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Functional analysis of GPI-anchored proteins by tissue specific gene targeting

通过组织特异性基因靶向对 GPI 锚定蛋白进行功能分析

基本信息

批准号：
09470063
负责人：
TAKEDA Junji
金额：
$ 8.32万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470063/
关键词：
GPI-anchor Cre loxp Pig-a Lethality Tissue specific gene targeting loxP

项目摘要

Glycosyiphosphatidylinositol (GPI)-anchored proteins are widely distributed on plasma membranes of eukaryotes. The core GPI-anchor that consists of phosphatidylinositol, glucosamine, three mannoses, and ethanolaminephosphate is synthesized in the endoplasmic reticulum and transferred to the C-terminus of precursor proteins to form GPI-anchored proteins. All GPI-anchored proteins share a common core GPI-anchor. A lack of GPI-anchor biosynthesis, therefore, would cause defective surface expression of many different GPI-anchored proteins and if it is caused by a germ line mutation, it would result in lethality at an early development of mouse embryos.In mice, over fifty GPI-anchored proteins are expressed in spatially and temporally different fashions. We examined the functional roles of GPI-anchored proteins in specific mouse tissues using the Cre/loxP system. We disrupted the Pig-a gene, an X-linked gene essential for GPI-anchor biosynthesis, in the epidermis and T-lymphocytes. Expression of GPI-anchored proteins was completely absent in the epidermis and T-lymphocytes of the mutant mice, demonstrating that Cre/loxP system worked very efficiently.The skin of the mutants looked wrinkled and more scaly than those of the wild-type mice.Histological examination of the mutant mice showed that the epidermal horny layer was tightly packed and thickened. Moreover, lipid reorganization in the horny layer was impaired. Trans-epidermal water loss (TEWL) was dramatically increased in the mutants at 8 hrs after birth. The mutant mice died within a few days after birth. Thus, GPI-anchor or GPI-anchored proteins are essential for proper skin maintenance.On the other hand, GPI-anchor deficient T lymphocytes respond to various stimulation same as GPI-anchor sufficient T lymphocytes. These results suggest that GPI-anchor play various roles in different tissues.

糖基磷脂酰肌醇（GPI）锚定蛋白广泛分布于真核生物的质膜上。核心GPI锚由磷脂酰肌醇、葡萄糖胺、三个甘露糖和乙醇胺磷酸组成，在内质网中合成并转移到前体蛋白的C-末端以形成GPI锚定蛋白。所有的GPI锚定蛋白都有一个共同的核心GPI锚。因此，缺乏GPI锚定蛋白的生物合成会导致许多不同GPI锚定蛋白的表面表达缺陷，并且如果它是由生殖系突变引起的，则会导致小鼠胚胎早期发育的致死性。我们使用Cre/loxP系统研究了GPI锚定蛋白在特定小鼠组织中的功能作用。我们破坏了表皮和T淋巴细胞中的Pig-a基因，这是一种对GPI锚生物合成至关重要的X连锁基因。突变小鼠表皮和T淋巴细胞中GPI锚定蛋白的表达完全缺失，表明Cre/loxP系统的作用非常有效，突变小鼠皮肤看起来比野生型小鼠更皱，更有鳞屑，组织学检查显示突变小鼠表皮角质层紧密堆积，增厚。此外，角质层中的脂质重组受损。在出生后8小时，突变体的经表皮水分损失（TEWL）显著增加。突变小鼠在出生后几天内死亡。因此，GPI锚定蛋白或GPI锚定蛋白对于适当的皮肤维护是必不可少的。另一方面，GPI锚定缺陷的T淋巴细胞与GPI锚定充足的T淋巴细胞一样对各种刺激作出反应。这些结果表明GPI锚在不同的组织中发挥不同的作用。