Identification And Characterization Of Genes Required For Glycosylphosphatidylinositol Inosito L Deacylation

Glycosylphosphatidylinositol(GPI) anchoring of proteins is a conserved post-translational modification carried out in the endoplasmic reticulum(ER). In mammalian cells, about 150 proteins have been found to be anchored to the cell surface by GPI, including complement regulators, cell adhesion molecules and tumor biomarkers. They are involved in important life processes such as cell recognition, growth, differentiation and programmed cell death. During GPI biosynthesis, an acyl-chain is transferred to 2-position of inositol on GPI intermediates, which is eliminated by a GPI-inositol deacylase PGAP1 immediately after GPI-attachment to proteins. In recent two years, clinical data showed PGAP1 loss of function would cause intellectual disability, encephalopathy and hypotonia. However, it hasn’t been clarified what genes are required for the GPI inositol deacylation and how it is regulated in the ER.To detect the acylation status of GPI-anchored proteins(GPI-APs), phosphatidylinositol(PI)-specific phospholipase C(PI-PLC), which cleaves GPI-anchors, but not inositol-acylated GPI-anchors, was used. Using the sensitivity to PI-PLC, genetic screen was performed to reveal the regulatory mechanisms of the GPI-inositol deacylation. Human haploid HAP1 cells were mutagenized by the integration of gene trap vectors into the genome, and mutant cells, in which GPI-APs showed resistance to PI-PLC, were enriched. Gene trap insertion sites in the non-selected population and in the enriched population were determined by next-generation sequencing. PGAP1 was listed up from the analysis, showing the screening was accurate. In addition, 7 genes including genes encoding factors involved in protein folding were enriched.The candidate genes were knocked out in HEK293FF6 cells mediated by CRISPR/Cas9 system. GPI-APs in the knockout cells partly showed resistance to PI-PLC. While overexpressing hPGAP1 in the knockout cells, all of them were rescued and showed decreased resistance to PI-PLC. It indicated that all these candidate genes are required for GPI inositol deacylation.The lectin activity of calnexin(CANX) was essential for PI-PLC resistance. And the deficiency of α-glucosidase caused deficiency of PGAP1. Based on MOGS-KO cell line, ALG6 and ALG8 were further knocked out. In these double mutant cells, GPI-APs showed decreased resistance to PI-PLC. Thus, a hypothesis was put forward and a new preliminary N-glycan dependent model was established. In this study, we would like to discuss the relationship between GPI inositol deacylation and candidate genes in the ER.