STRUCTURE & FUNCTION OF HUMAN RBC MEMBRANE PROTEIN 7.2B

结构

• 批准号: 3247800
• 负责人: William C. Mentzer
• 金额: $ 8.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3247800
• 关键词: antibody; congenital hemolytic anemia; erythrocyte membrane; erythrocytes; human tissue; immunofluorescence technique; ion transport; laboratory rabbit; liposomes; membrane channels; membrane proteins; molecular cloning; northern blottings; protein biosynthesis; protein structure function

The band 7 proteins (MW 26-30 kda) of the human erythrocyte constitute about 3.4% of the membrane protein mass. One of the proteins (protein 7.2b) is missing from the erythrocytes of all Caucasian cases of hereditary stomatocytosis, a disorder of monovalent cation permeability that gives rise to chronic hemolytic anemia. Although this finding suggests that protein 7.2b may be part of an erythrocyte ion channel, its actual function is currently unknown. The objective and specific aims of this proposal are 1) to understand the structure of protein 7.2b and its topography within the erythrocyte membrane, 2) to evaluate its putative function as a component of an ion channel, and 3) to explore the timing and mechanics of its biosynthesis in erythrocyte progenitors and of its insertion into the membrane lipid bilayer. To achieve these goals, protein 7.2b c DNA from erythroid tissues will be identified immunologically or by oligonucleotides using a bacteriophage expression library in E. Coli. Positive clones will be sequenced (Sanger) to determine the nucleotide sequence (and from this the amino acid sequence) of the entire coding region of protein 7.2b. The amino acid sequence will be verified by chemical methods and the sites of known structural modification (phosphorylation, palmitylation) determined. Parallel protein and nucleic acid studies in hereditary stomatocytes are expected to yield an explanation for the absence of protein 7.2b in these cells. The possible role of protein 7.2b in monovalent cation transport will be evaluated by 1) comparison of the structure of 7.2b to that of known transport proteins, 2) modification of protein 7.2b function by incorporation of polyvalent anti-7.2b antibody into intact red cells using the technique of osmotic lysis and resealing with subsequent evaluation of 86Rb transport, and 3) introduction of protein 7.2b into liposomes followed by evaluation of effects on cation transport. Protein 7.2b function will also be evaluated in non-erythroid cells. The time of appearance of protein 7.2b during erythroid maturation as well as its insertion into the plasma membrane will be determined by immunofluorescent microscopy and by Northern blot analysis, searching for the presence of 7.2b mRNA. The significance of this work lies in its potential for defining the molecular basis of a cation transport system in the erythrocyte and of a disease associated with hemolytic anemia in man.

人红细胞的带7蛋白（分子量26-30 kda）构成了 约占膜蛋白质量的3.4%。 蛋白质（Protein） 7.2b）在所有高加索人的红细胞中缺失， 遗传性口细胞增多症，一种单价阳离子渗透性疾病 导致慢性溶血性贫血 尽管该结果 表明蛋白7.2b可能是红细胞离子通道的一部分， 实际功能目前未知。 目标和具体目标 这一建议是：1）了解蛋白质7.2b的结构及其 红细胞膜内的地形，2）以评估其推定的 作为离子通道的组成部分，以及3）探索时间 及其在红细胞祖细胞中的生物合成机制， 插入膜脂质双层。 为了实现这些目标， 将鉴定来自红系组织的蛋白7. 2b-c DNA 免疫学上或通过使用噬菌体表达的寡核苷酸 图书馆在E.杆菌 将对阳性克隆进行测序（桑格）， 确定核苷酸序列（并由此确定氨基酸序列） 7.2b蛋白的整个编码区氨基酸序列将 通过化学方法和已知结构的位置进行验证 修饰（磷酸化、棕榈酰化）。 平行 遗传性口细胞的蛋白质和核酸研究是预期的 以解释这些细胞中蛋白质7.2b的缺失。 蛋白7.2b在单价阳离子转运中的可能作用将是 通过1）将7.2b的结构与已知的 转运蛋白，2）通过以下途径修饰蛋白7.2b的功能： 多价抗7.2b抗体掺入完整红细胞 使用渗透溶解和再密封的技术， 86 Rb转运的评估，和3）将蛋白7.2b引入 脂质体，然后评价对阳离子转运的影响。 蛋白 7.2b的功能也将在非红系细胞中进行评价。 时 在红系成熟过程中蛋白7.2b的出现以及其 插入质膜将由以下决定： 免疫荧光显微镜和北方印迹分析，寻找 7.2b mRNA的存在。 这项工作的意义在于它 定义阳离子转运系统分子基础的潜力 在红细胞和与溶血性贫血有关的疾病， 伙计