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

(1) GPI肌醇脱乙酰酶（PGAP1）的鉴定。我们利用我们建立的去乙酰化酶缺陷突变细胞，通过表达克隆鉴定了一种肌醇去乙酰化酶（PGAP1），并报道了该缺陷导致GPI-APs转运延迟。（II）建立PGAP1敲除小鼠，阐明其生物学意义。我们建立了PGAP1敲除小鼠。大多数PGAP1缺失小鼠在胚胎期死亡，出现耳头畸形。另一组小鼠生长迟缓，但寿命正常。虽然精子的数量和活力正常，但由于进入输卵管和附着卵细胞的缺陷，成年雄性小鼠是不育的。（三）GPI-anchor的脂质重塑及其对GPI-APs正确分选和筏式关联的影响我们建立了GPI-APs表面表达降低的突变细胞系，并通过表达克隆鉴定出了一个新的基因PGAP2，该基因是导致GPI-APs缺陷的原因。突变体的详细分析强烈表明，GPI-APs在从内质网转运到内质网的过程中，PI部分脂质重塑存在缺陷。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缺陷细胞恢复其表面GPI-APs的表达。在双突变细胞中，GPI在sn-2位置具有高度不饱和的脂链，而野生细胞中的GPI仅具有饱和脂链。此外，这种与筏相关的不饱和脂链的GPI-APs比野生型细胞弱得多。这些结果首次证明了哺乳动物细胞中GPI的脂质重塑发生，并且脂质重塑对于GPI- aps与筏的关联至关重要。

(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.