title: Exploring Nucleotide Sugar Transporter Structure and Function
Creators: name: Hadley, Barbara J
Publisher: name: Griffith University
Year: 2021
Subjects: subject: CMP-sialic acid transporter
subject: nucleotide sugar transporters

description: The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters (NSTs) to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this thesis, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters is described. At the time of commencing my research, little was known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins and no three-dimensional structure of any NST had been elucidated. What was known, was based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays, and phylogenetic analysis. In this regard the best-characterised NST was the CMP-sialic acid transporter (CST). The first structure of the first mammalian NST, specifically the mCST, was finally elucidated last year. The characterization of a membrane transport proteins mechanism of action, substrate interaction and transport rates are as essential as deducing its structure. SPR is now considered one of the most important biochemical techniques for studying biomolecular interactions between proteins and their binding partners. We have successfully utilised this label-free sensitive screening platform to determine CST kinetic and affinity data using the natural substrates CMP, CMP-Neu5Ac and a known CST inhibitor, UDP-Gal. Further to this, we tested the hypothesis raised from our past studies – can we measure transport of CMP and CMP-Neu5Ac by the CST using SPR? To do this, we have established a new SPR protocol, specifically addressing new transport parameters that have not previously been considered. Results indicate that binding and transport of substrates can be seen, and by data analysis, measurement can be made. The aims of my research included investigation of the structure/function of the murine CST (mCST) as a model for NST’s. This involved optimising the expression of functional mCST, so the transport protein was available for both functional and structural studies. Secondly, identification of detergent-mediated solubilisation and purification conditions to produce monodispersed CST, a critical pre-requisite for crystallisation. Thirdly, characterization of CST/ligand interactions at the molecular level using Surface Plasmon Resonance which allowed discovery of measurable differences between binding and transportation events.. The fourth and final objective was to crystallise the mCST within a lipid bilayer, using a combination of techniques including 2-dimensional and mesophase techniques, in order to investigate both its organisation and to obtain 3D structural information by means of electron crystallography.