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Single-molecule imaging of glycan–lectin interactions on cells with Glyco-PAINT

Abstract

Most lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT) super-resolution microscopy method to capture weak glycan–lectin interactions at the single-molecule level in living cells (Glyco-PAINT). Glyco-PAINT exploits weak and reversible sugar binding to directly achieve single-molecule detection and quantification in cells and is used to establish the relative kon and koff rates of a synthesized library of carbohydrate-based probes, as well as the diffusion coefficient of the receptor–sugar complex. Uptake of ligands correlates with their binding affinity and residence time to establish structure–function relations for various synthetic glycans. We reveal how sugar multivalency and presentation geometry can be optimized for binding and internalization. Overall, Glyco-PAINT represents a powerful approach to study weak glycan–lectin interactions on the surface of living cells, one that can be potentially extended to a variety of lectin–sugar interactions.

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Fig. 1: Scheme of the Glyco-PAINT procedure.
Fig. 2: Live cell imaging of the MR with the Glyco-PAINT approach.
Fig. 3: Lateral diffusion of the MR on the cell membrane.
Fig. 4: Dwell times and koff of mannoside probes binding to the MR.
Fig. 5: Cellular uptake of probes binding to the MR.
Fig. 6: Correlation of kinetic parameters with cellular uptake.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

R.R. and L.A. thank the European Research Council/Horizon 2020 for financial support (no. ERC-StG-757397). L.A. thanks NWO for support (VIDI grant no. 192.028). L.A. thanks the Barcelona Institute of Science and Technology for support. This work was funded by the NWO gravitation program 2013 granted to the Institute for Chemical Immunology (no. ICI-024.002.009) (T.P.H. and J.D.C.C.); NWO BBoL grant (W.D.); and the European Research Council (grant no. ERC-CoG-865175, S.I.v.K.).

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R.R., T.P.H., J.D.C.C., S.I.v.K. and L.A. conceived the experiments. R.R. performed microscopy. T.P.H. synthesized the probe library. W.D. synthesized propargyl GalNAc 33 and repeated synthesis of certain probes. Y.N. performed SPR experiments. E.B. provided the CHO-MR cell line. The manuscript was written by R.R. and T.H. in consultation with S.P., J.D.C.C., S.I.v.K. and L.A.

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Correspondence to Sander I. van Kasteren or Lorenzo Albertazzi.

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Peer review information Nature Chemical Biology thanks Khalid Salaita, Ben Schumann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Figs. 1–8 and note on the synthesis and characterization of reagents.

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Riera, R., Hogervorst, T.P., Doelman, W. et al. Single-molecule imaging of glycan–lectin interactions on cells with Glyco-PAINT. Nat Chem Biol 17, 1281–1288 (2021). https://doi.org/10.1038/s41589-021-00896-2

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