Engineering synthetic glycoproteins

In this multidisciplinary project we will develop new methods to introduce chemical modifications at specific sites in proteins. The modified proteins will be used as inhibitors of bacterial toxins and as pharmacological chaperones. The project falls under the EPSRC theme of chemical biology and biological chemistry and will lead to the development of novel healthcare technologies.

Protein-carbohydrate interactions at cell surfaces mediate many important processes in biology from fertilisation to adhesion of viruses, bacteria and their toxins. Individually, protein-sugar interactions are usually very weak, but both affinity and binding selectivity can be enhanced through a phenomenon called multivalency: multiple binding sites on the protein interact simultaneously with multiple copies of the sugar ligand to achieve a high avidity and enhance binding selectivity. A good example of this class of proteins is the cholera toxin which binds to five copies of a specific glycolipid ligand on the surface of cells that line the intestine. Binding to the cell surface leads to internalisation of the toxin, therefore, inhibitors of the protein-sugar interaction have the potential to be used as anti-toxin drugs.

We have recently developed methods to make chemically defined glycoproteins and demonstrated their enhanced binding to the cholera toxin protein. The cholera toxin B-subunit was converted to a non-binding mutant, to which the carbohydrate ligands were appended selectively at the N-termini of the pentameric protein. The glycoprotein inhibitor was around 14,000 times more potent as an inhibitor that the monovalent carbohydrate, but it is possible that we could enhance the binding affinity further.

Objectives.
In this project we will further develop the concept of tailored synthetic glycoprotein inhibitors by investigating the effect of the linker structure between the protein scaffold and the sugar ligand.
We will develop new chemical reagents for site-specific ligation of carbohydrates and glycosidase inhibitors to proteins.
We will evaluate the novel inhibitors using a combination of biochemical, biophysical and cell-based assays.

Potential outcomes.
The methods we develop for the synthesis of tailored glycoproteins will have future potential for the production of novel biopharmaceuticals for the treatment of infectious disease and chromic genetic conditions. Such methods will be of benefit to the growing UK biopharmaceutical industry.