Application of activity-based probes to the study of enzymes involved in cancer progression

Many tumor cells have elevated levels of hydrolytic and proteolytic enzymes, presumably to aid in key processes such as angiogenesis, cancer cell invasion, and metastasis. Functional roles of enzymes in cancer progression are difficult to study using traditional genomic and proteomic methods because the activities of these enzymes are often regulated by post-translational mechanisms. Thus, methods that allow for the direct monitoring of enzyme activity in a physiologically relevant environment are required to better understand the roles of specific players in the complex process of tumorigenesis. This review highlights advances in the field of activity-based proteomics, which uses small molecules known as activity-based probes (ABPs) that covalently bind to the catalytic site of target enzymes. We discuss the application of ABPs to cancer biology, especially to the discovery of tumor biomarkers, the screening of enzyme inhibitors, and the imaging of enzymes implicated in cancer.     

Use of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed in Complex Cell Extracts

Background

High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities.

Methodology and Principal Findings

Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)₂-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z’>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates.

Conclusions

We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.     

Tagging and detection strategies for activity-based proteomics

The field of activity-based proteomics is a relatively new discipline that makes use of small molecules, termed activity-based probes (ABPs), to tag and monitor distinct sets of proteins within a complex proteome. These activity-dependant labels facilitate analysis of systems-wide changes at the level of enzyme activity rather than simple protein abundance. While the use of small molecule inhibitors to label enzyme targets is not a new concept, the past ten years have seen a rapid expansion in the diversity of probe families that have been developed. In addition to increasing the number and types of enzymes that can be targeted by this method, there has also been an increase in the number of methods used to visualize probes once they are bound to target enzymes. In particular, the use of small organic fluorophores has created a wealth of applications for ABPs that range from biochemical profiling of diverse proteomes to direct imaging of active enzymes in live cells and even whole animals. In addition, the advent of new bioorthogonal coupling chemistries now enables a diverse array of tags to be added after targets are labeled with an ABP. This strategy has opened the door to new in vivo applications for activity-based proteomic methods.     

Specificity of aza-peptide electrophile activity-based probes of caspases

Activity-Based Probes (ABPs) are small molecules that form stable covalent bonds with active enzymes thereby allowing detection and quantification of their activities in complex proteomes. A number of ABPs that target proteolytic enzymes have been designed based on well-characterized mechanism-based inhibitors. We describe here the evaluation of a novel series of ABPs based on the aza-aspartate inhibitory scaffold. Previous in vitro kinetic studies showed that this scaffold has a high degree of selectivity for the caspases, clan CD cysteine proteases activated during apoptotic cell death. Aza-aspartate ABPs containing either an epoxide or Michael acceptor reactive group were potent labels of executioner caspases in apoptotic cell extracts. However they were also effective labels of the clan CD protease legumain and showed unexpected crossreactivity with the clan CA protease cathepsin B. Interestingly, related aza peptides containing an acyloxymethyl ketone reactive group were relatively weak but highly selective labels of caspases. Thus azapeptide electrophiles are valuable new ABPs for both detection of a broad range of cysteine protease activities and for selective targeting of caspases. This study also highlights the importance of confirming the specificity of covalent protease inhibitors in crude proteomes using reagents such as the ABPs described here.     

Activity-based protein profiling in bacteria: Applications for identification of therapeutic targets and characterization of microbial communities

Activity-based protein profiling (ABPP) is a robust chemoproteomic technique that uses activity-based probes to globally measure endogenous enzymatic activity in complex proteomes. It has been utilized extensively to characterize human disease states and identify druggable targets in diverse disease conditions. ABPP has also recently found applications in microbiology. This includes using activity-based probes (ABPs) for functional studies of pathogenic bacteria as well as complex communities within a microbiome. This review will focus on recent advances in the use of ABPs to profile enzyme activity in disease models, screen for selective inhibitors of key enzymes, and develop imaging tools to better understand the host–bacterial interface.     

Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes

The field of biochemistry is currently faced with the enormous challenge of assigning functional significance to more than thirty thousand predicted protein products encoded by the human genome. In order to accomplish this daunting task, methods will be required that facilitate the global analysis of proteins in complex biological systems. Recently, methods have been described for simultaneously monitoring the activity of multiple enzymes in crude proteomes based on their reactivity with tagged chemical probes. These activity based probes (ABPs) have used either radiochemical or biotin/avidin-based detection methods to allow consolidated visualization of numerous enzyme activities. Here we report the synthesis and evaluation of fluorescent activity based probes for the serine hydrolase super-family of enzymes. The fluorescent methods detailed herein provide superior throughput, sensitivity, and quantitative accuracy when compared to previously described ABPs, and provide a straight-forward platform for high-throughput proteome analysis.     

Localization of active endogenous and exogenous β‐glucocerebrosidase by correlative light‐electron microscopy in human fibroblasts

β‐Glucocerebrosidase (GBA) is the enzyme that degrades glucosylceramide in lysosomes. Defects in GBA that result in overall loss of enzymatic activity give rise to the lysosomal storage disorder Gaucher disease, which is characterized by the accumulation of glucosylceramide in tissue macrophages. Gaucher disease is currently treated by infusion of mannose receptor‐targeted recombinant GBA. The recombinant GBA is thought to reach the lysosomes of macrophages, based on the impressive clinical response that is observed in Gaucher patients (type 1) receiving this enzyme replacement therapy. In this study, we used cyclophellitol‐derived activity‐based probes (ABPs) with a fluorescent reporter that irreversibly bind to the catalytic pocket of GBA, to visualize the active enzymes in a correlative microscopy approach. The uptake of pre‐labeled recombinant enzyme was monitored by fluorescence and electron microscopy in human fibroblasts that stably expressed the mannose receptor. The endogenous active enzyme was simultaneously visualized by in situ labeling with the ABP containing an orthogonal fluorophore. This method revealed the efficient delivery of recombinant GBA to lysosomal target compartments that contained endogenous active enzyme.      

A comparison of the cholinesterases of an oyster (Crassostrea virginica) and a clam (Macrocallista nimbosa).

Cholinesterase activities in the hearts and ganglia of an oyster (Crassostrea virginica) and a venerid clam (Macrocallista nimbosa) were measured and compared. Tissue extracts were partially purified by ammonium sulfate fractionation followed by gel column chromatography. Enzymatic activity was assayed spectrophotometrically; substrates were acetyl-, butyryl-, and propionylthiocholine (ATC, BTC, PTC). Kinetic constants characterizing each enzyme were derived. At all substrate concentrations, the hydrolysis rates of both clam enzymes were in the order: BTC greater than PTC greater than ATC. With oyster enzymes the ranking was ATC greater than or equal to PTC greater BTC. The specific activities of oyster heart and ganglion enzymes were similar. In contrast, clam ganglion extracts were 75-100 times more active than clam heart extracts and, with any substrate, had greater activity than either oyster enzyme. All enzyme preparations proved to be homogeneous on the bases of constant substrate activity ratios in successive column fractions, and of intermediate velocities with mixed substrates. Six cholinesterase inhibitors were tested. The specific acetylcholinesterase antagonist, B.W. 62C47, WAS MUCH MORE EFFECTIVE AGAINST OYSTER ENZYMES, WHILE THE SPECIFIC ANTIBUTYRYLCHOLINESTERASE, ISO-OMPA, almost totally inhibited calm enzyme activity, but had little effect on oyster. Eserine was the most effective inhibitor of both enzymes. In conclusion, the enzymes in oyster tissues are acetylcholinesterases, while clam enzymes are butyrylcholinesterases. Nevertheless, clam ganglion esterase is sifficiently active to hydrolyze the physiological substrate, acetylcholine. These results explain the long-observed differences in isolated heart pharmacology between ostreid and venerid bivalves.     

Alkyne derivatives of isocoumarins as clickable activity-based probes for serine proteases

Activity-based probes (ABPs) have found increasing use in functional proteomics studies. Recently, ABPs that can be employed in combination with click chemistry gained particular attention due to their flexible application in vitro and in vivo. Moreover, there is a continuous need for new ABPs that target small subsets of enzymes. We here report novel clickable ABPs based on the 4-chloro-isocoumarin (IC) electrophile, a mechanism-based inhibitor scaffold that covalently binds serine proteases. We describe the synthesis of a small library of IC ABPs containing an alkyne function and a set of diverse selectivity elements. The different substituents on the IC structure determine which proteases are bound, showing good correlation with the preferred substrate preferences. The IC ABPs can detect their target proteases in a proteome background in a sensitive manner (down to 0.007% of total protein). Furthermore, we show activity-dependent and selective labeling of endogenous proteases in a tissue proteome. These ICs therefore represent a valuable extension to already existing ABPs for serine proteases and may be instrumental in future elucidation of serine protease functions.     

Use of monoclonal antibodies to quantify the dynamics of alpha-galactosidase and endo-1,4-beta-glucanase production by Trichoderma hamatum during saprotrophic growth and sporulation in peat

Trichoderma species are ubiquitous soil and peat-borne saprotrophs that have received enormous scientific interest as biocontrol agents of plant diseases caused by destructive root pathogens. Mechanisms of biocontrol such as antibiosis and hyperparasitism are well documented and the biochemistry and molecular genetics of these processes defined. An aspect of biocontrol that has received little attention is the ability of Trichoderma species to compete for nutrients in their natural environments. Trichoderma species are efficient producers of polysaccharide-degrading enzymes that enable them to colonize organic matter thereby preventing the saprotrophic spread of plant pathogens. This study details the use of monoclonal antibodies (mAbs) to quantify the production of two enzymes implicated in the saprotrophic growth of Trichoderma species in peat. Using mAbs specific to the hemicellulase enzyme alpha-galactosidase (AGL) and the cellulase enzyme endo-1,4-beta-glucanase (EG), the relationship between the saprotrophic growth dynamics of a biocontrol strain of Trichoderma hamatum and the concomitant production of these enzymes in peat-based microcosms was studied. Enzyme activity assays and enzyme protein concentrations derived by enzyme-linked immunosorbent assay (ELISA) established the precision and sensitivity of mAb-based assays in quantifying enzyme production during active growth of the fungus. Trends in enzyme activities and protein concentrations were similar for both enzymes, during a 21-day sampling period in which active growth and sporulation of the fungus in peat was quantified using an independent mAb-based assay. There was a sharp increase in active biomass of T. hamatum 3 days after inoculation of microcosms with phialoconidia. After 3 days there was a rapid decline in active biomass which coincided with sporulation of the fungus. A similar trend was witnessed with EG activities and concentrations. This showed that EG production related directly to active growth of the fungus. The trend was not found, however, with AGL. There was a rapid increase in enzyme activities and protein concentrations on day 3, after which they remained static. The reason for the maintenance of elevated AGL probably resulted from secretion of the enzyme from conidia and chlamydospores. ELISA, immunofluoresence and immunogold electron microscopy studies of these cells showed that the enzyme is localized within the cytoplasm and is secreted extracellularly into the surrounding environment. It is postulated that release of oligosaccharides from polymeric hemicellulose by the constitutive spore-bound enzyme leads to AGL induction and could act as an environmental cue for spore germination.     

A Sensitive Gel-based Method Combining Distinct Cyclophellitol-based Probes for the Identification of Acid/Base Residues in Human Retaining β-Glucosidases

Retaining β-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining β-glucosidases. Whereas β-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining β-glucosidases, β-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining β-glucosidases by the combined use of cyclophellitol β-epoxide- and β-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent β-aziridine but not β-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the β-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining β-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining β-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.     

Specific inhibition of post proline cleaving enzyme by benzyloxycarbonyl-Gly-Pro-diazomethyl ketone

N-Benzyloxycarbonyl-Gly-Pro-diazomethyl ketone (Z-Gly-Pro-CHN2) was synthesized and tested as inhibitor of the post proline cleaving enzyme from bovine brain. The compound was found to inactivate the enzyme completely and irreversibly at low concentrations (0.3 microM) without affecting other proteolytic enzymes such as post proline dipeptidyl aminopeptidase, pyroglutamate aminopeptidase or trypsin. Substrates of post proline cleaving enzymes such as luliberin (LH-RH; pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and Benzyloxycarbonyl-Gly-Pro-Ala protected the enzyme from the reaction with Z-Gly-Pro-CHN2. Thus, Z-Gly-Pro-CHN2 seems to be an active site directed, specific inhibitor of post proline cleaving enzyme. When administered intraperitoneally to rats, this inhibitor (8 mg/kg) completely inactivated the post proline cleaving enzyme in all tissues studied including brain. Therefore, Z-Gly-Pro-CHN2 should be a valuable tool for studies on the physiological function of this enzyme within the metabolism of neuropeptides.     

Cholesterol-Esterifying Enzymes in Developing Rat Brain

Abstract: A cholesterol-esterifying enzyme which incorporates exogenous fatty acids into cholesterol esters in the presence of ATP and coenzyme A was demonstrated in 15-day-old rat brain. This enzyme was maximally active at pH 7.4 and distinct from the cholesterol-esterifying enzyme reported earlier (Eto and Suzuki, 1971), which has a pH optimum at 5.2 and does not require cofactors. Properties of the two enzymes have been compared. Both the enzymes showed negligible esterification with acetate and were maximally active with oleic acid. The pH 5.2 enzyme esterified desmosterol, lanosterol and cholesterol at about the same rate, while the pH 7.4 enzyme was only 50% as active with lanosterol as it was with cholesterol and desmosterol. Phosphatidyl serine stimulated the pH 5.2 enzyme but not the pH 7.4 enzyme. Phosphatidyl choline and sodium taurocholate showed no effect on either of the enzymes. Both the enzymes were associated with particulate fractions, but the pH 7.4 enzyme was localized more in the microsomes. Purified myelin showed 2.6-fold and 1.5-fold higher specific activities of pH 5.2 and 7.4 enzymes respectively, when compared with homogenate. About 7–10% of total activity of both the enzymes was associated with purified myelin. Brain stem and spinal cord showed higher specific activity of pH 5.2 enzyme than cerebral cortex and cerebellum, while pH 7.4 enzyme specific activity was higher in cerebellum and brain stem than in cerebral cortex and spinal cord. Microsomal pH 7.4 activity showed progressive increase prior to the active period of myelination, reaching a maximum on the 15th day after birth and declined to 20% of the peak activity by 30 days. In contrast, pH 5.2 enzyme reached maximum activity about the 6th day after birth and remained at this level well into adulthood. In 15-day-old rat brain, pH 7.4 enzyme had five to six times higher specific activity than pH 5.2 enzyme, while in adults the activities were equal. The pH 7.4 enzyme showed a threefold higher specific activity than pH 5.2 enzyme in myelin from 15-day-old rats, but in adults the reverse was true.     

The presence of desulfo xanthine dehydrogenase in purified and crude enzyme preparations from rat liver

Crude and purified xanthine dehydrogenase preparations from rat liver were examined for the existence of a naturally occurring inactive form. Reduction of the purified enzyme by xanthine under anaerobic conditions proceeded in two phases. The enzyme was inactivated by cyanide, which caused the release of a sulfur atom from the molybdenum center as thiocyanate. The amount of thiocyanate released was almost in parallel with the initial specific activity. The active and inactive enzymes could be resolved by affinity chromatography on Sepharose 4B/folate gel. These results provided evidence that the purified enzyme preparation from rat liver contained an inactive form. A method for the determination of the active and inactive enzymes in crude enzyme preparations from rat liver was devised based on the fact that only active enzyme could react with [14C]allopurinol and both active and inactive enzymes could be immunoprecipitated quantitatively by excess specific antibody to xanthine dehydrogenase. The amount of [14C]alloxanthine (derived from [14C]allopurinol) bound to the active sulfo enzyme in crude rat liver extracts was about 0.5 mol/mol of FAD. As this content is closely similar to that in the purified enzyme, these results suggest the existence of an inactive desulfo form in vivo.     

Bacterial xanthine oxidase from Arthrobacter S-2.

Arthrobacter S-2, originally isolated by enrichment on xanthine, produced high levels of xanthine oxidase activity, requiring as little as a 20-fold purification to approach homogeneity with some preparations. Molecular oxygen, ferricyanide, and 2,6-dichlorophenol-indophenol served as electron acceptors, but nicotinamide adenine dinucleotide did not. The enzyme was relatively specific when compared with previously studied xanthine-oxidizing enzymes, but at least one purine was observed to be oxidized at each of the three positions of the purine ring that have been subject to oxidation by this type of enzyme. The enzyme had a relatively high Km for xanthine (1.3 X 10(-4) M), and substrate inhibition was not observed with this compound, in contrast to the enzyme from cow's milk. In fact, an opposite effect was observed, and double-reciprocal plots with xanthine as the variable substrate showed a concave downward deviation at high concentrations. At 2.5 mM xanthine the enzyme had a specific activity approximately 50 times that of the most active preparations of the milk enzyme. The spectrum of the Arthrobacter enzyme resembled that of milk xanthine oxidase, suggesting a similarity of the prosthetic centers of the two enzymes. The bacterial enzyme was relatively small and may be dimeric, with approximate native and subunit molecular weights of 146,000 and 79,000, respectively.     

The comparative characteristics of the biological and immunomodulating activities of enzyme preparations of microbial origin]

A number of enzyme preparations of microbial origin such as immunomodulators and immunostimulators have been studied in vitro according to reactions of rosette technique of cell (ROC) active and general, adhesion, activation of a complement and also the spontaneous activity and phagocytosis of neutrophils have been determined. The enzyme preparations of microbial origin have been shown to be capable of causing immunomodulation and of influencing the specific and nonspecific immune response. Unlike the enzymes of animal origin the enzymes of microbial origin are effective in smaller concentrations. The studied preparations modulate the immune response differently according to ROC, adhesion. Each of preparations has specific features of biological activity--bacterio-stationary in reaction to a certain group of microorganisms and some specific features of reaction with cell and levels of immune reactions.     

Active enzyme sedimentation, sedimentation velocity, and sedimentation equilibrium studies of succinyl-CoA synthetases of porcine heart and Escherichia coli

Succinyl-CoA synthetases from Escherichia coli and porcine heart muscle have been viewed as prototypes of two classes of the enzyme. The bacterial enzyme has been reported to be an alpha 2 beta 2 tetramer, with many suggestions in the literature for cooperative interactions between active sites that may contribute to its catalytic efficacy. In contrast, gel filtration experiments of others have indicated that the heart enzyme is a simple alpha beta dimer, with no evidence of dimerization or interaction between like sites. All previous estimates of molecular size of these enzymes have been carried out at concentrations that are much higher than those that are used during activity measurements. The present study was carried out to confirm the differences in the quaternary structures of these two species of succinyl-CoA synthetase and to extend our knowledge of these structures to very low concentrations to enable correlation of their subunit structures with their catalytic properties. Conventional sedimentation velocity centrifugation with both enzymes indicates behavior typical of noninteracting globular proteins with no evidence of size heterogeneity. The sedimentation coefficients at infinite dilution (s20,w) have been determined to be 7.04 S and 4.55 S for the E. coli and porcine heart enzymes, respectively. Sedimentation velocity measurements have been extended to very low enzyme concentrations (typical of those used in activity measurements) by active enzyme centrifugation experiments, in which we have determined the rate of sedimentation of a zone of active enzyme through a chromogenic substrate solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Examination of the role of arginine-143 in the human copper and zinc superoxide dismutase by site-specific mutagenesis

The active site arginine-143 of human Cu,Zn superoxide dismutase has been replaced by lysine or by isoleucine. The mutant proteins were expressed at high levels in yeast, purified, and the amino acid substitution explored through the use of group specific reagents. The specific activities of these enzymes, measured by the xanthine oxidase/cytochrome c method and by using dry weight determination to establish protein concentration, were: native enzyme, 6570 units/mg; Lys-substituted enzyme, 2840 units/mg, Ile-substituted enzyme, 708 units/mg. The active site arginine thus plays an important, but not an essential, role in the catalytic process.     

Comparative Assessment of Substrates and Activity Based Probes as Tools for Non-Invasive Optical Imaging of Cysteine Protease Activity

Recent advances in the field of non-invasive optical imaging have included the development of contrast agents that report on the activity of enzymatic targets associated with disease pathology. In particular, proteases have proven to be ideal targets for development of optical sensors for cancer. Recently developed contrast agents for protease activity include both small peptides and large polymer-based quenched fluorescent substrates as well as fluorescently labeled activity based probes (ABPs). While substrates produce a fluorescent signal as a result of processing by a protease, ABPs are retained at the site of proteolysis due to formation of a permanent covalent bond with the active site catalytic residue. Both methods have potential advantages and disadvantages yet a careful comparison of substrates and ABPs has not been performed. Here we present the results of a direct comparison of commercially available protease substrates with several recently described fluorescent ABPs in a mouse model of cancer. The results demonstrate that fluorescent ABPs show more rapid and selective uptake into tumors as well as overall brighter signals compared to substrate probes. These data suggest that the lack of signal amplification for an ABP is offset by the increased kinetics of tissue uptake and prolonged retention of the probes once bound to a protease target. Furthermore, fluorescent ABPs can be used as imaging reagents with similar or better results as the commercially available protease substrates.     

Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes

Gaucher disease is characterized by lysosomal accumulation of glucosylceramide due to deficient activity of lysosomal glucocerebrosidase (GBA). In cells, glucosylceramide is also degraded outside lysosomes by the enzyme glucosylceramidase 2 (GBA2) of which inherited deficiency is associated with ataxias. The interest in GBA and glucosylceramide metabolism in the brain has grown following the notion that mutations in the GBA gene impose a risk factor for motor disorders such as α-synucleinopathies. We earlier developed a β-glucopyranosyl-configured cyclophellitol-epoxide type activity based probe (ABP) allowing in vivo and in vitro visualization of active molecules of GBA with high spatial resolution. Labeling occurs through covalent linkage of the ABP to the catalytic nucleophile residue in the enzyme pocket. Here, we describe a method to visualize active GBA molecules in rat brain slices using in vivo labeling. Brain areas related to motor control, like the basal ganglia and motor related structures in the brainstem, show a high content of active GBA. We also developed a β-glucopyranosyl cyclophellitol-aziridine ABP allowing in situ labeling of GBA2. Labeled GBA2 in brain areas can be identified and quantified upon gel electrophoresis. The distribution of active GBA2 markedly differs from that of GBA, being highest in the cerebellar cortex. The histological findings with ABP labeling were confirmed by biochemical analysis of isolated brain areas. In conclusion, ABPs offer sensitive tools to visualize active GBA and to study the distribution of GBA2 in the brain and thus may find application to establish the role of these enzymes in neurodegenerative disease conditions such as α-synucleinopathies and cerebellar ataxia.