Stevens, ESL;
(2014)
An Investigation of Alpha-Dystroglycan Glycosylation in the Dystroglycanopathies: Implications for Diagnosis, Therapy and the Study of Novel Causative Genes.
Doctoral thesis , UCL (University College London).
Abstract
α-dystroglycan (α-DG) is a peripheral membrane protein that is an integral component of the dystrophin-glycoprotein complex (DGC). In an inherited subset of muscular dystrophies known as the dystroglycanopathies, α-DG has reduced glycosylation that results in lower affinity binding to a subset of extracellular matrix (ECM) proteins including laminins. The aim of this work was ultimately to increase the current understanding of α-DG glycosylation to better comprehend disease pathology and to aid in the development of targeted therapeutics. To achieve this, I first utilised flow cytometry to identify potential imino sugar compounds capable of increasing α-DG glycosylation, and six were found to have this effect. These six compounds significantly increased the level of α-DG glycosylation with sufficient selectivity and potency to make them attractive targets for drug development. Secondly, I optimised a flow cytometry method to determine the level of glycosylated α-DG in cell lines from confirmed and putative dystroglycanopathy patients to use as a complementary diagnostic aid alongside immunohistochemistry and immunoblotting. A total of twenty one dystroglycanopathy patient fibroblasts were assessed, as well as fibroblasts from three healthy controls and seven pathological controls. All cell lines assessed had detectable levels of glycosylated α-DG, and the level detected in dystroglycanopathy patient cell lines was significantly reduced (as measured as measured by the mean fluorescence intensity of an antibody recognising the epitope and the percentage of cells positive for the epitope) compared to that detected in healthy control fibroblasts (p<0.0001 for both values). Additionally, the results indicated that the amount of α-DG glycosylation in patient fibroblasts is comparable to that in patient skeletal muscle. Lastly, I was involved in the functional characterisation of variants in the glycan processing enzymes beta-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2) and GDP-mannose pyrophosphorylase B (GMPPB) to validate them as causative of dystroglycanopathy. The muscle biopsies and fibroblasts of patients with variants in these enzymes had reduced α-DG glycosylation compared to healthy controls. In some patient fibroblasts, complementation with the wild-type gene partially restored α-DG glycosylation. Additionally, several of the identified missense mutations in both genes resulted in a change in the subcellular localisation and/or aggregation of the recombinant enzyme compared to wild-type, suggestive of possible altered function. The results of this study provide insight into the complexity of α-DG glycosylation and pose questions that should help lay the foundation for future studies.
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