Biocatalysis with Endohexosaminidases: Access to Single Glycoforms of Glycoproteins

Approximately 70% of human proteins are post-translationally modified by glycosylation. It is clear that the precise carbohydrate structure attached to a glycoprotein peptide backbone can have a crucial biological role. The project aims to access a range of enzymes which are known to specifically cleave N-glycan structures from glycoproteins, and by using basic molecular biology techniques to create a variety of efficient catalysts which will allow the synthesis of particular defined glycoprotein structures. This is a particularly important area of research as glycoproteins are expressed as heterogeneous mixtures of structures, which are very difficult to separate, and so delineation of the role of particular carbohydrate structures on protein function is non-trivial. For example many glycoprotein therapeutics are currently administered as complex mixtures in which only a small fraction of the active material is present, or which contain particular carbohydrate structures which may result in toxic side effects. The project requires expertise in both molecular biology and synthetic carbohydrate chemistry.

Approximately 75% of human proteins are post-translationally modified by glycosylation. The precise carbohydrate structure attached to a glycoprotein peptide backbone can play a crucial role in protein folding, structure, and function. Since glycosylation is not under direct genetic control glycoproteins are biosynthesised as complex mixtures of glycoforms which are extremely difficult to separate. The precise roles of glycosylation on protein function have not yet been properly elucidated. Moreover currently used glycoprotein therapeutics are at the moment generally administered as complex mixtures, the individual components of which may display marked differences in bioactivity. The project aims to develop a range of biocatalysts which will allow glycoproteins to be remodeled to single homogeneous glycoforms, to greatly facilitate the further study of the role of glycosylation on protein function and also to allow the production of substantial quantities of improved glycoprotein therapeutics. A series of endo-hexosaminidases will be cloned and expressed. Site directed mutagenesis will allow access to 'synthase' enzymes which will be used for biocatalytic ligation experiments. Synthetic activated oligosaccharide coupling partners will be synthesised and used in biocatalytic experiments, with glycopeptide and protein glycosyl acceptors. Single homogeneous glycoforms of two remodeled glycoproteins will be produced, and their activity and stability investigated to test the feasibility and impact of such technological development.