Shishioh Nobue, Hong Yeongjin, Ohishi Kazuhito, Ashida Hisashi, Maeda Yusuke, Kinoshita Taroh
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
J Biol Chem. 2005 Mar 11;280(10):9728-34. doi: 10.1074/jbc.M413755200. Epub 2005 Jan 4.
Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). GPI is synthesized from phosphatidylinositol by stepwise reactions and attached en bloc to nascent proteins. In mammalian cells, the major GPI species transferred to proteins is termed H7. By attachment of an additional ethanolamine phosphate (EtNP) to the second mannose, H7 can be converted to H8, which acts as a minor type of protein-linked GPI and also exists as a free GPI on the cell surface. Yeast GPI7 is involved in the transfer of EtNP to the second mannose, but the corresponding mammalian enzyme has not yet been clarified. Here, we report that the human homolog of Gpi7p (hGPI7) forms a protein complex with PIG-F and is involved in the H7-to-H8 conversion. We knocked down hGPI7 by RNA interference and found that H7 accumulated with little production of H8. Immunoprecipitation experiments revealed that hGPI7 was associated with and stabilized by PIG-F, which is known to bind to and stabilize PIG-O, a protein homologous to hGPI7. PIG-O is a transferase that adds EtNP to the third mannose, rendering GPI capable of attaching to proteins. We further found that the overexpression of hGPI7 decreased the level of PIG-O and, therefore, decreased the level of EtNP transferred to the third mannose. Finally, we propose a mechanism for the regulation of GPI biosynthesis through competition between the two independent enzymes, PIG-O and hGPI7, for the common stabilizer, PIG-F.
许多真核细胞表面蛋白通过糖基磷脂酰肌醇（GPI）锚定在膜上。GPI由磷脂酰肌醇通过逐步反应合成，并整体连接到新生蛋白质上。在哺乳动物细胞中，转移到蛋白质上的主要GPI种类被称为H7。通过在第二个甘露糖上额外连接一个磷酸乙醇胺（EtNP），H7可以转化为H8，H8作为一种次要类型的蛋白质连接的GPI，也以游离GPI的形式存在于细胞表面。酵母GPI7参与将EtNP转移到第二个甘露糖上，但相应的哺乳动物酶尚未明确。在这里，我们报告Gpi7p的人类同源物（hGPI7）与PIG-F形成蛋白质复合物，并参与H7向H8的转化。我们通过RNA干扰敲低hGPI7，发现H7积累而H8产生很少。免疫沉淀实验表明，hGPI7与PIG-F相关并由其稳定，已知PIG-F能结合并稳定与hGPI7同源的蛋白质PIG-O。PIG-O是一种转移酶，它将EtNP添加到第三个甘露糖上，使GPI能够附着到蛋白质上。我们进一步发现，hGPI7的过表达降低了PIG-O的水平，因此也降低了转移到第三个甘露糖上的EtNP的水平。最后，我们提出了一种通过两种独立酶PIG-O和hGPI7对共同稳定剂PIG-F的竞争来调节GPI生物合成的机制。
