dc.contributor.advisor |
Atkinson, Paul |
|
dc.contributor.advisor |
Maass, David |
|
dc.contributor.author |
Yegambaram, Manivannan |
|
dc.date.accessioned |
2012-03-28T23:52:36Z |
|
dc.date.accessioned |
2022-11-01T21:03:26Z |
|
dc.date.available |
2012-03-28T23:52:36Z |
|
dc.date.available |
2022-11-01T21:03:26Z |
|
dc.date.copyright |
2012 |
|
dc.date.issued |
2012 |
|
dc.identifier.uri |
https://ir.wgtn.ac.nz/handle/123456789/27906 |
|
dc.description.abstract |
Congenital disorder of glycosylation disease type Ic (CDG-Ic) is a human inherited disorder characterised by mis-glycosylation in cells for which the enzyme defect has been well characterised. The CDG-Ic disorder results in an accumulation of dolichylpyrophosphate-linked Man₉GlcNAc₂ within the cells of affected patients owing to lack of an active α-1, 3-glucosyltransferase (ALG6) and leading to cellular defects because of protein under-glycosylation.
The N-glycosylation pathway is conserved from yeast to humans, making Saccharomyces cerevisiae, a useful model to study the genetics and basic cell biology of the CDG-Ic disorder. To characterise the phenotypic signatures of cells associated with mis-glycosylation, we constructed a specific alg6 deletion mutant (alg6Δ) and the human CDG-Ic mutant with C998T point mutation in yeast. Four phenotypic assays namely growth inhibition phenotype, carboxypeptidase Y (CPY) glycosylation patterns, localisation of green fluorescent protein tagged proteins, and induction of the unfolded protein response were developed to measure the effects of lack of the α-1, 3-glucosyltransferase in the alg6Δ and the CDG-Ic mutants. Difference in these properties when compared to the laboratory wildtype yeast strain provided quantifiable phenotypes of the glycosylation disorder.
The CDG-Ic strain was treated with 3000 individual compounds in a primary drug screen from two commercial libraries comprising bioactive compounds to select for compounds able to revert growth phenotype to wildtype. A small panel of compounds was obtained and one compound, Se-(methyl) selenocysteine hydrochloride (SMSC), in a secondary screen caused significant improvement in growth, CPY glycosylation and caused a mislocalised glycoprotein reporter to revert to normal subcellular localisation. SMSC also substantially reduced the activation of the unfolded protein response in the CDG-Ic mutant. Based on such phenotypic rescue assays, we propose that SMSC may assist the misfolded proteins to fold correctly. This approach comprises a good basis for future drug screens to rescue the CDG-Ic disorder in animal models.
Synthetic genetic analysis (SGA) is a method for determining networks of genes modifying particular genes of interest. Results from the SGAs identified networks of modifying genes that were similar between the CDG-Ic mutation and the alg6 deletion mutant. Such SGA analyses revealed a limited set of back-up genes assisting the ALG6 function. It is proposed that the different clinical presentations observed between CDG patients could be a direct reflection of the combination of mutations in one or more genes of the interacting networks. |
en_NZ |
dc.format |
pdf |
en_NZ |
dc.language |
en_NZ |
|
dc.language.iso |
en_NZ |
|
dc.publisher |
Te Herenga Waka—Victoria University of Wellington |
en_NZ |
dc.rights |
Access is restricted to staff and students only. For information please contact the library. |
en_NZ |
dc.subject |
Protein folding |
en_NZ |
dc.subject |
Misfold |
en_NZ |
dc.subject |
Drug rescue |
en_NZ |
dc.title |
A Study of Human Congenital Disorder of Glycosylation Type Ic (CDG-Ic) in Yeast |
en_NZ |
dc.type |
Text |
en_NZ |
vuwschema.contributor.unit |
School of Biological Sciences |
en_NZ |
vuwschema.subject.marsden |
270199 Biochemistry and Cell Biology not Elsewhere Classified |
en_NZ |
vuwschema.type.vuw |
Awarded Doctoral Thesis |
en_NZ |
thesis.degree.discipline |
Cell and Molecular Bioscience |
en_NZ |
thesis.degree.grantor |
Te Herenga Waka—Victoria University of Wellington |
en_NZ |
thesis.degree.level |
Doctoral |
en_NZ |
thesis.degree.name |
Doctor of Philosophy |
en_NZ |