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

描述（由申请人提供）：人体抵抗感染的能力必须精确调节，以对病原体敏感，但避免虚假激活，组织损伤和疾病。这种平衡在很大程度上是由促炎细胞Fc？受体(Fc ?Rs)，尽管存在关于这种关联的根本问题。阻断Fc-Fc？然而，R相互作用有望为自身免疫性疾病提供新的治疗方法。R，即必需的Fc Asn-297 n-聚糖的作用，尚未被描述。本研究旨在阐明n -聚糖在结合和扩展免疫系统激活中的作用。这对于理解人体调节免疫系统激活的机制具有重要意义。该项目的第一步是开发一种表达富含15n的天然n糖基化IgG1 Fc片段的结构生物学量的方法。纯化后，IgG n -聚糖将使用udp - 13c -半乳糖酶促重组至均匀性。接下来，我们将评估Fc？利用溶液核磁共振波谱法测量IgG1 Fc N-聚糖的运动和结构。Fc ?RIII将被制备并滴定成含有13c -半乳糖端接n -聚糖的IgG1 Fc片段。最后，我们将描述Fc的作用。iii细胞外结构域与总IgG1 Fc糖蛋白结构的结合。多肽的结构和运动变化将使用纳入多肽的15N探针来测量。这些数据将直接影响人类健康，并导致调节Fc-Fc的新方法。R相互作用和自身免疫性疾病，以及调整治疗性抗体以具有理想的免疫调节特性。目前的研究重点是描述IgG1 Fc n -聚糖仅在Fc上的结构和运动，以及与Fc结合肽复合物中的Fc。自身免疫性疾病是由免疫系统激活不当引起的，是一种使人衰弱的疾病，限制了受影响个体的健康和生产力。这一建议将利用最近的发现来研究免疫的分子细节

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.