Chemical tools for mechanistic insights into O-mannosyl glycan biosynthesis
Lead Research Organisation:
University of York
Department Name: Chemistry
Abstract
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.
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.