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

10 25 50
 
Description We have made the following interesting findings and discoveries from this BBSRC grants:
1) Displaying simple mannose and dimannose ligands polyvalently onto nanoparticle surfaces is a highly effective way to enhance their binding affinity with DC-SIGN, and to a lesser extent with DC-SIGNR. Their binding affinities in solution can be quantified via a new quantum dot based fluorescence resonance energy transfer (FRET) readout strategy and also a new gold nanoparticle base fluorescence quenching assay recently developed in this project.
2) Binding of DC-SIGN/R with glycan-nanoparticles produced distinct protein-nanoparticle assemblies: binding of DC-SIGN produces isolated single particles while binding of DC-SIGNR gives extensively inter-crosslinked nanoparticles as confirmed by hydrodynamic size measurement and capturing native dispersions of lectin-nanoparticle assemblies via S/TEM imaging. These results suggest that, despite close similarity, DC-SIGN/R actually bind to glycan-nanoparticles in distinct modes: with simultaneous tetravalent binding to one nanoparticle for DC-SIGN, and bis-divalent inter-crosslinking with different nanoparticles for DC-SIGNR.
3) The scaffold shape of glycan-nanoparticles has a significant impact on DC-SIGN/R multivalent binding affinity and binding mode. While DC-SIGN displays the same simultaneous tetravalent binding mode with very strong nM or to sub-nM binding Kd both spherical and rod-shaped glycan-nanoparticles, DC-SIGNR crosslinks with spherical glycan-nanoparticles in >100 fold lower affinity compared to DC-SIGN. However, for rod-shaped glycan-quantum rods, DC-SIGNR crosslinks at the spherical ends but bind simultaneously with all 4 binding sites at the central cylindrical region on the same quantum rod, leading to significantly higher affinity compared to spherical quantum dots and only limited cluster type assemblies.
4) The different binding modes and binding affinity of DC-SIGN/R give distinct binding thermodynamic parameters: while both lectins give similar binding enthalpy changes, a larger entropic penalty for DC-SIGNR (inter-crosslinking) is responsible for its weaker affinity with glycan-nanoparticles.
5) The size of the glycan-nanoparticles strongly affects their binding affinity with DC-SIGN/R, with larger particles giving stronger binding without changing their binding mode, suggesting that size is an important parameter in designing glycan-nanoparticle inhibitors against cell surface lectin receptor mediated viral infections .
6) Our glycan-nanoparticles can potently block DC-SIGN mediated pseudo-Ebola virus infection of host cells with IC50 values down to 95 pM for 5 nm nanoparticles and 24 pM for 27 nm nanoparticle. Such impressive IC50 values make them the most potent glycan-nanoparticle inhibitors against DC-SIGN mediated Ebola virus infections reported so far.
7) We have discovered that the binding mode between glycan-nanoparticles and cell surface lectin receptors is key to block virus infection, only those that bind simultaneously-, but not those inter-cross link-, with target lectin receptors can potently and robustly block viral infection.
8) The antiviral properties of glycan-nanoparticles are strongly dependent on scaffold size, while the small (5 nm) glycan-nanoparticles can only potently and robustly block DC-SIGN-, but not DC-SIGNR-, mediated virus infection, the large (27 nm) glycan-nanoparticles can potently and robustly block both DC-SIGN- and DC-SIGNR- mediated virus infections.
9) The binding environment can significantly affect the binding affinities between glycan-nanoparticles and target lectins: the solution affinity is significantly weaker than that on surface with lipid membrane immobilised lectins, implying that solution based binding data cannot be directly used to explain what happens on cell membrane surfaces .
10) Our glycan-nanoparticles can effectively modulate dendritic cell immune responses, and hence can be employed as a potential treatment of immune dysregulation diseases.
11) We have developed a new, general viral neutralisation strategy using nano-lectins with polyvalent, flexibly linked CRDs that can bind strongly and multivalently with virus surface conserved glycans, and demonstrated its great potential with both pseudotyped and authentic SARS-CoV-2 virus models.
Exploitation Route The glycan-nanoparticles can be exploited as potent entry inhibitors to block cell surface lectin receptor mediated virus and other pathogen infections. This anti-infection strategy can be advantageous as it can prevent viral mutation and develop resistance. Targeting virus surface conserved glycans using polyvalent nano-lectins can serve as a robust, and potentially long lasting antiviral strategy to fight against a wide range of virus infections. These new findings can potentially benefit the healthcare and pharmaceutical industries over the medium to longer terms.
The glycan-nanoparticles can potently modulate dendritic cell immune response, and hence can be employed for potential treatment of immune dysregulation diseases by exploit DC-SIGN's powerful immune modulation function.
Sectors Healthcare

Pharmaceuticals and Medical Biotechnology

URL https://pubs.acs.org/toc/jacsat/142/42
 
Description Developing glycan-nanoparticles for specific DC-SIGN targeting and dendritic cell immune modulation via picodroplet single cell analysis
Amount £103,000 (GBP)
Funding ID 2601051 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 08/2021 
End 03/2025
 
Description Elucidating mechanisms underlying multivalency modulating lectin-glycan binding and assembly properties-implications for lectin function regulation
Amount £831,281 (GBP)
Funding ID BB/Y005856/1 
Organisation University of Leeds 
Sector Academic/University
Country United Kingdom
Start 03/2024 
End 02/2027
 
Description Multifunctional quantum dot-small molecule ratiometric fluorescent nanoprobes for sensitive detection and targeted imaging of cancer cells
Amount £12,000 (GBP)
Funding ID IEC\NSFC\191397 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 03/2022
 
Title A multifunctional quantum dot-glycan probe for multivalent lectin-glycan interactions 
Description Developed a new glycan-quantum dot based multifunctional research tool for studying multivalent lectin-glycan interactions (MLGIs). It can quantitate the binding affinity for specific MLGIs via a ratiometric FRET readout strategy, revealing that a polyvalent display of simple mannose containing ligands on quantum dots can enhance its binding affinity with DC-SIGN by up to 1.5 million fold. By combining native-state electron microscopy imaging and hydrodynamic size analysis, it successfully dissected the exact binding modes of DC-SIGN and DC-SIGNR, a pair of closely related, almost identical tetrameric lectins, revealing that DC-SIGN binds tetravelently to one glycan while DC-SIGNR binds bis-divalently with two different QDs. The glycan-QDs potently inhibit DC-SIGN mediated pseudo-Ebola viral infection of host cells with sub-nM IC50 values, making it one of the most potent glycoconjugate based viral inhibitors. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact Our QD-glycan based probe can not only provide quantitative binding information for MLGIs, but also reveal key structural information (binding site orientation and binding mode) for some important multimeric lectins without known crystal structure, paving way for developing potent, specific antiviral reagents. As our method does not rely on any reagents of animal origin, its adaptation will contribute to the BBSRC's 3R agenda. 
URL https://pubs.acs.org/doi/abs/10.1021/bk-2020-1346.ch004
 
Title A new quantum dot-FRET method for analysing multivalent protein-sugar interactions 
Description A new FRET based method for analysing multivalent protein-carbohydrate interactions using glycan-conjugated quantum dot and dye-labelled lectins has been developed. It can quantify the binding affinity and distinguish the different binding modes involved in the multivalent protein-sugar interactions. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact The results have been published by Academic Press as a book chapter in Methods in Enzymology, 2018, volume 598, page 71-100. 
URL https://doi.org/10.1016/bs.mie.2017.06.012
 
Description Established a new international collaboration with Prof. Xiuqing Wang, South Dakota State University, USA 
Organisation South Dakota State University
Country United States 
Sector Academic/University 
PI Contribution The Leeds team has extensive expertise in carbohydrate synthesis, preparation and characterisation of novel glycan-nanoparticles as structural probes for lectins (DC-SIGN/R)-glycan multivalent interactions. We have prepared a range of glycan-nanoparticles coated with sugars of varied linker length, glycan valency and monovalent DC-SIGN/R binding affinity. We found that the glycan valency, linker length and monovalent affinity strongly affect their binding with DC-SIGN/R, with shorter linker and higher glycan valency showing stronger lectin binding.
Collaborator Contribution Our USA collaborator has expertise in dendritic cell immunology. She helps us investigate how different glycan-nanoparticle treatment of dendritic cells (via its surface DC-SIGN) can affect dendritic cell maturation and regulate immune function by monitoring DC surface maturation markers and cytokine production.
Impact The output from this collaboration has not yet published. We are in the process writing up joint research papers.
Start Year 2019
 
Description Interview for national news 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Our work on developing novel gold nanoclusters to address bacterial antibiotic resistance problem (Chemical Science 2021, 12, 14871) has been highlighted by a press release by University of Leeds. It has attracted significant public interests from the national and international news media as evidenced by numerous highlights from science news, websites and public social medium. Prof. Zhou was interviewed by a Dutch news media as well.
Year(s) Of Engagement Activity 2021
URL https://www.leeds.ac.uk/news-science/news/article/4956/going-for-gold-to-reduce-antibiotic-resistanc...
 
Description Invited research seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Prof. Zhou presented an invited research seminars about some of the research results obtained from this BBSRC grant (e.g. glycan nanoparticles as multifunctional research tools to address unmet biological and biomedical problems) at South University of Science and Technology in China. There was about ~50 seminar audiences, including many postgraduate and undergraduate students. The talk was well received with many questions and discussions afterwards.
Year(s) Of Engagement Activity 2019
URL https://bme.sustech.edu.cn/en/a-polyvalent-multifunctional-nanoparticle-strategy-to-ultrasensitive-b...
 
Description Invited research seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact Prof. Zhou was invited by the Chemical Physical Society of University College London to present an online research seminar. Some of the research results obtained from this BBSRC grant (e.g. glycan nanoparticles as multifunctional research tools to address biological and biomedical problems) were presented. There was about ~30 seminar audiences, mainly undergraduate students. The talk was well received, with quite some questions and discussions afterwards. Moreover, the talk was recorded and released on YouTube.
Year(s) Of Engagement Activity 2022
URL https://www.youtube.com/watch?v=sJZRa7epnbg
 
Description Invited research seminar (x 2) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Prof. Zhou presented two invited research seminars on some of the research results obtained from this BBSRC grant (e.g. glycan nanoparticles as new research tools to address unmet biological and biomedical problems) at two Universities in China, e.g. Soochow University (Suzhou) and Fudan University (Shanghai). Each seminar was attended by ~60 audiences, including many postgraduate and undergraduate students. The talk was well received with many questions and discussions afterwards.
Year(s) Of Engagement Activity 2019
URL http://funsom.suda.edu.cn/f1/a1/c13779a324001/page.htm
 
Description Leeds BeCurious Public Engagement online activity 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A short video describing how we use cryogenic electron microscopy in materials, physical and chemical sciences.
Year(s) Of Engagement Activity 2021
 
Description Shool visit x 2 (Leeds) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact One of the researchers employed on this grant (Dr Elizabeth Kalverda) gave a talk at the Kirkstall St Stephens Primary (~30 pupils, 9-10 years old) on a research scientist's daily life. The talk was part of a school topic on scientists and inventors, which was well received with lots of questions from the pupils.
Dr Elizabeth Kalverda also gave a talk at the Leeds City colleague (~40 access to higher education and foundation year students) about chemistry and biology and received very positive feedbacks, with students showing increased interest to the subject areas.
Year(s) Of Engagement Activity 2019
 
Description University of Leeds BeCurious Public Engagement Event 2019 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact As part of the University of Leeds public engagement festival 'BeCurious' members of the team led a stand called 'Tiny new world: Viewing nanomaterials'. Visitors could build models of atomic structures using craft supplies, and use a desktop scanning electron microscope to view real world items at high magnification. The purpose was to show the relevance of nanomaterials and the microscopes we use to examine these to the lives that we lead. The University believes more than 1200 people attended to the events, and dozens of people (including children) used the electron microscope. Feedback from people was positive (that it was enjoyable and interesting), with aspects of this included in school visits held later in the year and plans for the 2020 BeCurious event.
Year(s) Of Engagement Activity 2019
URL http://www.leeds.ac.uk/download/482/be_curious_programme