Date

Wednesday 08 Jul 2026

Time

15:15 - 15:45

Location

BW032

Supervisor

Rolf Boot

2nd reviewer

Hans Aerts

Jury

Marco van Eijk

One of the most common lysosomal storage disorders is Gaucher’s disease, which is caused by a deficiency in the enzyme glucocerebrosidase (GBA1). GBA1 is responsible for the degradation of glucosylceramide (GlcCer) in lysosomes and impaired GBA1 leads to an accumulation of GlcCer, and byproduct glucosylsphingosine (GlcSph), which results in hepatosplenomegaly, bone disease, and hematological abnormalities. C. elegans has proven to be a promising model organism to study GD, as it contains GBA1 like enzymes capable of degrading GlcCer. It has been shown that gba-3 deletion mutants lose glucocerebrosidase activity, suggesting that gba-3 encodes a GBA1-like enzymes. Previous research also showed that gba-4 was the most active of the C. elegans enzymes and was capable of hydrolyzing GlcCer, whereas for gba-3 no proof of GlcCer degradation has been found yet. Therefore, the aim of this project was to further characterize gba-3 and gba-4. A chitotriosidase signal peptide was attached to the enzymes. The gba-4 construct was successfully generated, whereas for gba-3 it was unsuccessful. Temperature-dependence activity assays were performed for the gba variants, showing an optimum similar to the human enzyme, which is similar to other metabolic enzymes of C. elegans. To further examine the activity of the gba-3 and gba-4 enzymes, chimeric human GBA1 constructs were made by replacing individual loops of hGBA1 with the corresponding loop sequences from C. elegans gba-3 or gba-4. The hydrolytic activity of the mutants was determined, and further quantification was done by activity-based protein profiling. It was found that replacement of the loops significantly reduced hydrolytic activity, indicating that these whole loops are important for maintaining enzymatic activity and that smaller mutations may be less disruptive and have a positive influence on enzymatic activity.