Biosynthesis pathway of GPI anchored proteins.

GPI锚定蛋白的生物合成途径。

• 批准号: 13480194
• 负责人: KINOSHITA Taroh
• 金额: $ 9.54万
• 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: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480194/
• 关键词: post trarnslational modification; endoplasmic reticulum; glycolipid; GPI anchor; biosynthesis; gene cloning; 塘脂質

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a prolacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GOI. It was known that the GPI transamidase is a complex containing GAAI and GPI8 To further characterize GPI transamidase, we affinity-purified the enzyme complex and found two new components PIG-S and PIG-T. To determinc roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins. We established Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the responsible gene, termed PIG-U, that encodes a 435-amino-acid hydrophobic protein. GPI transamidase complex affnity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U as well as four other known components. Cells lacking PIG-U normally formed complexes of four other components but had no ability to cleave the GPI attachment signal peptide. Therefore, these results demonstrated that GPI transamidase consists of five proteins. We also demonstrated that two subunits, GPI8 and GPI-T, from a functionally important disulfide bond through conserved cysteine residues.

许多真核细胞表面蛋白通过糖基磷脂酰肌醇（GPI）锚定在质膜上。GPI转酰胺酶通过用预组装的GOI替换蛋白质的C-末端GPI附着信号肽来介导GPI锚定在内质网中。众所周知，GPI转酰胺酶是一种含有GAAI和GPI 8的复合物。为了进一步表征GPI转酰胺酶，我们对该酶复合物进行了亲和纯化，发现了两个新组分PIG-S和PIG-T。为了确定PIG-S和PIG-T的作用，我们通过同源重组在小鼠F9细胞中破坏这些基因。PIG-S和PIG-T敲除细胞在GPI向蛋白质的转移方面有缺陷。我们建立了中国仓鼠卵巢细胞，代表一个新的互补组GPI锚定蛋白缺陷突变体，U类。U类细胞积累成熟和未成熟的GPI，并且不具有体外GPI转酰胺酶活性。我们克隆了负责基因，称为PIG-U，编码435个氨基酸的疏水蛋白。从表达表位标记的GPI 8的细胞亲和纯化的GPI转酰胺酶复合物含有PIG-U以及四种其它已知组分。缺乏PIG-U的细胞通常形成四种其他组分的复合物，但没有能力切割GPI附着信号肽。因此，这些结果表明GPI转酰胺酶由五种蛋白质组成。我们还证明了两个亚基，GPI 8和GPI-T，从一个功能上重要的二硫键通过保守的半胱氨酸残基。

项目成果

Maeda, Y.: "PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER."EMBOJ.. 20. 250-261 (2001)

Maeda, Y.：“PIG-M 将第一个甘露糖转移到 ER 腔侧的糖基磷脂酰肌醇上。”EMBOJ.. 20. 250-261 (2001)

Maeda, Yusuke: "PIG-M transfers the first mannose to glycosyiphosphatidylinositol on the lumenal side of the ER"EMBO J.. 20. 250-261 (2001)

Maeda, Yusuke：“PIG-M 将第一个甘露糖转移到 ER 腔侧的糖基磷脂酰肌醇”EMBO J.. 20. 250-261 (2001)

Ohishi, Kazuhito: "PIG-S and PIG-T, essential for GPI-anchor attachment to proteins, form a complex with GAA1 and GPI8"EMBO J.. 20. 4088-4098 (2001)

Ohishi、Kazuhito：“PIG-S 和 PIG-T 对于 GPI 锚定蛋白附着至关重要，与 GAA1 和 GPI8 形成复合物”EMBO J.. 20. 4088-4098 (2001)

Hong, Y.: "Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum a-toxin"EMBO J.. 21. 5047-5056 (2002)

Hong, Y.：“需要 GPI 锚定蛋白上的 N-聚糖才能有效结合气溶素，但不能有效结合败血梭菌 a-毒素”EMBO J.. 21. 5047-5056 (2002)

Hong, Y.: "Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins"Mol.Biol.Cell. (in press). (2003)

Hong, Y.：“人 PIG-U 和酵母 Cdc91p 是 GPI 转酰胺酶的第五个亚基，它将 GPI 锚附着到蛋白质上”Mol.Biol.Cell。