N-glycan-dependent Protein Folding And Endoplasmic Reticulum Retention Regulate The Maturation Of GPI-APS

N-glycosylation dependent ER quality control,the biosynthesis of GPI and its attachment to protein are all performed in the ER.Nascent GPI-APs synthesized by GPI transamidase are still immature and undergo remodeling reactions to become mature GPI-anchored proteins(GPI-APs).In the ER,two remodeling reactions occur in many GPI-APs.After GPI attachment to proteins,the first step is an acyl-chain on an inositol-ring of the GPI-anchor eliminated by the GPI-inositol deacylase post-GPI attachment protein 1(PGAP1).This remodeling reaction is crucial for the interaction of GPI-APs with p24 protein complexes,which are cargo receptors for GPI-APs,indicating that GPI-AP remodeling in the ER is required for their efficient sorting into transport vesicles at the ER exit sites.When proteins fail to fold correctly,they are recognized as misfolded proteins,the majority of which are retained in the ER and degraded through the ER-associated degradation(ERAD)pathway.However,misfolded GPI-APs do not appear to be suitable substrates for ERAD,possibly because of the presence of GPI-anchors.In mammalian cells,although misfolded GPI-APs are retained in the ER,they are rapidly released into the secretory pathway upon acute ER stress despite their misfolding and express on the plasma membrane,finally to lysosomes for degradation.Previous study showed that GPI-inositol deacylation is partially impaired in cells defective for calnexin(CANX).By directly binding with the N-glycan,calnexin can help the folding and maturation of nascent proteins.Although the function of calnexin in the ER quality control system is well understood,the mechanism of how calnexin contributes to the lipid remodeling and maturation of GPI-APs is still unclear.Also,a molecular mechanism is needed for the hypothesis that GPI inositol deacylation is the switch point of GPI-APs from protein folding to transport states.Based on that,in the present study,we investigated the roles of calnexin in the quality control and lipid remodeling of GPI-APs in the ER.By directly binding the N-glycan on proteins,calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded.In addition,sufficient ER retention time was crucial for GPI-inositol deacylation,which is mediated by PGAP1.The main results of the present study are summarized as follows:1)Calnexin&calreticulin-dependent GPI-anchor maturation is a commonly used pathway.In addition,calnexin-independent GPI-inositol deacylation occurs for non-N-glycosylated GPI-APs.Site-direct mutation results indicated that lectin activity of calnexin is necessary for efficient GPI-inositol deacylation.Calnexin was observed to associate with CD59 in an N-glycan-and GPI-dependent manner.2)Calnexin mediates the ER retention of misfolded GPI-APs.In HEK293 cells,EGFP-FLAG-tagged CD59(C94S)was primarily localized in the ER,in contrast,in CANX&CALR-DKO cells,CD59(C94S)was not retained in the ER but rather localized at the plasma membrane.In MOGS-KO and GANAB-KO cells,fractions of CD59(C94S)were expressed on the cell surface.For non-N-glycosylated misfolded CD59(C94S,N43Q)and CD55(C81A,N95Q),even in the WT cells,these proteins were not retained in the ER,but fractions of them were expressed on the cell surface.The above results suggest that calnexin retains misfolded GPI-APs in the ER through its binding to N-glycans on these proteins.3)Calnexin plays dual roles in the protein folding and inositol deacylation of GPI-APs.The small fraction of misfolded CD59 and CD55 on the surface of WT cells showed PI-PLC cleavage sensitivity,while in CANX&CALR-DKO cells,misfolded GPI-APs showed 2 times higher PI-PLC resistance.By ER stress induction,already inositol-deacylated misfolded GPI-APs can express on the cell surface.The difference in PI-PLC sensitivity of GPI-APs between CANX&CALR-DKO cells and ER stress induced WT cells suggests that ER retention time is important for efficient GPI-inositol deacylation.4)Sufficient ER retention time is required for efficient inositol deacylation by PGAP1.To confirm whether sufficient ER retention time is required for GPI-inositol deacylation,a doxycycline(DOX)-inducible VSVG^ts-FLAG-GFP-GPI(VFG-GPI)reporter system was used.VFG-GPI is a temperature-sensitive glycoprotein,and the reporter causes reversible misfolding at 40°C due to a mutation in the VSVG portion and is retained in the ER,whereas it is folded at 32°C and transported to the plasma membrane.When VFG-GPI expression was induced at 32°C,the PI-PLC resistance of VFG-GPI in CANX&CALR-DKO showed 3.6-fold higher than that measured for WT cells.A chase experiment was done like this,VFG-GPI was first expressed at 40°C to accumulate in the ER,after which the temperature was shifted to32°C to allow its transport to the plasma membrane.Under this condition,surface VFG-GPI in CANX&CALR-DKO became sensitive to PI-PLC cleavage,similar to that observed in WT cells.