Multivalent protein-sugar interactions initiate the 1st cell-pathogen contact and play a vital role in viral/bacterial infection. While individual protein-sugar interactions are typically weak and biologically inactive, by coating their surface with arrays of specific glycans, pathogens can simultaneously bind to multiple cell surface sugar binding proteins (lectins) to enhance affinity, allowing them to gain cell entry. Synthetic glycoconjugates can block such interactions and prevent infection, and the inhbition potency critically depends on the spatial-/orientation-match between such multivalent binding partners. The lack of structural information for some key lectins have hampered such development, due to challenges in solving the structure of such flexible, complex, and multimeric cell surface membrane proteins. To address this challenge, we are pursuing a polyvalent multifunctional nanoparticle (PMN) strategy to exploit multivalency and unique functions of nanoparticles. Here we report that fluorescent quantum dots (QDs) capped with a dense array of simple sugars (e.g. mannose, man-α-1,2-man) are powerful tools to dissect multivalent lectins (DC-SIGN/R)-sugar interactions. First, we report a highly-efficient cap-exchange method to prepare dense glycoslated QDs, allowing us to quantify the DC-SIGN/R-QD binding affinity via a FRET readout strategy. We find that a polyvalent display of glycans on the QD not only enhances its DC-SIGN binding affinity by 4-6 orders of magnitude, but also endows &gt;100 fold multivalent binding selectivity for DC-SIGN over DC-SIGNR, despite their almost identical tetrameric structures. We attribute this to the different orientation of their glycan-binding-domains: they point uprightly in DC-SIGN, but side-ways in DC-SIGNR, making the later unable to bind multivalently to one QD. Second, we verify their mode difference by TEM imaging: binding of DC-SIGN gives isolated QDs but binding of DC-SIGNR gives aggregated QDs. Third, we find the QDs potently inhibit pseudo-Ebola virus infection of DC-SIGN expressing cells with sub-nM IC50 values, matching well to their DC-SIGN binding affinity measured by FRET (e.g. 0.7 v.s. 0.6 nM). Finally, we show that gold nanoparticles coated with such glycan ligands can induce dendritic cells to produce immune tolerance signals, suggesting they may have potential application in allergy treatment.

Dissecting multivalent protein-carbohydrate interactions using polyvalent multifunctional glycan-nanopartocles

2019 Spring National Meeting

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