Selective biochemical labeling of C. jejuni cell-surface glycoconjugates
The display of cell-surface glycolipids and glycoproteins is essential for the motility, adhesion and colonization of pathogenic bacteria such as Campylobacter jejuni. Recently, the cell-surface display of C. jejuni glycoconjugates has been the focus of considerable attention; however, our understan...
| Main Authors: | , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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Oxford University Press
2015
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| Online Access: | http://hdl.handle.net/1721.1/98081 https://orcid.org/0000-0002-5749-7869 |
| _version_ | 1826208473463914496 |
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| author | Imperiali, Barbara Whitworth, Garrett |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Imperiali, Barbara Whitworth, Garrett |
| author_sort | Imperiali, Barbara |
| collection | MIT |
| description | The display of cell-surface glycolipids and glycoproteins is essential for the motility, adhesion and colonization of pathogenic bacteria such as Campylobacter jejuni. Recently, the cell-surface display of C. jejuni glycoconjugates has been the focus of considerable attention; however, our understanding of the roles that glycosylation plays in bacteria still pales in comparison with our understanding of mammalian glycosylation. One of the reasons for this is that carbohydrate metabolic labeling, a powerful tool for studying mammalian glycans, is difficult to establish in bacterial systems and has a significantly more limited scope. Herein, we report the development of an alternative strategy that can be used to study bacterial cell-surface glycoconjugates. Galactose oxidase (GalO) is used to generate an aldehyde at C-6 of terminal GalNAc residues of C. jejuni glycans. This newly generated aldehyde can be conjugated with aminooxy-functionalized purification tags or fluorophores. The label can be targeted towards specific glycoconjugates using C. jejuni mutant strains with N-glycan or lipo-oligosaccharides (LOS) assembly defects. GalO-catalyzed labeling of cell-surface glycoproteins with biotin, allowed for the purification and identification of known extracellular N-linked glycoproteins as well as a recently identified O-linked glycan modifying PorA. To expand the scope of the GalO reaction, live-cell fluorescent labeling of C. jejuni was used to compare the levels of surface-exposed LOS to the levels of N-glycosylated, cell-surface proteins. While this study focuses on the GalO-catalyzed labeling of C. jejuni, it can in principle be used to evaluate glycosylation patterns and identify glycoproteins of interest in any bacteria. |
| first_indexed | 2024-09-23T14:06:13Z |
| format | Article |
| id | mit-1721.1/98081 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T14:06:13Z |
| publishDate | 2015 |
| publisher | Oxford University Press |
| record_format | dspace |
| spelling | mit-1721.1/980812022-10-01T19:11:43Z Selective biochemical labeling of C. jejuni cell-surface glycoconjugates Selective biochemical labeling of Campylobacter jejuni cell-surface glycoconjugates Imperiali, Barbara Whitworth, Garrett Massachusetts Institute of Technology. Department of Biology Imperiali, Barbara Whitworth, Garrett E. Imperiali, Barbara The display of cell-surface glycolipids and glycoproteins is essential for the motility, adhesion and colonization of pathogenic bacteria such as Campylobacter jejuni. Recently, the cell-surface display of C. jejuni glycoconjugates has been the focus of considerable attention; however, our understanding of the roles that glycosylation plays in bacteria still pales in comparison with our understanding of mammalian glycosylation. One of the reasons for this is that carbohydrate metabolic labeling, a powerful tool for studying mammalian glycans, is difficult to establish in bacterial systems and has a significantly more limited scope. Herein, we report the development of an alternative strategy that can be used to study bacterial cell-surface glycoconjugates. Galactose oxidase (GalO) is used to generate an aldehyde at C-6 of terminal GalNAc residues of C. jejuni glycans. This newly generated aldehyde can be conjugated with aminooxy-functionalized purification tags or fluorophores. The label can be targeted towards specific glycoconjugates using C. jejuni mutant strains with N-glycan or lipo-oligosaccharides (LOS) assembly defects. GalO-catalyzed labeling of cell-surface glycoproteins with biotin, allowed for the purification and identification of known extracellular N-linked glycoproteins as well as a recently identified O-linked glycan modifying PorA. To expand the scope of the GalO reaction, live-cell fluorescent labeling of C. jejuni was used to compare the levels of surface-exposed LOS to the levels of N-glycosylated, cell-surface proteins. While this study focuses on the GalO-catalyzed labeling of C. jejuni, it can in principle be used to evaluate glycosylation patterns and identify glycoproteins of interest in any bacteria. National Institutes of Health (U.S.) (Grant GM-039334) Natural Sciences and Engineering Research Council of Canada 2015-08-13T19:48:23Z 2015-08-13T19:48:23Z 2015-03 2015-02 Article http://purl.org/eprint/type/JournalArticle 0959-6658 1460-2423 http://hdl.handle.net/1721.1/98081 Whitworth, G. E., and B. Imperiali. “Selective Biochemical Labeling of Campylobacter Jejuni Cell-Surface Glycoconjugates.” Glycobiology 25, no. 7 (March 11, 2015): 756–766. https://orcid.org/0000-0002-5749-7869 en_US http://dx.doi.org/10.1093/glycob/cwv016 Glycobiology Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Oxford University Press Imperiali |
| spellingShingle | Imperiali, Barbara Whitworth, Garrett Selective biochemical labeling of C. jejuni cell-surface glycoconjugates |
| title | Selective biochemical labeling of C. jejuni cell-surface glycoconjugates |
| title_full | Selective biochemical labeling of C. jejuni cell-surface glycoconjugates |
| title_fullStr | Selective biochemical labeling of C. jejuni cell-surface glycoconjugates |
| title_full_unstemmed | Selective biochemical labeling of C. jejuni cell-surface glycoconjugates |
| title_short | Selective biochemical labeling of C. jejuni cell-surface glycoconjugates |
| title_sort | selective biochemical labeling of c jejuni cell surface glycoconjugates |
| url | http://hdl.handle.net/1721.1/98081 https://orcid.org/0000-0002-5749-7869 |
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