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

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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.