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

Nature Chemical Biology volume 17, pages 1281–1288 (2021)Cite this article

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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.

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Data availability

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

References

  1. Ghazarian, H., Idoni, B. & Oppenheimer, S. B. A glycobiology review: carbohydrates, lectins and implications in cancer therapeutics. Acta Histochem. 113, 236–247 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Kumar, K. K. et al. Biological role of lectins: a review. J. Orofac. Sci. 4, 20–25 (2012).

    Article  Google Scholar 

  3. Holmskov, U., Thiel, S. & Jensenius, J. C. Collectins and ficolins: humoral lectins of the innate immune defense. Annu. Rev. Immunol. 21, 547–578 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Dam, T. K. & Brewer, C. F. Lectins as pattern recognition molecules: the effects of epitope density in innate immunity. Glycobiology 20, 270–279 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Lundquist, J. J. & Toone, E. J. The cluster glycoside effect. Chem. Rev. 102, 555–578 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Kiessling, L. L., Gestwicki, J. E. & Strong, L. E. Synthetic multivalent ligands in the exploration of cell-surface interactions. Curr. Opin. Chem. Biol. 4, 696–703 (2000).

    Article  CAS  PubMed  Google Scholar 

  7. Martinez‐Pomares, L. The mannose receptor. J. Leukoc. Biol. 92, 1177–1186 (2012).

    Article  PubMed  Google Scholar 

  8. Leteux, C. et al. The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated oligosaccharides of blood group Lewis A and Lewis X types in addition to the sulfated N-glycans of lutropin. J. Exp. Med. 191, 1117–1126 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Fiete, D. J., Beranek, M. C. & Baenziger, J. U. A cysteine-rich domain of the ‘mannose’ receptor mediates GalNAc-4-SO4 binding. Proc. Natl Acad. Sci. USA 95, 2089–2093 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Biessen, E. L. et al. Lysine-based cluster mannosides that inhibit ligand binding to the human mannose receptor at nanomolar concentration. J. Biol. Chem. 271, 28024–28030 (1996).

    Article  CAS  PubMed  Google Scholar 

  11. Blum, J. S., Stahl, P. D., Diaz, R. & Fiani, M. L. Purification and characterization of the d-mannose receptor from J774 mouse macrophage cells. Carbohydr. Res. 213, 145–153 (1991).

    Article  CAS  PubMed  Google Scholar 

  12. Taylor, M. E. & Drickamer, K. Structural requirements for high affinity binding of complex ligands by the macrophage mannose receptor. J. Biol. Chem. 268, 399–404 (1993).

    Article  CAS  PubMed  Google Scholar 

  13. Napper, C. E., Dyson, M. H. & Taylor, M. E. An extended conformation of the macrophage mannose receptor. J. Biol. Chem. 276, 14759–14766 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Burgdorf, S., Kautz, A., Bohnert, V., Knolle, P. A. & Kurts, C. Distinct pathways of antigen uptake and intracellular routing in CD4 and CD8 T cell activation. Science 316, 612–616 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Burgdorf, S., Schölz, C., Kautz, A., Tampé, R. & Kurts, C. Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation. Nat. Immunol. 9, 558–566 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. East, L. & Isacke, C. M. The mannose receptor family. Biochim. Biophys. Acta 1572, 364–386 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Garcia-Vallejo, J. J. & van Kooyk, Y. The physiological role of DC-SIGN: a tale of mice and men. Trends Immunol. 34, 482–486 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Hu, Z. et al. Structural insights into the pH-dependent conformational change and collagen recognition of the human mannose receptor. Structure 26, 60–71 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Frison, N. et al. Oligolysine-based oligosaccharide clusters: selective recognition and endocytosis by the mannose receptor and dendritic cell-specific intercellular adhesion molecule 3 (icam-3)-grabbing nonintegrin. J. Biol. Chem. 278, 23922–23929 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Feinberg, H. et al. Structural analysis of carbohydrate binding by the macrophage mannose receptor CD206. J. Biol. Chem. 296, 100368 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kéry, V., Křepinský, J. J. F., Warren, C. D., Capek, P. & Stahl, P. D. Ligand recognition by purified human mannose receptor. Arch. Biochem. Biophys. 298, 49–55 (1992).

    Article  PubMed  Google Scholar 

  22. Lee, S. J. et al. Mannose receptor-mediated regulation of serum glycoprotein homeostasis. Science 295, 1898–1901 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. DeSchoolmeester, M. L., Martinez‐Pomares, L., Gordon, S. & Else, K. J. The mannose receptor binds Trichuris muris excretory/secretory proteins but is not essential for protective immunity. Immunology 126, 246–255 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Dan, J. M., Kelly, R. M., Lee, C. K. & Levitz, S. M. Role of the mannose receptor in a murine model of Cryptococcus neoformans infection. Infect. Immun. 76, 2362–2367 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Dangaj, D. et al. Mannose receptor (MR) engagement by mesothelin GPI anchor polarizes tumor-associated macrophages and is blocked by anti-MR human recombinant antibody. PLoS ONE 6, e28386 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Martinez‐Pomares, L. et al. Analysis of mannose receptor regulation by IL-4, IL-10, and proteolytic processing using novel monoclonal antibodies. J. Leukoc. Biol. 73, 604–613 (2003).

    Article  PubMed  Google Scholar 

  27. Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl Acad. Sci. USA 103, 18911–18916 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Delcanale, P. et al. Aptamers with tunable affinity enable single-molecule tracking and localization of membrane receptors on living cancer cells. Angew. Chem. Int. Ed. Engl. 59, 18546–18555 (2020).

  30. Kiuchi, T., Higuchi, M., Takamura, A., Maruoka, M. & Watanabe, N. Multitarget super-resolution microscopy with high-density labeling by exchangeable probes. Nat. Methods 12, 743–746 (2015).

    Article  CAS  PubMed  Google Scholar 

  31. Giannone, G. et al. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys. J. 99, 1303–1310 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Winckler, P. et al. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells. Sci. Rep. 3, 2387 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Li, R.-J. E. et al. Systematic dual targeting of dendritic cell C-type lectin receptor DC-SIGN and TLR7 using a trifunctional mannosylated antigen. Front. Chem. 7, 650 (2019).

  34. Small, A. & Stahlheber, S. Fluorophore localization algorithms for super-resolution microscopy. Nat. Methods 11, 267–279 (2014).

    Article  CAS  PubMed  Google Scholar 

  35. Brockman, J. M. et al. Live-cell super-resolved PAINT imaging of piconewton cellular traction forces. Nat. Methods 17, 1018–1024 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Neumann, A. K., Thompson, N. L. & Jacobson, K. Distribution and lateral mobility of DC-SIGN on immature dendritic cells–implications for pathogen uptake. J. Cell Sci. 121, 634–643 (2008).

    Article  CAS  PubMed  Google Scholar 

  37. Liu, Y., Misulovin, Z. & Bjorkman, P. J. The molecular mechanism of sulfated carbohydrate recognition by the cysteine-rich domain of mannose receptor. J. Mol. Biol. 305, 481–490 (2001).

    Article  CAS  PubMed  Google Scholar 

  38. Clayton, A. H. A. Fluorescence-based approaches for monitoring membrane receptor oligomerization. J Biosci. 43, 463–469 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. Gambin, Y. et al. Lateral mobility of proteins in liquid membranes revisited. Proc. Natl Acad. Sci. USA 103, 2098–2102 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Naji, A., Levine, A. J. & Pincus, P. A. Corrections to the Saffman–Delbrück mobility for membrane bound proteins. Biophys. J. 93, L49–L51 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Chung, I. et al. Spatial control of EGF receptor activation by reversible dimerization on living cells. Nature 464, 783–787 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Low-Nam, S. T. et al. ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding. Nat. Struct. Mol. Biol. 18, 1244–1249 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Corzo, J. Time, the forgotten dimension of ligand binding teaching. Biochem. Mol. Biol. Educ. 34, 413–416 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Vigerust, D. J., Vick, S. & Shepherd, V. L. Stable expression and characterization of an optimized mannose receptor. J. Clin. Cell. Immunol. 6, 330 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Llorca, O. Extended and bent conformations of the mannose receptor family. Cell. Mol. Life Sci. 65, 1302–1310 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Gazi, U. & Martinez-Pomares, L. Influence of the mannose receptor in host immune responses. Immunobiology 214, 554–561 (2009).

    Article  CAS  PubMed  Google Scholar 

  47. Prigozy, T. I. et al. The mannose receptor delivers lipoglycan antigens to endosomes for presentation to T cells by CD1b molecules. Immunity 6, 187–197 (1997).

    Article  CAS  PubMed  Google Scholar 

  48. Mahnke, K. et al. The dendritic cell receptor for endocytosis, Dec-205, can recycle and enhance antigen presentation via major histocompatibility complex class II–positive lysosomal compartments. J. Cell Biol. 151, 673–684 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Jahagirdar, P., Lokhande, A. S., Dandekar, P. & Devarajan, P. V. in Mannose Receptor and Targeting Strategies (eds. Jahagirdar, P. et al.) 433–456 (Springer International Publishing, 2019).

  50. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).

    Article  CAS  PubMed  Google Scholar 

  51. Daly, R., Vaz, G., Davies, A. M., Senge, M. O. & Scanlan, E. M. Synthesis and biological evaluation of a library of glycoporphyrin compounds. Chem. Eur. J. 18, 14671–14679 (2012).

    Article  CAS  PubMed  Google Scholar 

  52. Wong, C. S., Hoogendoorn, S., van der Marel, G. A., Overkleeft, H. S. & Codée, J. D. C. Targeted delivery of fluorescent high-mannose-type oligosaccharide cathepsin inhibitor conjugates. ChemPlusChem 80, 928–937 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. Chan, W. and White, P. Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Oxford Scholarship Online, 1999); https://oxford.universitypressscholarship.com/view/10.1093/oso/9780199637256.001.0001/isbn-9780199637256

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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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  1. These authors contributed equally: Roger Riera, Tim P. Hogervorst.

Authors and Affiliations

  1. Department of Biomedical Engineering, Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands

    Roger Riera & Lorenzo Albertazzi

  2. Department of Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands

    Tim P. Hogervorst, Ward Doelman, Jeroen D. C. Codée & Sander I. van Kasteren

  3. Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands

    Yan Ni

  4. Nanoscopy for Nanomedicine, Institute for Bioengineering of Catalonia, Barcelona, Spain

    Silvia Pujals & Lorenzo Albertazzi

  5. Cellular and Molecular Immunology, Bioengineering Sciences Department, Vrije Universiteit Brussel, Brussels, Belgium

    Evangelia Bolli

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Contributions

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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