Project description:
Changes in the glycan structures of glycoproteins has been reported for patients and mutant cell lines, but for one category of glycoproteins, the proteoglycans (PGs) has no reports been published. A major defect in PG synthesis would lead to severe developmental defects, but altered PG synthesis could very well be the cause of some of the symptoms observed in patients. Alternatively, the Golgi enzymes involved in PG synthesis are organized and maintained by entirely different mechanisms than those operating for enzymes modifying glycoproteins. Therefore, the main aim is to initiate studies of PG synthesis in three cell lines with COG mutations and compare to normal, wild type fibroblasts.
- Study the overall synthesis of heparan sulfate and chondroitin sulfate PGs in cell culture, by metabolic labeling and by a HPLC method.
- Transfect the fibroblast cell lines to address the modification of model glycoproteins and proteoglycans in the presence of the different COG mutations and in control cells.
- If promising results, perform si-RNA knock down of the best candidate COG subunit(s) in epithelial cells, where glycoproteins and proteoglycans are transported towards two opposite membrane domains (the apical and basolateral).
- Investigate whether uptake of nucleotide sugars and nucleotide sulfate is affected in COG mutant cells. In collaboration with Department of Chemistry (Dr Steven R. Wilson), we attempt to establish a method for quantification of nucleotide sugars and nucleotide sulfate from cell and organelle fractions this autumn. The robustness will be tested with the cell lines mentioned above.
Typical techniques: Cell culture, molecular biology, confocal microscopy, Western blotting, immune precipitation, gel filtration, HPLC, additional analytical techniques are possible, for instance subcellular fractionation and mass spectrometry.
Possible outcome: Provide knowledge that will contribute to textbooks concerning how the Golgi apparatus works. Provide knowledge of importance for a diverse group of patients. Contribute to the understanding of how the Golgi apparatus functions in polarized cells.
Possible supervisors, pending direction of project: Kristian Prydz, Gunnar Dick, Heidi Tveit, Fr?y Gr?ndahl, Steven R. Wilson (Kjemisk institutt), Erik Eklund, pediatrician, University of Lund.
The Prydz group has grants from The Research Council of Norway (NFR), Blix and Jahre foundations and The Norwegian Cancer Society. The group is part of a team in the Center of Excellence final application round. The group has at present two master students, three PhD students, one postdoc., one scientist, one professor, one technician with PhD and two associated members.
The project described can accommodate two master students after discussion of how to share and divide projects.