Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis

Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.糖基磷脂酰肌醇（Glycosylphosphatidylinositol, GPI）在内质网（endoplasmic reticulum, ER）内合成并被转移至蛋白质分子。GPI锚定蛋白（GPI-anchored proteins）随后经高尔基体（Golgi apparatus）从内质网转运至细胞质膜。截至目前，学界已明确GPI生物合成通路至少需要17个步骤。 本研究旨在建立一套基于哺乳动物单倍体筛选的综合性方法，以鉴定参与GPI生物合成的相关基因。 研究人员通过将基因捕获载体（gene trap vectors）整合至基因组的方式，对人类单倍体细胞进行诱变处理；随后将诱变后的细胞暴露于细菌孔道形成毒素——气单胞菌溶素（aerolysin），该毒素可结合GPI锚定蛋白以靶向结合细胞膜；进而富集得到表面CD59（一种GPI锚定蛋白）表达量较低的细胞群体。通过深度测序（deep sequencing）技术，鉴定了未筛选群体与富集筛选群体中的基因捕获插入位点。 本次筛选在26个已知的GPI生物合成相关基因中，富集到23个基因区域；上述基因发生突变时，预期会降低GPI锚定蛋白的表面表达水平。 本研究结果表明，基于单倍体细胞的正向遗传学方法，是鉴定与目标表型相关因子的高效且有力的技术手段。