STUDIES ON DIPHTHERIA TOXIN AND ITS RECEPTOR

白喉毒素及其受体的研究

• 批准号: 3126850
• 负责人: LEON EIDELS
• 金额: $ 14.94万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3126850
• 关键词: antibody formation; diphtheria toxin; immunochemistry; membrane activity; microorganism immunology; monoclonal antibody; radiation detector; radiotracer; tissue /cell culture

Diphtheria is the prototype of an exotoxin-mediated infectious disease. Diphtheria toxin acts on toxin-sensitive cells by: 1) binding, via its B-fragment, to specific cell surface receptors, 2) translocation of the A-fragment into the cytoplasm, and 3) inhibition of protein synthesis by the fragment A-catalyzed ADP-ribosylation of elongation factor 2. The overall objective of this proposal is to characterize the receptor-binding domain(s) of the toxin molecule, and to fully characterize the specific diphtheria toxin-binding cell surface glycoproteins and their toxin-binding domain(s). The diphtheria toxin-binding glycoproteins are detected utilizing a glycoprotein-enriched fraction prepared from exogenously radiolabeled cells, followed by immunoprecipitation with diphtheria toxin plus antiserum to diphtheria toxin. By this method one major diphtheria toxin-binding glycoprotein is detected in hamster thymocytes while various (up to five) diphtheria toxin-binding glycoproteins are detected on the cell surface of toxin-sensitive cells grown in tissue culture (e.g. Vero, baby hamster kidney and Chinese hamster ovary cells). The nature of this multiple diphtheria toxin-binding glycoproteins will be investigated by both biochemical and immunological approaches. Specific monoclonal antibodies will be prepared against each of these glycoproteins and will be tested for their protective effect against toxin-mediated cytotoxicity in order to determine which one of the diphtheria toxin-binding glycoproteins represents the true physiological toxin receptor on cells. These antibodies will also be utilized to investigate the cell surface distribution and the internalization mechanism of the diphtheria toxin-binding glycoproteins. The results of this proposed research will significantly extend our knowledge of diphtheria toxin: receptor interactions and will further our understanding of the mechanisms involved in receptor-mediated internalization of such macromolecules as hormones, growth factors, and other exotoxins.

白喉是外毒素介导的传染病的原型。 白喉毒素作用于毒素敏感细胞，通过：1）结合，通过其结合 b-ragment，特定细胞表面受体，2） a将A碎片分解为细胞质，3）抑制蛋白质合成。 伸长因子的片段A催化的ADP-核糖基化2。 该建议的总体目的是表征受体结合 毒素分子的结构域，并充分表征特定 白喉毒素结合细胞表面糖蛋白及其毒素结合 域。 检测到白喉毒素结合糖蛋白 利用由外源制备的糖蛋白富集的分数 放射性标记的细胞，然后用白喉毒素免疫沉淀 加上抗​​白喉毒素的抗血清。 通过这种方法，一个主要的白喉 在仓鼠胸腺细胞中检测到毒素结合糖蛋白，而各种 （最多五个）在二脂毒素结合糖蛋白上被检测到 在组织培养中生长的毒素敏感细胞的细胞表面（例如Vero， 婴儿仓鼠肾脏和中国仓鼠卵巢细胞）。 这个的本质 多发性白喉毒素结合糖蛋白将通过 生化和免疫学方法。 特定的单克隆 将针对每种糖蛋白准备抗体，并将 测试了它们针对毒素介导的细胞毒性的保护作用 为了确定哪种白喉毒素结合糖蛋白 代表细胞上真正的生理毒素受体。 这些 抗体也将用于研究细胞表面 白喉的分布和内在化机制 毒素结合糖蛋白。 这项拟议研究的结果将 显着扩展了我们对白喉毒素的了解：受体 互动，将进一步了解我们对所涉及机制的理解 在受体介导的大分子等激素的内在化中， 生长因子和其他外毒素。