Study On Regulatory Mechanisms For The Processing Of Glycosylphosphatidylinositol Anchored Proteins

Glycosylphosphatidylinositol(GPI)anchoring of proteins is a conserved post-translational modification in eukaryotic cells.In mammalian cells,?150 proteins on the cell surface are modified by GPI anchors.GPI is biosynthesized and transferred to proteins in the endoplasmic reticulum(ER).Then,GPI-anchored proteins(GPI-APs)are transported from the ER to the plasma membrane through the Golgi apparatus.During GPI biosynthesis,an acyl-chain is attached to 2-position of inositol on GPI intermediates.Soon after GPI-attachment to proteins,this acyl-chain is eliminated by a GPI-inositol deacylase PGAP1,which is critical for efficient transport of GPI-anchored proteins(GPI-APs)from the ER.Phosphatidylinositol(PI)-specific phospholipase C(PI-PLC)can cleave the GPI-anchors,but can't cleave inositol-acylated GPI-anchors.In this study,Using reduction in the sensitivity to PI-PLC as a marker,I performed genetic screening to identify genes necessary for efficient GPI-inositol deacylation.I identified 8 genes in the screening,including PGAP1 which encodes the GPI inositol deacylase.Most glycan proteins are synthesized in the ER,where they are folded and oligomerized.Several molecular chaperones are required for proper protein folding in the ER.Before exiting from the ER,proteins are monitored by a quality control system that ensures correct folding.Asparagine(N)-linked protein glycosylation,one of the major posttranslational modifications occurring in the ER,plays key roles in quality control of newly synthesized glycoproteins.Among of 8 genes,MOGS,GANAB and CANX encoding proteins involved in the protein quality control.In study,I clarified functional relationship between GPI and N-glycan.My research found that N-glycan regulates the quality control and processing of GPI-anchored protein.Disruption of the calnexin cycle decreases the efficiency of GPI-inositol deacylation.The main results of this study are summarized as follows:(1)Using gene-trapping methods,PGAP1 and other 7 genes including SELT,CANX,C18orf32,CLPTM1,MOGS,SEC63 and GANAB show PI-PLC resistance in Human haploid HAP1 cells.The candidate genes were knocked out in HEK293 cells by CRISPR-Cas9 technique.The results were correlated to the HAP1 screening,PGAP1-KO Cells show completely resistance to PI-PLC treatment,other genes knockout show partial resistance.Overexpression the PGAP1 and other related genes can restore the phenotype.This results show these genes really involved in the GPI-inositol deacylation.The expression level,localization and the protein stability of PGAP1 were not changed.(2)Among those candidate genes,MOGS and GANAB encode ?-glucosidases ? and ?,respectively,and both are required in the ER for trimming glucose residues from the N-glycan moieties on newly synthesized proteins.CANX encodes calnexin,a molecular chaperone involved in folding and quality control of glycoproteins in the ER.Treated with ?-glucosidases inhibitor DNJ increased the resistance by PI-PLC treatment.This results were correlated to the knockout the MOGS and GANAB.Further,knockout out the glucosyltransferase ALG6 and ALG8 restored the PI-PLC resistance of GPI-APs,indicating that defects in the entering the calnexin cycle caused inefficient GPI-inositol deacylation of GPI-APs.Because calnexin possesses a lectin domain,we used two calnexin mutant constructs(Y164A or E216 A in human calnexin),each defective in lectin activity,to analyze whether the glycan binding ability is required for the phenotype.Compared with WT calnexin,PI-PLC sensitivity of GPI-APs was not rescued or only slightly rescued when calnexin mutant constructs were expressed,suggesting that lectin activity of calnexin was required for PI-PLC sensitivity.(3)Many genes involved in the unfolded protein response(UPR)were significantly up-regulated in MOGS-KO cells.To test the relationship between ER stress and GPI-inositol deacylation,cells were cultured with thapsigargin and tunicamycin.After 10 days treatment,the PI-PLC resistance of GPI-APs were increased.To confirm the results caused by ER stress inducers,the SERCA2,STT3 A and STT3 B were knocked out.The results were consistent with the effects of ER stress inducers.(4)Expression of misfolded GPI-APs accumulated in the ER and caused ER stress in the cells.The accumulation of misfolded GPI-APs inhibited the processing of normal GPI-APs,while expression of misfolded GPI-APs did not cause further PI-PLC resistance in CANX-KO cells.Further more,increasing PI-PLC resistance was not observed in cells expressing other types of misfolded proteins.These results suggested that the phenomenon caused by misfolded GPI-APs rely on the GPI and N-glycan part of the GPI-APs.Finally,we detected that calnexin can both interact with GPI-APs and PGAP1 by immunoprecipitation and immunoblotting to facilitate GPI-inositol deacylation.