Studies of the O-GlcNAc Modification

The sequencing of the human genome revealed a frightening observation. On the face of it, humans are not very complex at all: only 20-25,000 genes. Yet, as we all know, humans ARE complex organisms. This complexity is derived, in part, from chemical events that take place withing our cells which serves to expand the complexity of proteins - the products of these these 20,000 genes. One of the most important modifications that can take place withing cells is glycosylation of proteins with a sugar called N-acetylglucosamine. This is a dynamic modification: sugars can be put on and taken off in response to diverse stimuli including diet, hormones etc. These modifications are also very important in that it controls the function of the healthy cell. Furthermore, aberent states of modifications are linked to diseases such as cancer, and diabetes. The research proposed in this grant seeks to understand the chemistry and biochemistry of this N-acetylglucosamine modification. This will allow researchers to probe the biology of the healthy cell and also allow the design and exploitation of chemical molecules to inform our knowledge of disease processes.

The O-GlcNAc modification is probably the most important glycosylation reaction in higher organisms. By 'blocking' the serine and threonine sites that are the target for kinase / phosphatase action O-GlcNAc likely impacts on almost all of the phosphorylation-based cellular signalling pathways that are known. Furthermore, O-GlcNAc does this in an equally dynamic way, responding rapidly to cellular signals. O-GlcNAc levels also respond strongly to dietary factors and provide one of the key mechanisms by which diet impacts on human health. Unlike kinase and phosphatase action, for which there are several hundred proteins involved, onlty two proteins are incvolved the the addition and removal of O-GlcNAc: a glycosyltrabsferase termed OGTase and a hexosaminidase (hydrolase) termed the O-GlcNAc hydrolase. Both enzymes will be studied under the application. X-ray crystallography will be used to study the 3D structure of an OGTase; the Holy Grail of glycobiology. Diffracting crystals of an intact protein with its tetratrico-peptide repeats have been obtained. 3-D structure of the native protein will be followed by complexes both with speficically designed nucleotide-sugar analogues and putative inhibitors. The structure will inspire further ligand design and bump-and-hole experiments to provide tools for cell biology. In concert, we will expoit the 3-D structure of the hexosaminidase, solved in preliminary work. We will design inhibitors based both upon the 3-D stucture and the known neighbouring-group participation mechanism in order to provide tools for the analysis of glycosylation in living cells. Together, the Yin-Yan approach will provide a complete structural and enzymological description of what is arguably, the most important post-transaltional modification in glycobiology.