Dissecting Multivalent Lectin–Carbohydrate Recognition Using Polyvalent Multifunctional Glycan-Quantum Dots

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.

Microarray Analysis of Oligosaccharide‐Mediated Multivalent Carbohydrate–Protein Interactions and Their Heterogeneity

“…Manα(1–2)-linked oligosaccharides were selected because the high-mannose structure was expressed on the surface of the gp120 protein of HIV, which binds to human monoclonal antibody (mAb) 2G12. [10] In all glycodendrons, the fourth arm of the dendron was extended with an amine linker to allow them to be immobilized on the microarray epoxide surfaces. It is expected that the heterogeneity, nature of the carbohydrate ligand and multivalency of these glycodendrons could distinctly affect plant and animal lectin binding, and combining these effects would provide a unique recognition signature for differentiating the specific lectin recognition.…”

On‐Chip Screening of a Glycomimetic Library with C‐Type Lectins Reveals Structural Features Responsible for Preferential Binding of Dectin‐2 over DC‐SIGN/R and Langerin

“…The two homologues exhibit 73 % identity at the nucleic acid level and 77 % identity in the amino acid sequences, but display differences in the coiled‐coil neck domains that affect the spatial arrangement of the four CRDs. As a result, the two CLRs can show different avidity towards the same multivalent ligand . Similarly to DC‐SIGN, DC‐SIGNR is a pathogen receptor for HIV‐1, HCV, SARS‐coronavirus and Mycobacterium tuberculosis , recognizes influenza A and interacts with lymphocytes …”

Glycans in drug discovery

“…7B). 62 Recently, quantum dots surrounded by a dense array of mono- and disaccharides have been used as probes to gain insights into the binding mode of these multivalent interactions 63. Galectins, which comprise a large family of β- d -galactoside-binding lectins, are also involved in many biological functions with important implications in cancer progression, inflammation, immune responses, fibrosis and heart diseases 64.…”