Glycosphingolipids (GSLs) are produced by various GSL-synthesizing enzymes, but post-translational regulation of these enzymes is incompletely understood. To address this knowledge disparity, we focused on biosynthesis of globotriaosylceramide (Gb3), the Shiga toxin (STx) receptor, and performed a genome-wide CRISPR/CAS9 knockout screen in HeLa cells using STx1-mediated cytotoxicity. We identified various genes including sphingolipid-related genes and membrane-trafficking genes. In addition, we found two proteins, LAPTM4A and TM9SF2, for which physiological roles remain elusive. Disruption of either LAPTM4A or TM9SF2 genes reduced Gb3 biosynthesis, resulting in accumulation of its precursor, lactosylceramide. Loss of LAPTM4A decreased endogenous Gb3 synthase activity in a post-transcriptional mechanism, whereas loss of TM9SF2 did not affect Gb3 synthase activity but instead disrupted localization of Gb3 synthase. Furthermore, the Gb3-regulating activity of TM9SF2 was conserved in the TM9SF family. These results provide mechanistic insight into the post-translational regulation of the activity and localization of Gb3 synthase.

Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs). A TALEN pair targeting the human CERT gene (alternative name COL4A3BP) encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase), and B4GalT5 (encoding the major lactosylceramide synthase), and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a ‘sphingolipid-modified HeLa cell panel,’ which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background.

Globotriaosylceramide (Gb3) is a well known receptor for Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The expression of Gb3 also affects several diseases, including cancer metastasis and Fabry disease, which prompted us to look for factors involved in its metabolism. In the present study, we isolated two cDNAs that conferred resistance to Stx-induced cell death in HeLa cells by expression cloning: ganglioside GM3 synthase and the COOH terminus region of glutamate receptor, ionotropic, N-methyl-D-asparate-associated protein 1 (GRINA), a member of the transmembrane BAX inhibitor motif containing (TMBIM) family. Overexpression of the truncated form, named GRINA-C, and some members of the full-length TMBIM family, including FAS inhibitory molecule 2 (FAIM2), reduced Gb3, and lactosylceramide was accumulated instead. The change of glycolipid composition was restored by overexpression of Gb3 synthase, suggesting that the synthase is affected by GRINA-C and FAIM2. Interestingly, the mRNA level of Gb3 synthase was unchanged. Rather, localization of the synthase as well as TGN46, a trans-Golgi network marker, was perturbed to form punctate structures, and degradation of the synthase in lysosomes was enhanced. Furthermore, GRINA-C was associated with Gb3 synthase. These observations may demonstrate a new type of posttranscriptional regulation of glycosyltransferases.

Subtilase cytotoxin (SubAB) is a virulence factor produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains. The toxin recognizes sialoglycans for entry and cleaves an endoplasmic reticulum chaperon, binding immunoglobulin protein, to cause cell death. However, no systematic screening has yet been performed to identify critical host factors. Here, we performed a genome-wide CRISPR/Cas9 knockout screen for SubAB-induced cell death and identified various sialoglycan-related and membrane-trafficking genes. Analysis of glycan-deficient cells demonstrated that not only N-glycans but also O-glycans serve as SubAB receptors. In addition, SLC39A9, which is a predicted zinc transporter, as well as KDELRs and JTB, were required for SubAB to induce maximal cell death. Disruption of the SLC39A9 gene markedly reduced both complex-type N-glycans and core 1 O-glycans, and the O-glycan reduction was attributed to the reduction of core 1 synthase (C1GalT1). These results provide insights into the post-transcriptional regulation of glycosyltransferases by SLC39A9, as well as sialoglycan species as SubAB receptors.

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.