IMMUNOCHEMISTRY OF AN AGING ANTIGEN

老化抗原的免疫化学

• 批准号: 3121075
• 负责人: MARGUERITE M KAY
• 金额: $ 15.65万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1995-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3121075
• 关键词: affinity chromatography; aging; antigens; autoantibody; autoradiography; band 3 protein; cell senescence; chemical binding; erythrocytes; human subject; immunochemistry; immunoglobulin G; molecular site; mutant; nucleic acid sequence; phagocytosis; protein purification; protein sequence; protein structure function; site directed mutagenesis; synthetic peptide; tissue /cell culture; transfection; western blottings

Senescent cell antigen (SCA), an aging antigen, is a protein that appears on old cells and acts as a specific signal for the termination of that cell by initiating the binding of IgG autoantibody and subsequent removal by phagocytes. This appears to be a general physiologic process for removing senescent and damaged cells in mammals and other vertebrates. SCA is generated by degradation of the major anion transport protein of vertebrates, band 3. Because of its central role in respiration of CO2, band 3 is the most heavily used ion transport system in vertebrate animals. We have used synthetic peptides to identify antigenic sites on membrane protein band 3 recognized by the IgG that binds to old cells. Results indicate that crucial anion transport segments of the band 3 molecule carry the aging determinants and suggest that these may be the aging sites of the molecule. The purpose of this proposal is to further define the antigenic sites in senescent cell antigen and the aging vulnerable site(s) of the band 3 molecule which are involved in its demise. We will use purified IgG antibodies from old cells (SCIgG) to identify the antigenic site(s) on old cells. Three distinct assays will be used. These are a phagocytosis inhibition assay, a protein A/IgG binding assay, and immunoblotting. In the first aim, we will purify the specific IgG autoantibody to SCA from aged red blood cells. We will use this autoantibody to determine the amino acids critical to antigenicity using synthetic peptides. Peptides with single amino acid changes will be used to locate amino acid residues that are crucial to aging antigens. Results obtained in this manner will be tested using site directed mutagenesis. This information will be used to develop "designer" peptides for which SCIgG has a greater affinity and/or that are more resistant to degradation than the native aging antigen. Then, on the basis of this information, site-directed mutagenesis will be used to construct "mutant" band 3 proteins that contain amino acid substitutions at these positions. This allows assessment of the functional importance of these altered residues. Functional assays for anion transport will be added to the other assays for this part of the study. Molecular changes occurring during aging that initiate changes in the band 3 molecule will be defined using synthetic peptides and 980 antibodies which bind to aged band 3 before degradation to SCA. Site directed mutagenesis will be used as an independent approach to identifying the aging vulnerable site. These studies will allow us to identify and define the aging antigenic and aging vulnerable site, and to determine the critical amino acid residues for each site. We anticipate that the results of these studies will facilitate regulation of cellular lifespan and the development of peptides to alleviate cell destruction in hematologic diseases.

衰老抗原衰老细胞抗原（SCA）是一种出现的蛋白质 在旧细胞上，充当该细胞终止的特定信号 通过启动IgG自身抗体的结合，然后通过 吞噬细胞。 这似乎是去除的一般生理过程 哺乳动物和其他脊椎动物中的衰老和受损细胞。 SCA是 由主要阴离子转运蛋白的降解产生 脊椎动物，带3。由于其在CO2呼吸中的核心作用， 带3是脊椎动物动物中使用最严重的离子传输系统。 我们已经使用合成肽来识别膜上的抗原位点 IgG识别与旧细胞结合的蛋白质带3。 结果 表明频带3分子携带的关键阴离子传输段 老化的决定因素，并建议这些可能是 分子。 该提议的目的是进一步定义抗原 衰老细胞抗原和衰老脆弱部位的位置 带3个分子与其灭亡有关。 我们将使用纯化的IgG 来自旧细胞（SCIGG）的抗体以识别旧细胞的抗原位点 细胞。 将使用三种不同的测定法。 这些是吞噬作用 抑制测定，蛋白A/IgG结合测定和免疫印迹。 在 第一个目的，我们将净化特定的IgG自身抗体 老年红细胞。 我们将使用此自身抗体来确定氨基 使用合成肽对抗原性至关重要的酸。 肽与 单个氨基酸的变化将用于定位氨基酸残基 对于衰老抗原至关重要。 以这种方式获得的结果将是 使用位点定向诱变进行了测试。 此信息将用于 开发SCIGG具有更大亲和力和/或的“设计师”肽 比天然老化抗原更能抵抗降解。 然后，根据这些信息，将定为位置的诱变将是 用于构建含有氨基酸的“突变”条带3 在这些职位上取代。 这允许评估功能 这些改变的残留物的重要性。 阴离子的功能测定 该研究部分的运输将添加到其他测定中。 衰老期间发生变化的分子变化 3分子将使用合成肽和980抗定义 在降解为SCA之前，它与老年频带3结合。 站点定向 诱变将被用作识别的独立方法 老化脆弱的网站。 这些研究将使我们能够识别并定义 老化的抗原和老化脆弱部位，并确定 每个部位的关键氨基酸残基。 我们预计结果 这些研究将有助于调节细胞寿命和 肽的发展以减轻血液学的细胞破坏 疾病。