MEMBRANE PROTEIN DEFICIENCY OF PNH ERYTHROCYTES

衰变加速因子（decay accelerating factor，ERF）是一种完整的膜蛋白 在正常人红细胞表面表达。 抑制 经典和旁路补体途径C3转化酶通过加速 这些双分子酶的亚基解离。 一种基于 该活性允许层析分离出β-内酰胺。 产生的抗体的结果 针对分离的ESTA，用于评估ESTA抗原在 阵发性睡眠性血红蛋白尿症（PNH）红细胞表面。 II型 PNH-红细胞相对缺乏，III型PNH红细胞相对缺乏。 完全缺乏抗氧化剂。 异常敏感， 补体介导的裂解表达的PNH缺陷型红细胞， 强有力的证据表明，C3转化酶调节提供了一个 在保护正常红细胞和可能的其他细胞中的重要作用， 自体补体的损伤 建议的目标 研究的目的是：1）生产单克隆抗ESTA抗体，以促进 纯化及后续研究。 2)表征 亚基结构，组成，活性位点，结合 相关补体配体，膜中的拓扑位置，和 与其他膜部分的关系，如“最近邻”所定义 交叉研究。 3)评估了药物抑制 形成，以及C3转化酶的衰变，并作为辅因子 对于C4 b/C3 b灭活剂的（I）C4 b和C3 b的裂解。 4)延长 通过表征PNH细胞中的亚基来研究DAF缺陷 PNH患者的II型红细胞和白细胞及血小板中的血小板 捐助者。 异常亚基或缺少1或2个亚基将 表明结构基因突变，而正常的 亚基的出现表明调控基因发生了突变。 PNH细胞与抗PNH抗体反应的细胞荧光照相术将允许 第一次定量的异常细胞的比例， PNH捐献者血液中的细胞类型 建议的研究将提供（A）更多 全面了解网络如何执行其重要补充功能 正常细胞膜中的调节功能，以及（B）更好的 定义PNH细胞中β-内酰胺酶缺乏的分子基础。 这 知识将为未来的研究提供必要的背景， 导致PNH的体细胞突变的性质。 的试剂和 为拟议研究制定的方法可为以下方面提供基础： PNH的未来治疗干预。

The decay accelerating factor (DAF) is an integral membrane protein expressed on the surface of normal human erythrocytes. DAF inhibits the classical and alternative complement pathway C3 convertases by accelerating the subunit dissociation of these bimolecular enzymes. An assay based on this activity permitted chromatographic isolation of DAF. Antibody raised against isolated DAF was used to assess the quantity of DAF antigen on the surface of paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes. Type II PNH-erythrocytes are relatively deficient and type III PNH erythrocytes are totally deficient in DAF antigen. The abnormal sensitivity to complement-mediate lysis expressed by the DAF deficient PNH erythrocytes is strong evidence that the C3 convertase regulation provided by DAF has an important role in protecting normal erythrocytes and probably other cells, from damage by autologous complement. The objectives of the proposed studies are to: 1) Produce monoclonal anti-DAF antibodies to facilitate the purification and subsequent studies of DAF. 2) Characterize DAF's subunit structure, composition, active site(s), capacity to bind the relevant complement ligands, topographic position in the membrane, and relation to other membrane moieties as defined by "nearest neighbor" cross-linking studies. 3) Asses the capacity of DAF to inhibit the formation, as well as the decay of C3 convertases, and to act as a cofactor for the C4b/C3b inactivator's (I) cleavage of C4b and C3b. 4) Extend the studies of the DAF-deficiency in PNH cells by characterizing the subunits of DAF in type II PNH erythrocytes and in leukocytes and platelets from PNH donors. Abnormal subunit(s) or the absence of 1 or 2 subunits would suggest a structural gene mutation, whereas a reduced number of normal appearing subunits would suggest a mutation in regulatory gene. Cytofluorography of PNH cells reacted with anti-DAF antibody will allow for the first time the quantitation of the proportion of abnormal cells of each cell type in PNH donors' blood. The proposed studies will give (A) a more complete understanding of how DAF performs its important complement regulatory function in the membrane of normal cells, and (B) a better definition of the molecular basis of the DAF deficiency in PNH cells. This knowledge will provide the necessary background for future studies of the nature of the somatic mutation responsible for PNH. The reagents and methodologies developed for the proposed studies may provide the basis for future therapeutic intervention in PNH.