HEK293T cell lines defective for O-linked glycosylation

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Title: HEK293T cell lines defective for O-linked glycosylation
Author(s): Termini, JM
Silver, ZA
Connor, B
Antonopoulos, A
Haslam, SM
Dell, A
Desrosiers, RC
Item Type: Journal Article
Abstract: Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE), galactokinase 1 (GALK1), and galactokinase 2 (GALK2) genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc) to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2), O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.
Publication Date: 27-Jun-2017
Date of Acceptance: 7-Jun-2017
URI: http://hdl.handle.net/10044/1/50112
DOI: https://dx.doi.org/10.1371/journal.pone.0179949
ISSN: 1932-6203
Publisher: Public Library of Science
Journal / Book Title: PLOS One
Volume: 12
Issue: 6
Copyright Statement: © 2017 Terminiet al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricte d use, distribu tion, and reproduction in any medium, provided the original author and source are credited.https://creativecommons.org/licenses/by/4.0/
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (BBSRC)
Funder's Grant Number: BB/K016164/1
Keywords: Science & Technology
Multidisciplinary Sciences
Science & Technology - Other Topics
EPIMERASE-DEFICIENCY GALACTOSEMIA
MAMMALIAN-CELLS
LELOIR PATHWAY
MUTANTS
GLYCANS
ENZYMES
SYSTEMS
MOUSE
Science & Technology
Multidisciplinary Sciences
Science & Technology - Other Topics
EPIMERASE-DEFICIENCY GALACTOSEMIA
MAMMALIAN-CELLS
LELOIR PATHWAY
MUTANTS
GLYCANS
ENZYMES
SYSTEMS
MOUSE
MD Multidisciplinary
General Science & Technology
Publication Status: Published
Article Number: ARTN e0179949
Appears in Collections:Faculty of Natural Sciences



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