PATHOBIOLOGY OF PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

阵发性睡眠性血红蛋白尿症(PNH) 造血细胞不能放置或维持糖基磷脂酰肌醇 (GPI)-锚定在细胞表面的蛋白质。这可能是由于 有缺陷的GPI锚合成或附着或过度活跃 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。几种生物合成材料 已经确定了中间体，包括完整的GPI核心 使用代谢标记和化学/酶试验相结合的方法。 已经开发了一个分三步走的程序来提供 明确定义突变体中的生物合成缺陷。第一, 用甘露糖标记完整的细胞以鉴定甘露糖 GPI中间体。其次，用UDP-N-乙酰-[~3H]氨基葡萄糖 给无细胞系统贴上标签，以识别早期缺陷。第三，a 采用互补性检验对突变体进行分类。 补充课。 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 中间体的分析将采用层析和 光谱程序，利用积累的 在一些突变体中相对高浓度的中间体作为 生物合成障碍的结果。类似的分析将适用于PNH 中性粒细胞和PNH T细胞株及EBV转化的B细胞 针尖定位生物合成缺陷。我们的研究将导致更好的 了解GPI锚在健康和疾病中的生物学作用。

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.