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dc.contributor.authorKittelsen, Lene Aasland
dc.date.accessioned2015-09-07T22:02:22Z
dc.date.issued2015
dc.identifier.citationKittelsen, Lene Aasland. A Structural and Functional Characterisation of Human PGM1. Master thesis, University of Oslo, 2015
dc.identifier.urihttp://hdl.handle.net/10852/45590
dc.description.abstractCongenital disorders of glycosylation are a multifaceted group of disorders affecting mul- tiple organs with phenotypes ranging from mild to severe.Congenital disorders of glycosylation are a multifaceted group of disorders affecting mul- tiple organs with phenotypes ranging from mild to severe. PGM1 is recognised as one of the enzymes causing CDGs. PGM1 is crucial as a regulator of both glycosylation and glycolysis pathways. There are two isoforms of PGM1, both of which are highly conserved in mammals. Isoform 2 has an N-terminal extension that is not present in isoform 1. Previously the focus of attention has been on the ubiqui- tously expressed isoform 1. In this thesis isoform 2, expressed only in muscle cells, was investigated. During this thesis, human PGM1 isoform 2 wt with and without its substrate and prod- uct, G1P and G6P, as well as the mutants Thr485Ala/Thr were crystallised to obtain some insight into the structure of PGM1. The mutant Thr485Glu mimicks a phospho- rylation thought to increase the activity of PGM1. It is suggested that this phosphory- lation induces a conformational change in PGM1. Crystallisation of the PGM1 isoform 2-G1P/G6P complexes was done to investigate PGM1-substrate interactions. Data col- lection was undertaken at ESRF. Useable data sets for PGM1 isoform 2 wt with and without G1P or G6P were collected. The process of structure building was begun, but is not yet finished. However, some tentative insight into the structures was gained. The N-terminal extension looks like an α-helix. Based on observations of the structures, two residues assumed to be important for activity and substrate interaction were chosen for further activity analysis. These residues (Arg311 and Arg533) were mutated to alanine to eradicate their function in the protein. In addition a patient mutation (Asp80His) was selected for the activity assay. Asp80His could not be expressed, and further analysis of this residue was not achieved. Activity assays of Thr485Ala/E, Arg311Ala and Arg533Ala suggests that they have some impact on the activity. PGM1 is recognised as one of the enzymes causing CDGs. PGM1 is crucial as a regulator of both glycosylation and glycolysis pathways. There are two isoforms of PGM1, both of which are highly conserved in mammals. Isoform 2 has an N-terminal extension that is not present in isoform 1. Previously the focus of attention has been on the ubiqui- tously expressed isoform 1. In this thesis isoform 2, expressed only in muscle cells, was investigated. During this thesis, human PGM1 isoform 2 wt with and without its substrate and prod- uct, G1P and G6P, as well as the mutants Thr485Ala/Thr were crystallised to obtain some insight into the structure of PGM1. The mutant Thr485Glu mimicks a phospho- rylation thought to increase the activity of PGM1. It is suggested that this phosphory- lation induces a conformational change in PGM1. Crystallisation of the PGM1 isoform 2-G1P/G6P complexes was done to investigate PGM1-substrate interactions. Data col- lection was undertaken at ESRF. Useable data sets for PGM1 isoform 2 wt with and without G1P or G6P were collected. The process of structure building was begun, but is not yet finished. However, some tentative insight into the structures was gained. The N-terminal extension looks like an α-helix. Based on observations of the structures, two residues assumed to be important for activity and substrate interaction were chosen for further activity analysis. These residues (Arg311 and Arg533) were mutated to alanine to eradicate their function in the protein. In addition a patient mutation (Asp80His) was selected for the activity assay. Asp80His could not be expressed, and further analysis of this residue was not achieved. Activity assays of Thr485Ala/Glu, Arg311Ala and Arg533Ala suggests that they have some impact on the activity.eng
dc.language.isoeng
dc.subjectcrystallisation
dc.subjectbiochemistry
dc.subjectmolecular
dc.subjectbiology
dc.titleA Structural and Functional Characterisation of Human PGM1eng
dc.typeMaster thesis
dc.date.updated2015-09-07T22:06:55Z
dc.creator.authorKittelsen, Lene Aasland
dc.date.embargoenddate3015-06-01
dc.rights.termsDette dokumentet er ikke elektronisk tilgjengelig etter ønske fra forfatter. Tilgangskode/Access code A
dc.identifier.urnURN:NBN:no-49912
dc.type.documentMasteroppgave
dc.rights.accessrightsclosedaccess
dc.identifier.fulltextFulltext https://www.duo.uio.no/bitstream/handle/10852/45590/1/master.pdf


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