Elucidating mechanisms underlying multivalency modulating lectin-glycan binding and assembly properties-implications for lectin function regulation

Pathogen surfaces display high density carbohydrates to shield underneath elements from being recognized by antibodies for immune evasion. To recognize the unusually displayed carbohydrate, in immune systems, carbohydrate binding proteins (known as lectins) form multimeric structures where each lectin contains multiple carbohydrate recognition domains (CRDs). This also allows multiple contacts (multivalent binding) between them resulting in strong bindings similar to that observed with Velcro. Viruses and bacteria are much bigger than lectins, hence multiple lectins can bind to them, making the lectins cluster together. As many lectins are attached to immune cells, the cluster can be interpreted as signalling to initiate immune defence. However, some pathogens have developed strategies to exploit such strong binding to facilitate their infection. Currently, it is not clear whether they have changed the arrangement of carbohydrates to induce different lectin cluster patterns, and/or to make some CRDs unavailable for engagement which reduces binding strength and cluster stability, allowing more lectins to pack in to exclude other proteins from the area. These can be interpreted differently by immune cells.
Here, we employ a dendritic cell (DC) surface lectin DC-SIGN to address these questions. DC-SIGN contains 4 CRDs and binds to mannose (a type of carbohydrate) on pathogens including bacteria, viruses (such as HIV, SARS-COV-2) and fungi. Binding leads to virus being engulfed, digested inside DC and results in small pieces further being used to instruct other immune cell to produce antibodies for pathogen elimination. Binding also stimulates DC to produce some proteins through DC-SIGN cross-talking to a TLR 4 protein as part of defence actions. However, how communication is achieved is unknown and it is unclear if they have to be closely associated. But viruses such as HIV and SARS-COV-2 exploit binding to enhance their infection. In the case of HIV, it somehow avoids being digested inside DC and escapes later to infect other cells. This makes DC-SIGN an excellent model lectin for this study. We will also include another tetrameric lectin named DC-SIGNR which is almost identical to DC-SIGN with only difference being that their 4 CRDs have different orientations. This makes them an excellent pair to study CRDs availability in multivalent binding strength and cluster formation.
We will start with linking multiple pathogen glycans onto fluorescent quantum dot (QD) or rod (QR) surfaces with different densities to mimic possible displays on pathogen surfaces. We will develop a novel method to construct DC-SIGN or DC-SIGNR tetramers containing 4, 3 or 2 CRDs to investigate the effects of CRD engagement numbers on binding strength. To study cluster formation, we will exploit nanoparticles' high density to see their arrangement using electron microscopy to obtain information on lectin clusters: isolated particles mean lectins are assembled on the same particle and clustered particles are formed by proteins and particles cross-linking, these generate distinct cluster patterns. We will also label TLR 4 with a different shaped nanoparticle to see how it associates with DC-SIGN clusters. Proteins are invisible by electron microscope, by following nanoparticles we are able to gain their cluster information at nanometer level for the 1st time. Lectin binding can also be made to interfere with QD and QR's fluorescent property; we will follow the light signal by fluorescence microscopy to monitor the speed of signal change to gain information on cluster stability: the faster the change, the less stable the cluster. We will then use those QD/QR-glycans to stimulate DC to correlate observed cluster information with DC responses to explain how DC-SIGN instructs DC responses.
Information obtained here will provide guidance to design treatments against infection and to suppress immune overreaction to treat diabetes, arthritis and allergy.

The dendritic cell (DC) surface tetrameric lectin DC-SIGN recognizes mannose glycans on a wide range of pathogens. Binding internalizes viruses to lysosomes for degradation, but routes HIV to a different location for protection. It also modulates distinct cytokine production via cross-talking to TLR4. DC-SIGN forms clusters but how this modulates its function is unknown. We hypothesize DC-SIGN use different binding valency/modes to bind to differently presented pathogen glycans which results in difference in cluster dynamics and DC-SIGN packing, and alters TLR4 location hence outcomes. DC-SIGN and its almost identical tetramer DC-SIGNR bind to multivalent quantum dots (QD)-mannose using a different binding mode. We will compare them to reveal binding valency/mode in multivalent affinity enhancement.
We will conjugate mannoses onto QD or quantum rod (QR) surfaces at different densities to mimic different glycan displays. Extracellular segments of the lectin faithfully representing tetramer structure and multivalent binding feature will be used to construct tetramers with controlled mannose binding valency by mutagenesis and affinity purification. Binding thermodynamics/kinetics will be conducted in solution and by anchoring them on supported lipid bilayers via N-terminal his-tags. This reveals binding valency/mode on multivalent affinity enhancement, and how solution and surface bindings correlate to each other, which is currently unclear. We will develop novel TEM techniques to visualize binding induced QD/QR assembly to obtain information on lectin clustering and TLR4 location (TLR4 labelled with a different shaped goldparticle) at nm resolution for the 1st time. To reveal binding dynamics, we will label lectin with dye and also monitor QD/QR, and their FRET kinetic by TIRFM. This further confirms TLR4 and DC-SIGN location: QD/QR and dye fluorescence will be quenched if labelled TLR4 is co-locolized. We will stimulate DC with QD/QR-glycan to show DC responses.