ANALYSIS OF DAF IN AFFECTED LYMPHOCYTES

受影响淋巴细胞中 DAF 的分析

• 批准号: 3940937
• 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/3940937
• 关键词: acetylcholinesterase; affinity chromatography; autoradiography; cell membrane; complementary DNA; gel filtration chromatography; hemoglobinurias; high performance liquid chromatography; immunologic techniques; leukocyte activation /transformation; membrane activity; molecular cloning; 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（DAF）是一种表面糖蛋白， 保护宿主细胞免受自体补体的攻击。 阵发性贫血患者的补体敏感红细胞 夜间血红蛋白尿 (PNH) 缺乏 DAF。 受影响的细胞， 然而，也缺乏乙酰胆碱酯酶（AChE）以及其他 膜因素。 我们实验室最近的研究 揭示 DAF 通过 C 端糖脂锚定在细胞上 其结构与 AChE 非常相似。 这 非常规锚点类似于 Thy- 的 C 端结构 1 抗原和锥虫变体表面糖蛋白 (VSG) 被认为是在一个过程中添加到这些表面蛋白中的 翻译后修饰。 我们发现可溶性 DAF 类似于这些其他亲水形式的分子 蛋白质存在于许多体液中。 拟议的 实验针对1）DAF分布分析 在组织中，DAF 形式的进一步结构表征，以及 细胞外基质形成机制的研究 DAF 物种，2) 生物合成前体的鉴定以供研究 人体细胞中的糖脂锚定组装/附着 利用探针来表征所涉及的步骤 DAF 锚并入，3) DAF cDNA 的分离和使用 cDNA 以确定是否有 C 端延伸肽 预计 DAF 蛋白中不存在，如 VSG 和 Thy-1 cDNA 中所示 并确定 DAF 基因组 DNA 和 DAF 信息是否 在 PNH 中是正常的，并且 4）用 DAF 生物合成进行研究 特别注意受影响的糖脂锚附着 PNH 患者的淋巴细胞及其特征 异常。 从拟议研究中获得的信息 DAF 的研究不仅与 PNH 和分子生物学相关 解释宿主细胞自然能力的机制 抵抗自体效应系统的伤害，但也可以 提供有关其他糖脂锚定蛋白表达的见解 相关蛋白质。