Stabilization of Glucocerebrosidase by Active Site Occupancy

Fredj Ben Bdira, Wouter W. Kallemeijn*, Saskia V. Oussoren, Saskia Scheij, Boris Bleijlevens, Bogdan I. Florea, Cindy P. A. A. van Roomen, Roelof Ottenhoff, Marielle J. F. M. van Kooten, Marthe T. C. Walvoort, Martin D. Witte, Rolf G. Boot, Marcellus Ubbink, Herman S. Overkleeft, Johannes M. F. G. Aerts

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Glucocerebrosidase (GBA) is a lysosomal)beta-glucosidase that degrades glucosylceramide. Its deficiency results in Gaucher disease (GD). We examined the effects of active site occupancy of GBA on its structural stability. 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 demonstrate that cyclophellitol ABPs improve the stability of GBA in vitro, as revealed by thermodynamic measurements (T-m increase by 21 degrees C), and introduce resistance to tryptic digestion. 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. The same stabilization effect is observed for endogenous GBA in the liver of wild-type mice injected with cyclophellitol ABPs. Stabilization effects similar to those observed with ABPs were also noted at high concentrations of the reversible inhibitor isofagomine. 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. 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. Our study provides further rationale for the design of chaperones for GBA to ameliorate Gaucher disease.

Original languageEnglish
Pages (from-to)1830-1841
Number of pages12
JournalACS chemical biology
Volume12
Issue number7
DOIs
Publication statusPublished - Jul-2017

Keywords

  • ACID-BETA-GLUCOSIDASE
  • TYPE-1 GAUCHER-DISEASE
  • PHARMACOLOGICAL CHAPERONE
  • N370S MUTANT
  • INTRINSIC FLUORESCENCE
  • ISOFAGOMINE INCREASES
  • REPLACEMENT THERAPY
  • CIRCULAR-DICHROISM
  • IN-SITU
  • X-RAY

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