Lysosomal acid glucosylceramidase (GBA1) is a lysosomal enzyme that degrades glucolipids with its main substrate being glucosylceramide (GlcCer). Defects in the GBA1 gene lead to... Show moreLysosomal acid glucosylceramidase (GBA1) is a lysosomal enzyme that degrades glucolipids with its main substrate being glucosylceramide (GlcCer). Defects in the GBA1 gene lead to glycosphingolipidosis Gaucher disease (GD), in which the hydrolysis of GlcCer is impaired and therefore, it accumulates in the lysosome. GD has a wide range of phenotypes that reaches from asymptomatic to neuropathologic manifestations, which are lethal within the first years of life. Although various GBA1 mutations have been identified, they cannot be systematically matched with GD phenotypes. Therefore, this thesis aims to get a better understanding of GBA1 and its role in GD by identifying GBA1’s cellular interaction partners with the aid of an enrichment assay. For this assay, photocleavable activity-based probes (ABPs) were developed, which ought to be able to isolate GBA1 from a cell lysate together with its interaction partners with the aid of streptavidin-coated beads. By photocleaving the complex off the beads, the background of the GBA1-containing samples can be lowered. So far, GBA1 could successfully be isolated from a cell lysate with the developed ABPs. Next, the assay conditions need to be optimized for isolation of GBA1 together with its interaction partners. Show less
Within this thesis the central stage is taken by the discovery and investigation of transglycosylation of sterols. First, investigation focuses on the development of a method to accurately detect... Show moreWithin this thesis the central stage is taken by the discovery and investigation of transglycosylation of sterols. First, investigation focuses on the development of a method to accurately detect and quantify glucosylated metabolites in biological materials. Next, the studies concentrate on the formation and occurrence of specific glucosylated metabolites, in particular glucosyl-desmosterol (GlcDesm), glucosyl-7-dehydrocholesterol (Glc7DHC) and glucosylated vitamin D3 (GlcD3). Show less
The research described in this thesis combines the latest insights in lysosomal function with lysosome centred cell signalling. Novel imaging and labelling techniques are applied to provide in... Show moreThe research described in this thesis combines the latest insights in lysosomal function with lysosome centred cell signalling. Novel imaging and labelling techniques are applied to provide in depth characterization of lysosome function in health and disease. An integrative approach was used to study the physiological role of the lysosome, characterizing the function of lysosomal hydrolases and signalling on a cellular level as well as within the context of tissue. Show less
Lysosomal storage disorders (LSDs) are a group of orphan diseases characterized by lysosomal dysfunction or impaired lysosomal catabolism and affect collectively about 1 in 5000 live births. A... Show moreLysosomal storage disorders (LSDs) are a group of orphan diseases characterized by lysosomal dysfunction or impaired lysosomal catabolism and affect collectively about 1 in 5000 live births. A common LSD is Gaucher disease, which is characterized by a defect in glucocerebrosidase (GCase) degrading glucosylceramide (GlcCer) in lysosomes. In this thesis, the zebrafish is evaluated as vertebrate animal model for the investigation of lysosomal storage disorders, in particular Gaucher disease. Zebrafish are an appealing model organism to study genetic disorders with a high evolutionary conservation of genes and proteins compared to humans, easy maintenance and simple genetic and pharmacological manipulation. Zebrafish larvae are of particular use as zebrafish can generate hundreds of off-spring which have a rapid embryonal development, are transparent and fit in a 96-wells plate. In this thesis several biochemical and genetic techniques have been developed in order to 1) compare the catalytic features of zebrafish GCase with human GCase, 2) investigate the consequences of its defect in zebrafish larvae and adults as well as a concomitant defect in non-lysosomal GBA2 and 3) study the potential toxicity of excessive glucosylsphingosine during GCase deficiency as consequence of a defect in lysosomal acid ceramidase. GCase-deficient zebrafish showed similar symptoms and affected molecular mechanisms as patients and mouse models. Therefore the zebrafish offers exciting new possibilities to study molecular mechanisms underlying pathological processes during lysosomal hydrolase deficiencies. Show less
This thesis describes biochemical investigations of glucocerebrosidase (GBA), the lysosomal β- glucosidase that is deficient in Gaucher disease (GD). Central in the performed research was the... Show moreThis thesis describes biochemical investigations of glucocerebrosidase (GBA), the lysosomal β- glucosidase that is deficient in Gaucher disease (GD). Central in the performed research was the examination of factors influencing the intralysosomal stability and half-life of GBA. The investigations made use of new chemical biology tools such as activity based probes (ABPs) and photo-activatable and clickable (PAC) lipids.The Discussion reviews the present insights into GBA in health and disease. In this connection, the molecular basis and clinical manifestation of Gaucher disease and Action Myoclonus Renal Failure syndrome are discussed, including the metabolic adaptations to GBA deficiency. Particular attention is paid to the lysosomal structural stability of GBA and associated resistance against proteolytic degradation by cysteine cathepsins. Literature findings and novel own results on this topic are discussed. New technology to study GBA by labeling with GlcCer and cyclophellitol derived probes is introduced and the application is described. Unresolved research questions on GBA and related disease conditions are identified. As future research objective the translation of fundamental knowledge on GBA to effective therapy of neuronopathic Gaucher disease and other disease conditions caused by enzyme reduction are discussed. Show less
