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

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.

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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.

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).

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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.

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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.

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Correspondence to Mang Zhou or Wei Huang.

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Supplementary information

Supplementary Information

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

Reporting Summary

Supplementary Data

OPRD1 GenBank file.

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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

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