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

描述（由申请人提供）：必须精确调节人体对抗感染的能力，使其对病原体敏感，但避免假性激活、组织损伤和疾病。这种平衡是介导的，在很大程度上，在点的免疫球蛋白G（IgG）识别的促炎性Fc？受体（Fc？RS），尽管存在关于这种关联的基本问题。阻断Fc-Fc？R的相互作用有望提供新的治疗自身免疫性疾病，然而，Fc结合Fc？R，即必需Fc Asn-297 N-聚糖的作用尚未描述。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。这将对理解身体调节免疫系统激活的机制具有重要影响和关键。本项目的第一步是开发一种表达结构生物学量的15 N富集的天然N-糖基化IgG 1 Fc片段的方法。纯化后，使用UDP-13 C-半乳糖将IgG N-聚糖酶促重塑至均一。接下来，我们将评估Fc的影响。RIII对IgG 1 Fc N-聚糖构象的影响，使用聚糖运动和结构的溶液核磁共振光谱测量。FC？将制备RIII并滴定至含有13 C-半乳糖封端N-聚糖的IgG 1 Fc片段中。最后，我们将描述的作用的Fc？总IgG 1 Fc糖蛋白结构上的RIII胞外结构域结合。将使用掺入多肽中的15 N探针来测量多肽的结构和运动变化。这些数据将直接影响人类健康，并导致新的方法来调节Fc-Fc？R相互作用和因此自身免疫性疾病，以及调整治疗性抗体以具有期望的免疫调节性质。目前的研究重点是描述IgG 1 Fc N-聚糖仅在Fc上的结构和运动，以及Fc与Fc结合肽复合的结构和运动。自身免疫性疾病是由免疫系统激活不当引起的，是限制受影响个体健康和生产力的衰弱性疾病。这项提案将利用最近的发现来研究免疫的分子细节， 系统激活，并将指导未来开发更有效的治疗这些疾病。

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.