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

By 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. Codée, Gijsbert A. van der Marel and Herman S. Overkleeft

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 β-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.

Year: 2011
OAI identifier: oai:ub.rug.nl:dbi/5130a8bdf39f0
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