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

糖基磷脂酰肌醇锚对蛋白质功能和表达的调节

基本信息

批准号：
16570116
负责人：
MAEDA Yusuke
金额：
$ 2.24万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16570116/
关键词：
GPI-anchor transport remodeling de-acylation raft

项目摘要

(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.

（I）GPI的肌醇脱酰酶（PGAP 1）的鉴定我们利用我们建立的脱酰酶缺陷突变体细胞通过表达克隆鉴定了肌醇脱酰酶（PGAP 1），并报道了该缺陷导致GPI-AP的延迟转运。（二）PGAP 1基因敲除小鼠的建立为了阐明PGAP 1基因敲除小鼠的生物学意义，我们建立了PGAP 1基因敲除小鼠。大多数PGAP 1基因敲除小鼠在胚胎期是致死的，并表现出耳头畸形。另一组小鼠生长迟缓，但寿命正常。无效小鼠的成年雄性由于输卵管入口和卵细胞附着缺陷而不育，尽管精子的数量和活力正常。（III）GPI-anchor的脂质重构及其对GPI-AP正确分选和筏结合的影响我们建立了GPI-AP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基因PGAP 2，该基因是导致GPI-AP表面表达缺陷的原因。对突变体的详细分析表明GPI-AP的PI部分在从ER到PM的转运过程中存在脂质重构缺陷。为了更详细地了解缺陷，我们建立了双突变细胞，其GPI-AP的表面表达通过诱变PGAP 2缺陷细胞而恢复。在双突变细胞中，GPI在PI的sn-2位具有高度不饱和的脂质链，而野生型细胞中的GPI仅具有饱和的脂质链。此外，这种GPI-AP与不饱和脂链相关的筏比野生型细胞弱得多。这些结果首次证明GPI的脂质重构发生在哺乳动物细胞中，并且脂质重构对于GPI-AP与筏的缔合是至关重要的。