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

Abstract

16 17 18 Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with 19 each other in the mammalian plasma membranes, forming dynamic microdomains. How their 20interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 21 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi 22 apparatus, we report that b1,3-galactosyltransferase 4 (B3GALT4), also called GM1 23 ganglioside synthase, additionally functions in transferring galactose to the N-24 acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide 25 for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously 26 unexpected evolutionary and functional relationships between GPI-anchored proteins and 27 glycosphingolipids in the Golgi. Through the same screening, we also show that GPI 28 biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated 29 degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is 30 defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid 31 biosynthesis and the ER quality control system. 32 33CRISPR genome-wide screen. 36 37 Glycosylphosphatidylinositol (GPI) is a complex glycolipid for post-translational 38 modification of many cell-surface proteins in eukaryotic cells 1 . To date, more than 150 39 human proteins have been confirmed as GPI-anchored proteins (GPI-APs) 2 . The structure of 40 the core backbone of GPI, which is conserved in eukaryotic organisms, is EtNP-6Mana-41 2Mana-6Mana-4GlcNa-6Inositol-phospholipid (where EtNP, Man and GlcN are 42 ethanolamine phosphate, mannose and glucosamine, respectively) ( Fig. 1a). GPI is 43 synthesized in the endoplasmic reticulum (ER) followed by transfer of GPI to proteins that 44 have a C-terminal GPI-attachment signal peptide. The GPI-attachment signal peptide is 45 removed and replaced with GPI by the GPI-transamidase (GPI-Tase) complex to form 46 immature GPI-APs. Nascent GPI-APs undergo structural remodeling in the ER and the Golgi 47 apparatus. The inositol-linked acyl chain is deacylated and the EtNP side branch is removed 48 from the second Man (Man2) for efficient ER to Golgi transport 3-5 . In the Golgi, GPI fatty 49 acid remodeling occurs, in which an sn-2-linked unsaturated fatty acyl chain is removed and 50 reacylated with a saturated chain, usually stearic acid 6,7 . Fatty acid remodeling is crucial for 51 lipid-raft association of GPI, a feature of GPI-APs 8 . 52 53The structural variation of GPI anchors is introduced by side-chain modifications 1 . Structural 54 studies of GPIs from some mammalian GPI-APs indicated that the first mannose (Man1) is 55 often modified with β1,4-linked N-acetylgalactosamine (GalNAc) 9,10 . This modification is 56 mediated by PGAP4 (Post-GPI attachment to proteins 4 also known as TMEM246), a 57 recently identified Golgi-resident, GPI-specific GalNAc-transferase 11 . The GalNAc side-58 chain can be furth...

Biosynthesis and biology of mammalian GPI-anchored proteins

“…Recently, the Gal transferase was identified to be B3GALT4 [88], the Golgi-resident Gal transferase previously known to synthesize GM1 from GM2 [89,90]. B3GALT4 also synthesizes GA1 and GD1 from GA2 and GD2, respectively [89].…”

A phylogenetic view and functional annotation of the animal β1,3-glycosyltransferases of the GT31 CAZy family

“…The mammalian B3GALT4 enzyme determines the synthesis of glycosphingolipids of the ganglioseries, e.g., GD1b, GM1 and GA1 ( Figure 1) and therefore is also known as the GM1-synthase (Amado et al 1998;Daniotti et al 1999;Miyazaki et al 1997). Interestingly, it was suggested that it could affect lipid concentrations by modifying lipoprotein receptors (Willer et al 2008) and more recently it was shown to catalyze the transfer of a Gal residue from UDP-Gal to the β1,4GalNAc side chain of glycosylphosphatdylinositol (GPI)-anchored proteins (Wang et al 2020). Our data indicate that the B3GALT4 functions are restricted to a few vertebrate species (Supplemental Figure 5B).…”

α2,3 linkage of sialic acid to a GPI anchor and an unpredicted GPI attachment site in human prion protein

“…This implies that enzyme activities for GPIanchor galactosylation and sialylation are diverse in brain tissues of different organisms. We recently reported that the percentages of Gal-modified and Sia-Gal-modified GPIglycans in CD59, a GPI-AP, were only 10% and 1% respectively, in HEK293 cells (14). Overexpression of B3GALT4 in HEK293 cells increased these percentages, indicating that B3GALT4-mediated Gal transfer was ratelimiting.…”

“…We recently identified PGAP4 (also known as TMEM246 or C9orf125) as a GPIspecific GalNAc transferase (12). More recently, we reported that B3GALT4, which is also known as ganglioside GM1 synthase, also acts as a Gal transferase for the GPI-GalNAc sidechain (14). Although these achievements have progressed our understanding of GPI-GalNAc side-chain, the biosynthesis pathway of the GPIglycan structure is still incomplete.…”