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

描述(申请人提供)：人体抵抗感染的能力必须得到精确的调节，以对病原体敏感，但避免虚假的激活、组织损伤和疾病。这种平衡在很大程度上是通过促炎症的Fc？受体(Fc？Rs)，尽管存在关于这种联系的基本问题。阻断Fc-Fc？R的相互作用有望为自身免疫性疾病提供新的治疗方法，然而，Fc？R与Fc结合的因素，即基本的Fc ASN-297 N-聚糖的作用尚未描述。本研究旨在阐明N-糖链在结合免疫系统中的作用，进而激活免疫系统。这将对了解人体用来调节免疫系统激活的机制具有很大的影响和关键。这个项目的第一步是开发一种表达15N富含的天然N-糖基化的IgG1Fc片段的结构生物学数量的方法。纯化后，将用UDP-13C-半乳糖对Ig G N-糖链进行酶促改造，使其均一。接下来，我们将使用溶液核磁共振测量糖链的运动和结构来评估Fc？RIII对IgG1Fc N-糖链构象的影响。Fc？RIII将被制备并滴定为含有13C-半乳糖末端N-糖链的IgG1 Fc片段。最后，我们将表征Fc？RIII胞外区结合对总IgG1Fc糖蛋白结构的作用。多肽的结构和运动变化将使用加入到多肽中的15N探针来测量。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，从而调节自身免疫性疾病，以及调整治疗性抗体以具有理想的免疫调节特性。目前的研究主要集中在描述IgG1Fc N-糖链在Fc上的结构和运动，以及Fc与Fc结合肽的复合体中Fc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，是限制受影响个人的健康和生产力的衰弱疾病。这项提议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的方法。

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.