Activity-Based Profiling of Retaining beta-Glucosidases: A Comparative Study

Martin D. Witte, Marthe T. C. Walvoort, Kah-Yee Li, Wouter W. Kallemeijn, Wilma E. Donker-Koopman, Rolf G. Boot, Johannes M. F. G. Aerts, Jeroen D. C. Codee, Gijsbert A. van der Marel*, Herman S. Overkleeft

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Activity-based protein profiling (ABPP) is a versatile strategy to report on enzyme activity in vitro, in situ, and in vivo. The development and use of ABPP tools and techniques has met with considerable success in monitoring physiological processes involving esterases and proteases. Activity-based profiling of glycosidases, on the other hand, has proven more difficult, and to date no broad-spectrum glycosidase activity-based probes (ABPs) have been reported. In a comparative study, we investigated both 2-deoxy-2-fluoroglycosides and cyclitol epoxides for their utility as a starting point towards retaining beta-glucosidase ABP. We also investigated the merits of direct labeling and two-step bio-orthogonal labeling in reporting on glucosidase activity under various conditions. Our results demonstrate that 1) in general cyclitol epoxides are the superior glucosidase ABPs, 2) that direct labeling is the more efficient approach but it hinges on the ability of the glucosidase to be accommodated in the active site of the reporter (BODIPY) entity, and 3) that two-step bio-orthogonal labeling can be achieved on isolated enzymes but translating this protocol to cell extracts requires more investigation.

Original languageEnglish
Pages (from-to)1263-1269
Number of pages7
JournalChemBioChem
Volume12
Issue number8
DOIs
Publication statusPublished - 16-May-2011

Keywords

  • activity-based probes
  • bodipy fluorophores
  • carbohydrates
  • click chemistry
  • glucosidases
  • GLYCOSIDE SPLITTING ENZYMES
  • GAUCHER-DISEASE
  • MECHANISM
  • INHIBITORS
  • PROBES
  • CYCLOPHELLITOL
  • GLUCOCEREBROSIDASE
  • IDENTIFICATION
  • INTERMEDIATE
  • NUCLEOPHILE

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