bioRxiv preprint doi: https://doi.org/10.1101/2020.07.08.192955; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Calnexin mediates the maturation of GPI-anchors through ER retention 2 3 Xin-Yu Guo1, Yi-Shi Liu1, Xiao-Dong Gao1, Taroh Kinoshita2, 3, and Morihisa Fujita1,* 4 5 1Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, 6 School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China; 2Research 7 Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan; 3WPI 8 Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan 9 10 *Corresponding author: Morihisa Fujita, Ph.D. 11 E-mail: [email protected] 12 Tel & Fax: +86-510-851-97071 13 14 15 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.08.192955; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 16 Abstract 17 The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins 18 (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles 19 in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin 20 in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the 21 N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until 22 they were correctly folded. In addition, sufficient ER retention time was crucial for 23 GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once 24 the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could 25 not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin 26 deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell 27 surface, but its activity was significantly decreased. ER stress induced surface expression of 28 misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time 29 that they were retained in the ER. Our results indicate that calnexin-mediated ER quality 30 control systems for GPI-APs are necessary for both protein folding and GPI-inositol 31 deacylation. 32 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.08.192955; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 33 Introduction 34 Glycosylphosphatidylinositol (GPI) is a complex glycolipid that acts as a membrane 35 anchor for many cell surface proteins (Ikezawa, 2002; McConville & Menon, 2000; Tiede, 36 Bastisch, Schubert, Orlean, & Schmidt, 1999) and is a highly conserved post-translational 37 modification from yeast to mammals. In mammals, there are at least 150 GPI-anchored 38 proteins (GPI-APs), which serve as receptors, adhesion molecules, enzymes, protease 39 inhibitors, and so on (Kinoshita, 2020). The biosynthesis of GPI and its attachment to protein 40 occur in the endoplasmic reticulum (ER). Nascent GPI-APs synthesized by GPI transamidase 41 are still immature and undergo remodeling reactions to become mature GPI-APs. In the ER, 42 two remodeling reactions occur in many GPI-APs. First, an acyl-chain on an inositol-ring of 43 the GPI-anchor is eliminated by the GPI-inositol deacylase PGAP1 (Chen et al., 1998; Tanaka, 44 Maeda, Tashima, & Kinoshita, 2004). Second, a side-chain ethanolamine-phosphate (EtNP) 45 attached on the second mannose (Man2) of GPI-glycan is removed by the GPI-EtNP 46 phosphodiesterase PGAP5 (Fujita et al., 2009). These remodeling reactions are crucial for the 47 interaction of GPI-APs with p24 protein complexes, which are cargo receptors for GPI-APs 48 (Fujita & Kinoshita, 2012; Fujita et al., 2011), indicating that GPI-AP remodeling in the ER is 49 required for their efficient sorting into transport vesicles at the ER exit sites. Pathogenic 50 homozygous mutations in PGAP1 cause an inherited GPI deficiency, which results in 51 intellectual disability, encephalopathy and hypotonia (Granzow et al., 2015; Kettwig et al., 52 2016; Murakami et al., 2014). It has been reported that Pgap1 mutant mice exhibit abnormal 53 head development, such as otocephaly (Ueda et al., 2007; Zoltewicz et al., 2009) and 54 holoprosencephaly (McKean & Niswander, 2012), suggesting that Pgap1 function is required 55 for normal forebrain formation. In addition, male Pgap1-knockout mice are infertile (Ueda et 56 al., 2007). Taken together, these findings show that correct processing of GPI-anchors is 57 crucial for the proper functions of these proteins in vivo. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.08.192955; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 58 Protein asparagine (N)-glycosylation is another type of post-translational modification 59 occurring in the ER (Hammond, Braakman, & Helenius, 1994; Schwarz & Aebi, 2011). An 60 oligosaccharide consisting of Glc3Man9GlcNAc2 (Glc, glucose; Man, mannose, GlcNAc, 61 N-acetylglucosamine) is transferred to the amino group of asparagine within the motif NxS/T 62 (N, asparagine; S/T, serine or threonine; x, any amino acids except proline) of newly 63 synthesized proteins. The folding states of secretory proteins are monitored by the ER quality 64 control systems (Sun & Brodsky, 2019). N-glycans are processed in the ER, which contributes 65 to protein folding. First, two Glc residues on N-glycans are trimmed by -glucosidase I and II. 66 Processed monoglucosylated N-glycan structures are then recognized by calnexin and 67 calreticulin, which are molecular chaperones that possess a lectin domain. Calnexin has a 68 transmembrane domain, whereas calreticulin is a soluble protein (Helenius & Aebi, 2004), 69 and both proteins associate with protein disulfide isomerases such as ERp57 and ERp29, 70 prompting the folding of newly synthesized proteins (Ellgaard & Frickel, 2003; Kozlov, 71 Muñoz-Escobar, Castro, & Gehring, 2017). Once the remaining Glc residues on protein 72 N-glycans are trimmed by -glucosidase II, calnexin/calreticulin dissociate from the proteins. 73 However, proteins remaining in an unfolded state are re-glucosylated by UDP-Glc: 74 glycoprotein glucosyltransferase (UGGT), subsequently becoming bound again to calnexin 75 and calreticulin for refolding. This series of reactions is called the calnexin/calreticulin cycle 76 and is essential for glycoprotein folding. 77 In our previous study, we performed a genetic screening to identify factors that affect 78 GPI-inositol deacylation. In the screening, MOGS, GANAB, and CANX, as well as PGAP1, 79 were identified as candidates (Liu et al., 2018). MOGS, GANAB, and CANX encode 80 α-glucosidase I and II and calnexin, respectively (Tannous, Pisoni, Hebert, & Molinari, 2015). 81 These results suggested that N-glycan-dependent ER quality control systems participate in the 82 lipid remodeling of GPI-APs, whereas it was unclear how calnexin contributes to the 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.08.192955; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 83 processing. 84 When proteins fail to fold correctly, they are recognized as misfolded proteins, the majority 85 of which are retained in the ER and degraded through the ER-associated degradation (ERAD) 86 pathway (Vembar & Brodsky, 2008). However, misfolded GPI-APs do not appear to be 87 suitable substrates for ERAD, possibly because of the presence of GPI-anchors. A fraction of 88 misfolded GPI-APs are degraded through proteasomes (Fujita, Yoko-o, & Jigami, 2006; 89 Ishida et al., 2003), whereas the rest are exported from the ER and delivered to the vacuole for 90 degradation in yeast (Hirayama, Fujita, Yoko-o, & Jigami, 2008; Sikorska et al., 2016). In 91 mammalian cells, although misfolded GPI-APs are retained in the ER, they are rapidly 92 released into the secretory pathway upon acute ER stress despite their misfolding 93 (Satpute-Krishnan et al., 2014). Time-lapse imaging of live cells under acute ER stress 94 conditions showed misfolded prion proteins are transported from the ER to the Golgi and 95 plasma membrane, and subsequently to lysosomes for degradation. 96 In the present study, we investigated the roles of calnexin in the quality control and lipid 97 remodeling of GPI-APs in the ER. In wild-type (WT) cells, GPI-APs were folded and inositol 98 deacylated in the ER and expressed at the cell surface. In calnexin and calreticulin double 99 knockout (CANX&CALR-DKO) cells, protein folding and GPI-inositol deacylation 100 efficiencies were significantly decreased such that misfolded and inositol-acylated GPI-APs 101 were exposed on the plasma membrane. Thus, these results indicate that the 102 N-glycan-dependent calnexin/calreticulin cycle is responsible for the correct folding of 103 GPI-APs and provides sufficient ER retention time for efficient GPI-inositol deacylation. 104 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.08.192955; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 105 Materials and Methods 106 Cells, antibodies, and materials 107 All the cell lines, antibodies, and other reagents are listed in the key resource table. HEK293 108 and HEK293FF6 cells (Hirata et al., 2015) and their knockout (KO) derivatives were cultured 109 in Dulbecco’s Modified Eagle medium (DMEM) containing 10% (vol/vol) FCS (Biological 110 Industries).