Polyvalent glycan-nanoparticles as a powerful new biophysical platform for multivalent lectin glycan interactions Dejian Zhou 1 , Yuan Guo 1,2 , Darshita Budhadev 1 , James Hooper 2 , Emma Poole 1 , Bruce Turnbull 1 , Nicole Hondow 3 1 School of Chemistry and Astbury Centre for Structural Molecular Biology; 2 School of Food Science and Astbury Centre for Structural Molecular Biology; 3 School of Chemical and Process Engineering, University of Leeds, UK Multivalent lectin-glycan interactions (MLGIs) are central to many important biological processes, including viral infection, cell-cell communication and immune regulation. Understanding their underpinning biophysical mechanisms is thus critically important, allowing for developing multivalent glycans to target specific MLGIs for therapeutic interventions. [1] Despite extensive research, the biophysical mechanisms of some key lectins remain poorly understood, due to limited capacity of current biophysical methods in probing such complex yet flexible cell membrane lectins. To address this challenge, we have developed a new polyvalent glycan-nanoparticle (PGN) approach to fully exploit multivalent binding and unique size-dependent chemical-physical properties of nanomaterials. Here nanoparticles not only serve as scaffolds for displaying glycans to enhance affinity by exploiting multivalency, but also as sensitive fluorescence readouts ( e.g. FRET with strongly fluorescent quantum dots, QDs, and fluorescence quenching with gold nanoparticles, GNPs) MLGI affinity quantification. Using a pair of closely- related viral receptors lectins, DC-SIGN [2] and DC-SIGNR, [3] as model tetrameric lectins, we show that our QD based PGNs bind to DC-SIGN up to >1.5 million fold stronger than the corresponding monovalent binding, and effectively discriminate between DC-SIGN and DC-SIGNR based MLGIs despite of identical monovalent binding motifs. [4,5] By combining FRET and TEM analysis of binding induced nanoparticle assemblies, we reveal that the significant stronger binding between DC-SIGN and PGN over that of DC-SIGNR is due to different binding modes: DC-SIGN binds tetravalently to one PGN, whereas DC-SIGNR crosslinks with two different PGNs. [5,6] Further, by performing Van’t Hoff analysis of temperature dependent affinities, we reveal both DC-SIGN/R binding are enthalpy driven and involving all four bindings sites, however, a larger entropy penalty for DC-SIGNR is responsible for its weaker affinity. [7,8] Finally, we show PGNs only robustly inhibit (IC 50 down to sub-100 pM level) DC-SIGN-, but not DC-SIGNR-, mediated Ebola cellular infections, demonstrating the critical importance of MLGI binding modes in viral inhibition. [5,6] This project was supported by the Wellcome Trust (097354/Z/11/Z), BBSRC (BB/R007829/1), EPSRC (EP/M50807X/1), and University of Leeds. References 1. S. Bhatia et al . J. Am. Chem. Soc. , 2016, 138 , 8654. 2. T. B. Geijtenbeek et al. Cell , 2000, 100 , 587.
3. S. Pöhlmann et al. Proc. Natl. Acad. Sci. 2001, 98 , 2670. 4. Y. Guo et al . Angew. Chem. Int. Ed . 2016, 55 , 4738. 5. Y. Guo et al . J. Am. Chem. Soc . 2017, 139 , 11833. 6. D. Budhadev et al ., J. Am. Chem. Soc . 2020, 142 , 18022. 7. J. Hooper et al. ACS Appl. Mater. Interfaces , 2022, 14, 48. 8. J. Hooper et al. to be published .
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