Characterization of GPI anchor biosynthesis genes.

GPI 锚定生物合成基因的表征。

• 批准号: 08458181
• 负责人: KINOSHITA Taroh
• 金额: $ 4.35万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458181/
• 关键词: GPI Anchor; Glycogenes; Glycosyltransferases; Endoplasmic reticulum; 糖転移酵素

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.

我们的目标是克隆哺乳动物细胞中参与GPI锚定生物合成的所有基因，并对每个反应步骤进行表征。在本研究中，我们分析了第一步和第二步反应。我们鉴定了酵母GPI2的人类同源物，发现它是C类突变体PIG-C中的缺陷基因。PIG-C基因编码297个氨基酸的蛋白，在内质网中表达。PIG-C蛋白是一种疏水性蛋白，可能跨越膜六七次。它与其他已知功能的蛋白质没有显著的同源性。因此，从一级结构预测其功能是不可能的。我们发现，第一步涉及的另外两个基因产物，PIG-A和PIG-H，在内质网上形成了一个蛋白质复合体，它们都具有细胞质定向。由于PIG-A可能是介导第一步的N-乙酰氨基葡萄糖基转移酶的催化组分，这支持了第一步发生在内质网细胞质一侧的观点。我们产生了第二步缺陷的CHO细胞突变体，并使用该突变表达克隆了补充第二步缺陷的PIG-L基因。猪L基因编码252个氨基酸的蛋白，在内质网中表达。猪L蛋白具有胞质定位，支持第二步也发生在内质网的胞质侧的观点。

项目成果

Inoue, N., R.Watanabe, J.Takeda and T.Kinoshita: "PIG-C,one of the three human genes involved in the first step of glycosylphosphatidylinositol biosynthesis is a homologue of Saccharomyces cerevisiae GPI2" Biochem.Biophys.Res.Comm.226. 193-199 (1996)

Inoue, N.、R.Watanabe、J.Takeda 和 T.Kinoshita：“PIG-C 是参与糖基磷脂酰肌醇生物合成第一步的三个人类基因之一，是酿酒酵母 GPI2 的同源物”Biochem.Biophys.Res。

Takahashi, M., N.Inoue, K.Ohishi, Y.Maeda, N.Nakamura, Y.Endo, T.Fujita, J.Takeda and T.Kinoshita: "PIG-B,a membrane protein of the endoplasmic reticulum with a large lumenal domain, is involved in transferring the third mannose of the GPI anchor" EMBO J.

Takahashi, M., N.Inoue, K.Ohishi, Y.Maeda, N.Nakamura, Y.Endo, T.Fujita, J.Takeda 和 T.Kinoshita：“PIG-B，一种内质网膜蛋白，具有

Norimitsu Inoue et al.: "PIG-C,one of the three human genes involved in the first step of glycosylphospha-tidylinositol biosynthesis is a homologue of Saccharomyces cerevisiae GP12." Biochemical and Biophysical Research Communications. 226. 193-199 (1996)

Norimitsu Inoue 等人：“PIG-C 是参与糖基磷脂酰肌醇生物合成第一步的三个人类基因之一，是酿酒酵母 GP12 的同源物。”

Minoru Takahashiet al.: "PIG-B,a membrane protein of the endoplasmic reticulum with a large lumenal domain,is involved in transferring the third mannose of the GPI anchor." The EMBO Journal. 15・16. 4254-4261 (1996)

Minoru Takahashiet al.：“PIG-B 是一种具有大腔域的内质网膜蛋白，参与 GPI 锚定的第三个甘露糖的转移。” EMBO 杂志 15・16。

Reika Watanabe et al.: "PIG-A and PIG-H, which participate in glycosyl phsphatidyl-inositol anchor biosynthesis, form a protein complex in the endoplasmic reticulum." Journal of Biological Chemistry. 271・43. 26868-26875 (1996)

Reika Watanabe 等人：“PIG-A 和 PIG-H 参与糖基磷脂酰肌醇锚定生物合成，在内质网中形成蛋白质复合物。”《生物化学杂志》271·43（1996）。