Glycosphingolipid Metabolism and the Glycosphingolipid-Metabolizing Enzyme Beta-Glucosidase 2: Biochemical and Cell Biological Studies

Abstract

Glycosphingolipids (GSLs) are constituents of eukaryotic cell membranes and consist of a lipid (ceramide) linked to one or more sugar residues. GSLs occur in many structurally distinct forms, varying in both their lipid and oligosaccharide domains. Most cells contain multiple structurally different GSLs. Different cell types/animal tissues have distinct and consistent GSL profiles, indicating that cellular GSL homeostasis is under strict regulation. Deviations in GSL homeostasis have pathological consequences. Our understanding of the regulation of cellular GSL levels is very limited. In the first part of my project, I have attempted to assess the kinetics of GSL turnover by testing a mathematical model correlating the rates of GSL biosynthesis and degradation. Genetic and pharmacological interventions were made in cellular models to disrupt GSL biosynthetic enzymes. This approach, however, produced inconsistent results. In the second part of the project I investigated cellular and biochemical consequences of the mutations in β-glucosidase 2 (GBA2), a GSL-metabolizing enzyme reported being mutated in SPG46, a complex neurodegenerative disorder. SPG46 (SPastic Gait locus #46) patients present with a combination of spastic paraplegia and cerebellar ataxia and suffer from muscle weakness and spasticity in the upper and lower limbs along with other neurological symptoms. Currently, the cascade of events leading from mutations in the GBA2 gene to SPG46 is largely unexplored. I have characterized ten SPG46-associated GBA2 mutations, five nonsense, and five missense mutations. All ten GBA2 mutants were catalytically inactive. The lack of enzyme activity was not associated with decreased protein expression. Native conformations of all but Gly683Arg mutants were different from the WT GBA2, confirmed by native gel electrophoresis, indicative of different protein-binding behaviors of the mutant proteins. Further, nonsense GBA2 mutants caused mitochondrial depolarization and interfered with mitochondria fusion, resulting in the formation of fragmented mitochondria. I have identified a potential mitochondrial targeting site in GBA2 causing conditional import of GBA2 in the mitochondrial matrix. My research unveils the roles of GBA2 on mitochondrial forms and functions which have not been observed before.