STRUCTURAL BASIS FOR AUTOIMMUNE ANTI-DNA SPECIFICITY

自身免疫性抗 DNA 特异性的结构基础

• 批准号: 2070102
• 负责人: Marko Z Radic
• 金额: $ 10.15万
• 依托单位: ALLEGHENY UNIVERSITY OF HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2070102
• 关键词: DNA; DNA footprinting; affinity chromatography; antibody specificity; antinuclear autoantibody; gene expression; gene mutation; gene rearrangement; histones; molecular cloning; molecular pathology; nucleosomes; protein structure function; site directed mutagenesis; small nuclear ribonucleoproteins; systemic lupus erythematosus; transfection

Antigen selection and affinity maturation of autoantibodies leave a genetic imprint indicative of the type of antigen that is bound in vivo. Therefore, a detailed investigation of genetic mechanisms that generate the immunological diversity of autoantibodies should provide crucial information about the induction of autoimmunity. The principle of antigen selection has been tested with murine anti-DNA antibodies and recent studies suggest that it also applies to a wide range of autoantibodies that arise in human autoimmune disease. This application proposes to analyze by in vitro mutagenesis three autospecificities that are typical of Systemic Lupus Erythematosus: anti- double stranded DNA, anti-histone, and anti-snRNP. In particular, the effects of V gene use, rearrangement, and somatic mutation will be evaluated in three antibodies that have dual specificity and bind either histones or snRNP in addition to DNA. The focus of the studies will be to learn: a) the relative contributions of the different diversity-generating mechanisms, and b) the order in which the three specificities arise. It is hoped that conclusions from these studies will be relevant for the etiology and possible treatment of Systemic Lupus Erythematosus. The experiments described have the following aims: Specific Aim 1: Clone and express anti-DNA V genes. The heavy (H) and light (L) chain V genes used by three hybridomas 3H9, LG8-1, and 1-42 will be cloned and manipulated in vitro, in order to determine the structural basis for dsDNA, nucleosome, and SmD binding. These experiments will involve chain recombination, VHCDR3 grafting, and site-directed mutagenesis. Specific Aim 2: Test antibody specificity for dsDNA conformation. Purified mutant anti-dsDNA antibodies will be used to establish whether DNA curvature improves DNA binding or not. If conformational preference is found, then: a) The size and location of bound DNA sequences will be established using Exonuclease III and ethylation interference footprinting. b) Single chain antibody fragments (scFv) will be constructed to distinguish whether preference is intrinsic to each combining site or is a consequence of IgG structure. Specific Aim 3: Explore antibody specificity for dsDNA sequence. In order to evaluate the potential for DNA sequence specificity, oligonucleotides of initially random composition will be processed through several cycles of affinity enrichment and PCR purification. Affinity chromatography will be on anti-dsDNA antibodies or anti-dsDNA scFv linked to functional domains from the transcriptional factor GCN4 and from protein A. Substrates containing a palindromic core sequence will be used to reduce the lateral displacement of bound antibodies.

自身抗体的抗原选择和亲和力成熟留下了 指示体内结合的抗原类型的遗传印记。 因此，对产生的遗传机制进行详细研究 自身抗体的免疫学多样性应该提供至关重要的 有关诱导自身免疫的信息。 抗原原理 选择已经用鼠抗 DNA 抗体进行了测试，最近 研究表明它也适用于多种自身抗体 人类自身免疫性疾病中出现的。 本申请提出通过体外诱变分析三种 系统性红斑狼疮典型的自身特异性：抗 双链 DNA、抗组蛋白和抗 snRNP。 特别是， V基因使用、重排和体细胞突变的影响将是 在具有双重特异性并结合任一抗体的三种抗体中进行了评估 除 DNA 外，还包括组蛋白或 snRNP。研究的重点将是 了解：a）不同多样性生成的相对贡献 机制，以及 b) 三个特性出现的顺序。这是 希望这些研究的结论能够对 系统性红斑狼疮的病因学和可能的治疗方法。这 所描述的实验有以下目的： 具体目标 1：克隆并表达抗 DNA V 基因。重 (H) 和 三个杂交瘤 3H9、LG8-1 和 1-42 使用的轻 (L) 链 V 基因将 进行体外克隆和操作，以确定其结构 dsDNA、核小体和 SmD 结合的基础。 这些实验将 涉及链重组、VHCDR3 嫁接和定点 诱变。 具体目标 2：测试抗体的 dsDNA 构象特异性。 纯化的突变型抗 dsDNA 抗体将用于确定是否 DNA 曲率是否会改善 DNA 结合。如果构象偏好是 找到，然后： a) 结合 DNA 序列的大小和位置将是 使用核酸外切酶 III 和乙基化干扰建立 足迹。 b) 单链抗体片段 (scFv) 将 旨在区分偏好是否是每个人固有的 结合位点或者是 IgG 结构的结果。 具体目标 3：探索抗体对 dsDNA 序列的特异性。为了 评估 DNA 序列特异性、寡核苷酸的潜力 最初的随机组合将通过几个周期进行处理 亲和富集和PCR纯化。亲和层析将 是与功能性连接的抗 dsDNA 抗体或抗 dsDNA scFv 来自转录因子 GCN4 和蛋白 A 的结构域。 含有回文核心序列的底物将用于减少 结合抗体的横向位移。