New Spectroscopic Tools for Characterising Glycan Structure

Carbohydrates and sugars are found widely in all living systems, including humans, and they are important for maintaining our health and nutrition. They are also important for the economic prosperity of the UK as carbohydrates are major components of most foods and beverages. A principal way in which we develop new medicines, therapies, materials or other scientific advances in biology is by understanding the structure of molecules and how this structure determines their stability and interactions with other molecules. This approach has underpinned the last 50 years of developments in genetics, medicines, biomaterials etc., mainly through development of the tools for studying the structure of proteins and nucleic acids. Our understanding of the roles of carbohydrates in biology is much poorer than that of proteins, not because carbohydrates are less common than proteins or aren't involved in critical physiological processes (they clearly are), but because the main tools used in structural biology aren't easily applicable to carbohydrates. This long standing problem has resulted in our knowing little about the behaviour or function of the great majority of carbohydrates. Obviously, in order to better develop this level of understanding we need new tools for studying carbohydrate structure. For these tools to be generally useful they should be sensitive to a large amount of structural information, they should be able to differentiate between individual sugars and their various polymers, they should be able to inform on the stability and interactions of carbohydrates, and they should be able to do all this for all, or at least most, carbohydrates under a wide range of conditions. Raman spectroscopies fulfil all of these requirements and we will develop these laser-based techniques for studying carbohydrates in both solutions and the condensed phase (the two physiological conditions in which most carbohydrates are normally found). Both experimental Raman techniques and advanced forms of data analysis will be utilised to set up a complementary tool set for characterising the structure of any carbohydrate, from the smallest sugar to the largest polymer, and their functional behaviour. We have already developed analogous tools for studying other biomolecules and our preliminary studies verify that these tools will be highly useful to biologists studying carbohydrates. This pump priming project will establish a solid foundation for future developments and projects by, firstly, generating a spectral library for interpretation of carbohydrate data and, secondly, optimising specialised forms of data analysis for understanding the mechanisms of structural changes in carbohydrate polymers relevant to physiological processes. New tools generate new science and, ultimately, applications in medicine and industry. A great advantage of the Raman spectroscopies that we will develop is their ability to be used on many different problems, and particularly their ability to collect data on carbohydrates in the condensed phase, which is relevant to many biological examples but is not amenable to conventional glycobiology techniques. Therefore, our project will be the vital and large first step in designing a range of tools that will lead to many future developments in glycobiology, medicine and agriculture.

We will exploit the enhanced structural sensitivity of Raman and Raman optical activity (ROA) spectroscopies to establish new tools that are urgently needed for studying carbohydrate structure and conformational dynamics. As spectra can be collected for samples in a wide range of physiologically relevant conditions, and particularly the condensed phase which few other techniques can adequately probe, these tools will be able to structurally characterise most, or possibly all, carbohydrates; saccharides, glycoproteins, glycolipids and glycans. We will begin the development of these tools by training them on a number of biologically important saccharides and hyaluronan, a human glycosaminoglycan. Structure-spectra relationships will be elucidated as we establish the sensitivity of spectra to intra- and inter-molecular interactions, functional group identity and stereochemistry. In the second phase of the project we will combine spectra with chemometric techniques based on 2D correlation analysis to extend the studies to two important, but poorly understood, transitions of hyaluronan polymers; concentration-dependence and Ca2+-binding. Although structural changes are known to occur for these two perturbations, the natures of these structural changes are unknown, and they occur under experimental conditions which have prevented the application of conventional methods. We will use this coupled 2D correlation spectroscopy approach, previously successful for conformational studies on proteins and nucleic acids, to study the mechanisms underlying concentration-dependence and Ca2+-binding, and to test two conflicting hypotheses in the literature regarding hyaluronan structure. These proof of principle experiments will introduce a highly complementary set of tools to the glycobiology community that will be applicable to the many carbohydrates now being used in biology, medicine and food science.

This project will benefit the following groups: 1) Glycobiologists. The new tools we will develop are generic, and will be applicable to all types of carbohydrates including those of current scientific interest such as glycoproteins and proteoglycans. The current tools used to characterise biomolecular structure have performed poorly for most carbohydrates and, consequently, the structure and function of these carbohydrates is not well understood. The Raman tools evolving from this project will open many new opportunities to understand the complexities of carbohydrate structure and function as the principles of analysis and the Raman experiments will be as applicable to studies of, for example, glycan interactions. We will publicise these tools to glycobiologists first by publishing our work in relevant journals, e.g. Carbohydrate Research. The CI, Almond, has extensive links in both the academic community and industrial sectors of this community, and the Wellcome Trust Centre for Cell-Matrix Research is based in our Faculty at Manchester, so providing a direct pipeline to promote awareness of the Raman tools. 2) Unilever. We have a collaboration with Unilever UK to understand mucin structure and how this determines its interaction with foods. Please see the attached letter of support from Unilever. Although our project will not involve studies on mucins, as hyaluronan is better characterised so a more suitable model system, we expect to use these same methods to study mucins. We are discussing plans with Unilever for further studies and a follow-up grant application based on the tools to be developed in this project. Our collaboration with Unilever ensures that they will be kept informed about all findings from this project. As stated above, we and Unilever will capitalise on this project by utilising the techniques for planned studies on mucin function, and we plan to submit an IPA application to BBSRC in 2010 to this effect. 3) General Public and UK Industry. In the long term, both the general public and the UK food industry stand to benefit from the knowledge that will be revealed by these Raman tools. First, carbohydrates are major constituents of foodstuffs and beverages but their characterisation and even identification is often difficult. As Raman spectroscopy is well suited to studying foods and beverages (it is non-destructive, is label free, fast and can be used in remote sensing applications), the techniques we will develop also have the potential to identify and quantify carbohydrates in foods and beverages. Secondly, glycoproteins are involved in a wide range of physiological processes but the functionality of the glycans is not understood in most cases. These spectroscopic tools will be well suited to probing glycoprotein functionality, benefiting many areas of medical research. An examples is the respiratory mucins implicated in cystic fibrosis as the role of the glycan structure in disease progression is not understood, and our Raman techniques will be optimised to address this type of question. These benefits to UK industry and the general public will eventuate in the long term as the techniques will need to be developed, then publicised to potential interested partners before their application to the many potential areas of carbohydrate research. The potential long term impact on health (carbohydrates are important for nutrition, wellbeing and several diseases) and national wealth (aminoglycan healthcare products are worth many £millions each year in the UK) is great as there are so few other options currently available for characterising carbohydrates. The PDRA will learn a range of biological, spectroscopic and analytical skills they will be able to employ in either further academic research or in the commercial sector. Both Raman spectroscopy and analytical methods similar to those we will develop are used in the chemical and pharmaceutical industries.