PATHOBIOLOGY OF PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

糖基磷脂酰肌醇（GPI）作为一种新型的膜锚， 许多真核生物蛋白质。 我们的实验室已经确定了一系列的GPI 生物合成中间体，并提出了生物合成途径， 哺乳动物GPI锚。 这些进展有助于阐明 阵发性睡眠性血红蛋白尿症（PNH）的分子缺陷， 获得性造血障碍影响GPI锚生物合成。 最近，我们的实验室克隆了人类H类cDNA， 三个基因（H类、A类和C类）需要GPI锚的起始 在生物合成中，即在G1 cNAc从UDP-G1 cNAc转移到 肌醇磷脂（PI）受体。 同年，A类cDNA 是由另一个实验室独立克隆的 这些进步提供了 我们有一个极好的机会来学习GPI锚的第一步 在分子水平上的生物合成。 我们的目标是：1）表征H类蛋白。 抗体 将产生H类蛋白质。 蛋白质印迹分析， 免疫沉淀和免疫荧光显微镜将用于 确定其亚细胞定位、膜结合、膜 定向，并与其他蛋白质结合; 2）克隆小鼠 H类cDNA和人H类基因。 序列保守性 物种和外显子/内含子结构将提供结构上的线索 H类蛋白质的组织; 3）表征A类蛋白质 蛋白 H类和A类蛋白的潜在关联将是 探讨; 4）阐明在选定的PNH患者的分子缺陷。 由于大多数PNH患者有A类缺陷， A类cDNA中的移码突变将确定重要的 5）克隆C类cDNA。 为了充分了解转移酶的活性， C cDNA将通过表达克隆进行鉴定; 6）研究 UDP-G1 cNAc：PI转移酶的结构/功能关系。 一旦 已经鉴定了所有三种基因并且表达了它们的基因产物， 用叠氮基-UDP-G1 cNAc进行光亲和标记，固相ELISA将 以确定这些蛋白质的具体功能。 最后，纯化的蛋白质将用于重组转移酶 体外活性 这些研究将大大提高我们的 了解生物化学、分子生物学和细胞生物学 GPI锚生物合成在正常和PNH细胞。

Glycosylphosphatidylinositol (GPI) serves as a novel membrane anchor for many eukaryotic proteins. Our laboratory has identified a series of GPI biosynthetic intermediates and proposed a biosynthetic pathway for the mammalian GPI anchor. These advances contributed to the elucidation of the molecular defect in Paroxysmal Nocturnal Hemoglobinuria (PNH), an acquired hematopoietic disorder affecting GPI anchor biosynthesis. Recently, our laboratory has cloned the human class H cDNA, one of the three genes (Class H, A, and C) required the initiation of GPI anchor biosynthesis, i.e. in the transfer of G1cNAc from UDP-G1cNAc to an inositol phospholipid (PI) acceptor. In the same year, the class A cDNA was independently cloned by another laboratory. These advances provide us with an excellent opportunity to study the first step of GPI anchor biosynthesis in molecular terms. Our aims are: 1) to characterize the class H protein. Antibodies against the class H protein will be produced. Western blot analysis, immunoprecipitation, and immunofluorescent microscopy will be used to define its subcellular localization, membrane association, membrane orientation, and association with other proteins; 2) to clone the mouse class H cDNA and the human class H gene. Sequence conservation between species and exon/intron structure will provide clues on the structural organization of the class H protein; 3) to characterize the class A protein. The potential association of the class H and A protein will be explored; 4) to elucidate the molecular defects in selected PNH patients. Since most PNH patients have class A defect, knowledge of missense or frameshift mutations in the class A cDNA will pinpoint important functional domains in the class A protein; 5) to clone the class C cDNA. In order to fully understand the transferase activity, the human class C cDNA will be identified by expression cloning; 6) to study the structure/function relationship of the UDP-G1cNAc:PI transferase. Once all three genes have been identified and their gene products expressed, photoaffinity labeling with azido-UDP-G1cNAc and a solid phase ELISA will be carried out to determine the specific function of these proteins. Finally, purified proteins will be used to reconstitute the transferase activity in vitro. These studies will be significantly enhance our understanding of the biochemistry, molecular biology, and cell biology of GPI anchor biosynthesis in normal and PNH cells.