Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria

Nature Chemical Biology volume 16, pages 1062–1070 (2020)Cite this article

Subjects

Abstract

A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top down or construct non-natural ones from the bottom up for new and useful purposes. Here, we have developed a set of orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human O-glycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O-glycoproteins in a one-pot reaction. In addition, O-glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O-glycoforms for a range of important scientific and therapeutic applications.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Natural and synthetic mucin-type O-glycosylation pathways.
Fig. 2: Biosynthesis of O-glycoproteins bearing Tn and T antigens.
Fig. 3: Orthogonal biosynthesis of sialylated O-glycans.
Fig. 4: Cell-free O-glycosylation using glyco-enriched extracts.
Fig. 5: O-linked glycosylation of diverse protein targets.

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this Article (and its Supplementary Information) or are available from the corresponding authors on reasonable request. All unique materials used in this work are available from the authors. Source Data are provided with this paper.

References

  1. Khoury, G. A., Baliban, R. C. & Floudas, C. A. Proteome-wide post-translational modification statistics: frequency analysis and curation of the Swiss-Prot database. Sci. Rep. 1, 90 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Walsh, C. T., Garneau-Tsodikova, S. & Gatto, G. J. Jr Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem. Int. Ed. 44, 7342–7372 (2005).

    Article  CAS  Google Scholar 

  3. Abu-Qarn, M., Eichler, J. & Sharon, N. Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea. Curr. Opin. Struct. Biol. 18, 544–550 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Varki, A. Biological roles of glycans. Glycobiology 27, 3–49 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Sethuraman, N. & Stadheim, T. A. Challenges in therapeutic glycoprotein production. Curr. Opin. Biotechnol. 17, 341–346 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Rappuoli, R. Glycoconjugate vaccines: principles and mechanisms. Sci. Transl. Med. 10, eaat4615 (2018).

    Article  PubMed  Google Scholar 

  7. Valderrama-Rincon, J. D. et al. An engineered eukaryotic protein glycosylation pathway in Escherichia coli. Nat. Chem. Biol. 8, 434–436 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Meuris, L. et al. GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. Nat. Biotechnol. 32, 485–489 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hamilton, S. R. et al. Production of complex human glycoproteins in yeast. Science 301, 1244–1246 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Jaroentomeechai, T. et al. Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nat. Commun. 9, 2686 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Kightlinger, W. et al. A cell-free biosynthesis platform for modular construction of protein glycosylation pathways. Nat. Commun. 10, 5404 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Feldman, M. F. et al. Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli. Proc. Natl Acad. Sci. USA 102, 3016–3021 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tytgat, H. L. P. et al. Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies. Nat. Commun. 10, 5403 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Aumiller, J. J., Hollister, J. R. & Jarvis, D. L. A transgenic insect cell line engineered to produce CMP-sialic acid and sialylated glycoproteins. Glycobiology 13, 497–507 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Chang, M. M. et al. Small-molecule control of antibody N-glycosylation in engineered mammalian cells. Nat. Chem. Biol. 15, 730–736 (2019).

    Article  CAS  PubMed  Google Scholar 

  16. Yang, Z. et al. Engineering mammalian mucin-type O-glycosylation in plants. J. Biol. Chem. 287, 11911–11923 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Elliott, S. et al. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat. Biotechnol. 21, 414–421 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Huang, W., Giddens, J., Fan, S. Q., Toonstra, C. & Wang, L. X. Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions. J. Am. Chem. Soc. 134, 12308–12318 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Broecker, F. et al. Multivalent display of minimal Clostridium difficile glycan epitopes mimics antigenic properties of larger glycans. Nat. Commun. 7, 11224 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H. & Bailey, J. E. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat. Biotechnol. 17, 176–180 (1999).

    Article  CAS  PubMed  Google Scholar 

  21. Ilyushin, D. G. et al. Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo. Proc. Natl Acad. Sci. USA 110, 1243–1248 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schwarz, F. & Aebi, M. Mechanisms and principles of N-linked protein glycosylation. Curr. Opin. Struct. Biol. 21, 576–582 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Choi, B. K. et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc. Natl Acad. Sci. USA 100, 5022–5027 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Natarajan, A., Jaroentomeechai, T., Li, M., Glasscock, C. J. & DeLisa, M. P. Metabolic engineering of glycoprotein biosynthesis in bacteria. Emerg. Top. Life Sci. 2, 419–432 (2018).

    Article  CAS  PubMed  Google Scholar 

  25. Ollis, A. A., Zhang, S., Fisher, A. C. & DeLisa, M. P. Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity. Nat. Chem. Biol. 10, 816–822 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Henderson, G. E., Isett, K. D. & Gerngross, T. U. Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli. Bioconjug. Chem. 22, 903–912 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. Mueller, P. et al. High level in vivo mucin-type glycosylation in Escherichia coli. Microb. Cell Fact. 17, 168 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Du, T. et al. A bacterial expression platform for production of therapeutic proteins containing human-like O-linked glycans. Cell Chem. Biol. 26, 203–212 (2019).

    Article  CAS  PubMed  Google Scholar 

  29. Faridmoayer, A. et al. Extreme substrate promiscuity of the Neisseria oligosaccharyl transferase involved in protein O-glycosylation. J. Biol. Chem. 283, 34596–34604 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Pan, C. et al. Biosynthesis of conjugate vaccines using an O-linked glycosylation system. mBio 7, e00443–16 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Valentine, J. L. et al. Immunization with outer membrane vesicles displaying designer glycotopes yields class-switched, glycan-specific antibodies. Cell Chem. Biol. 23, 655–665 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Harding, C. M., Haurat, M. F., Vinogradov, E. & Feldman, M. F. Distinct amino acid residues confer one of three UDP-sugar substrate specificities in Acinetobacter baumannii PglC phosphoglycosyltransferases. Glycobiology 28, 522–533 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. Fisher, A. C. et al. Production of secretory and extracellular N-linked glycoproteins in Escherichia coli. Appl. Environ. Microbiol. 77, 871–881 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Tarp, M. A. & Clausen, H. Mucin-type O-glycosylation and its potential use in drug and vaccine development. Biochim. Biophys. Acta 1780, 546–563 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Yang, G. et al. Fluorescence activated cell sorting as a general ultra-high-throughput screening method for directed evolution of glycosyltransferases. J. Am. Chem. Soc. 132, 10570–10577 (2010).

    Article  CAS  PubMed  Google Scholar 

  36. Glasscock, C. J. et al. A flow cytometric approach to engineering Escherichia coli for improved eukaryotic protein glycosylation. Metab. Eng. 47, 488–495 (2018).

    Article  CAS  PubMed  Google Scholar 

  37. Yates, L. E. et al. Glyco-recoded Escherichia coli: recombineering-based genome editing of native polysaccharide biosynthesis gene clusters. Metab. Eng. 53, 59–68 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Hershewe, J. M. et al. Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles. Preprint at bioRxiv https://www.biorxiv.org/content/10.1101/2020.07.19.211201v2 (2020).

  39. Lai, P. H., Everett, R., Wang, F. F., Arakawa, T. & Goldwasser, E. Structural characterization of human erythropoietin. J. Biol. Chem. 261, 3116–3121 (1986).

    Article  CAS  PubMed  Google Scholar 

  40. Maier, A. G. et al. Plasmodium falciparum erythrocyte invasion through glycophorin C and selection for Gerbich negativity in human populations. Nat. Med. 9, 87–92 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Gendler, S., Taylor-Papadimitriou, J., Duhig, T., Rothbard, J. & Burchell, J. A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J. Biol. Chem. 263, 12820–12823 (1988).

    Article  CAS  PubMed  Google Scholar 

  42. Mazor, Y., Keydar, I. & Benhar, I. Humanization and epitope mapping of the H23 anti-MUC1 monoclonal antibody reveals a dual epitope specificity. Mol. Immunol. 42, 55–69 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Sorensen, A. L. et al. Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance. Glycobiology 16, 96–107 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Ju, T. & Cummings, R. D. A unique molecular chaperone Cosmc required for activity of the mammalian core 1 β3-galactosyltransferase. Proc. Natl Acad. Sci. USA 99, 16613–16618 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Skretas, G. et al. Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli. Micro. Cell Fact. 8, 50 (2009).

    Article  Google Scholar 

  46. Schulz, B. L. et al. Identification of bacterial protein O-oligosaccharyltransferases and their glycoprotein substrates. PLoS ONE 8, e62768 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. von Mensdorff-Pouilly, S. et al. Reactivity of natural and induced human antibodies to MUC1 mucin with MUC1 peptides and N-acetylgalactosamine (GalNAc) peptides. Int. J. Cancer 86, 702–712 (2000).

    Article  Google Scholar 

  48. Apostolopoulos, V. et al. A glycopeptide in complex with MHC class I uses the GalNAc residue as an anchor. Proc. Natl Acad. Sci. USA 100, 15029–15034 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ninkovic, T. & Hanisch, F. G. O-glycosylated human MUC1 repeats are processed in vitro by immunoproteasomes. J. Immunol. 179, 2380–2388 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Lakshminarayanan, V. et al. Immune recognition of tumor-associated mucin MUC1 is achieved by a fully synthetic aberrantly glycosylated MUC1 tripartite vaccine. Proc. Natl Acad. Sci. USA 109, 261–266 (2012).

    Article  CAS  PubMed  Google Scholar 

  51. Coyne, M. J. et al. Phylum-wide general protein O-glycosylation system of the Bacteroidetes. Mol. Microbiol. 88, 772–783 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Natarajan, A., Haitjema, C. H., Lee, R., Boock, J. T. & DeLisa, M. P. An engineered survival-selection assay for extracellular protein expression uncovers hypersecretory phenotypes in Escherichia coli. ACS Synth. Biol. 6, 875–883 (2017).

    Article  CAS  PubMed  Google Scholar 

  55. Shanks, R. M., Caiazza, N. C., Hinsa, S. M., Toutain, C. M. & O’Toole, G. A. Saccharomyces cerevisiae-based molecular tool kit for manipulation of genes from gram-negative bacteria. Appl. Environ. Microbiol. 72, 5027–5036 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Dykxhoorn, D. M., St Pierre, R. & Linn, T. A set of compatible tac promoter expression vectors. Gene 177, 133–136 (1996).

    Article  CAS  PubMed  Google Scholar 

  57. Apostolopoulos, V., Karanikas, V., Haurum, J. S. & McKenzie, I. F. Induction of HLA-A2-restricted CTLs to the mucin 1 human breast cancer antigen. J. Immunol. 159, 5211–5218 (1997).

    Article  CAS  PubMed  Google Scholar 

  58. Fierfort, N. & Samain, E. Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides. J. Biotechnol. 134, 261–265 (2008).

    Article  CAS  PubMed  Google Scholar 

  59. Cox, E. C. et al. Antibody-mediated endocytosis of polysialic acid enables intracellular delivery and cytotoxicity of a glycan-directed antibody–drug conjugate. Cancer Res. 79, 1810–1821 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Dodev, T. S. et al. A tool kit for rapid cloning and expression of recombinant antibodies. Sci. Rep. 4, 5885 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Jewett, M. C. & Swartz, J. R. Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnol. Bioeng. 86, 19–26 (2004).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank R. Lee and S. Murphy for their contributions working with GT enzymes, L. Yates for helpful discussions with glyco-recoding, D. Mills for helpful discussions regarding O-OSTs, M. Paszek, J. Hershewe, K. Warfel, J. Stark, and M. Jewett for helpful discussions and provision of reagents, M. Li for technical advice and J. Wilson, J. Brooks and J. Merritt for help with vector design and yeast-based recombineering. We are also grateful to R. Bhawal and S. Zhang of the Proteomics and Metabolomics Core Facility in the Cornell Institute of Biotechnology for assistance with LC-MS. This work was supported by the Defense Threat Reduction Agency (GRANT11631647 to M.P.D.), National Science Foundation (grant no. CBET-1605242 to M.P.D.) and National Institutes of Health (grant no. 1R01GM127578-01 to M.P.D.). Glycomics analysis was supported in part by the National Institutes of Health (grant no. 1S10OD018530 to P.A.). The work was also supported by seed project funding (to M.P.D.) through the National Institutes of Health-funded Cornell Center on the Physics of Cancer Metabolism (supporting grant no. 1U54CA210184-01). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. T.J. was supported by a Royal Thai Government Fellowship and also a Cornell Fleming Graduate Scholarship. E.C.C. was supported by a National Institutes of Health Chemical-Biology Interface (CBI) training fellowship (supporting grant no. T32GM008500).

Author information

Authors and Affiliations

  1. Department of Microbiology, Cornell University, Ithaca, NY, USA

    Aravind Natarajan, Marielisa Cabrera-Sánchez & Matthew P. DeLisa

  2. Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA

    Thapakorn Jaroentomeechai, Jody C. Mohammed, Olivia Young, Michael Vilkhovoy, Sandra Vadhin, Jeffrey D. Varner & Matthew P. DeLisa

  3. Biomedical and Biological Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY, USA

    Emily C. Cox & Matthew P. DeLisa

  4. Complex Carbohydrate Research Center, The University of Georgia, Athens, GA, USA

    Asif Shajahan & Parastoo Azadi

Authors
  1. Aravind Natarajan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thapakorn Jaroentomeechai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marielisa Cabrera-Sánchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jody C. Mohammed
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Emily C. Cox
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Olivia Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Asif Shajahan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael Vilkhovoy
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sandra Vadhin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jeffrey D. Varner
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Parastoo Azadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Matthew P. DeLisa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.N. designed and performed all research, analyzed all data and wrote the paper. T.J. designed and performed research related to cell-free glycosylation and analyzed data. M.C.-S. and O.Y. performed research related to constructing and testing glycan biosynthetic pathways. J.C.M. performed research related to testing different proteins for glycosylation. E.C.C. performed research related to antibody-based detection of different MUC1 glycoforms. A.S., M.V., S.V., J.D.V. and P.A. performed mass spectrometry analysis and aided in data interpretation. M.P.D. directed research, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Matthew P. DeLisa.

Ethics declarations

Competing interests

M.P.D. has a financial interest in Glycobia, Inc. and Versatope, Inc. M.P.D.’s interests are reviewed and managed by Cornell University in accordance with their conflict of interest policies. All authors declare no other competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 MS/MS fragmentation analysis of Tn-modified glycoprotein.

EThcD fragmentation analysis of glycosylated peptide 397NVGGDLDWPAAAS(HexNAc)APQPGKPPR418 derived from MBPMOOR by trypsin digestion. The spectrum identifies the neutral loss pattern of the single HexNAc monosaccharide, corresponding oxonium ions, and fragments of the glycopeptide (c and z ions), validating the glycosylation and the site of glycosylation at S409 within the 8-residue WPAAASAP core sequence of MBPMOOR.

Extended Data Fig. 2 Flow cytometric screening of Gal transferases for biosynthesis of T antigen.

(a) Schematic of flow cytometric screen to evaluate candidate Gal transferases (GalTs) for their ability to generate lipid-linked T antigen. Once formed, the T antigen is subsequently flipped to periplasm by the native E. coli flippase, Wzx, transferred to lipid A core by the promiscuous O-antigen ligase WaaL native to E. coli, and ultimately displayed on the cell surface. Cells are labeled with FITC-conjugated PNA that specifically binds the T antigen. (b) Flow cytometric analysis of PNA-labeled E. coli MC4100 ΔwecA (MCΔw) (yellow) or MC4100 ΔwecA ΔwaaL (MCΔww) (gray) carrying no plasmid, plasmid pOG-Tn, or plasmid pOG-Tn modified with one of the candidate GalT enzymes as indicated. (c) Flow cytometric analysis of PNA-labeled MCΔw (yellow) or MCΔww (gray) carrying no plasmid, plasmid pOG-T (producing T antigen glycan with EcWbwC), or plasmid pOG-TΔgne (encoding T antigen pathway but lacking CjGne epimerase). In (b) and (c), unlabeled MCΔw cells (white) were included as negative controls. Inset histograms show representative flow cytometric data used to generate mean fluorescence intensity data. See Supplementary Fig. 1 for flow cytometry gating strategy.

Extended Data Fig. 3 MS/MS fragmentation analysis of T-modified glycoprotein.

EThcD fragmentation analysis of glycosylated peptide 397NVGGDLDWPAAAS(HexHexNAc)APQPGKPPR418 derived from MBPMOOR by trypsin digestion. The spectrum identifies the neutral loss pattern of the HexHexNAc disaccharide, corresponding oxonium ions, and fragments of the glycopeptide (c and z ions), validating the glycosylation and the site of glycosylation at S409 within the 8-residue WPAAASAP core sequence of MBPMOOR.

Extended Data Fig. 4 Orthogonal biosynthesis of sialylated O-glycoforms in E. coli.

(a) Nano-LC-MS/MS analysis of purified acceptor protein generated by nanA-deficient E. coli cells carrying plasmid pConNeuDBAC for CMP-NeuNAc biosynthesis along with pOG-T-NgPglO and pEXT-spDsbA-MBPMOOR-EcWbwA. Sequence coverage of 88% was obtained for the MBPMOOR protein in the analysis. Spectrum reveals a predominant species (80% abundance) corresponding to the indicated peptide fragment bearing a single HexHexNAc modification as well as three less abundant species bearing a single NeuNAcHexHexNAc, a single HexNAc, and no modification (16%, 2%, and 2%, respectively). (b) Same as in (a) but with purified acceptor protein generated by nanA-deficient glyco-recoded cells carrying pOG-Tn-NgPglO and pEXT-spDsbA-MBPMOOR-PspST6. Sequence coverage of 92% was obtained for MBPMOOR in the analysis. Spectrum reveals a predominant species (90% abundance) corresponding to the indicated peptide fragment bearing a single HexNAc modification as well as two less abundant species bearing a single NeuNAcHexNAc and no modification (2% and 9%, respectively). Arrow denotes modified serine (bold underlined font) as determined by EThcD fragmentation analysis.

Extended Data Fig. 5 MS/MS fragmentation analysis of ST- and STn-modified glycoproteins.

EThcD fragmentation analysis of glycosylated peptide 397NVGGDLDWPAAAS(NeuNAcHexHexNAc)APQPGKPPR418 derived from (a) ST-modified MBPMOOR and (b) STn-modified MBPMOOR that were subjected to trypsin digestion. The spectrum identifies the neutral loss pattern of the single NeuNAc and Hex monosaccharides, corresponding oxonium ions, and fragments of the glycopeptide (c and z ions), validating the glycosylation and site of glycosylation at S409 within the 8-residue WPAAASAP core sequence of MBPMOOR.

Extended Data Fig. 6 Yield determination for MBPMOOR modified with different O-glycans.

(a) Coomassie-stained SDS-PAGE gel showing MBPMOOR proteins purified from different strains. MBPMOOR bearing Tn or T antigens was produced in CLM25 cells co-transformed with pEXT-based plasmid for acceptor protein and appropriate sialyltransferase expression and either pOG-Tn-NgPglO or pOG-T-NgPglO plasmids, respectively. MBPMOOR bearing STn or ST antigens was produced in glyco-recoded cells carrying the CMP-NeuNAc biosynthesis pathway in the genome and co-transformed with pEXT-based plasmid for acceptor protein expression and either pOG-Tn-NgPglO or pOG-T-NgPglO plasmids, respectively. CLM25 cells co-transformed with only the pEXT-based plasmid for expressing MBPMOOR (agly) and appropriate sialyltransferase served as the control. Molecular weight (Mw) marker included on the left. SDS-PAGE gel is representative of three biological replicates. See Source Data for uncropped version of the image. (b) Yield of each glycoprotein calculated by multiplying the total yield times the percentage glycosylated (% gly), the latter of which was determined from nano-LC-MS/MS analysis of each glycoprotein product. Yield values are the average of three biological replicates and the error is the standard deviation of the mean.

Source data

Extended Data Fig. 7 O-linked glycosylation of diverse protein targets.

(a) Immunoblot analysis of acceptor proteins purified from CLM25 cells co-transformed with pOG-T-NgPglO (+, top), pOG-T-NmPglL (+, bottom), or pOG-T without an O-OST (-) along with pEXT-based plasmid encoding each of the different protein targets as indicated. MBPMOOR and MBPMOORmut derived from the same cells served as positive and negative control, respectively. Blots were probed with anti-hexa-histidine antibody (6xHis) to detect acceptor proteins and PNA lectin to detect the T antigen. Molecular weight (Mw) markers are indicated on the left of each blot. All immunoblot results are representative of at least three biological replicates. (b, c) Same as in (a) with pOG-T-NmPglL (+) or pOG-T without NmPglL (-) along with pEXT-based plasmid encoding each of the different protein targets as indicated. See Source Data for uncropped versions of the images.

Source data

Extended Data Fig. 8 Secretion of O-glycoproteins in the culture supernatant.

Immunoblot analysis of culture supernatants derived from CLM24 ΔyaiW cells co-transformed with pOG-T-NgPglO or pOG-T-NmPglL along with pEXT-based plasmid encoding YebF-MBPMOOR or YebF-MBPMOORmut as indicated. Mutation of acceptor serine to glycine in YebF-MBPMOORmut served as negative control. Blots were probed with anti-hexa-histidine antibody (6xHis) to detect acceptor proteins and PNA lectin to detect the T antigen. Molecular weight (Mw) markers are indicated on the left of each blot. Immunoblot results are representative of at least three biological replicates. See Source Data for uncropped versions of the images.

Source data

Extended Data Fig. 9 Orthogonal biosynthesis of different MUC1 O-glycoforms in E. coli.

Nano-LC-MS/MS analysis of purified acceptor protein generated by CLM25 cells carrying plasmid pOG-T-NgPglO along with pEXT-based plasmid for expression of different MUC1 constructs including: (a) MUC1_8; (b) MUC1_20; (c) MUC1_24; and (d) MUC1_41. Sequence coverage of 77% was obtained for MUC1_8, 78% for MUC1_20, 88% for MUC1_24, and 75% for MUC1_41 in the analysis. All spectra reveal a predominant species corresponding to the indicated peptide fragments bearing a single HexHexNAc modification. Additional less abundant species bearing a single HexNAc and no modification were observed in all cases. For MUC1_41, several doubly glycosylated species were also identified as minor species. Arrow denotes modified serine (bold underlined font) as determined by EThcD fragmentation analysis.

Extended Data Fig. 10 MS/MS fragmentation analysis of MUC1 O-glycoforms bearing the T antigen.

EThcD fragmentation analysis of glycosylated peptides derived by trypsin digestion. The spectrum identifies the neutral loss pattern of HexHexNAc disaccharide, corresponding oxonium ions, and fragments of the glycopeptide (c and z ions), validating the glycosylation and the sites of glycosylation (S409 in MUC1_8; S415 in MUC1_20; S417 in MUC1_24 and S417 of MUC1_41) within relevant MUC1 peptides as indicated in the inset sequences.

Supplementary information

Supplementary Information

Supplementary Table 1 and Fig. 1.

Reporting Summary

Source data

Source Data Fig. 2

Unprocessed immunoblots.

Source Data Fig. 4

Unprocessed immunoblots.

Source Data Fig. 5

Unprocessed immunoblots.

Source Data Extended Data Fig. 6

Unprocessed immunoblots.

Source Data Extended Data Fig. 7

Unprocessed immunoblots.

Source Data Extended Data Fig. 8

Unprocessed immunoblots.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Natarajan, A., Jaroentomeechai, T., Cabrera-Sánchez, M. et al. Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria. Nat Chem Biol 16, 1062–1070 (2020). https://doi.org/10.1038/s41589-020-0595-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-020-0595-9

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research