Nakamura N, Inoue N, Watanabe R, Takahashi M, Takeda J, Stevens V L, Kinoshita T
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka 565, Japan.
J Biol Chem. 1997 Jun 20;272(25):15834-40. doi: 10.1074/jbc.272.25.15834.
Many eukaryotic cell surface proteins are bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Several genes involved in GPI anchor biosynthesis have been cloned using complementation of mutant mammalian cell lines and yeasts that are defective in its biosynthesis pathway. However, the gene involved in the second step of this pathway, in which N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) is N-deacetylated to form glucosaminyl (GlcN)-PI, has not been cloned. In this study, we established a GPI anchor-deficient mutant of Chinese hamster ovary (CHO) cells defective in the second step. Complementation analysis with the known GPI anchor mutant cells demonstrated that it belonged to the same complementation group as the CHO cell mutant G9PLAP.85. Using the new mutant, we cloned a rat gene termed PIG-L (for phosphatidylinositol glycan class L) that is involved in this step. PIG-L encodes a 252-amino acid, endoplasmic reticulum membrane protein, most of which is in the cytoplasmic side. This orientation of PIG-L protein is consistent with the notion that the second step of GPI anchor biosynthesis occurs on the cytoplasmic side of the endoplasmic reticulum.
许多真核细胞表面蛋白通过糖基磷脂酰肌醇（GPI）锚定在细胞膜上。利用在其生物合成途径中存在缺陷的突变哺乳动物细胞系和酵母的互补作用，已经克隆了几个参与GPI锚定生物合成的基因。然而，该途径第二步中涉及的基因，即N-乙酰葡糖胺基磷脂酰肌醇（GlcNAc-PI）被N-脱乙酰化形成葡糖胺基（GlcN）-PI的基因，尚未被克隆。在本研究中，我们建立了一个在第二步存在缺陷的中国仓鼠卵巢（CHO）细胞的GPI锚定缺陷突变体。与已知的GPI锚定突变体细胞进行互补分析表明，它与CHO细胞突变体G9PLAP.85属于同一互补群。利用这个新的突变体，我们克隆了一个参与这一步骤的大鼠基因，称为PIG-L（磷脂酰肌醇聚糖L类）。PIG-L编码一种252个氨基酸的内质网膜蛋白，其中大部分位于细胞质一侧。PIG-L蛋白的这种定位与GPI锚定生物合成第二步发生在内质网细胞质侧的观点一致。
