GPI ANCHOR BIOSYNTHESIS IN NORMAL, MUTANT, AND PNH CELLS

只有有限的信息是关于糖基- 哺乳动物细胞中的浆肌醇（GPI）锚预组装途径，其 与负责合成非- 锚GPIs和缺陷，这些缺陷是缺陷表面蛋白的基础 在实验性突变细胞和天然存在的 阵发性睡眠性血红蛋白尿（PNH）影响血液成分。 在 初步研究，我们有1）部分表征的氨基葡萄糖（GlcN） Pl前体（命名为GPI-A和-B）和甘露糖基（man）n和取代的 具有GPI锚定物性质的（X）-Man 3-GlcN-Pls（命名为GPI-C-G） 途径中间体，2）部分分析的异常产物， 在Thy-1淋巴瘤中合成，3）衍生的GPI-锚缺陷的人K562 表现出与淋巴瘤中的缺陷不同的缺陷的细胞突变体， 和4）获得关于生化病变部位的信息， 负责PNH。 根据现有的数据，哺乳动物 就以下方面而言，CPI与相应的布氏锥虫（Tryp）CPI相似 它们的聚糖核心结构，但不同之处在于它们是基于 烷基甘油并且均匀地含有酰化（酰基）肌醇（I）。 而 只有4个先前描述的Thy-1突变体合成GPIs， 这2种（E类和F类）产生异常产物，至少1 K562 突变体（IVEE）合成正常外观的前体。 8例PNH患者 迄今为止的检查显示，7例受影响的白细胞具有合成能力 GlcN-PI（GPI-B），而不是X-Man 3-GlcN-PI（GPI-G）。 拟议的实验 针对1）正常GPI锚的完整化学表征 途径中间体，2）亚细胞位点的定位， 与GPI-锚前体合成相关的酶活性，3） 衍生另外的K562细胞突变体（对于GPI结构研究， PNH缺陷的定位，以及通过基因重建的拯救尝试）， 4)开发用于单个GPI锚定合成反应的测定法（用于 定量和纯化GPI-锚定酶），和5）鉴定 编码GPI-锚定酶的基因通过使用反义和 正义RNA抑制和重建策略。 获得的数据 应该提供对GPI细胞内生物合成的见解， 一般属于哺乳动物GPI锚定蛋白， 以及基本的相关性。

Only limited information is available concerning the glycosyl- plasmanylinositol (GPI) anchor preassembly pathway in mammalian cells, its relationship to biochemical reactions responsible for synthesis of non- anchor GPIs, and defect(s) in it which underlie deficient surface protein expression in experimental mutant cells and in naturally occurring paroxysmal nocturnal hemoglobinuria (PNH) affected blood elements. In preliminary studies we have 1) partially characterized glucosaminyl (GlcN) Pl precursors (designated GPI-A and -B) and mannosyl (man)n and substituted (X)-Man3-GlcN-Pls (designated GPI-C-G) with properties of GPI-anchor pathway intermediates, 2) partially analyzed abnormal products which are synthesized in Thy-1 lymphomas, 3) derived GPI-anchor defective human K562 cell mutants which exhibit defects different from those in the lymphomas, and 4) obtained information on the site of the biochemical lesion(s) responsible for PNH. Based on the data available so far, the mammalian GPIs resemble corresponding Trypanosoma brucei (Tryp) GPIs with respect to their glycan core structures but differ in that they are based on alkylglycerol and uniformly contain acylated (acyl) inositol (I). While only 4 of the previously described Thy-1 mutants synthesize GPIs and of these 2 (classes E and F) generate abnormal products, at least 1 K562 mutant (IVEE) synthesizes normal appearing precursors. Of 8 PNH patients examined to date, affected leukocytes of 7 show an ability to synthesize GlcN-PI (GPI-B) but not X-Man3-GlcN-PI (GPI-G). The proposed experiments are directed at 1) complete chemical characterization of normal GPI-anchor pathway intermediates, 2) localization of the subcellular sites of enzymatic activities associated with synthesis of GPI-anchor precursors, 3) derivation of additional K562 cell mutants (for GPI structural studies, localization of PNH defects, and rescue attempts via gene reconstitution), 4) development of assays for individual GPI-anchor synthetic reactions (for quantitating and purifying GPI-anchor enzymes), and 5) identification of genes encoding GPI-anchor enzymes via transfections employing antisense and sense RNA inhibition and reconstitution strategies. The data obtained should provide insights into GPI intracellular biosynthesis which will pertain to mammalian GPI-anchored proteins in general and have clinical as well as basic relevance.