Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation

Yicheng Wang,Yoshiko Murakami,Yoko Takada,Yusuke Maeda,Yi-Shi Liu,Taroh Kinoshita,Akinori Ninomiya,Tetsuya Hirata,Morihisa Fujita

doi:10.1038/s41467-020-14678-2

Yicheng Wang, Yoshiko Murakami + Show 7 more

Open Access

https://doi.org/10.1038/s41467-020-14678-2

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Abstract

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 characterized 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 unexpected 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 accumulation when the transfer of synthesized GPI to proteins is defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system.

Highlights

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains
PIGS-KO HEK293 cells were only barely stained by T5-4E10 monoclonal antibody (T5 mAb), whereas PIGS and SLC35A2 double knockout (DKO) HEK293 cells, in which UDP-Gal is not available for Gal-Ts, were strongly stained by T5 mAb (Fig. 1b)
The PIGSSLC35A2 DKO cells were strongly stained by Griffonia simplicifolia lectin II (GS-II) because of exposure of N-acetylglucosamine (GlcNAc) on N-glycans and other oligosaccharides by defects in galactosylation (Fig. 1b, right, and Supplementary Fig. 1a)

Summary

Introduction

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. Structural studies of GPIs from some mammalian GPI-APs indicated that the first mannose (Man1) is often modified with β1,4-linked N-acetylgalactosamine (GalNAc)[3,4]. This modification is mediated by PGAP4 (Post-GPI attachment to proteins 4 known as TMEM246), a recently identified Golgi-resident, GPI-specific GalNAc-transferase[5]. An example is the role of a sialylated GPI side-chain in prion protein: It was shown by in vitro studies that lack of Sia in the GPI side-chain of PrPc slowed generation of pathogenic PrPsc after infection of a Protein +/– +/– +/– β1–4

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