Gaucher disease is one of the most common lipid storage disorders affecting 1 in 20,000 people worldwide. Patients suffer from liver and spleen enlargement and, in the most severe cases, impairment of the central nervous system function. The underlying cause is a deficiency in the lysosomal beta-glucosidase GBA1. GBA1 degrades the glycosphingolipid glucosylceramide (GlcCer) to glucose and ceramide. Gaucher disease patients suffer from a dramatic accumulation of GlcCer in the lysosomes, which causes enlargement of the cell and, thereby, enlargement of the whole organ. We could show that in patients with Gaucher disease not only the activity of GBA1 is diminished, but also the activity of a second beta-glucosidase, GBA2. This leads to an even more dramatic accumulation of GlcCer, suggesting that GBA2 contributes to the severity of Gaucher disease.
Complex glycosphingolipids are important components of the cell membrane. They are generated by the addition of sugar molecules to GlcCer, which serves as a precursor for more than 400 different glycosphingolipids. GlcCer itself is de novosynthesized from glucose and ceramide or it is generated from degradation of complex gycosphingolipids (Figure 1).
Figure 1: Glucosylceramide (Glccer) metabolism.
GBA1 degrades GlcCer in the lysosomes, the “stomach” of the cell. We could show that GBA2 is localized at the cytoplasmic site of the endoplasmic reticulum and the Golgi apparatus. Thus, degradation of GlcCer by GBA2 takes place close to where GlcCer is synthesized (Figure 2).
Figure 2: GBA2 is localized at the outside of the endoplasmic reticulum (ER) and Golgi. A. Using fluorescence microscopy, we localized GBA2 (red) at the outside of the ER (green, top) and Golgi (green, bottom) in neurons. Nucleus: blueB. GBA1 is localized in the lysosomes, GBA2 at the cytoplasmic site of the ER and Golgi.
We developed a new assay to distinguish between the activity of GBA1 and GBA2. An assay that has been used for more than 40 years was not suitable to reliably distinguish between the two activities. This assay is performed at a mildly acidic pH (5.5) in the presence of detergent. The lysosomes, where GBA1 is localized, are acidic (pH4), whereas GBA2 is localized in an environment with neutral pH. Detergents shift the pH dependence of GBA1 to higher and of GBA2 to lower pH values. Thereby, the pH profile of the two activities overlaps, making it almost impossible to discriminate between GBA1 and GBA2. Thus, we have developed a detergent-free assay that measures the activity of GBA1 and GBA2 at different pH values (Figure 3).
Figure 3: A new assay to discriminate between the activity of GBA1 and GBA2.
Using specific inhibitors, we could show that our assay reliably discriminates between GBA1 and GBA2 activity. Furthermore, we could show that the activity of GBA2 in the absence of detergent is augmented. Thus, previous assays significantly underestimated the GBA2 activity.
We used this assay to determine the activity of GBA1 and GBA2 in Gaucher disease patients. Gaucher disease is clinically heterogeneous and can be classified into three principal subtypes. However, there is no clear genotype-phenotype correlation that allows to predict the severity of Gaucher disease-pathology and the factors that contribute to the severity of the disease are ill defined. We hypothesized that in patients with Gaucher disease, also GBA2 activity is impaired, causing an augmented accumulation of GlcCer. We could show that in dermal fibroblasts from Gaucher disease patients, not only GBA1 activity is severely diminished, but also GBA2 activity is clearly reduced. Similarly, the activity of GBA2 was also reduced in embryonic fibroblasts from GBA1 knockout mice that lack any GBA1 activity (Figure 4).
Figure 4: Beta-glucosidase activity in fibroblasts from a Gaucher disease patient and from GBA1 knockout mice. A. GBA1 and GBA2 activity in fibroblasts from a Gaucher disease and a control patient. B. GBA1 and GBA2 activity in embryonic fibroblasts from wild-type and GBA1 knockout mice.
We hypothesize that there is cross-talk between GBA1 and GBA2. However, the underlying mechanism has to be elucidated in further studies. Thus, GBA2 seems to play a more prominent role in lipid metabolism than was previously described and could serve as a target for the therapy of Gaucher disease.
Körschen, H.G., Yildiz, Y., Raju, D.N., Schonauer, S., Bönigk, W., Jansen, V., Kremmer, E., Kaupp, U.B. & Wachten, D. (2013) "The non-lysosomal beta-glucosidase GBA2 is a non-integral membrane-associated protein at the ER and Golgi" J. Biol. Chem. 288, 3388-3393