Understanding the activity and function of hexosaminidase D

Carbohydrates, or sugars, are ubiquitous throughout nature and perform a number of important functions in our cells. Carbohydrates can exist in long chains, called polysaccharides, which is how energy is stored from the food we eat, why wood is strong and is responsible for the molecular glue that sticks our cells together. At the other end of the scale, single or a few sugars can be appended to other biomolecules such as proteins and lipids and are important in cell processes such as signalling and defence against pathogens. The structure and sequence of carbohydrates is complex and highly variable, but unlike DNA there is no genetic code that can be read to determine how it should exist. Instead, carbohydrate structure and sequence is defined only the enzymes, nature's catalysts, that make and break-down the carbohydrate molecules.

We are interested in an enzyme, called HexD, which cleaves a sugar called N-acetyl galactosamine from substrates. Little work has been done to characterise human HexD, and the substrate on which it acts in cells, and its function, are unknown. However, it has been shown that HexD is found in the synovial fluid of patients suffering from rheumatoid arthritis, and thus understanding HexD at the molecular level could have an important impact on the health of patients suffering from the disease in the longer term.

Our preliminary work on HexD suggests it may act on proteins in cells, but further investigations are needed to understand this fully. We have also revealed that HexD has some unprecedented activities, which we will dissect.

The over-arching aim of the project is to understand the biological role played by HexD, and we will do this by gaining fundamental insights into how HexD works at the molecular level. We will make HexD in the laboratory and study how it works, test various substrates in order to understand its catalytic activities, and identify proteins with which it interacts in cells. We will also develop specific inhibitors against HexD, which will significantly slow its activity. These inhibitors will be administered to cells, and we will examine the effect on how the cells grow and work, to aid our understanding of the role played by HexD. In addition, we will change (increase and decrease) the levels of HexD in cells and similarly monitor the effect.

Overall, these experiments will advance our understanding of the biological function of HexD.

Hexosaminidase D (HexD) is a human beta-hexosaminidase, located in the nucleocytoplasm, capable of hydrolysing substrates containing N-acetyl galactosamine (GalNAc) residues. However its physiological substrate(s) and function are unknown. HexD has been observed in the synovial fibroblast lysate and synovial fluid of patients suffering from rheumatoid arthritis, which affects around 1% of the world's population, meaning the research could impact on the health of an ageing population in the longer term. In addition, our preliminary work on HexD has revealed some unprecedented and surprising activities that need to be dissected and its relevance understood; these activities of HexD could be used to obtain biomolecules that are synthetically challenging to make. One of our goals is to identify and validate physiological substrates and/or interacting protein partners for HexD, using a number of different approaches including pull-down studies and a glycoarray screen. In addition, we will design, using insights from the structure and mechanism of HexD, and synthesize specific and potent inhibitors of HexD. These, together with genetic methods, will be used to modulate the activity or level of HexD in cells, to give insights into its function. Gaining a fundamental understanding of HexD at the molecular level is important in order to establish its role in biology.

Academic impact: The multi-disciplinary nature of the proposed research means that it will be of interest to academics from a range of backgrounds, including medicinal chemists, biologists, biochemists and medical researchers in the longer term (see Academic Beneficiaries). At present it is likely to be of most benefit to researchers in academic organizations, but once HexD is characterized at the molecular level and the connection with rheumatoid arthritis is better understood, then researchers in pharmaceutical or biotechnology companies may also be interested, especially given the burgeoning aging population. There is a strong carbohydrate enzymology community within the UK, as well as internationally, in particular in Canada, US and Japan, who will benefit from the scientific advances and tools developed. The project will benefit from collaborations with Prof. David Vocadlo, Simon Fraser University, Canada and Prof. Ten Feizi, Imperial College London.

The project will contribute to the research and professional training of two members of staff, Sr Stephen McMahon (PDRA) and Dr Verena Oehler (research technician). They, and the PI, will have access to the University's suite of courses and workshops for development of life skills and for research. Staff working on the proposed project will obtain diverse laboratory skills, which can be translated to a host of other biological questions and therefore important for future employment opportunities. In addition, staff will learn skills, such as oral presentations and time planning, which can be translated to other employment sectors. Both researchers are currently employed at the University of St Andrews on fixed term contracts due to end soon, meaning a successful outcome for the grant would enable the retention of talented and experienced staff at the University of St Andrews.

Economic and societal impact: The research will lead to insights into the biological role played by HexD at a fundamental level. It has been observed that HexD is present in the synovial fibroblast lysate and synovial fluid of rheumatoid arthritis patients, meaning in the longer term (beyond the grant period) there may be medical translational aspects to explore. Given the impact of rheumatoid arthritis on an aging population, it is possible the research could one day impact on the health of the nation by enhancing the quality of life for the elderly. Development of possible treatments for rheumatoid arthritis would take in the order of 10-15 years, but would bring economic prosperity to companies and in turn could lower the burden on UK government spending if new treatments are more financially viable than currently available. If intellectual property arises from discoveries during the course of the project, they will be patented as appropriate aided by the Knowledge Transfer Office at the University of St Andrews.

We aim to contribute societal impact through education of the public sector, in particular children and their families, by the development of a game (available as an app or offline) exploring a 'Carbohydrate world'. This is to educate the public that carbohydrates and sugars exist beyond the food that we eat, and will highlight that each carbohydrate has a different structure (represented by the standard glycan symbol nomenclature). The concept of specific enzymes targeting a specific carbohydrate substrate will be introduced, which will be used as a means to explain our work with HexD where we know the enzyme but are yet to identify the physiological substrate. The game will be developed with the local public engagement team at the University of St Andrews, in collaboration with artists and game developers; children will be consulted throughout the process. The game will be exhibited at Dundee Science Centre, and used for outreach activities with local science festivals, schools, in particular in under-privileged communities in Fife, and prisons.