Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Hennecke, K.
Cloning and expression of a novel GLB1 mutant discovered in GM1 gangliosidosis patient
Impact of GLB1 gene mutation on the GLB1 protein activity
Humanmedizin; [ Diplomarbeit ] Medizinische Universität Graz; 2024. pp.
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Hrzenjak Andelko
- Tokic Silvija
- Altmetrics:
- Abstract:
- Introduction: GM1 gangliosidosis is a lysosomal storage disorder leading to pro-gressive neurological symptoms. It is caused by pathogenic variants in the galac-tosidase beta 1 (GLB1) gene encoding β-galactosidase. This enzyme cleaves the terminal β-galactose from different substrates including gangliosides, glycopro-teins and glycosaminoglycans. Pathogenic variants in the GLB1 gene lead to ab-sent or reduced β-galactosidase activity resulting in an accumulation of the glyco-sphingolipid GM1 ganglioside in neuronal tissue. So far, around 300 pathogenic variants of the GLB1 gene have been described. Aim: The aim of this diploma thesis was to clone and express a new mutant form of the GLB1 gene that was recently discovered by our group. By measuring its activity in vitro, it was investigated whether this new mutation leads to the expres-sion of dysfunctional protein. Methods: A mutant and a wild-type variant of the GLB1 gene were cloned in competent E. coli cells. Recombinant plasmid-DNA was isolated with a maxiprep DNA isolation kit. To check successful DNA isolation, restriction analysis and DNA electrophoresis were performed. For expression of the GLB1 gene variants, HEK-293T cells were transfected with the recombinant plasmid-DNA. GLB1 expression was detected with a western blot analysis. Enzyme activity was measured with a photometric enzyme assay. Results: Both, wild-type and mutated enzyme were successfully expressed and the enzyme assay showed a significantly diminished activity of the mutated GLB1 protein in comparison to the wild-type (96.9±26.1 vs. 283.5±44.1 nmol/h/mg, p=0.0033). Discussion: The reduced enzyme activity suggests the novel mutation as a path-ogenic variant of the GLB1 gene, leading to the manifestation of GM1 gangli-osidosis.