UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology; Mass Spectrometry Facility

Publication Date

2018-11-27

Document Type

Article

Disciplines

Amino Acids, Peptides, and Proteins | Biochemistry, Biophysics, and Structural Biology | Cell Biology | Cellular and Molecular Physiology | Computational Biology | Genetic Phenomena

Abstract

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.

Keywords

Ricin, Cell binding, Toxins, Glycosylation, Sphingolipids, Cell viability testing, Glycolipids, Immunostaining

Rights and Permissions

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.

DOI of Published Version

10.1371/journal.pbio.2006951

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

Journal/Book/Conference Title

PLoS biology

Related Resources

Link to Article in PubMed

PubMed ID

30481169

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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