Ultrasensitive in situ visualization of active glucocerebrosidase molecules

Abstract

Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.

Spiro‐epoxyglycosides as Activity‐Based Probes for Glycoside Hydrolase Family 99 Endomannosidase/Endomannanase

“…The samples were then denatured with 5× Laemmli buffer (50 % (v/v) 1  m Tris‐HCl, pH 6.8, 50 % (v/v) 100 % (v/v) 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 electrophoresis on 10 % (w/v) SDS‐PAGE gel running continuously at 90 V 43. Wet slab‐gels were scanned on fluorescence using a Typhoon FLA 9500 (GE Healthcare at λ EX 532 nm and λ EM 575 nm for ABP TB340; and at λ EX 635 nm and λ EM 665 nm for 4 and 5 .…”

A Specific Activity‐Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

“…For this purpose, we used β-galactosidase ABP 3 and the previously reported β-glucosidase ABP 5 [28] ( Figure 1C) to label either recombinant GALC or recombinant glucocerebrosidase, a lysosomal retaining β-glucosidase. The two ABPs are functionalized with different Bodipy fluorophores and can therefore be visualized using different scanner settings for in-gel fluorescent readout.…”

“…[25][26][27] In our first forays into activity-based glycosidase profiling, however, we studied cyclophellitol derivatives modified at C6 (glucopyranose numbering) with a fluorophore. [28] These epoxide probes exhibit much higher selectivity than their aziridine analogues. Because of their high selectivity and potency for human lysosomal glucosylceramidase (GBA) -the enzyme deficient in Gaucher patients -we now routinely use the fluorescent cyclophellitol derivatives to monitor GBA activity in vitro, in situ and in vivo in healthy and Gaucher models.…”

“…Because of their high selectivity and potency for human lysosomal glucosylceramidase (GBA) -the enzyme deficient in Gaucher patients -we now routinely use the fluorescent cyclophellitol derivatives to monitor GBA activity in vitro, in situ and in vivo in healthy and Gaucher models. [28] We realized that this probe design might also hold potential for the development of a GALC ABP suitable for monitoring this enzyme in the context of Krabbe disease ( Figure 1B), the more so since GALC and GBA have related glycosylceramide substrates. Here we describe the evaluation of β-galactopyranose-configured epoxides 1-4 as inhibitors and ABPs for GALC ( Figure 1C).…”

“…The non-tagged inhibitor 1 was included in our studies as a galactose-configured isomer of the known retaining β-glucosidase inhibitor cyclophellitol. [24,28] For potential use in two-step activity based profiling studies, in ABP 2 the primary hydroxyl group is substituted with an azide that can be used for two-step labeling via copper(I)-catalyzed or copper-free strain promoted alkyne-azide [2+3] cycloaddition chemistry or Staudinger-Bertozzi ligation. In addition, this probe may serve as a control probe and an inhibitor.…”