Identification of host cell factors required for intoxication through use of modified cholera toxin
We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to...
| Main Authors: | , , , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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Rockefeller University Press
2012
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| Online Access: | http://hdl.handle.net/1721.1/70120 https://orcid.org/0000-0002-1090-6071 |
| _version_ | 1826211568067543040 |
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| author | Guimaraes, Carla P. Carette, Jan E. Varadarajan, Malini Antos, John Spooner, Eric Brummelkamp, Thijn R. Ploegh, Hidde Popp, Maximilian W.L. |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Guimaraes, Carla P. Carette, Jan E. Varadarajan, Malini Antos, John Spooner, Eric Brummelkamp, Thijn R. Ploegh, Hidde Popp, Maximilian W.L. |
| author_sort | Guimaraes, Carla P. |
| collection | MIT |
| description | We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ∼12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx. |
| first_indexed | 2024-09-23T15:08:03Z |
| format | Article |
| id | mit-1721.1/70120 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T15:08:03Z |
| publishDate | 2012 |
| publisher | Rockefeller University Press |
| record_format | dspace |
| spelling | mit-1721.1/701202022-10-02T00:47:44Z Identification of host cell factors required for intoxication through use of modified cholera toxin Guimaraes, Carla P. Carette, Jan E. Varadarajan, Malini Antos, John Spooner, Eric Brummelkamp, Thijn R. Ploegh, Hidde Popp, Maximilian W.L. Massachusetts Institute of Technology. Department of Biology Whitehead Institute for Biomedical Research Ploegh, Hidde Ploegh, Hidde Guimaraes, Carla P. Carette, Jan E. Varadarajan, Malini Antos, John Popp, Maximilian W. Spooner, Eric Brummelkamp, Thijn R. We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ∼12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx. Fundação para a Ciência e a Tecnologia (Fellowship) 2012-04-24T19:47:06Z 2012-04-24T19:47:06Z 2011-10 2011-08 Article http://purl.org/eprint/type/JournalArticle 0021-9525 1540-8140 http://hdl.handle.net/1721.1/70120 Guimaraes, C. P. et al. “Identification of Host Cell Factors Required for Intoxication Through Use of Modified Cholera Toxin.” The Journal of Cell Biology 195.5 (2011): 751–764. Web. https://orcid.org/0000-0002-1090-6071 en_US http://dx.doi.org/10.1083/jcb.201108103 Journal of Cell Biology Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported http://creativecommons.org/licenses/by-nc-sa/3.0/ application/pdf Rockefeller University Press Rockefeller UP |
| spellingShingle | Guimaraes, Carla P. Carette, Jan E. Varadarajan, Malini Antos, John Spooner, Eric Brummelkamp, Thijn R. Ploegh, Hidde Popp, Maximilian W.L. Identification of host cell factors required for intoxication through use of modified cholera toxin |
| title | Identification of host cell factors required for intoxication through use of modified cholera toxin |
| title_full | Identification of host cell factors required for intoxication through use of modified cholera toxin |
| title_fullStr | Identification of host cell factors required for intoxication through use of modified cholera toxin |
| title_full_unstemmed | Identification of host cell factors required for intoxication through use of modified cholera toxin |
| title_short | Identification of host cell factors required for intoxication through use of modified cholera toxin |
| title_sort | identification of host cell factors required for intoxication through use of modified cholera toxin |
| url | http://hdl.handle.net/1721.1/70120 https://orcid.org/0000-0002-1090-6071 |
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