Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.649191
Title: Identification of putative glycosyltransferases associated with N-glycan biosynthesis
Author: Davies, James William
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2004
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Abstract:
N-glycosylation of proteins plays an essential role in controlling protein folding; recognition and many other cellular interactions. The pathway in which N-glycans are formed is the Leloir pathway. Of this pathway one core feature is retained through all N-linked glycoproteins, the GlcNac2Man3 pentasaccharide core. This moiety if formed on the cytoplasmic face of the endoplasmic reticulum. A number of these enzymes have been identified but a single β-1, 4-mannosyltransferase cloned and expressed, ALG1. Although the enzymes are mannosyltransferases little similarity exists between them, this makes similarity searching to find other enzymes of this pathway impossible. A bioformatic method was employed to build profiles of the known enzymes to identify further enzymes from genome scanning to identify putative transferases of this pathway. A number of potential targets were identified, these ranged from known mannosyltransferases with precise function still undefined to undefined open reading frames. The target genes which most closely fitted the constructed profiles were cloned and expression attempted in a bacterial host. Expression was not observed for a majority of the clones even after modification to remove membrane spanning portions. Expression was observed for YGL047w; YBR070c, YBR095c. Although good expression was achieved, the purified protein showed no activity or affinity for GDP was observed. ALG2, the second known mannosyltransferase of the Leloir pathway, was successfully cloned and expressed. The expressed proteins were shown to have no activity towards the natural substrate (PPGn2Man1) or towards GDP. β-1,4-galactosyltransferase activity was also investigated with respect to modified activated sugar donor species. Although modified acceptor species is known to be tolerated investigations indicate that no modification in donor species is possible.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.649191  DOI: Not available
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