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dc.contributor.authorSoliman, Mohamed
dc.date.accessioned2014-05-10T22:00:33Z
dc.date.issued2014
dc.identifier.citationSoliman, Mohamed. Chondroitin polymerizing factor (ChPF) and proteoglycan synthesis in epithelial cells. Master thesis, University of Oslo, 2014
dc.identifier.urihttp://hdl.handle.net/10852/39172
dc.description.abstractChondroitin Sulfate (CS) glycosaminoglycan (GAG) is a modification carried by proteoglycans (PGs) that have important physiological functions in cartilage and bone formation, the immune system and the central nervous system. PGs are involved in biological processes during immune response and regulation of growth and body patterning during development. Chondroitin polymerizing factor (ChPF) is one of six glycosyltransferases (GTs) involved in CS biosynthesis and cooperates with CS synthases 1, 2 or 3 in hetero-oligomeric complexes, playing an important role by enhancing CS polymerization activity. The CS GAGs are modified by sulfotransferases (STs) adding sulfate groups to the polymerizing GAG. The sulfate is supplied by 3 -phosphoadenosine-5 -phosphosulfate (PAPS). In this thesis, I have investigated the effect of expressing ChPF on PG synthesis, especially CS GAG synthesis, in polarized epithelial cells. Furthermore, I have explored the connection between Golgi import of PAPS, sulfation of CS and ChPF. Firstly, detection of the endogenous canine ChPF in MDCK II epithelial cells using anti-human ChPF antibodies was established. Then, transfection of the human ChPF cDNA sequence and expression in MDCK II cells was carried out. Western blot (WB) analysis of the endogenous and overexpressed human ChPF indicated that variable isoforms of ChPF were present. Metabolic labelling analysis of [35S]-sulfate labelled PGs showed that overexpression of ChPF in polarized MDCK II cells induced a significant increase in the amount of sulfated PGs in the cell fraction, while the amount of secreted PGs appeared unchanged. When investigating a possible connection between PAPS uptake into the Golgi lumen through PAPS transporter 1 (PAPST1) and CS synthesis, the B22 sub-cell line of MDCK II cells, already overexpressing PAPST1-GFP was utilized. Overexpression of ChPF in B22 cells reduced the apical secretion of PGs, mainly CS, as measured by sulfate labelling. Finally, the lengths of CS GAGs carried by PGs secreted from MDCK II cells were investigated using gel filtration column chromatography, while the number of CS GAGs attached to cell surface PGs was investigated by immunofluorescence with confocal microscopy. Detached GAGs, released by alkaline β-elimination, from secreted PGs revealed increased CS GAG length and more homogenous structure, indicating that ChPF induced longer CS GAGs. Confocal imaging utilizing a CS GAG specific antibody indicated increased amounts of CS GAGs sites occupied at the cell surface after ChPF expression.eng
dc.language.isoeng
dc.subjectglycosamonoglycan
dc.subjectGolgi
dc.subjectsulfation
dc.subjectextracellular
dc.subjectmatrix
dc.titleChondroitin polymerizing factor (ChPF) and proteoglycan synthesis in epithelial cellseng
dc.typeMaster thesis
dc.date.updated2014-05-10T22:00:33Z
dc.creator.authorSoliman, Mohamed
dc.date.embargoenddate3014-01-31
dc.rights.termsDette dokumentet er ikke elektronisk tilgjengelig etter ønske fra forfatter. Tilgangskode/Access code A
dc.identifier.urnURN:NBN:no-44076
dc.type.documentMasteroppgave
dc.rights.accessrightsclosedaccess
dc.identifier.fulltextFulltext https://www.duo.uio.no/bitstream/handle/10852/39172/1/ChPFinMDCKcells_M_Soliman_master_thesis.pdf


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