Probing Multivalent DC-SIGN/R-Glycan Interactions Using Polyvalent Multifunctional Glycan-Quantum Dot

Lead Research Organisation: University of Leeds
Department Name: Sch of Chemistry

Abstract

All living cells and many viruses are coated with specific sugars, allowing them to interact with partners bearing specific sugar binding proteins (lectins). While each lectin-sugar interaction is often weak and biologically inactive, by coating their surfaces with arrays of specific sugars, viruses can interact with multiple cell surface lectins to strengthen the interaction, allowing them to gain cell entry which ultimately leads to infection. Despite new anti-viral and vaccine treatments, disease caused by virus infection remains high. For example, ~37 and 150 million people are living with HIV and HCV infections in 2015, causing annual global deaths of ~1.1 and 0.5 million, respectively. Fortunately, virus mimics with specific sugar coatings can block such interactions, thereby preventing infection. The inhibition potency depends critically on matching the spacing and orientation of individual interactions between the binding partners. Hence understanding how a lectin's multiple sugar binding sites (CRDs) are arranged is vital to design effective virus inhibitors. However, the advances in research have been hampered by the inability of current methods to reveal key structural information (e.g. binding site orientation, spacing and flexibility) of important cell surface lectins. For example, despite 20 years of extensive research worldwide, the structure of two critically important lectins, DC-SIGN and DC-SIGNR, remain unknown. They both contain four CRDs and bind to multiple sugars on the HIV and Ebola surface to enhance virus infection. However, why they have different binding preferences to multiple sugars and virus remain poorly understood.

We will address the capability gap of current methods by developing sugar coated tiny fluorescent particles called quantum dots (QDs) as virus mimics and study their interactions with DC-SIGN/R with single lectins in solution and multiple lectins on cell surface. We plan to achieve this goal by fully exploiting QD's unique properties: strong fluorescence for binding measurement; high contrast in electron microscopy for visualising binding induced particle arrangement to reveal binding site orientation; solid core for decorating with multiple sugars to enhance binding strength, and for adjusting sugar number and inter-sugar distance to probe lectin's CRD arrangement. We have assembled a team with extensive expertise in QD, sugar synthesis, electron microscopy and lectin biochemistry who will work together to address this significant challenge, each member contributing an essential expertise to this project.

We will first prepare a series of sugar-coated QDs with varying number and structure of sugars, inter-sugar distance and flexibility. We will then measure their interactions by fluorescence with individual DC-SIGN/R molecules in solution to find out how strong and how fast the molecules interact, what binding preference is for each QD-sugar-lectin partner. We will measure the particle arrangement after binding to different lectins by electron microscopy, and monitor their size changes upon each interaction. We will combine these results to find out how DC-SIGN/R CRDs are arranged and oriented, and how far apart their binding sites are spaced. We will also study why DC-SIGN/R CRDs are arranged in this particular way, which parts of the protein control such arrangement. We will further test the ability of the sugar-coated QDs to block Ebola virus infection of target cells and find out the link between individual QD-sugar-DC-SIGN/R binding strength and its virus blocking efficiency.
This study is extremely timely and important because it will develop a novel method to reveal key structural mechanisms of DC-SIGN/R-virus interactions, addressing an unmet technical challenge currently facing this important research area. It will also help to reveal the link between ligand binding strength and virus inhibition potency, and so guide the development new anti-viral strategies.

Technical Summary

Multivalent lectin-glycan interactions are widespread and play a key role in virus/bacterial infection and immune regulation. Understanding the structures and mechanisms involved is key to be able to design potent glycoconjugates to block such interactions. However, advances in research have been hampered by the inability of current methods to reveal key structural information (e.g. binding site orientation, spacing and flexibility) of some critically important cell surface lectins due to their flexible, complex and multimeric nature. For example, despite 20 years of extensive research worldwide, the structure of two critically important tetrameric lectins, DC-SIGN and DC-SIGNR, remain unknown. Both lectins bind to virus surface multivalent glycans and mediate deadly viral infections (e.g. HIV, HCV and Ebola).

This proposal will address the capability gap of current methods by developing a novel polyvalent glycan-quantum dot (QD) based multimodal readout strategy consisting of fluorescence resonance energy transfer, electron microscopy imaging and hydrodynamic size to dissect DC-SIGN/R-glycan multivalent interactions. By tuning QD surface glycan structure, valency, inter-glycan spacing and flexibility, we will create a perfect spatial and orientation match to those of CRDs in DC-SIGN/R, leading to greatly enhanced binding affinity and specificity. By studying the QD-glycan binding with DC-SIGN/R, we will reveal key structural information (e.g. CRD orientation, distance, binding mode) and the molecular basis of DC-SIGN/R CRD arrangements. We will verify the solution binding data by comparing them with native receptors on cell surfaces, and further correlate the QD-glycan-DC-SIGN/R binding affinity with virus inhibition potency. These studies are not only important to reveal the fundamental structure-property-function relationship of multimeric lectins, but also to design specific potent, multivalent inhibitors against virus infections.

Planned Impact

Academic
This project will develop a novel polyvalent glycan-quantum dot (QD) based multimodal readout strategy to elucidate multivalent protein-glycan interactions underpinning viral infections (e.g. HIV, HCV and Ebola). It directly addresses the capability gap of current methods in probing such complex, flexible and multimeric cell surface lectins. Given multivalent lectin-glycan interactions are wide-spread in biology and play a central role in many important biological events, this work will establish an effective new method to study such important, but under-explored research area, and impact the basic research in virus-cell interactions and infection. Moreover, it will reveal important structural information about DC-SIGN/R (e.g. CRD orientation, flexibility, binding site distance, binding mode). Given DC-SIGN/R play a critical role in recognising and facilitating many pathogen infections, this information can act as springboard to develop new glycoconjugates to specifically and potently block DC-SIGN/R mediated viral infections.

It will also address some fundamental issues about QD-bioconjugation, functionalisation and control over ligand valency and presentation. These are important to maximise ligand activity and exploit multivalency to enhance QD's sensing, imaging and diagnostic performance. The surface and bioconjugation chemistries can be extended to other nanomaterials to develop sensitive diagnostic assays for early disease diagnosis, multifunctional nanomedicine for targeted treatment of disease with greatly reduced side-effects; and controlled assembly & manipulation of novel nanostructures and nanodevices.

Importantly, this project will bring together a multidisciplinary team with highly complementary expertise to dissect DC-SIGN/R-glycan multivalent interactions where each member contributes a unique and essential expertise. This task can only be achieved by integrating the expertise of the whole team. Thus it will establish a multidisciplinary pipeline to tackle important biomedical challenges.

Our work will be presented at international and UK conferences, and published in leading, high-impact journals to reach the widest audience possible, allowing wide-spread and rapid adaptation of our method. Our method and findings will be shared and distributed throughout our collaborators and academics as well as knowledge transfer networks where the investigators are active members (e.g. directed assembly, crossing biological membranes, nanotechnology, biotechnology) to maximise its academic and potential commercial impact.

Staff Training
The PDRA and other students involved will be trained at the international forefront of polyvalent multifunctional nanoparticle development to reveal structural mechanisms of multivalent protein-glycan interactions. They will be trained in broad skills in organic and carbohydrate synthesis, nano-chemistry, bioconjugation and surface functionalisation, protein biochemistry and bioassays together with modern biophysical and the state of art electron microscopy techniques. Such training will be essential for the UK to remain at the international forefront of multivalent protein-glycan interaction research. This research will be available to larger community through our research websites and used as examples of cutting edge research in undergraduate and master's degree courses, allowing students to understand the frontiers of current research development.

Social & Economic
This work aims to address the capability gap of current methods in probing multivalent protein-glycan interactions underlying pathogen infections. While the current research mainly focuses on the fundamental mechanisms, the structural information of the virus receptors revealed here can serve as springboard to develop specific, potent antiviral reagents. This research will impact & benefit the treatment of virus infections, and improve healthcare of the general public over the medium to long terms.

Publications

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Description Despite the project only started 6 months ago, we have made several interesting discoveries:
1) Displaying polyvalent mannose and dimannose ligands onto the nanoparticle surface is highly effective in enhancing the multivalent binding affinity with DC-SIGN, and to a lesser extent with DC-SIGNR, which can be quickly quantified by a new FRET-based fluorescence readout strategy;
2) The resulting glycan-nanoparticles can potently block pseudo-Ebola virus infection of DC-SIGN expressing cells with sub-nM levels of IC50 values;
Exploitation Route The glycan nanoparticles can be potentially exploited as potent inhibitors against virus and other pathogen infections, which will benefit the healthcare and pharmaceutical industries.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology