GPI ANCHOR BIOSYNTHESIS IN NORMAL, MUTANT, AND PNH CELLS

关于糖基的信息有限- 哺乳动物细胞中的胞浆肌醇(GPI)锚定预组装途径，其 与生物化学反应的关系 锚定GPI及其缺陷(S)是缺乏表面蛋白的基础 在实验突变细胞和自然发生的细胞中的表达 阵发性睡眠性血红蛋白尿(PNH)影响血液成分。在……里面 初步研究：1)部分表征氨基葡萄糖(GlcN) PL前体(指定为GPI-A和-B)和甘露醇(MAN)n和取代物 (X)-Man3-GlcN-Pls(指定为GPI-C-G)，具有GPI-锚的性质 途径中间体，2)部分分析异常产物，这些异常产物 在Thy-1淋巴瘤中合成，3)来源的GPI锚定缺陷的人K562 细胞突变体表现出与淋巴瘤不同的缺陷， 4)获取生化损伤部位信息(S) 负责PNH。根据目前掌握的数据，这种哺乳动物 在以下方面，GPI与相应的布氏锥虫GPI相似 它们的葡聚糖核心结构不同，但它们的基础是 烷基甘油和均一含有酰化(酰基)肌醇(I)。而当 在前面描述的Thy-1突变体中，只有4个能合成GPI和 这2个(E类和F类)产生异常产品，至少有1个K562 突变体(常春藤)合成正常的外观前体。8名PNH患者中 到目前为止，7个受影响的白细胞显示出合成能力 GlcN-PI(GPI-B)，而不是X-Man3-GlcN-PI(GPI-G)。拟议中的实验 针对1)正常GPI-锚定的完整化学表征 途径中间体，2)亚细胞位置的定位 与合成GPI锚定前体相关的酶活性，3) 其他K562细胞突变体的衍生(用于GPI结构研究， PNH缺陷的定位，以及通过基因重组的拯救尝试)， 4)开发单独的GPI-锚合成反应的分析方法(用于 定量和纯化GPI锚酶)，以及5)鉴定 通过反义和反义基因导入编码GPI锚酶的基因 感知RNA抑制和重建策略。所获得的数据 应提供对GPI细胞内生物合成的见解，这将 一般属于哺乳动物的GPI锚定蛋白，具有临床应用价值 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.