Microarray bioassays for functional glycome screening

Complex sugars (glycans) are a highly diverse family of molecules with a broad range of functions in biological processes including cell recognition, adhesion, cell-cell communication and signalling. The study of the glycome - the entire set of glycans expressed by particular cells or tissues - is an emerging field which aims to understand how glycan functions underpin the complexity of human biology, and their involvement in disease processes. The selective interaction of proteins with glycans is the key to their biological functions. A number of new technologies have emerged to support such interaction studies, chief among these being microarray techniques ('glycoarrays'), which allow large numbers of individual glycans to be immobilised on surfaces for examination of their selective binding to protein partners. However, whilst allowing rapid large-scale screening, glycoarrays generate simple interaction data that do not directly reveal functional properties. In this project we propose to develop a novel glycoarray methodology in which live cell responses to large numbers of glycans arrayed on specially modified microscope slides are measured quantitatively. Responses such as cell growth or differentiation will be analysed by fluorescence measurements, using either antibodies or induced expression of fluorescent proteins by the cells themselves. This approach will provide a powerful and generic new tool for decoding the function of the glycome by allowing specific glycan structures to be matched to specific biological functions.

Glycans are a highly diverse class of molecules with myriad functions in biological processes including cell recognition, adhesion and communication. The study of the glycome - the entire set of glycans expressed by particular cells or tissues - is an emerging field which aims to understand how the functions of glycans underpins biological regulation and complexity. Decoding the glycome will be a critical facet of the post-genome era, in partnership with other post-translational modifications which modify with the functional proteome. A number of new technologies have emerged to support studies of the selective interactions of glycans with their cognate protein partners which underpins their functions. Chief among these are recent microarray techniques ('glycoarrays') which allow large numbers of individual glycans to be immobilised on surfaces for screening their selective binding to protein partners. However, whilst allowing rapid large-scale screening, glycoarrays generate simple interaction data that does not directly reveal functional properties. Here we propose to develop a novel glycoarray methodology in which live cell responses to large numbers of glycans arrayed as slide-based microarrays are measured quantitatively. Such data will provide high value functional information on glycan specificity. We will: 1. Develop a slide-based microarray methodology for large-scale analysis of live cell responses to immobilized glycans. 2. Test the application of this methodology to quantitative screening of cell responses to heparan sulphate saccharides (eg. signalling pathway activation; differentiation). 3. Evaluate various potential detection methods for cell responses (eg. immunofluorescence; induction of endogenous fluorescent proteins). 4. Explore the generic application of the methodology using a different class of glycans (sialyl N-glycans). This project will provide a powerful and generic new tool for decoding the functions of the glycome.