This proposal is designed to explore and establish the molecular basis of protein-carbohydrate interactions and an examination of some of the biochemical and physiological implications of these interactions. As models for these studies, we propose the use of carbohydrate binding proteins--- lectins. A search for new lectins with novel carbohydrate binding properties from a variety of sources will be undertaken. The techniques of X-ray crystallography, isothermal titration calorimetry, electron microscopy, proteomics, molecular cloning and cell agglutination and glycoconjugate precipitation and inhibition will be employed. The biomedical application of lectins will be pursued. These studies will include the use of the Marasmius oreades mushroom lectin which binds specifically to murine renal glomeruli causing protein-and hematuria as a model for human kidney disease; possible aberrant glycosylation of human serum glycoproteins by recombinant a1,3 galactosyltransferase for type B and a1,3-N-acetylgalactosaminyl transferase for Type A resulting in the synthesis of blood group-like antigens. This approach stems from our observation that small fractions of human serum glycoproteins bind to blood group-specific lectin affinity columns; mapping the cell surface carbohydrate present on the surface of murine embryonic stem cells and determining whether continuous contact with lectins that bind to these cells will direct them along developmental pathways leading to specific cell types: e.g., muscle, neuron and endothelial cells. The basis for lectin-carbohydrate binding specificity will be pursued by identifying the amino acid residues interacting with sugar hydroxyl groups. Based on X-ray crystal structures of lectins complexed with their sugar ligands, lectins will be cloned and mutated and their recombinant, expressed counterparts studied for their ability to bind sugars. Engineering lectins with altered specificity will also be pursued.
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