Calnexin mediates the maturation of GPI-anchors through ER retention

The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions 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 post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.

plasma membrane, and subsequently to lysosomes for degradation. 95 In the present study, we investigated the roles of calnexin in the quality control and lipid 96 remodeling of GPI-APs in the ER. In wild-type (WT) cells, GPI-APs were folded and inositol 97 deacylated in the ER and expressed at the cell surface. In calnexin and calreticulin double 98 knockout (CANX&CALR-DKO) cells, protein folding and GPI-inositol deacylation 99 efficiencies were significantly decreased such that misfolded and inositol-acylated GPI-APs 100 were exposed on the plasma membrane. Thus, these results indicate that the 7 were used as secondary antibodies. PE-conjugated anti-CD109 (HU17; eBioscience) was used 127 for flow cytometry. Thapsigargin (100 nM, TG, Sigma-Aldrich) and doxycycline (1 μg/ml, 128 631311; Clontech Laboratories) were used for drug treatments. All the plasmids and oligonucleotides used in this study are listed in Table S1 and Table S2, 132 respectively. For the CRISPR-Cas9 system used to KO target genes, guide RNA sequences 133 were designed using the E-CRISP website (   antibodies for endogenous CD59, CD55 and CD109 (10 μg/ml) for 25 min on ice and then 9 washed two times with cold FACS buffer (PBS containing 1% BSA and 0.1% NaN3).

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Subsequently, the cells were incubated with PE-conjugated goat anti-mouse IgG as the   175   secondary antibody for 25 min on ice, washed two times with FACS buffer and then analyzed   176 with an Accuri C6 instrument (BD). The data were analyzed using FlowJo, and the remaining 177 GPI-APs were calculated from mean values of PE-fluorescent intensity treated with or 178 without PI-PLC.

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For exogenous GPI-APs, pME-HA-GPI-AP (Table S1)   these proteins together with the plasmid pME-mRFP-KDEL as an ER marker. Then, 36 h 195 after transfection, cells were replated onto glass coverslips pretreated with 1% gelatin and 196 cultured for another 2 days. Subsequently, the cells were washed with PBS, fixed in 4% 197 paraformaldehyde for 10 min at roon temperature, washed again with PBS, and then 198 incubated with 40 mM ammonium chloride for 10 min at room temperature. Finally, the 199 coverslips were mounted onto slides using a mounting solution containing DAPI for 5 min.

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To assess the localization of VFG-GPI, HEK293FF6, CANX&CALR-DKO, and 201 CANX&CALR-DKO+CANX cells were transfected with pME-mRFP-KDEL. Subsequently, 202 36 h after transfection, the cells were harvested and replated onto glass coverslips pretreated 203 with 1% gelatin at 37°C for 1 day and then incubated with 1 µg/ml doxycycline at 40°C for 204 24 h to induce VFG-GPI expression. Then, the cells were quickly fixed with 4% 205 paraformaldehyde for 10 min before being washed with PBS and then 40 mM 206 ammonium chloride.

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To assess the localization of endogenous calnexin, the cells were fixed and permeabilized 208 with -20°C methanol for 5 min at 4°C before being blocked with PBS containing 5% FCS 209 (blocking buffer) for 1 h. Subsequently, the cells were incubated for 1 h with 210 mouse-anti-calnexin (M178-3; MBL) as primary antibody diluted in blocking buffer.  To assess the localization of HLA1-1-147-FLAG, pME-HLA1-1-147-FLAG and 216 pME-mRFP-KDEL were transfected together into WT cells. Then, 36 h after transfection, the 217 cells were harvested and replated onto glass coverslips pretreated with 1% gelatin and 218 incubated at 37°C for 1 day followed by an incubation at 37°C with 100 nM TG for 6 h. The   Substrate System for ELISA, P7998; Sigma) was added. Then, the plates were incubated at 250 37°C for 6.5 h, the reaction was stopped by adding 50 μl/well of 3 M NaOH, and the 251 absorbance at 405 nm was measured using an Enspire 2300 instrument. The calf intestinal 252 alkaline phosphatase (TaKaRa) was used as a standard for the reaction.

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Calnexin/calreticulin cycle is required for efficient GPI-inositol deacylation for both 257 endogenous and exogenous N-glycosylated GPI-APs 258 After GPI attachment to proteins, an acyl-chain linked to inositol on many GPI-APs is 259 removed by the GPI-inositol deacylase PGAP1. The inositol-deacylation status of GPI-APs

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Glycan binding of calnexin is required for efficient GPI-inositol deacylation 332 The structure of the ER-luminal portion of calnexin has two domains: a glycan binding suggesting that GPI-inositol deacylation occurred more efficiently in the absence of ERp57.

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Calnexin was observed to associate with CD59 in an N-glycan-and GPI-dependent         The above results suggest that calnexin retains misfolded GPI-APs in the ER through its    (C). mRFP-KDEL was used as an ER marker. Three days after transfection, images were obtained using confocal microscopy. DAPI staining is shown as blue in merged images. Scale bar, 10 μm.

(D) Pearson's correlation coefficient values between EGFP-FLAG-CD59 (C94S) or (C94S, N43Q) and
465 mRFP-KDEL were calculated using the ImageJ plugin JACoP. The data are presented as the means ± SD of the 466 measurements. "N=" represents cell number used for the calculation. P-values (two-tailed, student's t-test) are 467 shown.

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Misfolded and inositol-acylated GPI-APs are exposed on the plasma membrane in 470 CANX&CALR-DKO cells 471 The results of a previous study confirmed that misfolded prions resulting from acute ER  Figure 5E). ER stress induction by thapsigargin further increased the PI-PLC resistance of 497 CD59 and CD55 in CANX&CALR-DKO cells compared with that observed in WT cells 498 (11.2-and 4.7-fold higher, respectively) ( Figure 5C, bottom panels, gray peak and Figure 5F).

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These results indicate that misfolded GPI-APs in the ER of WT cells are already    including tissue non-specific, intestinal, placental, and placental-like typestypes in human.

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Among these different types, the expression of the placental alkaline phosphatase (ALPP, 538 contain 2 N-glycosylation sites) is the highest in HEK293 cells (Figure 6-figure supplement).

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The surface expression of endogenous ALPP was almost the same in WT and 540 CANX&CALR-DKO cells ( Figure 6A, heavy solid lines and Figure 6B). The ALPP showed 541 2.3-fold higher PI-PLC resistance in CANX&CALR-DKO cells compared to that observed in 542 WT cells ( Figure 6A, gray peaks and Figure 6C). We then assessed the ALP activity on the 543 cell surface and compared with WT cells, the activity in CANX&CALR-DKO cells was 544 significantly decreased by 60% ( Figure 6D). These results indicate that GPI-APs that are not 545 fully functional are expressed on the plasma membrane in CANX&CALR-DKO cells.

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The relative values were calculated and are presented as the means ± SD from three independent experiments.    Figure 7C, left), which is 576 consistent with that observed for other GPI-APs. 577 We next analyzed VFG-GPI using a chase experiment. VFG-GPI was expressed at 40°C 578 to accumulate in the ER, after which the temperature was shifted to 32°C to allow its transport 579 to the plasma membrane. Under these condition, surface VFG-GPI in CANX&CALR-DKO 580 became sensitive to PI-PLC cleavage, similar to that observed in WT cells ( Figure 7B, bottom 581 panels and Figure 7C, right). These results proved that sufficient ER retention time is 582 necessary for GPI-inositol deacylation.   (Figure 8, bottom). Since the GPI moiety of misfolded ER-resident GPI-APs signals 628 that they are ready to exit, they would be quickly transported from the ER via acute ER stress 629 induction.

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Top: Under normal conditions, N-glycans and GPI are transferred to the newly synthesized GPI-APs. After 678 glucose trimming by α-glucosidase I and II, the N-glycan structure becomes Glc1Man9GlcNAc2, which is 679 specifically recognized by calnexin/calreticulin. By directly binding with calnexin, immature GPI-APs enter the 680 ER quality control system and finally become mature GPI-APs. In addition, calnexin retains GPI-APs in the ER 681 and associates with PGAP1, which is required for the efficient GPI-inositol deacylation. After the GPI moiety is 682 remodeled by PGAP1 and PGAP5, and GPI-APs are transported from the ER to the Golgi by the cargo receptors, 683 p24 family of proteins.

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Middle: In CANX&CALR-DKO cells, there is no calnexin/calreticulin-dependent ER quality control system and 685 ER retention system. GPI-APs exit the ER with incomplete protein folding and GPI remodeling.

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Bottom: In WT cells, under basal conditions, misfolded GPI-APs are retained in the ER by calnexin. During the 687 long time being retained in the ER, an acyl-chain on the GPI-inositol is removed by PGAP1. Once acute ER 688 stress is induced, deacylated and misfolded GPI-APs are quickly transported by p24 family members and 689 exposed on the plasma membrane.

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Acknowledgements 692 We thank Drs Hideki Nakanishi, Ganglong Yang, Ning Wang (Jiangnan University) for their 693 critical reading of the manuscript and comments. This work was supported by grants-in-aid  703 The authors declare that they have no conflicts of interest with the contents of this article.  with an anti-calnexin antibody followed by an anti-mouse-Alexa Fluor 555, and detected by confocal microscopy.