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

糖酰基磷脂酰肌醇（GPI）锚定蛋白广泛分布于真核生物的质膜上。由磷脂酰肌醇、葡萄糖胺、三种甘露糖和乙醇胺磷酸组成的核心gpi锚在内质网中合成，并转移到前体蛋白的c端，形成gpi锚定蛋白。所有gpi锚定蛋白都有一个共同的核心gpi锚定。因此，缺乏gpi锚定蛋白的生物合成将导致许多不同gpi锚定蛋白的表面表达缺陷，如果是由种系突变引起的，将导致小鼠胚胎早期发育的致命性。在小鼠中，超过50种gpi锚定蛋白以空间和时间不同的方式表达。我们使用Cre/loxP系统检测了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gpi锚定生物合成所必需的x连锁基因Pig-a基因。gpi锚定蛋白在突变小鼠的表皮和t淋巴细胞中完全不表达，表明Cre/loxP系统非常有效。与野生型小鼠相比，突变小鼠的皮肤看起来皱巴巴的，而且鳞片更多。组织学检查显示，突变小鼠表皮角质层排列紧密，增厚。此外，角质层脂质重组受损。突变体出生后8小时表皮水分流失（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锚定蛋白对适当的皮肤维护至关重要。另一方面，GPI-anchor缺陷T淋巴细胞对各种刺激的反应与GPI-anchor充足T淋巴细胞相同。这些结果表明gpi锚蛋白在不同组织中发挥着不同的作用。

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.