Abstract
SummaryGlycosphingolipids (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.
Highlights
Glycosphingolipids (GSLs) are ubiquitously expressed in animals and are essential for embryonic development (Yamashita et al, 1999)
Identification of Genes Conferring Resistance to Shiga toxin (STx)-Induced Cell Death To identify host factors involved in the regulation of Gb3 biosynthesis by exploiting STx sensitivity as an indicator of cellular Gb3 levels, we performed a genome-wide CRISPR/CAS9 KO screen in HeLa cells
The single guide RNAs (sgRNAs) integrated into the cellular genomes of surviving cells were amplified by PCR and analyzed with high-throughput sequencing. sgRNAs enriched by STx in both independent cell libraries were selected as STx-resistance sgRNA candidates (Figure 1A, the full raw dataset is shown in Data S1 and S2)
Summary
Glycosphingolipids (GSLs) are ubiquitously expressed in animals and are essential for embryonic development (Yamashita et al, 1999). Mammalian cells produce a variety of GSLs, depending on the cell and tissue types. Various physiological roles of GSLs have been identified, including cell adhesion and cell signaling (Hakomori, 2008). Several GSLs are exploited as membrane receptors by toxins and infectious agents. Gb3 has other biological significance, especially under pathological conditions, including tumor metastasis (Kovbasnjuk et al, 2005) and Fabry diseases, caused by a-galactosidase A deficiency (Clarke, 2007). The regulatory mechanisms of GSL synthesis and degradation are important for understanding various physiological and pathological states
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