Abstract
Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin's binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection.
Highlights
Multivalent lectin-glycan interactions are widespread and mediate many important biological functions which include cell-cell communication, pathogen-host cell recognition, attachment and infection, and modulation of immune responses.[1−9] As most monovalent lectin-glycan binding events are often too weak to be biofunctional, many lectins form multimeric structures to cluster their carbohydrate-bindingdomains (CRDs) for efficient binding with spatially matched multivalent glycans to enhance binding affinity and specificity.[10−14] The overall multivalent affinity is directly linked to the monovalent affinity, and the glycan valency and the mode of binding
We have found previously that DC-SIGN binds more efficiently to quantum dots (QDs) capped with higher mannose densities.[34]
The branched ligands have the same dihydrolipoic acid (DHLA) anchoring group for gold nanoparticles (GNPs) binding as the monomeric glycans
Summary
Multivalent lectin-glycan interactions are widespread and mediate many important biological functions which include cell-cell communication, pathogen-host cell recognition, attachment and infection, and modulation of immune responses.[1−9] As most monovalent lectin-glycan binding events are often too weak to be biofunctional, many lectins form multimeric structures to cluster their carbohydrate-bindingdomains (CRDs) for efficient binding with spatially matched multivalent glycans to enhance binding affinity and specificity.[10−14] The overall multivalent affinity is directly linked to the monovalent affinity, and the glycan valency and the mode of binding. An inhibitor that intercross-links with target receptors would be much less effective in blocking virus infection than its simultaneous binding counterpart, making it almost impossible to achieve complete inhibition (Figure 5C/D) This effect was clearly demonstrated from a side-by-side comparison of virus inhibition data for two GNP-glycan-lectin pairs showing similar affinity but with distinct binding modes, e.g., GNPMan-DC-SIGN (Kd: 33 ± 2 nM) vs GNP-(DiMan)3-DCSIGNR (Kd: 42 ± 2 nM). The simultaneous binding pair (GNP-Man-DC-SIGN) clearly displayed higher inhibiting potencies over its cross-linking counterpart (GNP-(DiMan)3DC-SIGNR) across the whole range of concentrations studied (SI Figure S13A), even on a Kd normalized concentration (C/ Kd) term to eliminate the effect of small Kd differences (SI Figure S13B). The potential advantages of developing potent glycoconjugate viral inhibitors over other antiviral strategies are twofold: (1) it can reduce the chances of virus mutation by blocking its entry to host cells, and (2) its treatment potency is unlikely affected by virus mutation, allowing us to provide a potentially long lasting solution
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