SELECTIN MEDIATED CELL ADHESION UNDER HYDRODYNAMIC SHEAR
NEELAMEGHAM, SRIRAM Principal Investigator
MetadataShow full item record
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, theseglycoproteins control the rates of leukocyte adhesion in the human vasculature during normal immuneresponse, inflammatory diseases and certain types of cardiovascular disorders. It is widely believed thatcontrolling the rate of leukocyte adhesion in vascular disorders can lead to new therapies to combat theseailments. 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 disaccharidecarbohydrate structure (GalNAc(31,3GalNAca-O-Methyl) to competitively inhibit selectin binding interactionswith its ligand. Our preliminary data suggests that this disaccharide alone can bind P- selectin. We alsodemonstrate that appropriate modification of this unit can dramatically enhance the binding affinity of theresulting carbohydrate for selectins, when compared with the prototypic selectin ligand sialyl Lewis-X. In Aim2, we test an approach where small-molecule metabolic inhibitors are designed based on the structure ofmonosaccharides that compose natural selectin ligands. These modified monosaccharidesare fed to cells inorder to interfere with the biosynthesis of specific carbohydrate epitopes on the glycoprotein ligands ofselectins. More specifically, these molecules are directed to alter either the core or terminal residues ofglycans 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 andmodify glycan biosynthetic pathways and inhibit cell adhesion. In Aim 3, to complement the experimentalwork above, a Systems Biology based mathematical model is developed to simulate biochemical networksthat regulate O-glycan biosynthesis in leukocytes. Many of the assumptions in this mathematical model areexperimentally 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 acuteinflammation, western blot analysis, molecular biology based approaches, flow cytometry, surface plasmonresonance and liquid chromatography. In the long run, we anticipate that small-molecule selectin-antagonistswill be identified from this work that may aid future drug design. Mathematical models developed willenhance the application of metabolic engineering principles in the area of biological chemistry. Such analysiscan also provide the rationale for the chemical synthesis of new inhibitors and for interpretation ofexperimental observations.