Abstract
Multivalent protein–carbohydrate interactions initiate the first contacts between virus/bacteria and target cells, which ultimately lead to infection. Understanding the structures and binding modes involved is vital to the design of specific, potent multivalent inhibitors. However, the lack of structural information on such flexible, complex, and multimeric cell surface membrane proteins has often hampered such endeavors. Herein, we report that quantum dots (QDs) displayed with a dense array of mono-/disaccharides are powerful probes for multivalent protein–glycan interactions. Using a pair of closely related tetrameric lectins, DC-SIGN and DC-SIGNR, which bind to the HIV and Ebola virus glycoproteins (EBOV-GP) to augment viral entry and infect target cells, we show that such QDs efficiently dissect the different DC-SIGN/R-glycan binding modes (tetra-/di-/monovalent) through a combination of multimodal readouts: Förster resonance energy transfer (FRET), hydrodynamic size measurement, and transmission electron microscopy imaging. We also report a new QD-FRET method for quantifying QD-DC-SIGN/R binding affinity, revealing that DC-SIGN binds to the QD >100-fold tighter than does DC-SIGNR. This result is consistent with DC-SIGN’s higher trans-infection efficiency of some HIV strains over DC-SIGNR. Finally, we show that the QDs potently inhibit DC-SIGN-mediated enhancement of EBOV-GP-driven transduction of target cells with IC50 values down to 0.7 nM, matching well to their DC-SIGN binding constant (apparent Kd = 0.6 nM) measured by FRET. These results suggest that the glycan-QDs are powerful multifunctional probes for dissecting multivalent protein–ligand recognition and predicting glyconanoparticle inhibition of virus infection at the cellular level.
Highlights
Multivalent protein−carbohydrate interactions are widespread in biology and play a central role in many important biological events, including viral and bacterial infection, cell−cell communication, and host immune response regulation.[1−5] Such interactions initiate the first contact between pathogens and target cells that leads to infection
Using a pair of closely related tetrameric lectins, DC-SIGN and DC-SIGNR, which bind to the HIV and Ebola virus glycoproteins (EBOV-GP) to augment viral entry and infect target cells, we show that such quantum dots (QDs) efficiently dissect the different DC-SIGN/R-glycan binding modes through a combination of multimodal readouts: Förster resonance energy transfer (FRET), hydrodynamic size measurement, and transmission electron microscopy imaging
The FRET signal (Figure 5B), suggesting no binding competition occurred. These results indicate that wild-type and labeled DC-SIGN molecules must bind to the same sugar sites on the QD surface, whereas DC-SIGNR
Summary
Multivalent protein−carbohydrate interactions are widespread in biology and play a central role in many important biological events, including viral and bacterial infection, cell−cell communication, and host immune response regulation.[1−5] Such interactions initiate the first contact between pathogens (e.g., viruses and bacteria) and target cells that leads to infection. 100% glycan-QDs under high PQRs. Using the FRET efficiency obtained from the QD quenching (e.g., E = 1 − I/I0, where I and I0 are the fluorescence intensities of the QD with and without the protein, respectively) and a single QD in FRET interaction with N identical acceptor model, E = 1/[1 + (r/R0)6/N],34 the average QD-dye distance r was calculated to be ∼5.2 and ∼5.7 nm for DC-SIGN binding to QD-EG3-DiMan and QD-EG11-DiMan, respectively (Figure S3C and D). These results indicate that wild-type and labeled DC-SIGN molecules must bind to the same sugar sites (same binding mode) on the QD surface, whereas DC-SIGNR may be too weak to displace the labeled DC-SIGN from binding to the QD Their different competition efficiencies were clearer in the normalized I626/I554 versus WLR plots (Figure 5C), where DC-SIGNR gave no apparent changes but DC-SIGN yielded significantly reduced FRET ratios. The toxic cadmium content can prevent the current QD-glycans from being used for treatment and prevention of EBOV infection, replacing the CdSe/ZnS QD with other biocompatible, nontoxic nanoparticles (e.g., gold, Cd-free QD) should overcome this problem, where nanoparticles displayed with similar polyvalent glycan ligands could be used as potent, specific virus inhibitors and therapeutic reagents
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