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

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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.