PATHOBIOLOGY OF PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

在阵发性睡眠性血红蛋白尿症（PNH）中， 造血细胞不能放置或维持糖基磷脂酰肌醇 （GPI）锚定蛋白在细胞表面上。 这可能是由于 GPI锚合成或附着缺陷或过度活跃 磷脂酶 我们的实验室已经开始对GPI进行表征 哺乳动物细胞中的生物合成途径。 几种生物合成 中间体，包括完整的GPI核心，已经确定 使用代谢标记和化学/酶测试的组合。 已制定了一个三步程序， 突变体中生物合成缺陷的明确定义。 第一、 [3 H]甘露糖用于标记完整细胞以鉴定甘露糖基化的 GPI中间体。 第二，使用UDP-N-乙酰基-[3 H]葡糖胺， 标记无细胞系统以识别早期缺陷。 三是 互补试验用于将突变体分为不同的 互补类 在这里，我们建议将这项技术应用于一组小鼠， 和人类突变细胞系。 关键GPI的详细结构 中间体将采用色谱和 光谱程序，利用积累的 在某些突变体中， 生物合成阻滞的结果。 类似的分析将适用于PNH 嗜中性粒细胞和PNH T细胞系和EBV转化的B细胞， 找出生物合成缺陷 我们的研究会带来更好的 了解健康和疾病中GPI锚的生物学。

In Paroxysmal Nocturnal Hemoglobinuria (PNH) a number of hematopoietic cells cannot place or maintain glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface. This could be a result of defective GPI anchor synthesis or attachment or an overactive phospholipase. Our laboratory has begun to characterize the GPI biosynthetic pathway in mammalian cells. Several biosynthetic intermediates, including the complete GPI core, have been identified using a combination of metabolic labeling and chemical/enzymatic tests. A three-step procedure has been developed to provide unequivocal definition of biosynthetic defects in the mutants. First, [3H]mannose was used to label intact cells to identify the mannosylated GPI intermediates. Second, UDP-N-acetyl-[3H]glucosamine was used to label a cell-free system to identify earlier defects. Third, a complementation test was used to classify the mutants into different complementation classes. Here, we propose to apply this technology to a panel of murine and human mutant cell lines. The detailed structures of key GPI intermediates will analyzed with a combination of chromatographic and spectroscopic procedures, taking advantage of the accumulation of relatively high concentrations of intermediates in some mutants as a result of biosynthetic block. Similar analysis will be applied to PNH neutrophils and to PNH T cell lines and EBV-transformed B cells to pin-point the biosynthetic defect. Our research will lead to a better understanding of the biology of the GPI anchor in health and disease.