Proteins on the surface of mammalian cells are often modified with diverse, complex carbohydrate structures. These carbohydrates, named glycans, play key roles in a range of cellular processes including cell-cell and host-pathogen interactions. Glycans are synthesized in the ER and Golgi by networks of multistep enzymatic pathways. In this project, we will focus on a specific type of glycan, named O-mannosyl glycan, which is unique to mammals and plays an essential role in neuromuscular tissue by linking the cell’s cytoskeleton with components of the extracellular matrix. The importance of these specific cell-matrix interactions to the proper function of muscle tissue is reflected by their role in human disease: loss of the interactions causes a range of congenital muscular dystrophies. The underlying pathology results from problems with the correct assembly of the O-mannosyl glycan due to mutations in one of the responsible biosynthetic enzymes. While the involvement of most of these enzymes in disease pathology has long been known from a genetics point of view, the exact roles and function of some of the proteins have long remained elusive. Recent findings have started to address this issue, but many aspects regarding the molecular details of enzyme function are still poorly characterised. As a consequence, it is unclear what the effect of specific genetic mutations is on the functioning of the corresponding enzymes. In order to better understand the roles of the enzymes in disease pathology, we need to better understand the function of these enzymes on a cellular and molecular level.
This project is part of a recently awarded EU grant that enables us to explore some of the key steps in the O-mannosyl glycan biosynthetic pathway through the design, synthesis and implementation of novel chemical tools. Here, we aim to isolate some of the newly identified carbohydrate-active enzymes to study their 3-D structure, gain a better understanding of their activity and unravel the mechanism by which the enzymes add carbohydrate substrates onto the glycan chain. Ultimately the objective is to elucidate how specific mutations, that are known to be causative of muscular dystrophy, affect the enzyme’s properties and how this leads to specific disease phenotypes. The project will involve the expression of genes in a range of expression hosts, purification of the protein products, biochemical assays and X-ray structure determination. In the second stage of the project, mutant proteins will be generated for analysis of the resulting effects on protein activity/structure.
Together, these approaches will enhance our fundamental understanding of the molecular mechanisms underlying the pathway of O-mannosylation. This will have significant potential to drive further cutting-edge research into the functioning of the pathway, both in healthy conditions and in the context of muscular dystrophy. This work will also have a great impact on clinical research by helping us better understand how specific mutations in our target enzymes contribute to disease. This will lay the groundwork for new approaches to treat these diseases.
Working on this project, you will benefit from a truly interdisciplinary training environment and will be exposed to methods and techniques in synthetic chemistry, biochemistry, cell biology, structural biology and glycomics. You will be working closely together with other group members to integrate your work with the synthesis of substrates and substrate analogues. Attendance at group meetings, departmental and YSBL seminars and relevant conferences will further strengthen the training of the student in their multidisciplinary approach to research.
All Chemistry research students have access to our innovative Doctoral Training in Chemistry (iDTC): cohort-based training to support the development of scientific, transferable and employability skills: https://www.york.ac.uk/chemistry/postgraduate/idtc/
In addition, the project will strengthen the students’ knowledge and skills in a variety of techniques covering biochemistry, molecular biology, cell biology and structural biology, combined with the use of small molecule inhibitors and probes generated in-house. Specifically, the student will be exposed to project specific training for the production and purification of recombinant proteins and their characterisation through spectroscopic techniques, activity assays and crystallography. Techniques include cloning and mutagenesis, gene expression in bacterial, insect and mammalian hosts, protein/affinity purification, SDS-PAGE and immunoblotting, enzyme assays and X-ray diffraction. Depending on the evolution of the project, CryoEM approaches may also be considered as the York facility is established.
The Department of Chemistry holds an Athena SWAN Gold Award and strives to provide a working environment which allows all staff and students to contribute fully, to flourish, and to excel: https://www.york.ac.uk/chemistry/ed/
It is anticipated that this PhD will start on 1 October 2020. However, there is flexibility with the start date.