High-Throughput Luminescence Assay for Sulfotransferase Activity

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Living things develop and grow, adapt to constant changes in their environment and energy sources, reproduce and resist disease. These things they achieve through communication between organisms and between cells within an organism, often by changing the chemical make-up of molecules. A common route is to add or remove a small charged chemical such as phosphate or sulfate, which act as switches, altering the information that is communicated. This dictates the biological outcome, which may be as varied as cell movement, cell death, and successful (or not) infection. The addition of a negative sulfate group is performed by specific enzymes called sulfotransferases, but we lack the technology to easily measure the activity of these enzymes.Sulfotransferases catalyse the transfer of sulfate to alcohols (-OH) on proteins, large sugars (glycans) and many small molecules, including hormones. There has been focussed research on small selective subsets of these enzymes and on some of their sulfated products. Glycan sulfation in animals plays key roles in development, wound repair and is associated with ageing, neurodegenerative diseases and infection by viruses. Sulfation of proteins on the amino acid tyrosine results in changes in protein-protein interactions related to immune function and virus infectivity. In plants, tyrosine sulfation produces hormones, and can be used by pathogens as part of their infection strategy. Bacterial sulfotransferases can sulfate a wider range of small molecule targets than animals, and some of these enzymes have potential as replacements of currently unsustainable chemical production of personal care and food products.Efforts to understand the global significance of sulfation and to harness it have faced an unsurmountable obstacle: there is no simple and accessible means to measure the sulfation of a target by a sulfotransferase. The consequence is piecemeal research, which is largely unable to deliver the depth and breadth of fundamental knowledge and advances necessary to harness sulfation for our benefit in animal and plant health, and in product synthesis.Our proposal aims to develop a simple, cheap and versatile assay technology that will allow all researchers to measure the addition of a sulfate to any biological or indeed man-made molecule by a sulfotransferase. Such assays have proven to be the key to understanding and exploiting related chemical modifications to biological molecules, such as phosphorylation.Sulfotransferases use a universal sulfate donor, PAPS, which upon transfer of its sulfate group becomes PAP. Measuring PAP provides a direct measure of sulfation. We will develop molecular probes based on a rare earth element, Europium, which emit red light upon bind reversibly to PAP. Measurement of the emitted light is very sensitive and provides a real-time readout of the activity of the sulfotransferase. This will be independent of the molecule the sulfotransferase is attaching the sulfate to. In this way the assay will be fast, real-time and eliminate the need for chemically modified reagents, radioactivity, antibodies and expensive equipment not generally available. Our proposed molecular probes have enormous potential to make the measurement of sulfotransferase activity routine and reduce the cost and time required to conduct high-throughput screening assays. These probes will provide a vital step towards the rapid, accurate determination of sulfotransferase kinetics and mechanism. This will enable better selection and validation of sulfotransferase inhibitors (drug leads), and of mutant enzymes for industrial use. The new technology will pave the way for understanding biological sulfation and exploiting it in the contexts of health and disease, and of product manufacture.