A Structure-Function Investigation of Cytidine 5'-Monophosphate N-Acetylneuraminic Acid Transport Protein

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Author(s)
Primary Supervisor
Itzstein, Mark von
Other Supervisors
Tiralongo, Joe
Year published
2011
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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 ...
View more >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.
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View more >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.
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Thesis Type
Thesis (PhD Doctorate)
Degree Program
Doctor of Philosophy (PhD)
School
Institute for Glycomics
Copyright Statement
The author owns the copyright in this thesis, unless stated otherwise.
Item Access Status
Public
Note
In order to comply with copyright some diagrams have been removed from this copy.
Subject
Cytidine 5'-monophosphate N-Acetylneuraminic acid transport protein
Tumor cells
Glycoproteins
Glycolipids
Golgi enriched Fractions (GeF)
Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR)