(15-19) Notably, the intralysosomal stability of N370S GBA is also reduced. This amino acid substitution is in a loop close to the catalytic pocket and was found to affect the enzyme’s pH optimum and its kinetic parameters such as affinity for substrates and inhibitors. (14) An exception is the N370S GBA substitution, which is the most prevalent Gba1 mutation among caucasian GD patients. In fact, many of the documented mutations in GBA lead to defective folding and reduced transport to lysosomes. (11) Premature degradation may also occur in the case of GBA molecules with mutations in the catalytic domain. (13) Homozygosity for L444P GBA nearly always leads to a severe neuronopathic course of GD, albeit with great individual variability in onset and progression. For instance, the L444P substitution in GBA causes faulty folding of most of its molecules in the ER, followed by subsequent proteasomal degradation. (12) Substitutions in the GBA polypeptide chain distant from the catalytic site destabilized GBA’s structure, decreasing its half-life in the cell. (11) At present, more than 200 mutations in the Gba1 gene have been linked with GD, and next to truncations and splicing defects, several hundred amino acid substitutions in GBA have been shown to cause GD. (10)ĭeficiency of GBA results in Gaucher disease (GD, OMIM #203800). Also, an aglycon binding pocket is present, formed by a narrow hydrophobic channel in which the aliphatic tail of the inhibitor forms a cluster of hydrophobic interactions with residues Leu241, Phe246, Tyr313, Leu314, and Tyr244 ( Figure 2d). (9) Inspection of the GBA crystal structure in complex with N-nonyldeoxynojirimycin (NN-DNJ) (PDB code: 2V3E) shows that the binding site of the enzyme is formed by a hydrophilic glycon binding pocket, where the sugar ring of the inhibitor is accommodated, forming multiple hydrogen bonds with its surrounding residues. (8) These seem to adopt multiple conformations, indicating their structural flexibility and presumably reflecting their crucial role in the enzyme’s conformational stability and/or its substrate turnover. The catalytic domain contains several loops located in the proximity of the enzyme’s active site. GBA belongs to family 30 of glycoside hydrolase clan A ( its structural topology displays a typical (α/β) 8TIM barrel fold that forms the catalytic domain, a β-sheets domain, and an immunoglobulin-like domain. Our study provides further rationale for the design of chaperones for GBA to ameliorate Gaucher disease. These effects are more pronounced in the case of the amphiphilic ABPs, presumably due to their high lipophilic potential, which may promote further structural compactness of GBA through hydrophobic interactions. In conclusion, we provide evidence that the increase in cellular levels of GBA by ABPs and by the reversible inhibitor is in part caused by their ability to stabilize GBA folding, which increases the resistance of GBA against breakdown by lysosomal proteases. Stabilization effects similar to those observed with ABPs were also noted at high concentrations of the reversible inhibitor isofagomine.

The same stabilization effect is observed for endogenous GBA in the liver of wild-type mice injected with cyclophellitol ABPs.

The stabilizing effect of cell-permeable cyclophellitol ABPs is also observed in intact cultured cells containing wild-type GBA, N370S GBA (labile in lysosomes), and L444P GBA (exhibits impaired ER folding): all show marked increases in lysosomal forms of GBA molecules upon exposure to ABPs. We demonstrate that cyclophellitol ABPs improve the stability of GBA in vitro, as revealed by thermodynamic measurements ( T m increase by 21 ☌), and introduce resistance to tryptic digestion.

For this, we made use of cyclophellitol-derived activity-based probes (ABPs) that bind irreversibly to the catalytic nucleophile (E340), and for comparison, we used the potent reversible inhibitor isofagomine. We examined the effects of active site occupancy of GBA on its structural stability. Its deficiency results in Gaucher disease (GD). Glucocerebrosidase (GBA) is a lysosomal β-glucosidase that degrades glucosylceramide.

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