Mechanism of biosynthesis of protein GPI anchors.

蛋白质 GPI 锚的生物合成机制。

• 批准号: 11480166
• 负责人: KINOSHITA Taroh
• 金额: $ 8.13万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480166/
• 关键词: GPI anchor; post translational modification; endoplasmic reticulum; GIPアンカー; 生合成経路; 糖転移酵素

We aimed to clarify molecular mechanisms of GPI anchor biosynthesis and obtained following results.1. We purified GPI-GlcNAc transferase that mediates the first step in GPI biosynthesis pathway. The enzyme consisted of six proteins : four previously reported and two new components. One of the new components was PIG-P that is essential for the enzyme because mutant mouse cell line defective in PIG-P is completely deficient in GPI biosynthesis. The other new component was DPM2 that is known as a regulatory component of dolichol-phosphate-mannose synthase. GPI-GlcNAc transferase activity of DPM2-deficient cells was one-third that of wild-type cells, indicating that GPI-GlcNAc transferase is also regulated by DPM2.2. We found that human dolichol-phosphate-mannose synthase consists of DPM1, DPM2 and DPM3, that DPM2 stabilizes DPM3 and that DPM3, in turn, stabilizes catalytic DPM1.3. We cloned PIG-M cDNA and demonstrated that PIG-M encodes a mannosyltransferase that transfers the first mannose to GPI.PIG-M had a functionally important DXD motif within a lumenal domain, suggesting that the first mannose is transferred on the lumenal side of the endoplasmic reticulum.4. We demonstrated that PIG-O is involved in transfer of phosphoethanolamine to the third mannose. PIG-O that is most likely a catalytic component is associated with PIG-F that is also involved in the same step. Stable exprrssion of PIG-O was dependent upon PIG-F.5. We characterized mammalian genes for GAA1, a component of GPI transamidase. Human and mouse GAA1 genes had 12 exons and spanned 4 kb. Human and mouse GAA1 genes are present in 8q24.3 and 15E, respectively. Intron 8's of those GAA1 genes were of minor class. GPI8, the other component of GPI transamidase, had a functionally essential cysteine that is conserved among GPI8 from other organisms and members of a cystein protease family, suggesting that this cystein is active site of GPI transamidase.

本研究旨在阐明GPI锚定生物合成的分子机制，并取得了以下结果。我们纯化了介导GPI生物合成途径第一步的GPI-GlcNAc转移酶。该酶由六种蛋白质组成：四种先前报道的蛋白质和两种新的成分。其中一个新的成分是PIG-P，它对这种酶是必不可少的，因为PIG-P缺陷突变小鼠细胞系在GPI生物合成方面完全缺乏。另一个新的成分是DPM2，它被认为是二羟甲基-磷酸-甘露糖合成酶的调节成分。DPM2基因缺陷细胞的GPI-GlcNAc转移酶活性是野生型细胞的三分之一，说明GPI-GlcNAc转移酶也受DPM2.2的调控。我们发现人多酚-磷酸-甘露糖合成酶由DPM1、DPM2和DPM3组成，DPM2稳定DPM3，DPM3又稳定催化的DPM1.3。我们克隆了PIG-M基因，并证明PIG-M编码一个甘露糖转移酶，将第一个甘露糖转移到GPI。PIG-M在管腔结构域中有一个重要的DXD基序，这表明第一个甘露糖转移到内质网的管腔一侧。我们证明了PIG-O参与了磷酸乙醇胺向第三甘露糖的转移。最有可能是催化成分的PIG-O与也参与同一步骤的PIG-F相关。PIG-O的稳定表达依赖于PIG-F.5。我们鉴定了哺乳动物的GAA1基因，它是GPI转氨酶的一种成分。人和小鼠GAA1基因有12个外显子，大小为4kb。人和小鼠的GAA1基因分别存在于8q24.3和15E。内含子8‘S的GAA1基因属于小类。GPI转氨酶的另一个组分GPI8含有一个功能必需的半胱氨酸，该半胱氨酸在其他生物的GPI8和半胱氨酸蛋白酶家族成员中是保守的，这表明该半胱氨酸是GPI转氨酶的活性部位。

项目成果

Ohishi,Kazuhito.: "Gaalp and Gpi8p are components of a glycosylphosphatidylinositol (GPI). transamidase that mediates attachment of GPI to proteins."Mol.Biol.Cell. 11. 1523-1533 (2000)

Ohishi, Kazuhito.：“Gaalp 和 Gpi8p 是糖基磷脂酰肌醇 (GPI) 的组成部分。转酰胺酶可介导 GPI 与蛋白质的附着。”Mol.Biol.Cell。

Hong, Y.: "GPI1 stabilizes an enzyme essential in the first step of glycosylphosphatidylinositol biosynthesis."J.Biol.Chem.. 274. 18582-18588 (1999)

Hong, Y.：“GPI1 稳定糖基磷脂酰肌醇生物合成第一步中必需的酶。”J.Biol.Chem.. 274. 18582-18588 (1999)

Ohishi, K.: "Gaalp and Gpi8p are components of a glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of GPI to proteins."Mol.Biol.Cell. 11. 1523-1533 (2000)

Ohishi, K.：“Gaalp 和 Gpi8p 是糖基磷脂酰肌醇 (GPI) 转酰胺酶的组成部分，可介导 GPI 与蛋白质的附着。”Mol.Biol.Cell。

Maeda,Yusuke: "Human dolichol-phosphate-mannose synthase consists of three subunits, DPM1,DPM2 and DPM3."EMBO J.. 19. 2475-2482 (2000)

Maeda,Yusuke：“人多甘醇-磷酸-甘露糖合酶由三个亚基组成，DPM1、DPM2 和 DPM3。”EMBO J.. 19. 2475-2482 (2000)

Hong, Y.et al.: "GPI1 stabilizes an enzyme essential in the first step of glycosylphosphatidylinositol biosynthesis"J.Biol.Chem.. 274. 18582-18588 (1999)

Hong, Y.等人：“GPI1 稳定糖基磷脂酰肌醇生物合成第一步中必需的酶”J.Biol.Chem.. 274. 18582-18588 (1999)