The regulation of functions and expressions of proteins by glycosylphosphatidylinositol-anchor

(I)GPI的肌醇脱酰酶(PGAP1)的鉴定：我们利用我们建立的脱酰酶缺陷突变细胞，通过基因克隆的方法鉴定了一个肌醇脱酰酶(PGAP1)，并报道了该缺陷导致GPI-APs转运延迟。(Ii)建立了PGAP1基因敲除小鼠，以阐明其生物学意义。PGAP1基因缺失的小鼠在胚胎期大部分死亡，并表现为耳头畸形。其余空白组小鼠生长迟缓，但寿命正常。(3)GPI锚定的脂质重塑及其对GPI-APs正确分选和筏结合的影响我们建立了GPI-APs表面表达降低的突变细胞系，并通过表达克隆鉴定了一个新的与GPI-APs缺陷相关的基因PGAP2。突变体的详细分析有力地表明，GPI-APs的PI部分在从ER到PM的运输过程中存在脂质重塑缺陷为了更详细地了解这一缺陷，我们建立了双突变细胞，通过诱变PGAP2缺陷细胞来恢复其表面GPI-APs的表达。在双突变细胞中，GPI在PI的sn-2位具有高度的不饱和脂链，而在野生细胞中，GPI仅具有饱和脂链。此外，与筏相关的这种带有不饱和脂链的GPI-AP比野生型细胞弱得多。这些结果首次证明GPI的脂质重塑发生在哺乳动物细胞中，并且脂质重塑是GPI-AP与RAFT关联的关键。

(I)Identification of inositol deacylase for GPI (PGAP1).We identified an inositol deacylase (PGAP1) by expression cloning using deacylase-deficient mutant cells we established and reported that the defect caused the delayed transport of GPI-APs.(II)Establishment of PGAP1 knockout mice to clarify the biological significance.We established PGAP1 knockout mice. The majority of PGAP1 null mice was lethal in embryonic stage and showed otocephaly. The other null mice had growth retardation but normal life span. The adult males of null mice were sterile due to defective entrance to oviducts and attachment to egg cells, although the number and motility of sperm was normal.(III)Lipid remodeling of GPI-anchor and its effects upon the correct sorting and raft association of GPI-APsWe established mutant cell line in which the surface expression of GPI-APs was decreased and identified a new gene, PGAP2, responsible for the defect by expression cloning. The detail analysis of mutants strongly indicated the defect of lipid remodeling of PI portion of GPI-APs during the transport from ER to PM. To understand the defect more in detail, we established double mutant cells whose surface expression of GPI-APs was restored by mutagenizing PGAP2-deficient cells. In double mutant cells, GPI had highly unsaturated lipid chains at sn-2 position of PI, whereas GPI in wild cells exclusively had saturated lipid chain. Moreover such GPI-APs with unsaturated lipid chains associated with rafts much weaker than wild-type cells. These results were first evidences proving that lipid remodeling of GPI takes place in mammalian cells and that lipid remodeling is critical for association of GPI-APs with rafts.