Characterization of GPI anchor biosynthesis genes.

我们的目标是克隆哺乳动物细胞中参与GPI锚生物合成的所有基因，并表征每个反应步骤。在本研究中，我们分析了第一和第二反应步骤。我们鉴定了酵母GPI 2的人类同源物，并表明它是C类突变体中有缺陷的基因，称为PIG-C。PIG-C cDNA编码一种在内质网中表达的297个氨基酸的蛋白质。PIG-C蛋白质是疏水性的，可能跨越膜六或七次。它与其他已知功能的蛋白质没有明显的同源性。因此，不可能从一级结构预测其功能。我们发现参与第一步的另外两个基因产物PIG-A和PIG-H在内质网中形成蛋白质复合物，并且它们都具有细胞质取向。由于PIG-A可能是N-乙酰葡糖胺转移酶介导的第一步的催化成分，这支持了第一步发生在内质网的胞质侧的概念。我们产生了一个CHO细胞突变体，这是有缺陷的第二步，并使用这种突变体表达克隆的PIG-L cDNA的缺陷的第二步。PIG-L cDNA编码一个252个氨基酸的蛋白质，在内质网中表达。PIG-L蛋白质具有细胞质取向，支持第二步也发生在内质网的细胞质侧的观点。

Our goal is to clone all genes involved in GPI anchor biosynthesis in mammalian cells and to characterize each reaction steps. In the present study, we analyzed first and second reaction steps. We identified human homologue of yeast GPI2 and showed that it is the gene defective in class C mutant termed PIG-C.PIG-C cDNA encoded a 297 amino-acid protein that is expressed in the endoplasmic reticulum. PIG-C protein is hydrophobic, probably spanning the membrane six or seven times. It has no significant homology to other proteins with known functions. So, it is not possible to predict its function from the primary structure. We found that two other gene products involved in the first step, PIG-A and PIG-H,form a protein complex in the endoplasmic reticulum and that they both have cytoplasmic orientation. Since PIG-A may be a catalytic component of N-acetylglucosaminyl transferase that mediates the first step, this supports the notion that the first step occurs on the cytoplasmic side of the endoplasmic reticulum.We generated a CHO cell mutant that is defective in the second step and using this mutant expression cloned PIG-L cDNA that complements the defective second step. PIG-L cDNA encoded a 252 amino-acid protein that is expressed in the endoplasmic reticulum. PIG-L protein had cytoplasmic orientation, supporting the notion that the second step also occurs on the cytoplasmic side of the endoplasmic reticulum.