ANALYSIS OF DECAY ACCELERATING FACTOR (DAF) IN AFFECTED LYMPHOCYTES

受影响淋巴细胞中腐烂加速因子 (DAF) 的分析

• 批准号: 3855022
• 负责人: M EDWARD MEDOF
• 金额: --
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3855022
• 关键词: acetylcholinesterase; affinity chromatography; autoradiography; cell membrane; complementary DNA; enzyme mechanism; gel filtration chromatography; glycolipids; glycoproteins; hemoglobinurias; high performance liquid chromatography; immunologic techniques; laboratory rabbit; leukocyte activation /transformation; membrane activity; membrane proteins; molecular cloning; nucleic acid biosynthesis; posttranslational modifications; radioimmunoassay; scintillation spectrometry

Decay accelerating factor (DAF) is a surface glycoprotein which protects host cells from attack by autologous complement. Complement-sensitive erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH) lack DAF. The affected cells, however, also lack acetylcholinesterase (AChE) as well as other membrane factors. Recent studies in our laboratory have revealed that DAF is anchored to cells by a C-terminal glycolipid structure which closely resembles that in AChE. This unconventional anchor is similar to C-terminal structures of Thy- 1 antigen and of trypanosome variant surface glycoproteins (VSGs) which are thought to be added to these surface proteins during a post-translational modification. We have found that soluble DAF molecules which resemble hydrophilic forms of these other proteins are present in numerous bodily fluids. The proposed experiments are directed at 1) analysis of the distribution of DAF in tissues, further structural characterization of DAF forms, and investigation of the mechanism of formation of the extracellular DAF species, 2) identification of biosynthetic precursors to study glycolipid anchor assembly/attachment in human cells and exploitation of the probes to characterize the steps involved in DAF anchor incorporation, 3) isolation of DAF cDNA and use of the cDNA to establish whether a C-terminal extension peptide absent from DAF protein is predicted as in VSG and Thy-1 cDNA and to determine whether DAF genomic DNA and DAF message are normal in PNH, and 4) investigation of DAF biosynthesis with specific attention to glycolipid anchor attachment in affected lymphocytes of PNH patients and characterization of any abnormalities. The information gained from the proposed studies of DAF could not only have relevance for PNH and molecular mechanisms accounting for the natural ability of host cells to resist injury from autologous effector systems, but could also provide insights about the expression of other glycolipid anchor- associated proteins.

衰变加速因子（Decay Accelerating Factor，简称EGF）是一种表面糖蛋白， 保护宿主细胞免受自体补体的攻击。 阵发性血小板减少性紫癜患者的补体敏感性红细胞 夜间血红蛋白尿（PNH）缺乏特异性。 受影响的细胞， 然而，也缺乏乙酰胆碱酯酶（AChE）以及其他 膜因子 我们实验室最近的研究表明， 揭示了C18通过C-末端糖脂锚定在细胞上， 结构与乙酰胆碱酯酶非常相似。 这 非常规锚类似于Thy的C-末端结构， 1抗原和锥虫变体表面糖蛋白（VSG） 这些蛋白质被认为是在 翻译后修饰 我们发现可溶性的 类似于这些其他分子的亲水形式的分子 蛋白质存在于许多体液中。 拟议 实验针对1）DAF分布分析 在组织中，进一步表征了组织型的结构，以及 细胞外基质形成机制的探讨 2）鉴定生物合成前体以进行研究 糖脂锚组装/附着在人细胞中， 利用探针来表征涉及的步骤， 3）cDNA锚分离和使用 cDNA以确定C-末端延伸肽 与VSG和Thy-1 cDNA中一样， 并确定基因组DNA和DNA信息是否 PNH中的β-淀粉样蛋白的生物合成是正常的; 特别注意受影响糖脂锚附着 PNH患者的淋巴细胞和任何 异常 从拟议研究中获得的信息 PNH的发生不仅与PNH的发生有关， 解释宿主细胞的天然能力的机制， 抵抗来自自体效应系统的损伤，但也可以 提供关于其他糖脂锚的表达的见解- 相关蛋白质