bioRxiv preprint doi: https://doi.org/10.1101/743914; this version posted August 24, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis 2 and ER-associated degradation 3 4 Yicheng Wang1,2, Yusuke Maeda1, Yishi Liu3, Yoko Takada2, Akinori Ninomiya1, Tetsuya 5 Hirata1,2,4, Morihisa Fujita3, Yoshiko Murakami1,2, Taroh Kinoshita1,2,* 6 7 1Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan 8 2WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, 9 Japan 10 3Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, 11 School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China 12 4Current address: Center for Highly Advanced Integration of Nano and Life Sciences (G- 13 CHAIN), Gifu University, 1-1 Yanagido, Gifu-City, Gifu 501-1193, Japan 14 15 *Correspondence and requests for materials should be addressed to T.K. (email: 16 [email protected]) 17 18 19 Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with 20 each other in the mammalian plasma membranes, forming dynamic microdomains. How their 21 interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 22 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi 23 apparatus, we report that b1,3-galactosyltransferase 4 (B3GALT4), also called GM1 24 ganglioside synthase, additionally functions in transferring galactose to the N- 25 acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide 26 for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously 27 unexpected evolutionary and functional relationships between GPI-anchored proteins and 28 glycosphingolipids in the Golgi. Through the same screening, we also show that GPI 29 biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated 30 degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is 31 defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid 32 biosynthesis and the ER quality control system. 33 1 bioRxiv preprint doi: https://doi.org/10.1101/743914; this version posted August 24, 2019. 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. 34 Keywords: Golgi apparatus, glycosylphosphatidylinositol, glycosphingolipids, 35 glycosyltransferases, glucosylceramide, lactosylceramide, gangliosides, biosynthesis, ERAD, 36 CRISPR genome-wide screen. 37 2 bioRxiv preprint doi: https://doi.org/10.1101/743914; this version posted August 24, 2019. 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. 38 Glycosylphosphatidylinositol (GPI) is a complex glycolipid for post-translational 39 modification of many cell-surface proteins in eukaryotic cells1. To date, more than 150 40 human proteins have been confirmed as GPI-anchored proteins (GPI-APs)2. The structure of 41 the core backbone of GPI, which is conserved in eukaryotic organisms, is EtNP-6Mana- 42 2Mana-6Mana-4GlcNa-6Inositol-phospholipid (where EtNP, Man and GlcN are 43 ethanolamine phosphate, mannose and glucosamine, respectively) (Fig. 1a). GPI is 44 synthesized in the endoplasmic reticulum (ER) followed by transfer of GPI to proteins that 45 have a C-terminal GPI-attachment signal peptide. The GPI-attachment signal peptide is 46 removed and replaced with GPI by the GPI-transamidase (GPI-Tase) complex to form 47 immature GPI-APs. Nascent GPI-APs undergo structural remodeling in the ER and the Golgi 48 apparatus. The inositol-linked acyl chain is deacylated and the EtNP side branch is removed 49 from the second Man (Man2) for efficient ER to Golgi transport3–5. In the Golgi, GPI fatty 50 acid remodeling occurs, in which an sn-2-linked unsaturated fatty acyl chain is removed and 51 reacylated with a saturated chain, usually stearic acid6,7. Fatty acid remodeling is crucial for 52 lipid-raft association of GPI, a feature of GPI-APs8. 53 54 The structural variation of GPI anchors is introduced by side-chain modifications1. Structural 55 studies of GPIs from some mammalian GPI-APs indicated that the first mannose (Man1) is 56 often modified with β1,4-linked N-acetylgalactosamine (GalNAc)9,10. This modification is 57 mediated by PGAP4 (Post-GPI attachment to proteins 4 also known as TMEM246), a 58 recently identified Golgi-resident, GPI-specific GalNAc-transferase11. The GalNAc side- 59 chain can be further modified with β1,3 galactose (Gal) by an unknown galactosyltransferase 60 (Gal-T) and then with sialic acid (Sia) (Fig. 1a)10. Some GPI-APs have forth Man (Fig. 1a), 61 which is transferred by an ER-resident mannosyltransferase PIGZ to the third Man before 62 transfer of the GPI to proteins12. These side-chains of GPI do not seem to affect cell surface 63 expression of GPI-APs in cultured cells, but might affect the properties of some GPI-APs. An 64 example is the role of a sialylated GPI side-chain in prion protein: It was shown by in vitro 65 studies that lack of Sia in the GPI side-chain of PrPc slowed generation of pathogenic PrPsc 66 after infection of cultured cells, suggesting involvement of the sialylated GPI side-chain in 67 conversion of PrPc to PrPsc13,14. A recent in vivo study showed that the presence of PrPc, 68 which lacks Sia in the GPI, was protective against infection by PrPsc15. However, another 69 report suggested that GPI sialylation of PrPc is regulated in a host-, cell- or tissue-specific 70 manner and that PrPc proteins with both sialo- and asialo-GPI were converted to PrPsc16. 3 bioRxiv preprint doi: https://doi.org/10.1101/743914; this version posted August 24, 2019. 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. 71 Because current understanding of GPI sialylation is still incomplete, identification of the 72 relevant GPI-Gal-T would be helpful for further understanding of the GPI-GalNAc side-chain 73 modification pathway, which is required for understanding of the significance of GPI 74 structural variation in vivo. 75 76 Glycosphingolipids (GSLs) are glycolipids consisting of ceramide and an oligosaccharide 77 chain17. GPI-APs were proposed to associate with “lipid rafts”, microdomains enriched in 78 cholesterol and sphingolipids, from the trans-Golgi network (TGN) to the plasma 79 membrane18–21. Evidence suggests that lipid rafts are highly dynamic and heterogeneous22, 80 and GPI-APs are thought to be organized in cholesterol- and sphingomyelin-dependent 81 submicron-sized domains in live cell membranes23–26. The current view of the functional 82 microdomains is that they might be present in the Golgi27–29. Interactions between GPI-APs 83 and GSLs are rather transient30,31. Although both GPIs and GSLs are synthesized in the ER 84 and the Golgi, evidence for their functional interactions in the Golgi has not been reported, 85 and whether GPI-APs associate with specific GSLs in the Golgi remains unclear. 86 87 Here, we report genome-scale CRISPR-Cas9 knockout (GeCKO) screening for genes 88 involved in galactosylation of the GalNAc side-chain of GPI, particularly the GPI-Gal-T. 89 Through the screening, we defined key enzymes and regulators of the GPI galactosylation 90 pathway. Of particular interest, b1,3-galactosyltransferase 4 (B3GALT4, also known as GM1 91 synthase), a Gal-T thought to be limited to the GSL pathway, was the only candidate for GPI- 92 Gal-T identified by our GeCKO screen. B3GALT4 transfers Gal to a β1,4-linked GalNAc 93 side-chain of GPI. We also demonstrate the requirement for lactosylceramide (LacCer) for 94 efficient galactosylation of GPI-GalNAc. In addition, we identified components of an ER- 95 associated degradation (ERAD) pathway through the same screening: we show that the 96 ERAD is not involved in regulation of GPI-GalNAc galactosylation but instead negatively 97 regulates GPI biosynthesis when too much GPI is accumulated. 98 99 Results 100 101 CRISPR-based genetic screen identified genes involved in galactosylation of GPI. 102 To identify the GPI-Gal-T that transfers Gal to GPI-GalNAc, we established a forward 103 genetic screening system. For enrichment of mutant cells defective in galactosylation of GPI- 4 bioRxiv preprint doi: https://doi.org/10.1101/743914; this version posted August 24, 2019. 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. 104 GalNAc, a probe that detects the galactosylation status of GPI-GalNAc is required. T5-4E10 105 monoclonal antibody (T5 mAb) recognizes non-protein-linked, free GPI when it has the 106 GalNAc side-chain but only when the GalNAc is at the non-reducing end (Fig. 1a, bottom), 107 i.e., T5 mAb does not bind free GPI when GalNAc is galactosylated (Fig. 1a, middle)11,32,33. 108 Therefore, T5 mAb is useful to identify mutant cells defective in galactosylation of GPI- 109 GalNAc. As a parental cell that receives a genome-wide guide RNA library, we used PIGS- 110 knockout (KO) HEK293 cells34. PIGS is one of the subunits of GPI-Tase and essential for 111 transfer of GPI to proteins; therefore, all GPIs become free, non-protein-linked GPI in PIGS- 112 KO cells. PIGS-KO HEK293 cells were only barely stained by T5 mAb, whereas PIGS and 113 SLC35A2 double knockout (DKO) HEK293 cells, in which UDP-Gal is not available for 114 Gal-Ts, were strongly stained by T5 mAb (Fig.