ANALYSIS OF DAF IN AFFECTED LYMPHOCYTES

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。 发作性疾病患者补体敏感红细胞的检测 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表明 揭示DAF通过C-末端糖脂锚定在细胞上 与AChE中的结构非常相似的结构。这 非常规锚类似于Thy-Ty的C端结构- 1抗原和锥虫变异表面糖蛋白(VSGs) 它们被认为在这些表面蛋白中被添加到 翻译后修饰。我们发现，可溶性DAF 类似于这些化合物的亲水形式的分子 蛋白质存在于许多体液中。建议数 实验的目的是1)分析DAF的分布 在组织中，DAF形式的进一步结构特征，以及 细胞外膜形成机制的研究 DAF种，2)待研究生物合成前体的鉴定 糖脂锚定在人体细胞中的组装/附着 利用探头来表征所涉及的步骤 DAF锚定掺入，3)DAF基因的分离及应用 确定一个C-末端延伸肽是否存在于 与VSG和Thy-1基因一样，预测DAF蛋白缺失 并确定DAF基因组DNA和DAF消息 在PNH中是正常的，以及4)DAF生物合成的研究 受影响的患者应特别注意糖脂锚定附着 PNH患者外周血淋巴细胞及其亚群的特征 异常现象。从拟议研究中获得的信息 DAF不仅与PNH和分子水平有关 解释宿主细胞天然能力的机制 抵抗自体效应器系统的伤害，但也可以 提供关于其他糖脂锚定表达的见解- 相关蛋白质。

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.