Abstract 2885 Supramolecular Glyco-Assemblies for Investigating Lectin-Glycan Interactions

Glycans play a pivotal role in essential biological processes including cell-cell adhesion, host-pathogen defense, and immune recognition.Proteins that bind glycans are known as lectins.Each lectin evolved with exquisite selectivity and stereospecificity for specific glycotypes.While many lectins have been characterized structurally, including their glycan bound states, little is known about the biophysical mechanisms behind these interactions.Here we present supramolecular glyco-assemblies as an approach to investigate lectin-glycan interactions.These glyco-assemblies display three terminal mannoses per Fe scaffold with the options of one, two, or four Fe scaffolds.We tested the binding of these assemblies to a well characterized lectin, Griffithsin (GRFT), an algal lectin known for its high-affinity binding to terminal mannoses.Utilizing biolayer interferometry (BLI), thermodynamic parameters KD and kinetic parameters ka and kd were determined.These data demonstrated that the binding affinity of GRFT is highly sensitive to the number and spatial arrangement of the mannoses, providing similar results to a native Man-9 glycan.Uniquely, these limitations could be overcome by the addition of polyethylene glycol (PEG) linker between the Fe scaffold and mannose sugar resulting in a 1,000-fold increase in binding affinity.Using 2D 1H-15N HSQC NMR spectroscopy, differential binding modes between various glyco-assemblies were observed.Specifically, the initial mannoses displays exhibited weaker transient interactions with GRFT while the displays containing a PEG extended linker allowed for stronger and more stable interactions with all three mannoses.In addition, doubling the number of mannose sugars displayed resulted in a 100-fold increase in binding affinity likely due to avidity effects from the two displays of mannoses.Further combinations of increased valency and increased linker length led to synergistic effects and resulted in binding events too strong to quantify.These findings suggest that the arrangement and flexibility of glycans are critical in mediating strong and stable lectin-glycan interactions, offering insights into the design of glycan-based therapeutic strategies, including antiviral agents.