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:

Selective N-glycan editing on living cell surfaces to probe glycoconjugate function

Nature Chemical Biology volume 16, pages 766–775 (2020)Cite this article

Subjects

Abstract

Cell surfaces are glycosylated in various ways with high heterogeneity, which usually leads to ambiguous conclusions about glycan-involved biological functions. Here, we describe a two-step chemoenzymatic approach for N-glycan-subtype-selective editing on the surface of living cells that consists of a first ‘delete’ step to remove heterogeneous N-glycoforms of a certain subclass and a second ‘insert’ step to assemble a well-defined N-glycan back onto the pretreated glyco-sites. Such glyco-edited cells, carrying more homogeneous oligosaccharide structures, could enable precise understanding of carbohydrate-mediated functions. In particular, N-glycan-subtype-selective remodeling and imaging with different monosaccharide motifs at the non-reducing end were successfully achieved. Using a combination of the expression system of the Lec4 CHO cell line and this two-step glycan-editing approach, opioid receptor delta 1 (OPRD1) was investigated to correlate its glycostructures with the biological functions of receptor dimerization, agonist-induced signaling and internalization.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: Cell-surface N-glycan editing in a subtype-selective manner by a two-step strategy.
Fig. 2: Selective editing and imaging of core-fucosylated N-glycans on a living cell surface by the two-step strategy using Endo-F3 and its mutant.
Fig. 3: Selective editing and imaging of non-core-fucosylated N-glycans on a living cell surface by the two-step strategy using Endo-M and its mutant.
Fig. 4: Less heterogeneous to homogeneous N-glycan editing on Lec4 CHO cells.
Fig. 5: N-glycan editing of the OPRD1 receptor on Lec4 CHO cell membrane deciphers the role of glycosylation.
Fig. 6: OPRD1 internalization assay by time-lapse monitoring on confocal imaging with an anti-HA antibody.

Similar content being viewed by others

Data availability

All of the data are available in the paper and its Supplementary Information. Synthetic procedures and the characterization data of the new N-glycan substrates are also provided. Supporting data and processing protocols of cell imaging, FRET, FACS and western blot gels are available in the Supplementary files. The sequence data for the OPRD1 plasmid have been deposited to GenBank (accession code MN922300).

References

  1. Varki, A. & Kornfeld, S. in Essentials of Glycobiology (eds. Cummings, R. D. et al.) 1–18 (Cold Spring Harbor, 2015).

  2. Moremen, K. W., Tiemeyer, M. & Nairn, A. V. Vertebrate protein glycosylation: diversity, synthesis and function. Nat. Rev. Mol. Cell Biol. 13, 448–462 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pinho, S. S. & Reis, C. A. Glycosylation in cancer: mechanisms and clinical implications. Nat. Rev. Cancer 15, 540–555 (2015).

    Article  CAS  PubMed  Google Scholar 

  4. Stanley, P. Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity. Mol. Cell Biol. 9, 377–383 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Weinstein, J. et al. A point mutation causes mistargeting of Golgi GlcNAc-TV in the Lec4A Chinese hamster ovary glycosylation mutant. J. Biol. Chem. 271, 27462–27469 (1996).

    Article  CAS  PubMed  Google Scholar 

  6. Chen, W. & Stanley, P. Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N-acetylglucosaminyltransferase I. Glycobiology 13, 43–50 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Yang, Z. et al. Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nat. Biotechnol. 33, 842–844 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Yamane-Ohnuki, N. et al. Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol. Bioeng. 87, 614–622 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Li, H. et al. Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat. Biotechnol. 24, 210–215 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. North, S. J. et al. Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity. J. Biol. Chem. 285, 5759–5775 (2010).

    Article  CAS  PubMed  Google Scholar 

  11. Mahal, L. K., Yarema, K. J. & Bertozzi, C. R. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276, 1125–1128 (1997).

    Article  CAS  PubMed  Google Scholar 

  12. Prescher, J. A., Dube, D. H. & Bertozzi, C. R. Chemical remodelling of cell surfaces in living animals. Nature 430, 873–877 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Vocadlo, D. J., Hang, H. C., Kim, E.-J., Hanover, J. A. & Bertozzi, C. R. A chemical approach for identifying O-GlcNAc-modified proteins in cells. Proc. Natl Acad. Sci. USA 100, 9116–9121 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hang, H. C., Yu, C., Kato, D. L. & Bertozzi, C. R. A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation. Proc. Natl Acad. Sci. USA 100, 14846–14851 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kayser, H. et al. Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-d-hexosamines as precursors. J. Biol. Chem. 267, 16934–16938 (1992).

    CAS  PubMed  Google Scholar 

  16. Zeng, Y., Ramya, T. N. C., Dirksen, A., Dawson, P. E. & Paulson, J. C. High-efficiency labeling of sialylated glycoproteins on living cells. Nat. Methods 6, 207–209 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hui, J. et al. Localized chemical remodeling for live cell imaging of protein-specific glycoform. Angew. Chem. Int. Ed. 56, 8139–8143 (2017).

    Article  CAS  Google Scholar 

  18. Mbua, N. E. et al. Selective exo-enzymatic labeling of N-glycans on the surface of living cells by recombinant ST6Gal I. Angew. Chem. Int. Ed. 52, 13012–13015 (2013).

    Article  CAS  Google Scholar 

  19. Sun, T. et al. One-step selective exoenzymatic labeling (SEEL) strategy for the biotinylation and identification of glycoproteins of living cells. J. Am. Chem. Soc. 138, 11575–11582 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wen, L. et al. Two-step chemoenzymatic detection of N-acetylneuraminic acid-α(2-3)-galactose glycans. J. Am. Chem. Soc. 138, 11473–11476 (2016).

    Article  CAS  PubMed  Google Scholar 

  21. Wang, W. et al. Chemoenzymatic synthesis of GDP-l-fucose and the Lewis X glycan derivatives. Proc. Natl Acad. Sci. USA 106, 16096–16101 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Briard, J. G., Jiang, H., Moremen, K. W., Macauley, M. S. & Wu, P. Cell-based glycan arrays for probing glycan–glycan binding protein interactions. Nat. Commun. 9, 880 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Rabuka, D., Hubbard, S. C., Laughlin, S. T., Argade, S. P. & Bertozzi, C. R. A chemical reporter strategy to probe glycoprotein fucosylation. J. Am. Chem. Soc. 128, 12078–12079 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zheng, T. et al. Tracking N-acetyllactosamine on cell surface glycans in vivo. Angew. Chem. Int. Ed. 50, 4113–4118 (2011).

    Article  CAS  Google Scholar 

  25. Li, Q., Li, Z., Duan, X. & Yi, W. A tandem enzymatic approach for detecting and imaging tumor-associated Thomsen–Friedenreich antigen disaccharide. J. Am. Chem. Soc. 136, 12536–12539 (2014).

    Article  CAS  PubMed  Google Scholar 

  26. Wang, L. X. & Huang, W. Enzymatic transglycosylation for glycoconjugate synthesis. Curr. Opin. Chem. Biol. 13, 592–600 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Fairbanks, A. J. The ENGases: versatile biocatalysts for the production of homogeneous N-linked glycopeptides and glycoproteins. Chem. Soc. Rev. 46, 5128–5146 (2017).

    Article  CAS  PubMed  Google Scholar 

  28. Huang, W., Yang, Q., Umekawa, M., Yamamoto, K. & Wang, L. X. Arthrobacter endo-β-N-acetylglucosaminidase shows transglycosylation activity on complex-type N-glycan oxazolines: one-pot conversion of ribonuclease B to sialylated ribonuclease C. Chembiochem 11, 1350–1355 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 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 

  30. Giddens, J. P., Lomino, J. V., Amin, M. N. & Wang, L. X. Endo-F3 glycosynthase mutants enable chemoenzymatic synthesis of core-fucosylated triantennary complex type glycopeptides and glycoproteins. J. Biol. Chem. 291, 9356–9370 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Tang, F., Wang, L. X. & Huang, W. Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody-drug conjugates. Nat. Protoc. 12, 1702–1721 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Tang, F. et al. One-pot N-glycosylation remodeling of IgG with non-natural sialylglycopeptides enables glycosite-specific and dual-payload antibody-drug conjugates. Org. Biomol. Chem. 14, 9501–9518 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Tang, Y. et al. Real-time analysis on drug–antibody ratio of antibody–drug conjugates for synthesis, process optimization and quality control. Sci. Rep. 7, 7763 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Yang, Q. et al. Glycan remodeling of human erythropoietin (EPO) through combined mammalian cell engineering and chemoenzymatic transglycosylation. ACS Chem. Biol. 12, 1665–1673 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Takegawa, K., Nakoshi, M., Iwahara, S., Yamamoto, K. & Tochikura, T. Induction and purification of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae grown in ovalbumin. Appl. Environ. Microbiol. 55, 3107–3112 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yamamoto, K., Kadowaki, S., Fujisaki, M., Kumagai, H. & Tochikura, T. Novel specificities of Mucor hiemalis endo-β-N-acetylglucosaminidase acting complex asparagine-linked oligosaccharides. Biosci. Biotech. Biochem. 58, 72–77 (1994).

    Article  CAS  Google Scholar 

  37. Tarentino, A. L. & Plummer, T. H. Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum.Methods Enzymol. 230, 44–57 (1994).

    Article  CAS  PubMed  Google Scholar 

  38. Muramatsu, H. et al. Molecular cloning and expression of endo-β-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides. J. Biochem. 129, 923–928 (2001).

    Article  CAS  PubMed  Google Scholar 

  39. Plummer, J. T. H., Phelan, A. W. & Tarentino, A. L. Porcine fibrinogen glycopeptides: substrates for detecting endo-β-N-acetylglucosaminidases F2 and F3. Anal. Biochem. 235, 98–101 (1996).

    Article  CAS  PubMed  Google Scholar 

  40. Lin, W., Du, Y., Zhu, Y. & Chen, X. A cis-membrane FRET-based method for protein-specific imaging of cell-surface glycans. J. Am. Chem. Soc. 136, 679–687 (2014).

    Article  CAS  PubMed  Google Scholar 

  41. Huang, W. et al. Glycosynthases enable a highly efficient chemoenzymatic synthesis of N-glycoproteins carrying intact natural N-glycans. J. Am. Chem. Soc. 131, 2214–2223 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Eshima, Y., Higuchi, Y., Kinoshita, T., Nakakita, S. & Takegawa, K. Transglycosylation activity of glycosynthase mutants of endo-β-N-acetylglucosaminidase from Coprinopsis cinerea. PLoS ONE 10, e0132859 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Murakami, S. et al. Identification and characterization of endo-β-N-acetylglucosaminidase from methylotrophic yeast Ogataea minuta. Glycobiology 23, 736–744 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Whittaker, J. W. Free radical catalysis by galactose oxidase. Chem. Rev. 103, 2347–2364 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Waddling, C. A., Plummer, T. H., Tarentino, A. L. & Van Roey, P. Structural basis for the substrate specificity of endo-β-N-acetylglucosaminidase F3. Biochemistry 39, 7878–7885 (2000).

    Article  CAS  PubMed  Google Scholar 

  46. Hamouda, H. et al. Rapid analysis of cell surface N-glycosylation from living cells using mass spectrometry. J. Proteome Res. 13, 6144–6151 (2014).

    Article  CAS  PubMed  Google Scholar 

  47. Burford, N. T. et al. Identification of selective agonists and positive allosteric modulators for micro- and delta-opioid receptors from a single high-throughput screen. J. Biomol. Screen. 19, 1255–1265 (2014).

    Article  CAS  PubMed  Google Scholar 

  48. Xie, R., Hong, S., Feng, L., Rong, J. & Chen, X. Cell-selective metabolic glycan labeling based on ligand-targeted liposomes. J. Am. Chem. Soc. 134, 9914–9917 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Xiao, H., Woods, E. C., Vukojicic, P. & Bertozzi, C. R. Precision glycocalyx editing as a strategy for cancer immunotherapy. Proc. Natl Acad. Sci. USA 113, 10304–10309 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Tong, X., Li, T., Li, C. & Wang, L. X. Generation and comparative kinetic analysis of new glycosynthase mutants from Streptococcus pyogenes endoglycosidases for antibody glycoengineering. Biochemistry 57, 5239–5246 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Umekawa, M. et al. Mutants of Mucor hiemalis endo-β-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities. J. Biol. Chem. 283, 4469–4479 (2008).

    Article  CAS  PubMed  Google Scholar 

  52. Fujita, K. et al. Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-β-N-acetylglucosaminidase A. Biochem. Biophys. Res. Commun. 267, 134–138 (2000).

    Article  CAS  PubMed  Google Scholar 

  53. Sun, B. et al. A simplified procedure for gram-scale production of sialylglycopeptide (SGP) from egg yolks and subsequent semi-synthesis of Man3GlcNAc oxazoline. Carbohydr. Res. 396, 62–69 (2014).

    Article  CAS  PubMed  Google Scholar 

  54. Dell, A. et al. Mass spectrometry of carbohydrate-containing biopolymers. Methods Enzymol. 230, 108–132 (1994).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 21877116, to W.H.), the National Science & Technology Major Project ‘Key New Drug Creation and Manufacturing Program’ of China (no. 2018ZX09711002-006, to W.H.) and the China Postdoctoral Science Foundation (no. BX20180339, to F.T., and no. 2018M642120, to F.T.). We thank R. Huang’s colleagues for their kind help with imaging studies. We also thank P. Wu of the Scripps Research Institute for providing us with Lec4 CHO cells.

Author information

Author notes

  1. These authors contributed equally: Feng Tang, Mang Zhou.

Authors and Affiliations

  1. CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Center for Biotherapeutics Discovery Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Pudong, Shanghai, China

    Feng Tang, Mang Zhou, Ken Qin, Wei Shi, Ansor Yashinov, Yang Yang, Liyun Yang, Dongliang Guan, Lei Zhao, Yubo Tang, Yujie Chang, Lifen Zhao, Hu Zhou, Ruimin Huang & Wei Huang

  2. University of Chinese Academy of Sciences, Beijing, China

    Feng Tang, Ken Qin, Wei Shi, Ansor Yashinov, Hu Zhou, Ruimin Huang & Wei Huang

  3. School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Feng Tang & Wei Huang

  4. Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China

    Huaiyu Yang

Authors
  1. Feng Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mang Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ken Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wei Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ansor Yashinov
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yang Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Liyun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dongliang Guan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Lei Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yubo Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yujie Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Lifen Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Huaiyu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Hu Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Ruimin Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Wei Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.T. prepared the N-glycan substrates, performed cell-surface N-glycan editing by Endo-F3, conducted the N-glycan profiling by MALDI-TOF and carried out the OPRD1 internalization assay. M.Z. contributed to initial condition optimization and the proof of concept, performed N-glycan editing by Endo-M, constructed the OPRD1-expressing Lec4 CHO cell line and finished the cAMP assay. K.Q. provided the Endo-F3 and its mutant enzymes and performed Lec4 cell N-glycan editing. W.S. prepared the azido-complex-type glycan substrate (Az-CT-Ox). A.Y. and F.T. performed the FRET experiments. Y.Y. provided the Endo-A, Endo-M and its mutant enzymes, and PNGase F. L.Y. helped with western blots and FACS data acquisition and discussion. D.G., L.Z. and Y.T. helped with synthesis of chemical compounds and intermediates. L.Z. and H.Y. helped with OPRD1 expression. Y.C. and H.Z. assisted with MALDI-TOF determination. R.H. helped with imaging experiments. W.H. conceived the original idea and supervised the research. F.T., M.Z. and W.H. wrote the manuscript.

Corresponding authors

Correspondence to Mang Zhou or Wei Huang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–40 and Notes 1 and 2.

Reporting Summary

Supplementary Data

OPRD1 GenBank file.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tang, F., Zhou, M., Qin, K. et al. Selective N-glycan editing on living cell surfaces to probe glycoconjugate function. Nat Chem Biol 16, 766–775 (2020). https://doi.org/10.1038/s41589-020-0551-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-020-0551-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing