Tanaka Satoshi, Maeda Yusuke, Tashima Yuko, Kinoshita Taroh
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
J Biol Chem. 2004 Apr 2;279(14):14256-63. doi: 10.1074/jbc.M313755200. Epub 2004 Jan 20.
The inositol moiety of mammalian glycosylphosphatidylinositol (GPI) is acylated at an early step in GPI biosynthesis. The inositol acylation is essential for the generation of mature GPI capable of attachment to proteins. However, the acyl group is usually absent from GPI-anchored proteins (GPI-APs) on the cell surface due to inositol deacylation that occurs in the endoplasmic reticulum (ER) soon after GPI-anchor attachment. Mammalian GPI inositol-deacylase has not been cloned, and the biological significance of the deacylation has been unclear. Here we report a GPI inositol-deacylase-deficient Chinese hamster ovary cell line established by taking advantage of resistance to phosphatidylinositol-specific phospholipase C and the gene responsible, which was termed PGAP1 for Post GPI Attachment to Proteins 1. PGAP1 encoded an ER-associated, 922-amino acid membrane protein bearing a lipase consensus motif. Substitution of a conserved putative catalytic serine with alanine resulted in a complete loss of function, indicating that PGAP1 is the GPI inositol-deacylase. The mutant cells showed a clear delay in the maturation of GPI-APs in the Golgi and accumulation of GPI-APs in the ER. Thus, the GPI inositol deacylation is important for efficient transport of GPI-APs from the ER to the Golgi.
哺乳动物糖基磷脂酰肌醇（GPI）的肌醇部分在GPI生物合成的早期阶段被酰化。肌醇酰化对于生成能够连接到蛋白质上的成熟GPI至关重要。然而，由于GPI锚定连接后在内质网（ER）中很快发生的肌醇脱酰作用，细胞表面的GPI锚定蛋白（GPI-APs）上通常不存在酰基。哺乳动物GPI肌醇脱酰酶尚未被克隆，脱酰作用的生物学意义也不清楚。在这里，我们报告了一种利用对磷脂酰肌醇特异性磷脂酶C的抗性建立的GPI肌醇脱酰酶缺陷型中国仓鼠卵巢细胞系以及相关基因，该基因被命名为PGAP1（Post GPI Attachment to Proteins 1，GPI连接到蛋白质后）。PGAP1编码一种与内质网相关的、含有922个氨基酸的膜蛋白，带有一个脂肪酶保守基序。将一个保守的假定催化丝氨酸替换为丙氨酸导致功能完全丧失，表明PGAP1就是GPI肌醇脱酰酶。突变细胞在高尔基体中GPI-APs的成熟明显延迟，并且GPI-APs在内质网中积累。因此，GPI肌醇脱酰作用对于GPI-APs从内质网到高尔基体的有效转运很重要。
