NMR Studies on the Role of the Essential IgG N-Glycan in Immune System Activation

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DESCRIPTION (provided by applicant): The ability of the human body to fight infection must be precisely regulated to be sensitive to pathogens but avoid spurious activation, tissue damage and disease. This balance is mediated, in large part, at the point of immunoglobulin G (IgG) recognition by the pro-inflammatory Fc? receptors (Fc?Rs), though fundamental questions about this association exist. Blocking the Fc-Fc?R interaction promises to offer new treatments for autoimmune disorders, however, a description of the factors contributing to Fc binding by Fc?R, namely the role of the essential Fc Asn-297 N-glycan, has not been described. This study aims to elucidate the role of the N-glycan in binding, and by extension, immune system activation. This will be of high impact and critical to understanding the mechanism used by the body to regulate immune system activation. The first step in this project is to develop a method to express structural-biology quantities of 15N-enriched, natively N-glycosylated IgG1 Fc fragment. Following purification, the IgG N-glycans will be remodeled enzymatically to homogeneity using UDP-13C-galactose. Next, we will assess the affect of Fc?RIII on the conformations of the IgG1 Fc N- glycan using solution nuclear magnetic resonance spectroscopy measurements of glycan motion and structure. Fc?RIII will be prepared and titrated into the IgG1 Fc fragment containing 13C-galactose terminated N-glycans. Finally, we will characterize the role of the Fc?RIII extracellular domain binding on the structure of the total IgG1 Fc glycoprotein. Structural and motional alterations in the polypeptide will be measured using the 15N probes incorporated into the polypeptide. These data will directly impact human health and lead to new approaches to modulate the Fc-Fc?R interaction and thus autoimmune diseases, as well as tuning of therapeutic antibodies to have desirable immunoregulatory properties. Current research focuses on describing the structure and motion of the IgG1 Fc N-glycan on the Fc only and the Fc in a complex with a Fc-binding peptide. Autoimmune disorders result from improper immune system activation and are debilitating diseases that limit the health and productivity of affected individuals. This proposal will utilize recent discoveries to study the molecular details of immune system activation and will guide the future development of more effective treatments for these disorders.