Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation
Authors
Tian, SonghaiMuneeruddin, Khaja
Choi, Mei Yuk
Tao, Liang
Bhuiyan, Robiul H.
Ohmi, Yuhsuke
Furukawa, Keiko
Furukawa, Koichi
Boland, Sebastian
Shaffer, Scott A.
Adam, Rosalyn M.
Dong, Min
UMass Chan Affiliations
Mass Spectrometry FacilityDepartment of Biochemistry and Molecular Pharmacology
Document Type
Journal ArticlePublication Date
2018-11-27Keywords
RicinCell binding
Toxins
Glycosylation
Sphingolipids
Cell viability testing
Glycolipids
Immunostaining
Amino Acids, Peptides, and Proteins
Biochemistry, Biophysics, and Structural Biology
Cell Biology
Cellular and Molecular Physiology
Computational Biology
Genetic Phenomena
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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.Source
PLoS Biol. 2018 Nov 27;16(11):e2006951. doi: 10.1371/journal.pbio.2006951. eCollection 2018 Nov. Link to article on publisher's site
DOI
10.1371/journal.pbio.2006951Permanent Link to this Item
http://hdl.handle.net/20.500.14038/40874PubMed ID
30481169Related Resources
Rights
Copyright: © 2018 Tian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Distribution License
http://creativecommons.org/licenses/by/4.0/ae974a485f413a2113503eed53cd6c53
10.1371/journal.pbio.2006951
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Except where otherwise noted, this item's license is described as Copyright: © 2018 Tian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.