A Structure-Function Investigation of Cytidine 5'-Monophosphate N-Acetylneuraminic Acid Transport Protein
Mark von Itzstein
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One of the main phenotypic changes associated with malignant transformation of tumor cells is the over-sialylation of cell surface glycoproteins and glycolipids. This post- translational modification takes place in the lumen of the Golgi apparatus where CMP-sialic acid, the activated form of sialic acid, is translocated from the cell cytosol by a member of the Nucleotide Sugar Transporter proteins family, the CMP-sialic acid transport protein (CST). Due to its terminal position and its negative charge at physiological pH, sialic acids play a crucial role in the physiopathology of the cancer cell. In particular, cell surface over-sialylation plays a crucial role in all the steps involved in metastasis formation, ranging from detachment of cells from the primary lesion and survival in the blood or lymphatic streams, to extravasation and implantation in a different body district to generate a secondary lesion (metastasis). Several reports detail how cell surface over-sialylation and metastatic potential of cancer cells can be reduced without exerting cytotoxic effects, by targeting CST activity, making the transporter an ideal candidate for drug design. Here we describe the over-expression of murine CST (mCST) in E. coli, L. lactis and the methylotropic yeast P. pastoris. In particular, the over-expression of the mCST in P. pastoris has allowed to set up a purification protocol that retrieves amounts of the transporter protein amenable for structural studies, following detergent solubilisation and affinity chromatography purification.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
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Cytidine 5'-monophosphate N-Acetylneuraminic acid transport protein
Golgi enriched Fractions (GeF)
Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR)