外分析开发、凝胶电泳和免疫印迹、细菌和哺乳动物蛋白质的表达、感兴趣的酶的纯化以及结晶技术。这项工作将加深我们对α-DG O-甘露糖化的分子机制的理解，并将为分析这些酶的分子突变的影响奠定基础。糖基转移酶探针和抑制剂的开发是化学生物学领域的一个反复出现的挑战，因此这种工具的开发将对该领域具有重要价值。

Proteins on the surface of cells are often modified with diverse, complex carbohydrate structures (glycans) that play key roles in a range of cellular processes including cell-cell and host-pathogen interactions. This project focuses on enzymes that are involved in the biosynthesis of O-mannosyl glycans, a type of glycan that is O-linked to threonine or serine residues and initiated by an aplha-linked mannose residue. This type of glycan is relatively poorly characterized, yet it is known to play fundamental roles in the interactions of cells with the extracellular matrix and with pathogens. Over the past decade there has been growing interest in O-mannosylation of the protein alpha-dystroglycan (alpha-DG), which plays essential roles in muscle tissue and the nervous system by linking the cytoskeleton with the extracellular matrix. Failure to assemble the correct O-mannosyl glycans on alpha-DG - due to a deficiency in one of the enzymes involved in their biosynthesis - causes a range of congenital muscular dystrophies and can also promote metastatic properties of cancer cells. However, the biosynthetic pathway of the O-mannosyl glycans on alpha-DG has only recently been elucidated (2016/2017), and although cellular roles for most enzymes have been assigned, much remains unclear regarding substrate specificity, mechanism of action, and factors regulating their localisation, stability and activity. Unfortunately, few tools are available to study the activity and mechanism of the enzymes involved. Using a chemical biology approach, this project aims to enhance our understanding of the mechanism of alpha-DG O-mannosylation, by focusing on two enzymes that play key roles in the biosynthesis of the glycan structure. Defects in these proteins impair the proper functioning of alpha-DG and thereby lead to specific types of congenital muscular dystrophies. We aim to combine structural studies with the use of novel chemical tools, which will be designed to act as substrate analogues for one or both of the enzymes of interest, to help improve our understanding of substrate binding and mechanism of the target enzymes. These results will also guide the design of novel inhibitors and probes that enable the functional analysis of the target enzymes. In order to reach these goals, we will use a cross-disciplinary approach that combines organic synthesis with biochemistry and structural biology. The student will initially focus on generating sufficient amounts of soluble protein to enable kinetic and structural studies. To enhance progress with the crystallography studies, the student will be supervised by Prof. Davies, and will benefit from the resources and expertise available in his laboratory. These experiments will further be facilitated by using substrate analogues synthesised by a second PhD student who will be starting at the same time. The mechanistic insights gained from these initial studies will then be used to design a new set of inhibitors and probes tailored to bind specifically within the enzyme active site. The student will synthesise these compounds and use them to study enzyme activity and mechanism in more detail. The student will thus receive a highly interdisciplinary training that includes chemical synthesis, in vitro assay development, gel electrophoresis and immunoblotting, bacterial and mammalian protein expression, purification of the enzymes of interest, and crystallisation techniques. This work will enhance our understanding of the molecular mechanisms underlying alpha-DG O-mannosylation and will lay the foundation for analysing the effects of molecular mutations in these enzymes. The development of probes and inhibitors for glycosyltransferases is a recurring challenge in the field of chemical biology and the development of such tools would thus be of significant value to the field.