Shotgun functional glycomics of heparan sulphate saccharides: generating diverse libraries to decode biological selectivity

Complex sugars (glycans) are a highly diverse family of molecules with a broad range of functions in biological processes including cell recognition, adhesion, cell-cell communication and signalling. The study of the glycome - the entire set of glycans expressed by particular cells or tissues - is an emerging field which aims to understand how glycan functions underpin the complexity of human biology, and their involvement in disease processes. The selective interaction of proteins with glycans is one of the keys to their biological functions. A number of new technologies have emerged to support the development of 'glycomics' studies - large-scale studies of glycan structural diversity and the selective interactions of glycans with their matching protein partners which underpins their functions. However, these approaches do not directly reveal functional properties, especially for some complex classes of glycans like the heparan sulphate (HS) family of sulphated glycans which are responsible for regulation of a wide range of biological processes including growth factor signalling, enzyme activity and cell adhesion. We now need to have the tools to ask how specific structures control specific proteins to control biological systems. In this project we propose to develop a new approach to address this question by generating a large library of novel HS glycans with a wide range of structures, and screening them in biological assays, followed by analysis of selected structures. This will permit us to evaluate the structure-activity relationships of HS at higher throughput for the first time. This 'shotgun' library approach will provide a powerful and generic new tool for decoding the function of the HS glycome by allowing specific glycan structures to be matched to specific biological functions. In the future this strategy could provide new information that could be translated into applications in biotechnology and drug development.

Decoding post-translational modifications of proteins, including the glycome, is a critical facet of the post-genome era, since they modify the functional proteome. Strategies have emerged to support 'glycomics' (large-scale) studies of glycan structure diversity and their selective interactions with cognate proteins. However to date these approaches have lacked the ability to directly generate functional data, particularly in the case of the complex heparan sulphate (HS) glycans. There is growing evidence that functional specificity exists in HS-dependent control of cognate protein activities, but technologies to truly address this question have been a bottleneck. The aim of this project is to develop a 'shotgun' functional glycomics approach for exploring the structure-activity relationships of HS. Generating a large random (unbiased) library of novel and diverse saccharides within HS chemical space, and screening them in test-bed bioassays, will permit detailed evaluation of biological specificity at higher throughput for the first time. We will: 1. Extend the chemical space of natural HS saccharide libraries covered by our existing pilot library (using a wider range of starting materials and saccharide generation methods). 2. Enhance the diversity of the HS library using enzymic approaches (recombinant sulfotransferases in concert with novel natural, semi-synthetic and synthetic substrates). 3. Screen the HS libraries to identify hits in test-bed bioassays (including FGF growth factor and Slit/Robo signalling). 4. Initiate structural analysis of HS library components and screening hits (to confirm structural diversity and identify novel structure-activity relationships, using state-of-the-art MS methods). This project will generate unique resources and a powerful new strategy for decoding the functions of the HS glycome. Such data will provide high value functional information on structure-activity relationships for the HS family.

Expected Beneficiaries: This project is expected to have wide impact in many areas of biomedicine and biotechnology related to medicine in particular (eg. applications in diagnostics, drug discovery and regenerative medicine). This is due firstly to the relevance of HS biology to many disease processes (eg. cancer, inflammation, neurodegeneration, wound repair) and also fundamental biological processes that are critical for stem cell control and tissue engineering. New methods for determining specific HS targets will open up opportunities for breakthroughs in identification of novel information on HS specifity of biological action which could underpin commercial exploitation. More widely, the glycomics strategies will be of broad interest in view of the wide application potential of glycans in general. Better understanding of HS biology is also relevant to societal impacts. For example, Prof. Turnbull has met with families involved in the UK Hereditary Multiple Exostoses (HME) Support group (www.hmesg.org.uk). HME is caused by genetic deficiencies in HS biosynthesis that result in a multifactorial clinical problems including growth deficiency, bone tumours and premature death. The HME group are interested in promoting better understanding of the disease, current research into its causes and symptoms, and potential new treatments. Communications and Engagement: Commercial: The Turnbull lab has a number of active collaborations with Industry both in the UK and overseas, including IRL Ltd (a partner on this project), and research collaborations with SpheriTech Ltd (Runcorn; BBSRC CASE) and Summit (Dextra) Ltd (Reading). All these projects will benefit from the project and we will actively develop these partnerships as described in the Impact plan statement. Prof. Turnbull is also actively involved in discussions with the NorthWest Development Agency regarding the establishment of a Centre of Excellence in Glycosciences, aimed at networking of the high level of academic expertise in this field in the northwest of England with commercial partners. Societal: The UK Hereditary Multiple Exostoses (HME) Support group has a Liverpool group contact, Tina Read, who is developing web-based resources and information for families with children affected by this disease. The Turnbull group plan to assist them with information on the molecular basis of the disease and how future research might help with new disease treatments, and hosting of a meeting of the national organisation in Liverpool in 2010. General dissemination: We will actively seek to disseminate information about our research efforts to both industry and the general public, through websites (Liverpool Centre for Glycobiology, and University Business Gateway); press releases and opportunities for public speaking. Collaboration: The principal commercial partnership within the proposed project is with IRL Ltd (Wellington, NZ). We have an existing research agreement initiated in 2009 with IRL Ltd on identification of synthetic targets with potential commercial applications in Alzheimers disease and cancer. IRL will have an active interest in outputs regarding new targets and enzymic modification of their synthetic saccharides. Exploitation and Application: There is considerable potential for commercial exploitation of outputs from this project, for example in drug development, stem cell exploitation and tissue engineering. The tools and strategy development aspect of the project may also yield new intellectual property of commercial potential. We will actively and regularly monitor our research output and potential publications with ULive Ltd (the University of Liverpool IP and tech transfer company), protect by patenting, and exploit via out-licencing or development of a spin-out company. For full details see the Impact Plan appended