Decoding heparan sulfate-protein interactions: from single molecules to cell mimics

Single-molecule fluorescence methods have revolutionized the study of biomolecules in vitro and in vivo. By detecting individual molecules, one by one, unique information about molecules can be revealed, information that is obscured when averaging over many molecules. This includes insight into the shape and motion of single biomolecules and how they interact with their environment. Such tools have already become essential for the study of DNA-protein interactions. Many critical biological interactions and signaling events involve interactions of carbohydrate molecules, specifically as oligosaccharide molecules. Amongst the most diverse of these are the roles played by the glycosaminoglycan (GAG) type of oligosaccharides. The GAG oligosaccharide heparan sulfate (HS) is known to interact with over a hundred proteins and plays ubiquitous regulatory roles in many processes, including angiogenesis, inflammation and stem-cell differentiation, with relevance to cancer, and also many pathogenic infections by viruses and bacteria. Thus, new molecular-level tools that can add to our understanding of such interactions would have significant impact in future pharmaceutical development. Unlike nucleic acids and proteins, however, the chemical synthesis of structurally-defined carbohydrates is extremely challenging, which explains why they have not been studied previously using these methods. New methods to study the interactions of HS-related oligosaccharides are at the forefront of carbohydrate chemical biology and future therapeutics. We recently demonstrated the chemical synthesis and fluorescent labeling of one of the major disaccharide units of HS and studied it using state-of-the-art single-molecule fluorescence methods. We were able to detect the disaccharide both free in solution and when encapsulated in surface-immobilized vesicles. Importantly, the fluorescent dye was not perturbed by attachment to the sugar. This suggested that fluorescently-labelled and structurally-pure synthetic oligosaccharides could be utilised for studying their biological interactions at the single-molecule level.

In this work we aim to demonstrate single-molecule fluorescence as a powerful method for probing the structure, dynamics and behavior of carbohydrates. We will take advantage of our proof-of-principle demonstration that specific oligosaccharides can be employed for single-molecule spectroscopy to develop a new platform technology to study HS-protein interactions at the single-molecule level. We will use these to reveal unique information on the interactions of a number of key proteins whose interactions with GAGs in biology are believed to be critical in regulating a number of medically-relevant processes, thus maximizing the potential for new insights of value to biomedicine.

The GAG oligosaccharide heparan sulfate (HS) interacts with numerous proteins and plays ubiquitous regulatory roles in many processes, including angiogenesis, inflammation and stem-cell differentiation, with relevance to cancer, and also many pathogenic infections. This project will exploit the application of single-molecule (SM) fluorescence methods to study the interactions of homogenous, pure synthetic HS fragments and high-importance target proteins. New molecular-level tools that can add to our understanding of such interactions would have significant impact in the future analysis of the wider field of HS-protein interactions and in subsequent glycopharmaceutical development. The project will focus on interrogating interactions of HS with prototypical cytokines, FGF1 and FGF2, and two key chemokines CXCL8 (IL8) and SDF1a (CXCL12), shedding light on the essential role of HS in protein regulation.

The project will develop a designed library of mutant proteins in which additional residues are included at different sites, allowing labelling, attachment and immobilization. We have previously demonstrated this is approach viable for a mutant of IL8 and the present project will extend this to the development of specific mutant libraries. This will involve generating an array of labelled variants (3-4 per protein) with site-defined labels, enabling a virtual mapping of HS-interactions using SM fluorescence methods. Building on our proven capacity to generate a diversity of pure, defined HS fragments, we will apply cutting-edge synthetic carbohydrate chemistry to make a range of novel HS probes, which will be used to study biocatalysis in solution. This will further be exploited by investigating such interactions in cell-mimic systems both as models of membrane embedded proteoglycans (vis synthesis of HS-lipid constructs in vesicles) and also to use SM methods to interrogate HS-protein interactions in encapsulated systems.

Methodology for carbohydrate synthesis: This project will develop synthetic methods for diversity-applicable labeling/conjugation of GAG fragments of biological importance. Efficient selection of end-groups and methods for routine labeling will have potentially wide impact for other applications of labeled, structurally-pure, GAG fragments. The project will also include scope to develop improved synthetic methods for fragments, including investigating supportable chemistry, which offers potentially significant output impact, such as array applications, re-tasking to supported synthesis and other high-value bioconjugations.

Carbohydrate-protein single molecule platform:
This work will add to the existing UK base at the forefront of oligosaccharide science, adding to this a new direction in single-molecule biophysical methodology. This is anticipated to provide an internationally-leading approach to enhance understanding of GAG-protein interactions. GAG interactions are involved in many pathogenic infections, as well as involvement in many host diseases where the facility to provide a single-molecule approach to protein interaction interrogation would be expected to offer a new and significant long term generic approach to accelerating and expanding structure-specific understanding.

Applications for cutting-edge biophotonics instrumentation: Direct visualisation of events in real time at the single-molecule level has transformed our understanding of biological systems and consequently there is a growing interest in these advanced imaging techniques. Our proposed experiments will highlight novel applications of these technologies and may aid the development of commercial biophotonics instrumentation.

Glycotherapeutics: Insight into the binding of HS-type ligands and biomedically-relevant signaling proteins will have impact beyond this grant in the design and development of potential therapeutic saccharides. It would be anticipated that this could lead to direct exploitation through existing biomedical translational collaborations for example in relation to anti-cancer therapeutic GAG development, promising anti-angiogenic lead structures and combination therapy potential, where we have strong links within Manchester. More broadly, a platform for evaluating GAG-protein interactions can be envisaged as being applicable to other therapeutically-relevant targets outside the cancer area.

Commercialization: Structure-specific labeled or 'click-labelable' modified GAG fragments may offer potential for commercial impact as a resource to commercialize a tool-box approach to GAG fragment investigations as these would have applications in a number of areas outside of single molecule work. Commercial potential will be reviewed regularly and the Manchester and Glasgow University commercialization companies (see Pathways to Impact document) will be engaged.

Output and Education: The project would be expected to lead to high impact publications across oligosaccharide chemistry and chemical biology, biophysics and at the carbohydrate biomaterials interface. Developments from this project will be disseminated by presentations at international conferences and other speaking opportunities. Postdoctoral staff will gain advanced multidisciplinary training. The project will also be allied to future directions of new PhDs and this will be expected to provide a significant educational benefit in PhD training and cross-disciplinary knowledge exchange within these labs. All researchers will be encouraged to also engage with Outreach activities at both Universities.