Bacterial N-Glycosylation Efficiency is Dependent on the Structural Context of Target Sequons
Site selectivity of protein N-linked glycosylation is dependent on many factors, including accessibility of the modification site, amino acid composition of the glycosylation consensus sequence, and cellular localization of target proteins. Previous studies have shown that the bacterial oligosacchar...
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| Format: | Article |
| Language: | en_US |
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American Society for Biochemistry and Molecular Biology (ASBMB)
2018
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| Online Access: | http://hdl.handle.net/1721.1/117180 https://orcid.org/0000-0003-1432-0438 https://orcid.org/0000-0002-5749-7869 |
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| author | Silverman, Julie Imperiali, Barbara |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Silverman, Julie Imperiali, Barbara |
| author_sort | Silverman, Julie |
| collection | MIT |
| description | Site selectivity of protein N-linked glycosylation is dependent on many factors, including accessibility of the modification site, amino acid composition of the glycosylation consensus sequence, and cellular localization of target proteins. Previous studies have shown that the bacterial oligosaccharyltransferase, PglB, of Campylobacter jejuni favors acceptor proteins with consensus sequences ((D/E)X[subscript 1]NX[subscript 2](S/T), where X[subscript 1,2] ≠ proline) in flexible, solvent-exposed motifs; however, several native glycoproteins are known to harbor consensus sequences within structured regions of the acceptor protein, suggesting that unfolding or partial unfolding is required for efficient N-linked glycosylation in the native environment. To derive insight into these observations, we generated structural homology models of the N-linked glycoproteome of C. jejuni. This evaluation highlights the potential diversity of secondary structural conformations of previously identified N-linked glycosylation sequons. Detailed assessment of PglB activity with a structurally characterized acceptor protein, PEB3, demonstrated that this natively folded substrate protein is not efficiently glycosylated in vitro, whereas structural destabilization increases glycosylation efficiency. Furthermore, in vivo glycosylation studies in both glyco-competent Escherichia coli and the native system, C. jejuni, revealed that efficient glycosylation of glycoproteins, AcrA and PEB3, depends on translocation to the periplasmic space via the general secretory pathway. Our studies provide quantitative evidence that many acceptor proteins are likely to be N-linked-glycosylated before complete folding and suggest that PglB activity is coupled to general secretion-mediated translocation to the periplasm. This work extends our understanding of the molecular mechanisms underlying N-linked glycosylation in bacteria. Keywords: N-linked glycosylation, oligosaccharyltransferase, protein folding, protein translocation, substrate specificity |
| first_indexed | 2024-09-23T13:54:17Z |
| format | Article |
| id | mit-1721.1/117180 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T13:54:17Z |
| publishDate | 2018 |
| publisher | American Society for Biochemistry and Molecular Biology (ASBMB) |
| record_format | dspace |
| spelling | mit-1721.1/1171802022-10-01T17:55:18Z Bacterial N-Glycosylation Efficiency is Dependent on the Structural Context of Target Sequons Silverman, Julie Imperiali, Barbara Massachusetts Institute of Technology. Department of Biology imperiali b Silverman, Julie Imperiali, Barbara Site selectivity of protein N-linked glycosylation is dependent on many factors, including accessibility of the modification site, amino acid composition of the glycosylation consensus sequence, and cellular localization of target proteins. Previous studies have shown that the bacterial oligosaccharyltransferase, PglB, of Campylobacter jejuni favors acceptor proteins with consensus sequences ((D/E)X[subscript 1]NX[subscript 2](S/T), where X[subscript 1,2] ≠ proline) in flexible, solvent-exposed motifs; however, several native glycoproteins are known to harbor consensus sequences within structured regions of the acceptor protein, suggesting that unfolding or partial unfolding is required for efficient N-linked glycosylation in the native environment. To derive insight into these observations, we generated structural homology models of the N-linked glycoproteome of C. jejuni. This evaluation highlights the potential diversity of secondary structural conformations of previously identified N-linked glycosylation sequons. Detailed assessment of PglB activity with a structurally characterized acceptor protein, PEB3, demonstrated that this natively folded substrate protein is not efficiently glycosylated in vitro, whereas structural destabilization increases glycosylation efficiency. Furthermore, in vivo glycosylation studies in both glyco-competent Escherichia coli and the native system, C. jejuni, revealed that efficient glycosylation of glycoproteins, AcrA and PEB3, depends on translocation to the periplasmic space via the general secretory pathway. Our studies provide quantitative evidence that many acceptor proteins are likely to be N-linked-glycosylated before complete folding and suggest that PglB activity is coupled to general secretion-mediated translocation to the periplasm. This work extends our understanding of the molecular mechanisms underlying N-linked glycosylation in bacteria. Keywords: N-linked glycosylation, oligosaccharyltransferase, protein folding, protein translocation, substrate specificity National Institutes of Health (U.S.) (GM-039334) National Institutes of Health (U.S.) (GM-007270) 2018-07-27T20:39:50Z 2018-07-27T20:39:50Z 2016-08 2016-07 Article http://purl.org/eprint/type/JournalArticle 0021-9258 1083-351X http://hdl.handle.net/1721.1/117180 Silverman, Julie Michelle, and Barbara Imperiali. “Bacterial N -Glycosylation Efficiency Is Dependent on the Structural Context of Target Sequons.” Journal of Biological Chemistry, vol. 291, no. 42, Oct. 2016, pp. 22001–10. https://orcid.org/0000-0003-1432-0438 https://orcid.org/0000-0002-5749-7869 en_US http://dx.doi.org/10.1074/jbc.M116.747121 Journal of Biological Chemistry Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf American Society for Biochemistry and Molecular Biology (ASBMB) Prof. Imperiali via Courtney Crummett |
| spellingShingle | Silverman, Julie Imperiali, Barbara Bacterial N-Glycosylation Efficiency is Dependent on the Structural Context of Target Sequons |
| title | Bacterial N-Glycosylation Efficiency is Dependent on the Structural Context of Target Sequons |
| title_full | Bacterial N-Glycosylation Efficiency is Dependent on the Structural Context of Target Sequons |
| title_fullStr | Bacterial N-Glycosylation Efficiency is Dependent on the Structural Context of Target Sequons |
| title_full_unstemmed | Bacterial N-Glycosylation Efficiency is Dependent on the Structural Context of Target Sequons |
| title_short | Bacterial N-Glycosylation Efficiency is Dependent on the Structural Context of Target Sequons |
| title_sort | bacterial n glycosylation efficiency is dependent on the structural context of target sequons |
| url | http://hdl.handle.net/1721.1/117180 https://orcid.org/0000-0003-1432-0438 https://orcid.org/0000-0002-5749-7869 |
| work_keys_str_mv | AT silvermanjulie bacterialnglycosylationefficiencyisdependentonthestructuralcontextoftargetsequons AT imperialibarbara bacterialnglycosylationefficiencyisdependentonthestructuralcontextoftargetsequons |