This research project examines the function of carbohydrate chains that are O-linked to leukocyte cell- surface glycoproteins. By acting as the natural ligands of the selectin family of adhesion molecules, these glycoproteins control the rates of leukocyte adhesion in the human vasculature during normal immune response, inflammatory diseases and certain types of cardiovascular disorders. It is widely believed that controlling the rate of leukocyte adhesion in vascular disorders can lead to new therapies to combat these ailments. Thus, in the current proposal, we evaluate two mechanisms for controlling selectin-ligand binding.
In Aim 1, we develop and test the ability of unique molecules based on an unusual disaccharide carbohydrate structure (GalNAc(31,3GalNAca-O-Methyl) to competitively inhibit selectin binding interactions with its ligand. Our preliminary data suggests that this disaccharide alone can bind P- selectin. We also demonstrate that appropriate modification of this unit can dramatically enhance the binding affinity of the resulting carbohydrate for selectins, when compared with the prototypic selectin ligand sialyl Lewis-X.
In Aim 2, we test an approach where small-molecule metabolic inhibitors are designed based on the structure of monosaccharides that compose natural selectin ligands. These modified monosaccharides are fed to cells in order to interfere with the biosynthesis of specific carbohydrate epitopes on the glycoprotein ligands of selectins. More specifically, these molecules are directed to alter either the core or terminal residues of glycans expressed by an important leukocyte selectin-ligand called PSGL-1 (P-selectin glycoprotein ligand- 1). We evaluate the ability and mechanism by which these chemical inhibitors permeate cells, engage and modify glycan biosynthetic pathways and inhibit cell adhesion.
In Aim 3, to complement the experimental work above, a Systems Biology based mathematical model is developed to simulate biochemical networks that regulate O-glycan biosynthesis in leukocytes. Many of the assumptions in this mathematical model are experimentally validated. Diverse experimental methods are applied to accomplish the above three aims. These include cell adhesion studies under controlled flow, in vivo experiments in a mouse model of acute inflammation, western blot analysis, molecular biology based approaches, flow cytometry, surface plasmon resonance and liquid chromatography. In the long run, we anticipate that small-molecule selectin-antagonists will be identified from this work that may aid future drug design. Mathematical models developed will enhance the application of metabolic engineering principles in the area of biological chemistry. Such analysis can also provide the rationale for the chemical synthesis of new inhibitors and for interpretation of experimental observations. ? ?
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