Journal Article

Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation

Tao, Liang, Tian, Songhai, Furukawa, Keiko, Adam, Rosalyn M., Choi, Mei Yuk, Boland, Sebastian, Dong, Min, Bhuiyan, Robiul H., Furukawa, Koichi, Shaffer, Scott A., Muneeruddin, Khaja, Ohmi, Yuhsuke

2018

Overview

Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific \"activator\" for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Bacterial toxins / Bacterial Toxins - metabolism / Binding sites / Biochemistry / Biology / Biology and Life Sciences / Biosynthesis / Cell adhesion & migration / CRISPR / CRISPR-Cas Systems / Crystal structure / Defects / DNA sequencing / Domains / Endoplasmic reticulum / Endosomes - metabolism / Genetic aspects / Genome-wide association studies / Genome-Wide Association Study - methods / Genomes / Globotriaosylceramide / Glycolipids / Glycolipids - metabolism / Glycosphingolipids / Glycosylation / Golgi apparatus / Golgi Apparatus - metabolism / Golgi Apparatus - physiology / Health sciences / HEK293 Cells / HeLa Cells / Homology / Hospitals / Humans / Insects / Lipids / Loss of Function Mutation - genetics / Mass spectrometry / Mass spectroscopy / Medical schools / Medicine and Health Sciences / Membrane proteins / Membrane Proteins - metabolism / Membrane Transport Proteins - metabolism / Membrane Transport Proteins - physiology / Methods / Methods and Resources / Nucleotide sequencing / Oncogene Proteins - metabolism / Physiological aspects / Polysaccharides / Protein Transport / Proteins / Receptors / Research and Analysis Methods / Ricin / Ricin - genetics / Ricin - metabolism / Scientific imaging / Screens / Shiga toxin / Shiga Toxins - genetics / Shiga Toxins - metabolism / Surgery / Therapeutic applications / Toxicity / Toxins / Trihexosylceramides - metabolism / Trihexosylceramides - physiology / University colleges / Urology