Elucidating the molecular basis of nucleotide sugar transport in health and disease.

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

Glycosylation, the process by which proteins are coated with sugars, occurs in specialised compartments in the cell. However, the sugars themselves are manufactured elsewhere, and are called nucleotide sugars. They must be transported into the specialised compartments across an impermeable barrier, called a membrane. Evolution has solved this conundrum through the use of integral membrane proteins, called transporters, which act as canal locks, allowing molecules, such as sugars, to pass across the membrane barrier. How these membrane transporters work is currently of intense interest, as they hold the key to understanding how the raw materials for glycosylation, the nucleotide sugars, gain access to the machinery that uses them to glycosylate the cell.

This research seeks to understand how sugar molecules are transported within human, fungal and parasite cells. Several pathogenic organisms use sugary coats to evade our immune system causing widespread diseases. Several fungal species, in particular Candida albicans and Aspergillus fumigatus are able to establish infections in patients undergoing organ transplant or chemotherapy. Yeast infections cause several million deaths each year worldwide and can establish chronic yeast infections in healthy patients, known as thrush. In the developing world, several species of parasites, called trypanosomes, use similar sugar coats to hide from immune cells in humans and cattle, causing devastating diseases in both. Fortunately, the type of sugar that these organisms need to create these sugary defence systems are not present in human cells, making them attractive targets for drug development.

This proposal seeks to understand how the specific membrane proteins for nucleotide sugar transport work. Using state of the art facilities in the UK and abroad, we use X-rays to probe the atomic structure of the transport proteins. This information will provide a blue print that will tell us how these proteins are made and importantly how we can design drugs that stop them. This is particularly important for the development of new antifungal and anti-trypanosome drug molecules. This work will also reveal the basis for several developmental and immune diseases caused by mutations in the transporters responsible for nucleotide sugar transport in the human body. Our research will help to improve our fundamental knowledge of glycosylation in the cell, impacting several areas of human, parasite and fungal cell biology.

Technical Summary

This project builds on the technical expertise of the Newstead group on studying the structural and functional basis of transport across cellular membranes. In particular the application of recent developments in eukaryotic expression systems, lipid-based crystallisation and nanobody technology have enabled the advancement of the study of eukaryotic transporters. We will use in house expression systems to produce milligram amounts of human, fungal and parasitic nucleotide sugar transporters. Using our cubic phase crystallisation platform, we will screen these proteins for x-ray crystallisation providing insight into the molecular basis for ligand recognition. Crystals of the CMP-sialic acid transporter have already been grown and diffract to medium resolution. We will experimentally phase this new structure using heavy atom and long wavelength methods.

Further biophysical characterisation of ligand binding and the role of specific residues identified through the structural approaches will be validated through the use of differential scanning fluorimetry, nanoDSF. Facilitating the structural and functional studies we will generate libraries of single chain antibodies, nanobodies to both native and conformationally locked variants.

Insights into lipid specificity will be gained through the study of reconstituted protein in defined lipid environments and monitoring transport through the use of radioactive nucleotide-sugar derivatives. Identification of specific lipids binding events will be facilitated through the use of native mass spectrometry including an analysis of lipid mediated dimerization.

Planned Impact

The main impact of this research will be the generation of new fundamental insights into nucleotide sugar transporters, proteins essential for both eukaryotic glycosylation and the pathogenicity of fungal and trypanosomatid microbes. Little is currently known regarding the molecular basis of nucleotide sugar transport, including how different nucleotide sugars are recognised, the implications for their different oligomeric states and their regulation within the dynamic lipid environment in these organelles. Several Congenital Disorders of Glycosylation have been mapped to SLC35 genes, including CDGIIf, resulting from the abnormal function of the human CMP-sialic acid transporter and Leukocyte adhesion deficiency type II (LADII) disease, resulting from defects in the GDP-fucose transporter. From disease perspective, pathogenic microbes, including Candida albicans and Aspergillus fumigatus, as well as protozoan parasites, such as Leishmania donovani and Tyrpanosoma brucei, require functional GDP-mannose transporters for virulence. These microbes are the cause of major diseases in both the new and old worlds, resulting in several million deaths each year. However, targeting these transporters for therapeutic intervention is hampered though the lack of detailed biochemical studies into this family of proteins.

The main impact of this research will be the reporting of new crystal structures for disease relevant members of the SLC35 transporter family, facilitating a molecular understanding of their role in these diseases. Beneficiaries will be research groups and clinicians studying the diseases caused by nucleotide sugar transporter disfunction. Impact will also be made through our functional and structural studies on the pathogenic GDP-mannose transporters from fungi and protozoan parasites. Our comparison with the human transporters, in particular our studies into substrate recognition, will enable their potential as new drug targets to be evaluated. The development of transport and binding assays for these proteins will have significant impact with research groups and biotechnology companies seeking to develop new families of antifungal and anti-trypanosome drugs.

A third major impact of our research will be the generation of new fundamental insights into this important part of eukaryotic glycosylation and organellular transport. To date very few studies have been reported in the literature on the biochemical characterisation, using purified and reconstituted proteins, from the ER or Golgi apparatus. Our study will address several pertinent issues related to these organelles, such as the role of lipids, membrane fluidity and curvature on these transporters. These insights will have important ramifications for understanding how membrane transporters function in these unusual internal membranes. Our methods will provide impact by serving as a reference for future studies into this family and facilitate other research groups studying similar systems from plant, mammalian and pathogenic microbes.

Membrane protein structural biology is an expanding area of biomedical research. An important impact of our cutting-edge research is the opportunity it provides for undergraduate students to learn how these important proteins are studied at an atomic level. Undergraduates are engaged in the Newstead group in original research projects, often using our yeast-based system and trained in cubic phase crystallisation, allowing them to develop key laboratory experience in this field. We expect to continue this important aspect of research led teaching and training.

Publications

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