The overall objectives of this research project are to ascertain the principles that determine binding affinity and specificity in the recognition of the tetrasaccharide sialyl-Lewis x by the E-selectin receptor and to generate molecules that inhibit this interaction. Sialyl-Lewis x is found on the surface of leukocytes, and the E-selectin receptor is expressed on endothelial cells. Mounting evidence suggests that the formation of a protein-complex between these molecules, mediates call adhesion between leukocytes and the vascular endothelium. This intercellular interaction is an early event in an inflammatory or immune response. Inhibitors of this adhesive interaction could be used to develop new strategies for treatment of inflammatory conditions such as rheumatoid arthritis, multiple sclerosis, and reperfusion injury. These cell adhesion processes represent fundamentally new therapeutic targets. The generation of molecules to interfere with carbohydrate function offers a new frontier in inhibitor design. The proposed approach involves combining concepts and synthetic methods from organic chemistry with techniques from biochemistry and molecular and structural biology to generate the component molecules and to study the structure-function relationships in this system.
The specific aims of this research are as follows: (1) To use organic synthesis to generate the naturally-occurring carbohydrate sialyl-Lewis x and to investigate the folding properties of this carbohydrate by multi- dimensional NMR spectroscopy, (2) To produce the region of the E-selectin receptor that is responsible for carbohydrate-binding using a fusion expression system, which will allow for the generation of the free protein for NMR studies and immobilized protein for carbohydrate-binding studies, (3) To study the conformation of the oligosaccharide bound to the carbohydrate recognition domain of the E-selectin protein by multi- dimensional NMR spectroscopy, (4) To synthesize polymers containing carbohydrate residues in defined locations as potential multivalent inhibitors of cell adhesion. The information that is gained from the studies of the complex will be incorporated into the design of anti-adhesive compounds. The proposed research will generate insight into the molecular recognition and biological role for oligosaccharides in cell adhesion. It is anticipated that these studies will lead to the development of new methodologies for the modulation of cell-cell and carbohydrate-receptor interactions.
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