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

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.

Comparing Cyclophellitol N‐Alkyl and N‐Acyl Cyclophellitol Aziridines as Activity‐Based Glycosidase Probes

“…Inhibitory constants k i and K i ′ were determined by calculating the K obs. per ABP concentration and curve‐fitting the data to a one‐phase exponential association function (GraphPad Prism 5.0) 8a. 9–11…”

“…The eluted sample was separated, with 50 % being snap‐frozen in liquid nitrogen and stored at −20 °C, whereas the remaining 50 % was chased for 8 h, including hourly washing with the appropriate McIlvaine buffer, over a new Zeba column. Samples were denatured with 5× Laemmli buffer (50 % (v/v) 1 M Tris‐HCl, pH 6.8, 50 % (v/v) 100 % glycerol, 10 % (w/v) DTT, 10 % (w/v) SDS, 0.01 % (w/v) bromophenol blue), boiled for 4 min at 100 °C, and separated by gel electrophoresis on 10 % (w/v) SDS‐PAGE gels running continuously at 90 V 8a. 9–11 Wet slab‐gels were then scanned for ABP‐emitted fluorescence using a Typhoon TRIO variable mode imager (Amersham Biosciences) using λ EX =532 nm and λ EM =610 n M (band pass filter 30 nm) for red fluorescent ABPs 1 – 6 .…”

From Covalent Glycosidase Inhibitors to Activity‐Based Glycosidase Probes

“…Considering the successful application of both cyclitol epoxides and fluorinated glycosides as retaining glycosidase inhibitors, each class of compounds would in principle provide a good starting point for the development of ABPs. Although little in‐depth comparative studies have been reported62 regarding the selectivity and reactivity of these inhibitor classes, it is apparent that they differ considerably in binding mechanism and kinetics. The epoxide warhead in cyclophellitol and conduritol epoxides can be protonated by the general acid/base residue in the active site of a target enzyme, thereby promoting reaction with the catalytically active nucleophile.…”

Synthesis of Cyclophellitol, Cyclophellitol Aziridine, and Their Tagged Derivatives

“…[6] We have previously reported on our work on activitybased protein profiling of human retaining β-exoglucosidases. [7][8][9] In those studies we capitalized on the remarkable ability of compounds 1 and 2 to select and inactivate these glycosidases in complex biological samples, ranging from cell extracts to cells and animal models. Grafting a reporter group at C8 of cyclophellitol, through an azide group installed for this purpose at C8 [as in 8-deoxy-8-azidocyclophellitol (4), see Figure 2], allowed us to detect and image human acid glucosylceramidase, or GBA, with high selectivity and efficiency in the context of healthy and (Gaucher) diseased tissue.…”

“…Grafting a reporter group at C8 of cyclophellitol, through an azide group installed for this purpose at C8 [as in 8-deoxy-8-azidocyclophellitol (4), see Figure 2], allowed us to detect and image human acid glucosylceramidase, or GBA, with high selectivity and efficiency in the context of healthy and (Gaucher) diseased tissue. [7,8] Placing a reporter group on the cyclophellitol aziridine nitrogen, through its acyl congener 5, enabled us to achieve broad profiling of all human retaining β-glucosidase activities: in addition to GBA, nonlysosomal β-glucosidase (GBA2), cytosolic (broad-specificity) β-glucosidase (GBA3), and the β-glucosidase activity presented by the intestinal lactase-phlorizin hydrolase (LPH). [9] Figure 2.…”