COMPLEMENT ACTIVATION BY ANTIBODY N-GLYCANS

描述（改编自研究者摘要）： 免疫球蛋白（Ig）可以发挥深刻的和迄今未被认识到的作用， 其生物功能。这项研究是由矛盾的 观察到同种型匹配的单克隆抗体（Abs）在其 激活经典补体途径的能力。一对IgG 2a Ab 具有相似的单糖组成，但与凝集素的结合不同。 当Abs变性时，这些性质丧失，表明 天然抗体之间的差异反映了N-聚糖取向，而不是 聚糖本身的凝集素结合能力。实验中使用 糖基转移酶和糖苷酶来探测支持的N-聚糖可及性 这个假设。对糖基转移酶和 糖基化酶和凝集素的可及性与细胞的能力呈负相关， 结合补体的初始反应成分C1 q 系统的经典路径。携带更多糖基的Ab的去糖基化 可及聚糖增强了其激活补体的能力，这表明， 更易接近的聚糖可能抑制C1 q结合。相反，去除 其它Ab聚糖降低其补体激活潜力。有 两种抗体的可变结构域中存在许多序列差异， 编码其重链恒定区的序列是相同的，表明 可变结构域序列影响恒定结构域中的聚糖方向， 地区一对具有相似特征的同种型匹配的人Ab具有 也被识别。该提案旨在定义IG的影响 可变（V）结构域对N-聚糖方向的影响， 然后是突变的人类和小鼠抗体聚糖的详细结构分析 还将执行与功能有关结构。额外 实验将确定是否使用同种型匹配对的观察结果 Abs作为初始模型系统代表了一个更普遍的生物学模型， 现象，并确定聚糖调节的补体激活是否影响 免疫球蛋白的其他生物学特性，如清除免疫复合物 肾小球内的循环和沉积。初步意见和 拟议的研究代表了我们理解的根本性范式转变， 抗体激活补体系统，将提供有价值的见解， 自身免疫性疾病和其他炎性疾病的发病机制以及 重组治疗性Ab的合理设计。

DESCRIPTION (Adapted from Investigator's Abstract): N-glycans on immunoglobulins (Igs) can play a profound and hitherto unappreciated role in their biological function. This study was prompted by the paradoxical observation that isotype-matched monoclonal antibodies (Abs) differed in their capacities to activate the classical complement pathway. A pair of IgG2a Abs had similar monosaccharide composition yet differed in binding to lectins. These properties were lost when the Abs were denatured, suggesting that differences between the native Abs reflected N-glycan orientation rather than the lectin binding capacity of the glycans themselves. Experiments using glycosyltransferases and glycosidases to probe N-glycan accessibility supported this hypothesis. The relative susceptibility to glycosyltransferases and glycosidases and accessibility to lectins was inversely related to the ability of the two Abs to bind C1q, the initial reactive component of the complement system's classical pathway. Deglycosylation of the Ab bearing the more accessible glycan enhanced its capacity to activate complement, suggesting that the more accessible glycan might inhibit C1q binding. In contrast, removal of the other Abs glycan reduced its complement activation potential. There were many sequence differences in the variable domains of the two Abs but the sequence encoding their heavy chain constant regions was identical, suggesting that variable domain sequence affects glycan orientation in the constant region. An isotype-matched pair of human Abs with similar characteristics has also been identified. This proposal seeks to define the influence of Ig variable (V) domains on the orientation of the N-glycan using recombinant and then mutated human and mouse Abs. A detailed structural analysis of glycan structure in relation to function will also be performed. Additional experiments will determine if the observations using the isotype-matched pairs of Abs as an initial model system represent a more general biological phenomenon and determine if glycan-modulated complement activation affects other biological properties of Igs such as clearance of immune complexes from the circulation and deposition in glomeruli. The preliminary observations and proposed studies represent a fundamental paradigm shift in our understanding of Abs activate the complement system and will provide valuable insights into the pathogenesis of autoimmune and other inflammatory diseases as well as in the rational design of recombinant therapeutic Abs.