ARTICLE https://doi.org/10.1038/s41467-020-14678-2 OPEN Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation Yicheng Wang 1,2, Yusuke Maeda 1, Yi-Shi Liu3, Yoko Takada 2, Akinori Ninomiya 1, Tetsuya Hirata1,2,4, ✉ Morihisa Fujita3, Yoshiko Murakami 1,2 & Taroh Kinoshita 1,2 1234567890():,; Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that β1,3-galactosyltransferase 4 (B3GALT4), the previously char- acterized GM1 ganglioside synthase, additionally functions in transferring galactose to the N- acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unex- pected functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accu- mulation when the transfer of synthesized GPI to proteins is defective. Our data demon- strates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system. 1 Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. 2 WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan. 3 Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China. 4Present address: Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, 1-1 ✉ Yanagido, Gifu-City Gifu 501-1193, Japan email: [email protected] NATURE COMMUNICATIONS | (2020) 11:860 | https://doi.org/10.1038/s41467-020-14678-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14678-2 lycosylphosphatidylinositol (GPI) is a complex glycolipid usually stearic acid. Fatty acid remodeling is crucial for lipid-raft for post-translational modification of many cell-surface association of GPI, a feature of GPI-APs. G 1 proteins in eukaryotic cells . To date, more than 150 The structural variation of GPI anchors is introduced by side- human proteins have been confirmed as GPI-anchored proteins chain modifications1. Structural studies of GPIs from some mam- (GPI-APs)2. The structure of the core backbone of GPI, which is malian GPI-APs indicated that the first mannose (Man1) is often conserved in eukaryotic organisms, is EtNP-6Manα−2Manα modified with β1,4-linked N-acetylgalactosamine (GalNAc)3,4. −6Manα−4GlcNα−6Inositol-phospholipid (where EtNP, Man This modification is mediated by PGAP4 (Post-GPI attachment and GlcN are ethanolamine phosphate, mannose and glucosa- to proteins 4 also known as TMEM246), a recently identified mine, respectively) (Fig. 1a). GPI is synthesized in the endo- Golgi-resident, GPI-specific GalNAc-transferase5.TheGalNAc plasmic reticulum (ER) followed by transfer of GPI to proteins side-chain can be further modified with β1,3 galactose (Gal) by an that have a C-terminal GPI-attachment signal peptide. The GPI- unknown galactosyltransferase (Gal-T) and then with sialic acid attachment signal peptide is removed and replaced with GPI by (Sia) (Fig. 1a)4. Some GPI-APs have forth Man (Fig. 1a), which is the GPI-transamidase (GPI-Tase) complex to form immature transferred by an ER-resident mannosyltransferase PIGZ to the GPI-APs. Nascent GPI-APs undergo structural remodeling in the third Man before transfer of the GPI to proteins. These side-chains ER and the Golgi apparatus. The inositol-linked acyl chain is of GPI do not seem to affect cell surface expression of GPI-APs in deacylated and the EtNP side branch is removed from the second cultured cells, but might affect the properties of some GPI-APs. An Man (Man2) for efficient ER to Golgi transport. In the Golgi, GPI example is the role of a sialylated GPI side-chain in prion protein: It fatty acid remodeling occurs, in which an sn-2-linked unsaturated was shown by in vitro studies that lack of Sia in the GPI side-chain fatty acyl chain is removed and reacylated with a saturated chain, of PrPc slowed generation of pathogenic PrPsc after infection of ab Protein nd 2 Ab PIGS-KO PIGS-SLC35A2-DKO 5 105 10 105 +/– 4 104 10 104 +/– +/– β1–4 P 3 3 3 β1–3 10 0%10 1.31%10 99.2% Ethanolamine phosphate GS-II 2 2 2 Mannose 10 10 10 0 0 0 2 –102 –10 –102 +/– β1–4 Glucosamine P 3 4 5 0 103 104 105 0 10 10 10 0 103 104 105 β 1–3 P Phosphatidylinositol T5 mAb N-acetylgalactosamine β1–4 P Galactose Sialic acid c sgRNA library- Next generation sequencing to PIGS-KO HEK293 cells T5 mAb positive-staining cells expressing cells analyze sgRNA abundance FACS Whole genome sgRNA library d e 12.5 TMEM165 2nd Ab Starting clone After sort 3 10.0 UBE2J1 5 5 105 10 10 B3GALT4 SLC35A2 GOSR1 4 4 4 7.5 10 10 10 3 UGCG 3 103 10 10 UNC50 5.0 SYVN1 GS-II UBE2G2 2 2 STX5 102 10 10 0 0.32% 0 6.73% 0 86.8% 2 2 2 –10 –10 –10 Significance [-Log10 (RRA)] 2.5 3 4 5 3 4 5 0 103 104 105 0 10 10 10 0 10 10 10 T5 mAb 0.0 All targeted genes Glycosyltransferase ERAD protein UDP-Gal transporter SNARE complex Golgi Mn2+ homeostasis Regulator of endosome-to-Golgi transport Fig. 1 CRISPR-based genetic screen identified genes involved in galactosylation of GPI. a GPI-APs and free GPIs with side-chain modifications. Monosaccharide symbols are drawn according to the symbol nomenclature for glycans77. b Flow cytometry analysis of PIGS knockout (PIGS-KO) and PIGS-SLC35A2-DKO HEK293 cells. Cells were double stained with T5 mAb for free GPI-GalNAc and GS-II lectin. c Schematic depicting a FACS-based genome-wide CRISPR screen for GPI-galactosylation using PIGS-KO HEK293 cells. d Flow cytometry analysis of starting clone and sort3 of PIGS-KO cells stained with T5 mAb and GS-II lectin. e Gene scores in unsorted versus sort3 PIGS-KO cells. Ten top-ranking ER or Golgi protein-coding genes are shown. Bubble size shows the number of active sgRNAs per gene. See also Supplementary Fig. 1e, f. 2 NATURE COMMUNICATIONS | (2020) 11:860 | https://doi.org/10.1038/s41467-020-14678-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-14678-2 ARTICLE cultured cells, suggesting involvement of the sialylated GPI side- cells (shown schematically in Supplementary Fig. 1a). The PIGS- chain in conversion of PrPc to PrPsc6. A recent in vivo study SLC35A2 DKO cells were also strongly stained by Griffonia showed that the presence of PrPc, which lacks Sia in the GPI, was simplicifolia lectin II (GS-II) because of exposure of N-acet- protective against infection by PrPsc7. However, another report ylglucosamine (GlcNAc) on N-glycans and other oligosaccharides suggested that GPI sialylation of PrPc is regulated in a host-, cell- or by defects in galactosylation (Fig. 1b, right, and Supplementary tissue-specific manner and that PrPc proteins with both sialo- and Fig. 1a). These results indicate that if GPI-Gal-T is lost, PIGS-KO asialo-GPI were converted to PrPsc8. Because current understanding HEK293 cells would be positively stained by T5 mAb but not by of GPI sialylation is still incomplete, identification of the relevant GS-II. In contrast, if the general galactosylation ability is lost, the GPI-Gal-T would be helpful for further understanding of the PIGS-KO cells would be positively stained by both T5 mAb and GPI-GalNAc side-chain modification pathway, which is required GS-II. for understanding of the significance of GPI structural variation Based on this principle, we transduced PIGS-KO HEK293 cells in vivo. with a lentiviral short guide RNAs (sgRNA) library (GeCKO v2)20. Glycosphingolipids (GSLs) are glycolipids consisting of cer- We then cultured the cells for 2 weeks, and cells positively stained amide and an oligosaccharide chain9. GPI-APs were proposed to by T5 mAb were enriched by cell sorting (Fig. 1c). After three associate with lipid rafts, microdomains enriched in cholesterol rounds of cell sorting and culture (sort3 cells), most cells became and sphingolipids, from the trans-Golgi network (TGN) to the positively stained by T5 mAb, and some were also positively plasma membrane10,11. Evidence suggests that lipid rafts are stained by GS-II lectin (Fig. 1d). The sgRNA abundance in the highly dynamic and heterogeneous12, and GPI-APs are thought sorted and unsorted cells was determined by deep sequencing and to be organized in cholesterol- and sphingomyelin-dependent analyzed by the Model-based Analysis of Genome-wide CRISPR- submicron-sized domains in live cell membranes13. The current Cas9 Knockout (MAGeCK) method21. We calculated significance view of the functional microdomains is that they might be pre- scores based on the robust ranking aggregation (RRA) algorithm sent in the Golgi14,15. Interactions between GPI-APs and GSLs as the fold-change in the abundance of multiple sgRNAs targeting are rather transient16. Although both GPIs and GSLs are syn- the same gene. We observed hits that were highly enriched for thesized in the ER and the Golgi, evidence for their functional some genes in sort3 cells, including the expected SLC35A2 interactions in the Golgi has not been reported, and whether (Fig. 1e), and a significant enrichment of individual sgRNAs for GPI-APs associate with specific GSLs in the Golgi remains those genes (Supplementary Fig.