Integrated workflows for glycan analysis: tagging strategies to facilitate structural and functional characterisation of carbohydrates

Proteins and other important biological molecules such as lipids are often highly decorated with complex chains of carbohydrates. These carbohydrates are involved in many processes, such as cell-cell adhesion and the recognition of pathogens.

The types of carbohydrate structures involved in these interactions, however, are often not easily accessible in quantity by standard synthetic routes. This is largely due to their structural complexity and their diversity. In addition, the analysis of these complex and diverse sugars species itself poses many analytical challenges and is often also additionally hampered by the lack of available material. Although chemical synthesis of these sugars is challenging, large carbohydrate structures can be isolated directly from biological molecules and they can be tagged with a fluorescent label to aid their separation. Such technology is exploited commercially in the analysis of biopharmaceuticals, which (as of 2014) made up eight of the ten top selling drugs in Europe. An understanding of the sugars decorating the surface of such therapeutics is an important aspect of quality control, as changes in the sugars displayed on the surface can affect the storage stability, the efficacy and the safety of biologic drugs. Despite the use of this technique in the field of analysis, the labelling strategies commonly employed in this process result in the isolated carbohydrates being of little use for downstream applications and they are routinely discarded. The isolation of sufficient unlabelled material for use in biological assays often requires repeated "blind preparation" where unlabelled material is isolated from a complex mixture of sugars cleaved from the surface of a protein, and a sample of this isolate then labelled and checked for fidelity and purity. Such approaches to isolating carbohydrates require comparatively large quantities of starting material and multiple time-consuming post-processing steps.

This project describes the preparation of an alternative fluorescent label exhibiting comparable properties in the separation system, whilst bearing additional functionality which can be exploited to facilitate downstream uses directly. It is the intention that this label will allow for labelled sugars isolated by procedures applied in industrial carbohydrate analysis to be utilised in the production of synthetic glycolipids. These synthetic glycolipids will be layered onto a surface to create artificial cell membranes and also inserted into droplets to mimic whole cells. Using these types of methods to present sugars, we can create systems where the sugars are displayed on a surface in such a way that they mimic the correct orientation, closely modelling how they would be displayed in a natural system.

By using different combinations of synthetic glycolipids bearing different fluorescent labels and carrying different defined carbohydrates, it is envisaged that these artificial membrane can be used to investigate how proteins and pathogenic species bind to the sugars displayed on the cell surface. Creating artificial membranes in this way will allow us to investigate not only how different carbohydrates bind to protein partners, but also how combinations of glycolipids bearing different sugars are recruited by pathogens. It will allow us to investigate how such species arrange themselves to maximise binding efficiency when these recognition events take place. A deeper understanding of the nature of these events and how these molecules behave and interact can provide important insights into biological processes, and have the potential to inform the development of diagnostic tools and novel therapeutics.

It is also hoped that the same tagged sugars can be made available to the wider scientific community to provide access to hitherto difficult to obtain reagents which can be used directly in a variety of different assays, analytical techniques and synthetic applications.

This project has been designed to have an impact beyond the academic environment. In particular, it has been designed to be of direct relevance to a number of sectors within the commercial glycomics markets, both nationally and internationally. The commercial glycomics markets encompass end products such as specialty chemicals, biocatalysts and analysis kits. It also includes such applications as the development of instrumentation, analytical workflows, novel therapeutics and healthcare technologies. The global glycomics market is expected to be valued at US$930 million by 2019, increasing from over US$510 million in 2014. Growth has been driven in part by the shift towards biopharmaceuticals and an increase in the awareness of the importance of carbohydrates, carbohydrate containing biomolecules and their interaction partners by the healthcare technologies and biomedical sector. It is further supported by advancements in analytical sciences and uplift in investment, both from government and industry sources.

This project seeks to develop a tool-kit which can be used to probe the interactions of proteins and carbohydrate binding partners. The ultimate end goal to contribute to the development of diagnostic platforms capable of distinguishing pathogens by virtue of their binding to model cell membranes. These model cell membranes will bear structurally complex carbohydrates not currently easily accessible in quantity by synthetic methodology. It is anticipated that there will be economic beneficiaries in the biopharmaceutical sector, the specialty chemical sector and the healthcare technologies sector. In addition, this project will have measurable societal impact through a contribution to quality of life metrics related to economic growth, as well as through its contribution to health and wellbeing. As the project is interdisciplinary and of industrial relevance, it will also contribute to the knowledge base by helping to bridge the skills gap between educational training and specialist skills required by employers.

Eight of the top ten drugs in Europe (as of 2014) are biologics, and for the biopharmaceutical sector, an understanding glycosylation is increasingly important. Changes in glycosylation can influence the stability, efficiency and safety of such therapeutics. The tagging strategies employed in this project have the potential to contribute to analytical workflows employed in the biopharmaceutical production, whilst the validation techniques have the potential beyond the lifecycle of this project to contribute to therapeutic design and product pipelines.

The glycomics specialty chemicals sector will benefit from the production of a series of easy-to-handle reagents becoming available. By providing supporting data, the validation techniques employed in this project will further encourage the use and uptake of such materials in research and development environments. There is additional scope for exploitation through licensing, partnership or kit production as well as through supply; hence there exists multiple significant and varied routes for the work to deliver quantifiable impact in this area.

This project seeks to develop methodology which can be ultimately developed into diagnostic platforms. Within healthcare technologies sector, it should be noted that diagnostics also have the potential to effect significant societal impact through contributing to health and wellbeing. This can impact through precision personal medicine provision as well as contributing to global health by enhancing access to such tools and their benefits. The enablement of rapid early detection of disease allows the concomitant rapid appliance of appropriate treatment regimes. It should also be noted the lead-time for development of diagnostic tools is also significantly less than that associated with pharmaceutical development; hence applications in this area have the opportunity to have timely societal impact.