STUDIES ON DIPHTHERIA TOXIN AND ITS RECEPTOR

白喉毒素及其受体的研究

• 批准号: 3126851
• 负责人: LEON EIDELS
• 金额: $ 15.89万
• 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 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3126851
• 关键词: affinity chromatography; bacterial toxicology; chemical binding; crosslink; diphtheria toxin; gene expression; genetic manipulation; immunochemistry; laboratory mouse; laboratory rabbit; membrane activity; membrane proteins; microorganism immunology; molecular cloning; monoclonal antibody; mutant; nucleic acid sequence; protein structure; radiotracer; receptor; receptor binding; receptor mediated endocytosis; tissue /cell culture; transfection

The overall objective of this grant proposal is to characterize the receptor binding domain(s) of diphtheria toxin, and to characterize fully and purify the specific diphtheria toxin-binding cell surface protein(s) from highly toxin-sensitive Vero cells. The diphtheria toxin-binding proteins are detected by i) prebinding toxin to extrinsically radiolabeled Vero cells, followed by non- ionic detergent lysis and immunoprecipitation of the formed (receptor-toxin) complexes with anti-toxin coupled to Sepharose, and by ii) chemical cross-linking of radioiodinated diphtheria toxin bound to its receptor on the surface of Vero cells. Monoclonal antibodies will be prepared against the isolated diphtheria toxin- binding protein(s), as well as to the toxin receptor on Vero cells, to determine whether the diphtheria toxin-binding protein(s) corresponds to the toxin receptor. These monoclonal antibodies will also be utilized to investigate the cell surface distribution and mechanism of internalization of the toxin receptor on Vero cells, as well as to investigate the location and physiological state of the receptor under conditions where it lacks toxin-binding capacity (e.g. NaF, vanadate, or TPA treatment). The gene that codes for the toxin receptor will be cloned, sequenced, and will be employed to transfect toxin-resistant mouse L-cells in order to determine whether toxin resistance is due to lack of receptor expression. The results of this proposed project will provide an insight into the possible nature of the physiological cell surface receptor that diphtheria toxin utilizes to gain illicit access to the cell cytosol, will significantly extend our knowledge of toxin: receptor interactions, and will further our understanding on receptor- mediated internalization of such macromolecules as growth factors, hormones, and other exotoxins.

该赠款提案的总体目的是表征 毒素的受体结合结构域，以及 充分表征并纯化特定的白喉毒素结合 来自高度毒素敏感的Vero细胞的细胞表面蛋白。 i）预先固定检测到白喉毒素结合蛋白 毒素至外内标记的Vero细胞，然后是非 - 离子洗涤剂裂解和形成的免疫沉淀 （受体毒素）与抗毒素偶联的络合物，偶联， 并由ii）放射性白喉毒素的化学交联 与其在Vero细胞表面的受体结合。 单克隆 将制备针对分离的白喉毒素 - 结合蛋白以及Vero细胞上的毒素受体， 确定白喉毒素结合蛋白是否（S）是否 对应于毒素受体。 这些单克隆抗体 还将用于研究细胞表面分布 和Vero上毒素受体内在化的机理 细胞以及研究位置和生理状态 在缺乏毒素结合的条件下受体的 容量（例如NAF，钒酸盐或TPA治疗）。 那个基因 毒素受体的代码将被克隆，测序，并且将为 用于转染抗毒素的小鼠L细胞的使用 确定毒素耐药性是否由于缺乏受体 表达。 该提议的项目的结果将提供有关 生理细胞表面受体的可能性质 白喉毒素用来非法进入细胞细胞质， 将大大扩展我们对毒素的了解：受体 相互作用，并将进一步我们对受体的理解 介导的大分子作为生长的内在化 因素，激素和其他外毒素。