Abstract

Gaucher disease (GD) is a rare lysosomal storage disorder caused by mutations in the GBA1 gene, encoding the lysosome-resident glucocerebrosidase enzyme involved in the hydrolysis of glucosylceramide. The discovery of an association between mutations in GBA1 and the development of synucleinopathies, including Parkinson disease, has directed attention to glucocerebrosidase as a potential therapeutic target for different synucleinopathies. These findings initiated an exponential growth in research and publications regarding the glucocerebrosidase enzyme. The use of various commercial and custom-made glucocerebrosidase antibodies has been reported, but standardized in-depth validation is still not available for many of these antibodies. This work details the evaluation of several previously reported glucocerebrosidase antibodies for western blot analysis, tested on protein lysates of murine gba+/+ and gba−/− immortalized neurons and primary human wild-type and type 2 GD fibroblasts.

Introduction

The lysosome-resident glucocerebrosidase (GCase) enzyme (EC 3.2.1.45), encoded by the glucocerebosidase 1 (GBA1) gene, catalyzes hydrolysis of its substrates glucosylsphingosine and glucosylceramide. Gaucher disease (GD; OMIM #606463) is a recessive lysosomal storage disorder caused by pathologic GBA1 mutations resulting in glucocerebrosidase enzyme deficiency and consequential lysosomal accumulation of substrate in cells of the reticulo-endothelial system [1].

After the publication of numerous smaller studies, in 2009, an association between mutations in GBA1 and Parkinson's disease was definitively established by a multicenter international collaborative study, showing that GBA1 mutations are now the most common genetic risk factor for the development of Parkinson's disease [2]. This finding launched an exponential growth in the number of studies researching the cellular mechanism underlying this association, as well as the potential of GCase as a therapeutic target for synucleinopathies [3]. Distinct commercial and custom-made antibodies to GCase have been used in various GCase-related studies, but in-depth antibody validation is often missing. For years, there has been a considerable discourse about and effort towards improving antibody reporting, validation, and reproducibility, as many fields are suffering from irreproducible and false discoveries due to poorly reported or validated antibodies [417]. Particularly, five strategies for antibody validation have been proposed, including genetic, orthogonal, independent antibody, tagged protein, and immunocapture/mass spectrometric strategies. Antibody validation through at least one of these pathways has been highlighted as the minimum criterion for claiming an antibody has been validated; the usage of multiple of these proposed techniques is thought to increase confidence in the conclusions drawn [4].

Here, we present the first comprehensive GCase antibody validation study based on western blot analyses of human and murine cells, making use of genetic, orthogonal, and independent antibody strategies. Specifically, we evaluated nine GCase antibodies for western blot analysis, seven commercially available and two custom-made (Table 1), on lysates prepared from previously described immortalized murine gba/ and gba+/+ neurons [18]. Furthermore, the antibodies were also tested on fibroblast cell lines derived from an infant with the most severe Gaucher phenotype, type 2 GD, that was known to have negligible GCase protein expression, as well as a control fibroblast line. This work should not only be of value for research on GD, but also for the synucleinopathies.

Table 1
The nine tested antibodies and recent publications clearly reporting their use in western blotting
Antibody Manufacturer Catalogue number RRID Recent uses 
β-Glucosidase (C-2) Santa Cruz Biotechnology sc-365745 AB_10846851 [22,23
β-Glucosidase (H-300) Santa Cruz Biotechnology sc-32883 AB_2109070 [29,47
Anti-glucocerebrosidase antibody Sigma–Aldrich G4046 AB_1078957 [25,27
Anti-glucocerebrosidase (C-terminal) antibody Sigma–Aldrich G4171 AB_1078958 [23,28,3037
Anti-GBA antibody [EPR5143(3)] Abcam Ab128879 AB_11144121 [3840
Anti-GBA antibody Abcam Ab55080 AB_2109076 [41,42
GBA antibody Origene TA325083 NA  
Monoclonal 8E4 GBA antibody Custom-made NA NA [4347
R386 polyclonal GBA antibody Custom-made NA NA [4850
Antibody Manufacturer Catalogue number RRID Recent uses 
β-Glucosidase (C-2) Santa Cruz Biotechnology sc-365745 AB_10846851 [22,23
β-Glucosidase (H-300) Santa Cruz Biotechnology sc-32883 AB_2109070 [29,47
Anti-glucocerebrosidase antibody Sigma–Aldrich G4046 AB_1078957 [25,27
Anti-glucocerebrosidase (C-terminal) antibody Sigma–Aldrich G4171 AB_1078958 [23,28,3037
Anti-GBA antibody [EPR5143(3)] Abcam Ab128879 AB_11144121 [3840
Anti-GBA antibody Abcam Ab55080 AB_2109076 [41,42
GBA antibody Origene TA325083 NA  
Monoclonal 8E4 GBA antibody Custom-made NA NA [4347
R386 polyclonal GBA antibody Custom-made NA NA [4850

Materials and methods

Antibody details

The epitope for the mouse monoclonal anti-β-glucosidase antibody (C-2) (Santa Cruz Biotechnology, cat. No. sc-365745) maps between amino acids 67–95 in the N-terminal region of the human GCase enzyme. This antibody is described as reacting with mouse, rat, and human GCase, and was used for western blot analysis at a concentration of 0.4 μg/ml (1/500 dilution). The recently discontinued rabbit polyclonal anti-β-glucosidase antibody (H-300) (Santa Cruz Biotechnology, cat. no. sc-32883) recognizes an epitope corresponding to amino acids 237–536 at the C-terminus of the human GCase enzyme. This antibody was used for western blot analysis at a concentration of 0.2 μg/ml (1/1000 dilution) and was expected to react with human and mouse GCase. The rabbit polyclonal anti-GCase antibody (Sigma, cat. no. G4046) was raised against a specific peptide corresponding to amino acids 83–100 of the human GCase enzyme. Based on sequence conservation, it is predicted to interact with human, mouse, and rat GCase. This antibody was used for western blot analysis at a concentration of 1 μg/ml (1/1000 dilution). The rabbit polyclonal anti-GCase antibody (Sigma, cat. no. G4171) was raised against a specific peptide corresponding to amino acids 517–536 of the human GCase enzyme. This sequence is identical in rat and differs by only one amino acid from mouse GCase. This antibody was used for western blot analysis at a concentration of 1 μg/ml (1/1000 dilution). The rabbit monoclonal anti-GCase antibody (Abcam, cat. no. ab128879) was raised against a nonspecified synthetic fragment corresponding to human GCase. This antibody, predicted to interact with human and rat, was used for western blot analysis at a 1/1000 dilution; the concentration of this antibody was not available on the datasheet. The rabbit monoclonal anti-GCase antibody (Abcam, cat. no. ab55080) was raised against a recombinant fragment corresponding to amino acids 146–236 of human GCase. This antibody is predicted to interact with human and was used for western blot analysis at a concentration of 1 μg/ml (1/1000 dilution). The rabbit polyclonal anti-GCase antibody (Origene, cat. no. TA325083) was raised against a synthetic peptide corresponding to amino acids 337–365 of human GCase. This antibody, predicted to interact with human, mouse, bovine, and pig, was used for western blot analysis at a concentration of 0.345 μg/ml (1/1000 dilution). The 8E4 mouse monoclonal anti-GCase antibody was raised against purified placental antibody to produce monoclonal antibodies directed against single antigenic determinants on GCase using a hybridoma technique. Myeloma cells were fused with spleen cells from a hyperimmunized mouse screened by ELISA, and the specificity was confirmed by immunoblotting and binding experiments [19]. This antibody was used for western blot analysis at a 1/1000 dilution. The rabbit polyclonal anti-GCase antibody R386 was raised in rabbit by injecting a preparation of human placental GCase and was previously described in detail [20]. The 8E4 antibody was used for western blot analysis at a 1/10 000 dilution.

The mouse monoclonal anti-actin antibody (Abcam, ab20272) conjugated to horseradish peroxidase (HRP) was raised against a synthetic peptide corresponding to amino acids 1–100 of human β-actin. This antibody was used for western blot analysis at a 1 : 5000 dilution; the concentration of this antibody was not available on the datasheet. For western blotting on the Odyssey LI-COR platform, we used the following secondary antibodies: IRDye 680RD goat anti-mouse IgG (LI-COR, 925-68070) and IRDye 680RD goat anti-rabbit IgG (LI-COR, 925-68071) at a concentration of 0.066 μg/ml (dilution of 1 : 15 000).

Cell culture

The establishment of immortalized gba+/+ and gba/ cortical mouse neurons was described in detail in recent work [6]. Briefly, the cells were maintained in poly-l-lysine-coated flasks in medium consisting of neurobasal growth medium (ThermoFisher Scientific, 21103-049), B27 (ThermoFisher Scientific, 17504-044, 1 : 50), 200 mM glutamine (ThermoFisher Scientific, 25030-081, 1 : 200), and penicillin/streptomycin (ThermoFisher Scientific, 15140-122, 1 : 100). 50% of the neurobasal growth medium was changed every 3 days.

Human wild-type (WT) primary fibroblasts and type 2 Gaucher fibroblasts (genotype IVS2 + 1G > A/L444P) were cultured in Dulbecco's modified Eagle high glucose Glutamax medium (ThermoFisher Scientific, 10566016), supplemented with 10% fetal bovine serum (ThermoFisher, 16000044) and penicillin/streptomycin (ThermoFisher Scientific, 15140-122, 1 : 100) at 37°C in a humidified 5% CO2.

Preparation of protein samples

Protein lysate was prepared using two different buffers, RIPA buffer or citrate phosphate buffer at pH 4.8 [6]. The RIPA lysis and extraction buffer were bought as a ready-to-use solution from ThermoFisher Scientific (cat. no. 89901). For citrate phosphate buffer, a 0.1 M citrate solution and a 0.2 M solution of dibasic sodium phosphate (Na2HPO4) were made in water. About 24.8 ml of 0.2 M Na2HPO4 was mixed with 25.2 ml of 0.1 M citrate. The pH of this solution was measured and adjusted to 4.8 using the above solutions. Triton X-100 [0.25% (v/v)] was added as well as a protease inhibitor cocktail (Sigma–Aldrich). Cells were scraped with a cell scraper in RIPA or citrate phosphate buffer. Cell lysates were transferred to an 1.5 ml Eppendorf tube, followed by sonication. Sonicated cell lysates were centrifuged at 5000g for 10 min at 4°C. We evaluated two widely used platforms for western blot read-out, namely HRP with ECL (Enhanced Chemiluminescence) and near-infrared fluorescence. The amount of protein for each sample was determined with the DC™ protein assay (Bio-Rad Laboratories, Hercules, CA, U.S.A.).

Expression analysis with GCase-specific chemical probe

For validation of GCase expression in murine and human cell lines, we used a previously described GCase-specific chemical probe (MDW933) (synthesized by the Imaging Probe Development Center, NHLBI, NIH) [21]. Total cell lysate (20 µg) made with citrate phosphate buffer, pH 4.8, was mixed with 3 µl of an 1 µM stock solution of MDW933 probe followed by the addition of NaAc buffer (pH 5.0) to obtain a total reaction volume of 30 µl. The mixture was incubated while shaking at 500 rpm for 90 min at 37°C. As a technical control, we used 60 nM imiglucease, which is recombinant GCase used for enzyme replacement therapy (Genzyme, Cambridge, MA, U.S.A.). After incubation, the samples were mixed with 10 µl 4× Laemmli sample buffer (Bio-Rab Laboratories) and loaded on an SDS–PAGE 4–20% Mini-PROTEAN® Precast Gel (Bio-Rad Laboratories). In-gel imaging of fluorescent signal was performed with a Typhoon Variable Mode Imager (Amersham Biosciences, Piscataway, NJ, U.S.A.) with λex at 488 nm and λem at 520 nm. Afterwards, protein from the gel was transferred to a PVDF membrane with the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories). The membrane was air dried for 30 min at RT and re-activated with 100% methanol for 1 min, followed by an 1 min wash in dH2O. The PVDF membrane was incubated for 90 min at RT with blocking solution [1× PBS, 0.5% (v/w) milk, 0.1% Tween 20 (Sigma–Aldrich, St. Louis, MO, U.S.A.)]. This was followed by an overnight incubation with the blocking solution containing anti-β-actin conjugated with HRP (Cat# 20272, Sigma, 1 : 4000) overnight at 4°C. The PVDF membrane was washed (3 × 5 min) with blocking solution followed by washing (3 × 5 min) with 1× PBS, 0.1% Tween 20. The β-actin was detected with an ECL Kit (Amersham Biosciences) using the ChemiDoc XRS+ Imager System (Bio-Rad).

Western blotting: chemiluminescence platform

For each sample, 20 µg of protein was loaded onto a 4–20% Mini-PROTEAN® TGX™ gel (Bio-Rad Laboratories). After blotting with the Trans-Blot Turbo transfer system (Bio-Rad Laboratories), PVDF membranes (Bio-Rad Laboratories) were blocked for 1 h at RT with blocking solution [1× PBS, 0.5% (v/w) milk, 0.1% Tween]. PVDF membranes were probed overnight with primary antibodies in blocking solution at 4°C. PVDF membranes were washed 3 × 5 min at RT with blocking solution. This was followed by incubation with HRP-coupled secondary anti-mouse or anti-rabbit antibodies (Amersham Biosciences, Piscataway, NJ, U.S.A., NA931 and NA934, respectively, 1 : 4000). PVDF membranes were washed 3 × 5 min with blocking solution followed by 3 × 5 min with 1× PBS plus 0.1% Tween. The antigen–antibody complexes were detected with an ECL kit (Amersham Biosciences) using the ChemiDoc XRS+ Imager System (Bio-Rad). Blots were exposed for 30 s and images were generated every 2 min.

Western blotting: Odyssey LI-COR platform

For each sample, 20 µg of protein was loaded onto a 4–20% Mini-PROTEAN® TGX™ gel (Bio-Rad Laboratories). After blotting with the Trans-Blot Turbo transfer system (Bio-Rad Laboratories), PVDF membranes (Bio-Rad Laboratories) were dried for 30 min, and this was followed by activation of the PVDF membrane with 100% methanol for 30 s, washing with dH2O and blocking in Odyssey buffer for 1 h at room temperature (LI-COR Biosciences, Lincoln, NE, U.S.A.). PVDF membranes were probed overnight with primary antibodies in Odyssey blocking buffer + 0.1% Tween-20 (Sigma–Aldrich) at 4°C. PVDF membranes were washed 3 × 5 min at RT with 1× PBS + 0.1% Tween-20. This was followed by incubation with IRDye 680RD-labeled secondary anti-mouse or anti-rabbit antibodies (LI-COR Biosciences, 1 : 15 000) for 1 h at room temperature. PVDF membranes were washed 3 × 5 min with 1× PBS plus 0.1% Tween (Sigma–Aldrich) followed by 3 × 5 min with 1× PBS. The antigen–antibody complexes were detected on an Odyssey CLx imaging system with an IR 680 laser (LI-COR Biosciences).

Western blotting: actin protein detection

For detection of actin protein on both western blotting platforms, the PVDF membranes were stripped with Restore Western Blot Stripping Buffer (ThermoFisher Scientific) for 10 min at room temperature. This was followed by an 1-h incubation with anti-β-actin conjugated with HRP (Sigma–Aldrich, cat. no. 20272, 1 : 4000) in 1× PBS, 0.5% (v/w) milk, 0.1% Tween overnight at 4°C. The PVDF membrane was washed (6 × 5 min) with 1× PBS, 0.1% Tween 20. The β-actin was detected with an ECL Kit (Amersham Biosciences) under the ChemiDoc XRS+ Imager System (Bio-Rad).

Each western blot experiment was performed in duplicate.

Results

Validation of GCase expression in murine and human cells

In the present study, we used previously described gba+/+ and gba/ immortalized murine cortical neurons as well as control and GD type 2 human fibroblasts to establish the specificity and species reactivity of a variety of anti-GCase antibodies for western blotting. We used two different buffers to prepare protein lysates, RIPA and citrate phosphate, the latter being frequently used in GCase enzyme activity assays. First, we validated GCase expression in the murine and human cell lines with a previously established GCase-specific inhibody (MDW933) [21]. GCase protein was detected in citrate phosphate protein lysates from gba+/+ mouse neurons (Figure 1A, lane 1) and control human fibroblasts (Figure 1B, lane 1), while no signal could be detected in protein lysates from immortalized gba−/− mouse neurons (Figure 1A, lane 2) and GD type 2 human fibroblasts (Figure 1B, lane 2). Imiglucerase, a commercially available recombinant GCase enzyme, was used as a positive control for GCase detection with MDW933 (Figure 1A,B, lanes 3). Inhibody experiments established the absence of GCase expression in gba/ mouse neurons and GD type 2 human fibroblasts, which was used as a strict criterion for antibody specificity in the antibody validation experiments.

GCase expression in cell lysates from immortalized mouse neurons and human fibroblasts detected with inhibody MDW933.

Figure 1.
GCase expression in cell lysates from immortalized mouse neurons and human fibroblasts detected with inhibody MDW933.

(A) MDW933 incubated with citrate phosphate cell lysates from immortalized mouse gba+/+ neurons (lane 1) and gba−/− neurons (lane 2). (B) MDW933 incubated with citrate phosphate cell lysates from human WT fibroblast (lane 1) and GD type 2 fibroblasts (lane 2). Recombinant Imiglucerase was used as an experimental control (lanes 3A and 3B). Actin detection with an anti-actin antibody was used as a protein loading control.

Figure 1.
GCase expression in cell lysates from immortalized mouse neurons and human fibroblasts detected with inhibody MDW933.

(A) MDW933 incubated with citrate phosphate cell lysates from immortalized mouse gba+/+ neurons (lane 1) and gba−/− neurons (lane 2). (B) MDW933 incubated with citrate phosphate cell lysates from human WT fibroblast (lane 1) and GD type 2 fibroblasts (lane 2). Recombinant Imiglucerase was used as an experimental control (lanes 3A and 3B). Actin detection with an anti-actin antibody was used as a protein loading control.

Validation of GCase antibodies

Blots of mouse and human samples, loaded with 20 micrograms of protein for each sample, were probed with nine-different anti-GCase antibodies (Table 1), many of them used in previous studies [2250]. The specificity of each antibody was tested in protein extracts from cells made in RIPA and citrate phosphate buffer and on two different western blot platforms, ECL and near-infrared fluorescence (LI-COR).

Antibody #1: anti-β glucosidase C-2 mouse monoclonal antibody Santa Cruz sc-365745

The anti-β glucosidase C-2 monoclonal antibody did not show specificity for GCase, typically present at ∼60 kDa, in RIPA and citrate phosphate protein lysates from immortalized mouse gba+/+ and gba/ neurons on both ECL and LI-COR western blot platforms (Figure 2A). However, this antibody specifically detected human GCase in RIPA and citrate phosphate protein lysates from WT human fibroblasts on both ECL and LI-COR western blot platforms, while no signal was detected in protein lysates from GD type 2 human fibroblasts (Figure 3A). Nonspecific bands showed in RIPA buffer protein lysates on the ECL platform, in RIPA buffer protein lysates in mouse samples visualized by ECL, and citrate phosphate protein lysates on the LI-COR platform. While the anti-β glucosidase C-2 antibody does not detect mouse GCase, it does detect human GCase in RIPA and citrate phosphate buffers on both ECL and LI-COR.

GCase expression in cell lysates from immortalized mouse neurons detected with the nine antibodies anti-β glucosidase C-2 mouse monoclonal antibody from Santa Cruz.
Figure 2.
GCase expression in cell lysates from immortalized mouse neurons detected with the nine antibodies anti-β glucosidase C-2 mouse monoclonal antibody from Santa Cruz.

The four blots shown include from left to right: Blot 1: 20 µg of RIPA protein lysate from immortalized mouse gba+/+ and gba/ neurons detected with ECL. Blot 2: 20 µg of RIPA protein lysate from immortalized mouse gba+/+ and gba/ neurons detected with LI-COR. Blot 3: 20 µg of citrate phosphate protein lysate from immortalized mouse gba+/+ and gba/ neurons detected with ECL. Blot 4: 20 µg of citrate phosphate protein lysate from immortalized mouse gba+/+ and gba/ neurons detected with LI-COR. Actin detection with an anti-actin antibody was used as a protein loading control. For (AI), each PVDF membrane was probed with: (A) anti-β glucosidase C-2 mouse monoclonal antibody from Santa Cruz (dilution factor 1 : 500). (B) Anti-β glucosidase H-300 rabbit polyclonal antibody from Santa Cruz (dilution factor 1 : 1000). (C) Anti-glucocerebrosidase rabbit polyclonal antibody from Sigma–Aldrich (G4046, dilution factor 1 : 1000). (D) Anti-glucocerebrosidase (C-terminal) rabbit polyclonal antibody from Sigma–Aldrich (G4171, dilution factor 1 : 1000). (E) Anti-GBA rabbit monoclonal antibody from Abcam (128879, dilution factor 1 : 1000). (F) Anti-GBA mouse monoclonal antibody from Abcam (55080, dilution factor 1 : 1000). (G) Anti-GBA rabbit polyclonal antibody from Origene (TA325083). (H) Custom-made anti-GCase 8E4 mouse monoclonal antibody (dilution factor 1 : 1000). (I) Custom-made anti-GCase R386 rabbit polyclonal antibody (dilution factor 1 : 2000).

Figure 2.
GCase expression in cell lysates from immortalized mouse neurons detected with the nine antibodies anti-β glucosidase C-2 mouse monoclonal antibody from Santa Cruz.

The four blots shown include from left to right: Blot 1: 20 µg of RIPA protein lysate from immortalized mouse gba+/+ and gba/ neurons detected with ECL. Blot 2: 20 µg of RIPA protein lysate from immortalized mouse gba+/+ and gba/ neurons detected with LI-COR. Blot 3: 20 µg of citrate phosphate protein lysate from immortalized mouse gba+/+ and gba/ neurons detected with ECL. Blot 4: 20 µg of citrate phosphate protein lysate from immortalized mouse gba+/+ and gba/ neurons detected with LI-COR. Actin detection with an anti-actin antibody was used as a protein loading control. For (AI), each PVDF membrane was probed with: (A) anti-β glucosidase C-2 mouse monoclonal antibody from Santa Cruz (dilution factor 1 : 500). (B) Anti-β glucosidase H-300 rabbit polyclonal antibody from Santa Cruz (dilution factor 1 : 1000). (C) Anti-glucocerebrosidase rabbit polyclonal antibody from Sigma–Aldrich (G4046, dilution factor 1 : 1000). (D) Anti-glucocerebrosidase (C-terminal) rabbit polyclonal antibody from Sigma–Aldrich (G4171, dilution factor 1 : 1000). (E) Anti-GBA rabbit monoclonal antibody from Abcam (128879, dilution factor 1 : 1000). (F) Anti-GBA mouse monoclonal antibody from Abcam (55080, dilution factor 1 : 1000). (G) Anti-GBA rabbit polyclonal antibody from Origene (TA325083). (H) Custom-made anti-GCase 8E4 mouse monoclonal antibody (dilution factor 1 : 1000). (I) Custom-made anti-GCase R386 rabbit polyclonal antibody (dilution factor 1 : 2000).

GCase expression in cell lysates from human fibroblasts detected with the nine antibodies.
Figure 3.
GCase expression in cell lysates from human fibroblasts detected with the nine antibodies.

The four blots shown include from left to right: Blot 1: 20 µg of RIPA protein lysate from control and type 2 fibroblasts detected with ECL. Blot 2: 20 µg of RIPA protein lysate from control and GD type 2 detected with LI-COR. Blot 3: 20 µg of citrate phosphate protein lysate from control and type 2 fibroblasts detected with ECL. Blot 4: 20 µg of citrate phosphate protein lysate from control and GD type 2 fibroblasts detected with LI-COR. Actin detection with an anti-actin antibody was used as a protein loading control. For (AI), each PVDF membrane was probed with: (A) anti-β glucosidase C-2 mouse monoclonal antibody from Santa Cruz (dilution factor 1 : 500). (B) Anti-β glucosidase H-300 rabbit polyclonal antibody from Santa Cruz (dilution factor 1 : 1000). (C) Anti-glucocerebrosidase rabbit polyclonal antibody from Sigma–Aldrich (G4046, dilution factor 1 : 1000). (D) Anti-glucocerebrosidase (C-terminal) rabbit polyclonal antibody from Sigma–Aldrich (G4171, dilution factor 1 : 1000). (E) Anti-GBA rabbit monoclonal antibody from Abcam (128879, dilution factor 1 : 1000). (F) Anti-GBA mouse monoclonal antibody from Abcam (55080, dilution factor 1 : 1000). (G) Anti-GBA rabbit polyclonal antibody from Origene (TA325083). (H) Custom-made anti-GCase 8E4 mouse monoclonal antibody (dilution factor 1 : 1000). (I) Custom-made anti-GCase R386 rabbit polyclonal antibody (dilution factor 1 : 2000).

Figure 3.
GCase expression in cell lysates from human fibroblasts detected with the nine antibodies.

The four blots shown include from left to right: Blot 1: 20 µg of RIPA protein lysate from control and type 2 fibroblasts detected with ECL. Blot 2: 20 µg of RIPA protein lysate from control and GD type 2 detected with LI-COR. Blot 3: 20 µg of citrate phosphate protein lysate from control and type 2 fibroblasts detected with ECL. Blot 4: 20 µg of citrate phosphate protein lysate from control and GD type 2 fibroblasts detected with LI-COR. Actin detection with an anti-actin antibody was used as a protein loading control. For (AI), each PVDF membrane was probed with: (A) anti-β glucosidase C-2 mouse monoclonal antibody from Santa Cruz (dilution factor 1 : 500). (B) Anti-β glucosidase H-300 rabbit polyclonal antibody from Santa Cruz (dilution factor 1 : 1000). (C) Anti-glucocerebrosidase rabbit polyclonal antibody from Sigma–Aldrich (G4046, dilution factor 1 : 1000). (D) Anti-glucocerebrosidase (C-terminal) rabbit polyclonal antibody from Sigma–Aldrich (G4171, dilution factor 1 : 1000). (E) Anti-GBA rabbit monoclonal antibody from Abcam (128879, dilution factor 1 : 1000). (F) Anti-GBA mouse monoclonal antibody from Abcam (55080, dilution factor 1 : 1000). (G) Anti-GBA rabbit polyclonal antibody from Origene (TA325083). (H) Custom-made anti-GCase 8E4 mouse monoclonal antibody (dilution factor 1 : 1000). (I) Custom-made anti-GCase R386 rabbit polyclonal antibody (dilution factor 1 : 2000).

Antibody #2: anti-β glucosidase H-300 rabbit polyclonal antibody Santa Cruz sc-32883

The anti-β glucosidase H-300 polyclonal antibody did not show specificity for GCase in RIPA and citrate phosphate protein lysates from immortalized mouse gba+/+ and gba/ neurons on both ECL and LI-COR western blot platforms. In fact, both RIPA and citrate phosphate protein lysates displayed a band at the expected size of GCase at the same intensity in the gba/ protein lysates (Figure 2B). The human fibroblast protein lysates showed similar MW bands in both the WT and GD type 2 cells on the near-infrared Odyssey LI-COR platform and no signal on the ECL platform (Figure 3B). These results demonstrate the need for inclusion of a negative control (gba/ or GD type 2 cells) to confirm specificity of an antibody.

Antibody #3: anti-glucocerebrosidase rabbit polyclonal antibody Sigma G4046

The Sigma G4046 polyclonal anti-GCase antibody did not show specificity for GCase on protein lysates from immortalized mouse neurons. When imaged by fluorescence and when lysed in RIPA and visualized by ECL, nonspecific bands were observed in both gba/ and gba+/+ protein samples. No signal was observed at all in samples lysed in citrate phosphate (Figure 2C). In protein lysates from human fibroblasts, a prominent GCase band was observed in WT samples and a faint band of the same molecular mass as GCase could be observed in GD type 2 human fibroblast protein lysates made in RIPA and citrate phosphate (Figure 3C). Since no GCase expression could be detected with a previously established GCase-specific inhibody (MDW933) in GD type 2 human fibroblasts (Figure 1B, lane 2), we speculate that the detected faint band might be nonspecific instead of being a remnant of L444P GCase (Figure 3C). Therefore, we labeled this antibody as non-efficacious (Table 2).

Table 2
Reactivity of tested antibodies to GCase in mice and humans
Antibody ECL platform LI-COR platform 
RIPA Citrate phosphate RIPA Citrate phosphate 
Mouse Human Mouse Human Mouse Human Mouse Human 
Santa Cruz sc-365745 − − − − 
Santa Cruz sc-32883 − − − − − − − − 
Sigma G4046 − − − − − − − − 
Sigma G4171 − − − − − − 
Abcam 128879 − − − − − − − − 
Abcam 55080 − − − − − − 
Origene TA325083 − − − − − − − − 
8E4 − − − − − − 
R386 − − − − 
Antibody ECL platform LI-COR platform 
RIPA Citrate phosphate RIPA Citrate phosphate 
Mouse Human Mouse Human Mouse Human Mouse Human 
Santa Cruz sc-365745 − − − − 
Santa Cruz sc-32883 − − − − − − − − 
Sigma G4046 − − − − − − − − 
Sigma G4171 − − − − − − 
Abcam 128879 − − − − − − − − 
Abcam 55080 − − − − − − 
Origene TA325083 − − − − − − − − 
8E4 − − − − − − 
R386 − − − − 

Antibody #4: anti-glucocerebrosidase (C-terminal) rabbit polyclonal antibody Sigma G4171

The polyclonal G4171 anti-GCase antibody from Sigma showed specificity on protein lysates from immortalized mouse neurons made in both RIPA and citrate phosphate buffers on the ECL platform. A specific band for GCase was observed in gba+/+ protein lysates, while no band for GCase was detected in gba/ protein lysates. However, using the LI-COR platform, both gba/ and gba+/+protein lysates showed bands at the same molecular mass of GCase (Figure 2D). On protein lysates of human fibroblasts, the Sigma G4171 anti-GCase antibody showed similar results to the Sigma G4046, with a prominent GCase band observed in WT samples and a faint band of the same molecular mass as GCase in GD type 2 human fibroblast protein lysates made in RIPA and citrate phosphate (Figure 3D). Since the GCase-specific inhibody established that GD type 2 human fibroblasts lacked GCase expression, we labeled this antibody as non-efficacious for detection of human GCase (Table 2).

Antibody #5: anti-GBA rabbit monoclonal antibody Abcam 128879

Across all buffer and detection platforms, the anti-GCase rabbit monoclonal antibody 128879 from Abcam was not able to detect GCase. No signal was seen in any blot involving mouse tissues, except a nonspecific band visible in the gba/ sample lysed in RIPA and visualized by fluorescence (Figure 2E). In protein lysates of human fibroblasts, this antibody showed similar results to the Sigma G4046, with a prominent GCase band observed in WT samples and a faint band of the same molecular mass as GCase in GD type 2 human fibroblast protein lysates (Figure 3E). Since the GCase-specific inhibody established that GD type 2 human fibroblasts and gba/ mouse neurons lacked GCase expression, we labeled this antibody as non-efficacious (Table 2).

Antibody #6: anti-GBA mouse monoclonal antibody Abcam 55080

This anti-GBA mouse monoclonal antibody did not show specificity for GCase on protein lysates from immortalized mouse neurons. Moreover, many nonspecific bands were observed in both gba/ and gba+/+ protein samples on the LI-COR platform, while no signal was detected on the ECL platform (Figure 2F). However, this antibody showed specificity on the ECL platform on protein lysates from human fibroblasts made in both RIPA and citrate phosphate buffer. Indeed, a specific band for GCase was observed in WT human fibroblast protein lysates, while no band for GCase was detected in GD type 2 fibroblast protein lysates. On the LI-COR platform, a prominent GCase band was observed in WT fibroblasts. However, a faint band at the same molecular mass as GCase was observed in GD type 2 human fibroblast protein lysates made in RIPA and citrate phosphate (Figure 3F). Since the GCase-specific inhibody established that GD type 2 human fibroblasts lacked GCase expression, we labeled this antibody as non-efficacious for human fibroblasts on the LI-COR platform in both RIPA and citrate phosphate buffer (Table 2).

Antibody #7: anti-GBA rabbit polyclonal antibody Origene TA325083

On all detection platforms and buffer combinations across both mouse and human protein lysates, no specific GCase bands were observed when blots were probed with this antibody (Figures 2G and 3G).

Antibody #8: custom-made anti-GCase 8E4 mouse monoclonal antibody

A band with a similar molecular mass to GCase was observed in gba+/+ and gba/ protein lysates at similar intensity levels (Figure 2H). However, this antibody showed specificity on the ECL platform on protein lysates from human fibroblasts made in both RIPA and citrate phosphate buffer with a specific band for GCase in WT human fibroblast protein lysates but not in GD type 2 fibroblast protein lysates. On the LI-COR platform, which is more sensitive, a prominent GCase band was observed in WT fibroblasts, but a faint band at the same molecular mass as GCase was observed in GD type 2 human fibroblast protein lysates (Figure 3H). Since GCase expression was completely absent from GD type 2 fibroblast lysates, this antibody was listed as efficacious for RIPA and citrate phosphate lysates on the ECL platform (Table 2).

Antibody #9: custom-made anti-GCase R386 rabbit polyclonal antibody

In protein lysates from immortalized mouse neurons, all blots probed with the R386 antibody did not show specificity for GCase. No signal was observed with ECL, and many nonspecific bands were seen with LI-COR (Figure 2I). On both ECL and LI-COR, a specific band for GCase was observed in protein lysates from WT human fibroblasts, while no band for GCase was detected in GD type 2 fibroblast protein lysates made in RIPA and citrate phosphate (Figure 3I). Since GCase expression was completely absent from GD type 2 fibroblast lysates, this antibody was listed as efficacious for RIPA and citrate phosphate lysates on the ECL and LI-COR platform (Table 2).

Discussion

Vast differences in GCase detection were observed with the different protein lysate preparation methods, murine versus human protein lysates, western blot detection platforms, and antibodies. The previously validated fluorescent GCase inhibody MDW933 provided a robust control for antibody validation. Based on this inhibody, we established that protein lysates from immortalized murine gba/ neurons and GD type 2 fibroblasts were true negative controls. Antibodies that detected GCase in mouse gba/ neurons or human GD type 2 fibroblasts were considered not to be useful in the current study.

One limitation of the present study was that it was not feasible to test multiple conditions for each antibody, and we have not attempted to reproduce all conditions used in all published reports. We believe that our selection of a uniform set of conditions is a strength of the study. However, the cellular model tested could be utilized in the future for antibody validation using other assay conditions if desired.

For protein lysates from immortalized mouse neurons, only the 4171 anti-glucocerebrosidase (C-terminal) rabbit polyclonal antibody from Sigma could specifically detect murine GCase, seen using the ECL platform. For protein lysates from human fibroblasts, both the anti-GCase R386 rabbit polyclonal antibody and the anti-β glucosidase C-2 mouse monoclonal antibody from Santa Cruz specifically detected human GCase in RIPA and citrate phosphate buffer with the ECL and LI-COR detection platforms. The anti-GBA mouse monoclonal antibody from Abcam (55080) and the anti-GCase 8E4 mouse monoclonal antibody could specifically detect human GCase in both RIPA and citrate phosphate buffers on the ECL platform. The 8E4 antibody has also previously been used to immunoprecipitate GCase [51]. The anti-β glucosidase H-300 rabbit polyclonal antibody from Santa Cruz (sc-32883), the Sigma anti-rabbit polyclonal G4046, the Abcam anti-mouse monoclonal 55080, and the anti-rabbit polyclonal antibody from Origene (TA325083) could not detect GCase in any of the tested conditions. These results are summarized in Table 2.

With the growing interest in the GCase enzyme as a possible therapeutic target for Parkinson's disease and related synucleinopathies, there is an increased need for validated and reliable antibodies. This examination of commonly used GCase antibodies shows that many antibodies being sold to target this protein are not useful for western blotting and likely other antibody-dependent methodologies. These results, made clear by our usage of genetic knockouts, orthogonal antibody-independent studies, and independent antibodies, are robust. It is our hope that these uniformly performed analyses with appropriate controls will prove useful to the scientific community for review of past publications and antibody selection for future experiments. Furthermore, the immortalized mouse gba/ neurons and human type 2 GD fibroblasts may provide important negative controls critical for the ongoing validation of anti-GCase antibodies.

Abbreviations

     
  • ECL

    enhanced chemiluminescence

  •  
  • GBA1

    glucocerebosidase 1

  •  
  • GCase

    glucocerebrosidase

  •  
  • GD

    Gaucher disease

  •  
  • HRP

    horseradish peroxidase

  •  
  • WT

    wild type

Author Contribution

W.Q., M.N., R.J.G. and R.B. performed experiments. B.A.D. helped with writing the manuscript and designing the figures. T.L. helped with literature searches and writing of the manuscript. E.A. supervised students and designed experiments. E.S. designed the study and wrote the manuscript. W.W. designed the study, supervised students, and wrote the manuscript.

Funding

This work was supported by the Intramural Research Program of the National Human Genome Research Institute and the National Institutes of Health. Antibodies R386 and 8E4 were gifts from Dr Edward Ginns.

Acknowledgements

We thank the Imaging Probe Development Center (IPDC, NHLBI, NIH) for the GCase-specific inhibody (MDW933).

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Rosenbloom
,
B.E.
and
Weinreb
,
N.J.
(
2013
)
Gaucher disease: a comprehensive review
.
Crit. Rev. Oncog.
18
,
163
175
2
Sidransky
,
E.
,
Nalls
,
M.A.
,
Aasly
,
J.O.
,
Aharon-Peretz
,
J.
,
Annesi
,
G.
,
Barbosa
,
E.R.
et al.  (
2009
)
Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease
.
N. Engl. J. Med.
361
,
1651
1661
3
Siebert
,
M.
,
Sidransky
,
E.
and
Westbroek
,
W.
(
2014
)
Glucocerebrosidase is shaking up the synucleinopathies
.
Brain
137
,
1304
1322
4
Uhlen
,
M.
,
Bandrowski
,
A.
,
Carr
,
S.
,
Edwards
,
A.
,
Ellenberg
,
J.
,
Lundberg
,
E.
et al.  (
2016
)
A proposal for validation of antibodies
.
Nat. Methods
13
,
823
827
5
Bourbeillon
,
J.
,
Orchard
,
S.
,
Benhar
,
I.
,
Borrebaeck
,
C.
,
de Daruvar
,
A.
,
Dübel
,
S.
et al.  (
2010
)
Minimum information about a protein affinity reagent (MIAPAR)
.
Nat. Biotechnol.
28
,
650
653
6
Weller
,
M.G.
(
2018
)
Ten basic rules of antibody validation
.
Anal. Chem. Insights
13
,
117739011875746
7
Weller
,
M.G.
(
2016
)
Quality issues of research antibodies
.
Anal. Chem. Insights
11
,
21
27
8
Baker
,
M.
(
2015
)
Antibody anarchy: a call to order
.
Nature
527
,
545
551
9
Baker
,
M.
(
2015
)
Reproducibility crisis: blame it on the antibodies
.
Nature
521
,
274
276
10
Vasilevsky
,
N.A.
,
Brush
,
M.H.
,
Paddock
,
H.
,
Ponting
,
L.
,
Tripathy
,
S.J.
,
LaRocca
,
G.M.
et al.  (
2013
)
On the reproducibility of science: unique identification of research resources in the biomedical literature
.
PeerJ
1
,
e148
11
Voskuil
,
J.L.A.
(
2017
)
The challenges with the validation of research antibodies
.
F1000Res.
6,
161
12
Voskuil
,
J.
(
2014
)
Commercial antibodies and their validation
.
F1000Res.
3
,
232
13
Bradbury
,
A.
and
Plückthun
,
A.
(
2015
)
Reproducibility: standardize antibodies used in research
.
Nature
518
,
27
29
14
Freedman
,
L.P.
(
2015
)
Antibodies: validate recombinants too
.
Nature
518
,
483
483
15
Polakiewicz
,
R.D.
(
2015
)
Antibodies: the solution is validation
.
Nature
518
,
483
483
16
Andersson
,
S.
,
Sundberg
,
M.
,
Pristovsek
,
N.
,
Ibrahim
,
A.
,
Jonsson
,
P.
,
Katona
,
B.
et al.  (
2017
)
Insufficient antibody validation challenges oestrogen receptor beta research
.
Nat. Commun.
8
,
15840
17
Älgenäs
,
C.
,
Agaton
,
C.
,
Fagerberg
,
L.
,
Asplund
,
A.
,
Björling
,
L.
,
Björling
,
E.
et al.  (
2014
)
Antibody performance in western blot applications is context-dependent
.
Biotechnol. J.
9
,
435
445
18
Westbroek
,
W.
,
Nguyen
,
M.
,
Siebert
,
M.
,
Lindstrom
,
T.
,
Burnett
,
R.A.
,
Aflaki
,
E.
et al.  (
2016
)
A new glucocerebrosidase-deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease
.
Dis. Model. Mech.
9
,
769
778
19
Aerts
,
J.M.
,
Donker-Koopman
,
W.E.
,
Murray
,
G.J.
,
Barranger
,
J.A.
,
Tager
,
J.M.
and
Schram
,
A.W.
(
1986
)
A procedure for the rapid purification in high yield of human glucocerebrosidase using immunoaffinity chromatography with monoclonal antibodies
.
Anal. Biochem.
154
,
655
663
20
Ginns
,
E.I.
,
Brady
,
R.O.
,
Pirruccello
,
S.
,
Moore
,
C.
,
Sorrell
,
S.
,
Furbish
,
F.S.
et al.  (
1982
)
Mutations of glucocerebrosidase: discrimination of neurologic and non-neurologic phenotypes of Gaucher disease
.
Proc. Natl Acad. Sci. U.S.A.
79
,
5607
5610
21
Witte
,
M.D.
,
Kallemeijn
,
W.W.
,
Aten
,
J.
,
Li
,
K.-Y.
,
Strijland
,
A.
,
Donker-Koopman
,
W.E.
et al.  (
2010
)
Ultrasensitive in situ visualization of active glucocerebrosidase molecules
.
Nat. Chem. Biol.
6
,
907
913
22
Squillaro
,
T.
,
Antonucci
,
I.
,
Alessio
,
N.
,
Esposito
,
A.
,
Cipollaro
,
M.
,
Melone
,
M.A.B.
et al.  (
2017
)
Impact of lysosomal storage disorders on biology of mesenchymal stem cells: evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes
.
J. Cell. Physiol.
232
,
3454
3467
23
Kim
,
M.J.
,
Jeon
,
S.
,
Burbulla
,
L.F.
and
Krainc
,
D.
(
2018
)
Acid ceramidase inhibition ameliorates α-synuclein accumulation upon loss of GBA1 function
.
Hum. Mol. Genet.
27
,
1972
1988
24
Jian
,
J.
,
Zhao
,
S.
,
Tian
,
Q.-Y.
,
Liu
,
H.
,
Zhao
,
Y.
,
Chen
,
W.-C.
et al.  (
2016
)
Association between progranulin and Gaucher disease
.
EBioMedicine
11
,
127
137
25
Collins
,
L.M.
,
Drouin-Ouellet
,
J.
,
Kuan
,
W.-L.
,
Cox
,
T.
and
Barker
,
R.A.
(
2018
)
Dermal fibroblasts from patients with Parkinson's disease have normal GCase activity and autophagy compared to patients with PD and GBA mutations
.
F1000Res.
6
,
1751
26
Zheng
,
J.
,
Chen
,
L.
,
Skinner
,
O.S.
,
Ysselstein
,
D.
,
Remis
,
J.
,
Lansbury
,
P.
et al.  (
2018
)
β-Glucocerebrosidase modulators promote dimerization of β-glucocerebrosidase and reveal an allosteric binding site
.
J. Am. Chem. Soc.
140
,
5914
5924
27
Kurzawa-Akanbi
,
M.
,
Hanson
,
P.S.
,
Blain
,
P.G.
,
Lett
,
D.J.
,
McKeith
,
I.G.
,
Chinnery
,
P.F.
et al.  (
2012
)
Glucocerebrosidase mutations alter the endoplasmic reticulum and lysosomes in Lewy body disease
.
J. Neurochem.
123
,
298
309
28
Kim
,
S.
,
Yun
,
S.P.
,
Lee
,
S.
,
Umanah
,
G.E.
,
Bandaru
,
V.V.R.
,
Yin
,
X.
et al.  (
2018
)
GBA1 deficiency negatively affects physiological α-synuclein tetramers and related multimers
.
Proc. Natl Acad. Sci. U.S.A.
115
,
798
803
29
Papadopoulos
,
V.E.
,
Nikolopoulou
,
G.
,
Antoniadou
,
I.
,
Karachaliou
,
A.
,
Arianoglou
,
G.
,
Emmanouilidou
,
E.
et al.  (
2018
)
Modulation of β-glucocerebrosidase increases α-synuclein secretion and exosome release in mouse models of Parkinson's disease
.
Hum. Mol. Genet.
27
,
1696
1710
30
Taguchi
,
Y.V.
,
Liu
,
J.
,
Ruan
,
J.
,
Pacheco
,
J.
,
Zhang
,
X.
,
Abbasi
,
J.
et al.  (
2017
)
Glucosylsphingosine promotes α-synuclein pathology in mutant GBA-associated Parkinson's disease
.
J. Neurosci.
37
,
9617
9631
31
Tayebi
,
N.
,
Parisiadou
,
L.
,
Berhe
,
B.
,
Gonzalez
,
A.N.
,
Serra-Vinardell
,
J.
,
Tamargo
,
R.J.
et al.  (
2017
)
Glucocerebrosidase haploinsufficiency in A53T α-synuclein mice impacts disease onset and course
.
Mol. Genet. Metab.
122
,
198
208
32
Mazzulli
,
J.R.
,
Zunke
,
F.
,
Isacson
,
O.
,
Studer
,
L.
and
Krainc
,
D.
(
2016
)
α-Synuclein–induced lysosomal dysfunction occurs through disruptions in protein trafficking in human midbrain synucleinopathy models
.
Proc. Natl Acad. Sci. U.S.A.
113
,
1931
1936
33
Choi
,
S.
,
Kim
,
D.
,
Kam
,
T.-I.
,
Yun
,
S.
,
Kim
,
S.
,
Park
,
H.
et al.  (
2015
)
Lysosomal enzyme glucocerebrosidase protects against Aβ1-42 oligomer-induced neurotoxicity
.
PLoS ONE
10
,
e0143854
34
Akiyama
,
H.
,
Kobayashi
,
S.
,
Hirabayashi
,
Y.
and
Murakami-Murofushi
,
K.
(
2013
)
Cholesterol glucosylation is catalyzed by transglucosylation reaction of β-glucosidase 1
.
Biochem. Biophys. Res. Commun.
441
,
838
843
35
Bendikov-Bar
,
I.
,
Rapaport
,
D.
,
Larisch
,
S.
and
Horowitz
,
M.
(
2014
)
Parkin-mediated ubiquitination of mutant glucocerebrosidase leads to competition with its substrates PARIS and ARTS
.
Orphanet. J. Rare Dis.
9
,
86
36
Bendikov-Bar
,
I.
,
Maor
,
G.
,
Filocamo
,
M.
and
Horowitz
,
M.
(
2013
)
Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase, blood cells
.
Mol. Dis.
50
,
141
145
37
Berger
,
Z.
,
Perkins
,
S.
,
Ambroise
,
C.
,
Oborski
,
C.
,
Calabrese
,
M.
,
Noell
,
S.
et al.  (
2015
)
Tool compounds robustly increase turnover of an artificial substrate by glucocerebrosidase in human brain lysates
.
PLoS ONE
10
,
e0119141
38
Liu
,
G.
,
Chen
,
M.
,
Mi
,
N.
,
Yang
,
W.
,
Li
,
X.
,
Wang
,
P.
et al.  (
2015
)
Increased oligomerization and phosphorylation of α-synuclein are associated with decreased activity of glucocerebrosidase and protein phosphatase 2A in aging monkey brains
.
Neurobiol. Aging
36
,
2649
2659
39
Samarani
,
M.
,
Loberto
,
N.
,
Soldà
,
G.
,
Straniero
,
L.
,
Asselta
,
R.
,
Duga
,
S.
et al.  (
2018
)
A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest
.
FASEB J.
,
fj201701512RR
40
Straniero
,
L.
,
Rimoldi
,
V.
,
Samarani
,
M.
,
Goldwurm
,
S.
,
Di Fonzo
,
A.
,
Krüger
,
R.
et al.  (
2017
)
The GBAP1 pseudogene acts as a ceRNA for the glucocerebrosidase gene GBA by sponging miR-22-3p
.
Sci. Rep.
7
,
12702
41
McNeill
,
A.
,
Magalhaes
,
J.
,
Shen
,
C.
,
Chau
,
K.-Y.
,
Hughes
,
D.
,
Mehta
,
A.
et al.  (
2014
)
Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells
.
Brain
137
,
1481
1495
42
Lu
,
J.
,
Chiang
,
J.
,
Iyer
,
R.R.
,
Thompson
,
E.
,
Kaneski
,
C.R.
,
Xu
,
D.S.
et al.  (
2010
)
Decreased glucocerebrosidase activity in Gaucher disease parallels quantitative enzyme loss due to abnormal interaction with TCP1 and c-Cbl
.
Proc. Natl Acad. Sci. U.S.A.
107
,
21665
21670
43
Mazzulli
,
J.R.
,
Zunke
,
F.
,
Tsunemi
,
T.
,
Toker
,
N.J.
,
Jeon
,
S.
,
Burbulla
,
L.F.
et al.  (
2016
)
Activation of glucocerebrosidase reduces pathological α-synuclein and restores lysosomal function in Parkinson's patient midbrain neurons
.
J. Neurosci.
36
,
7693
7706
44
Ben Bdira
,
F.
,
Kallemeijn
,
W.W.
,
Oussoren
,
S.V.
,
Scheij
,
S.
,
Bleijlevens
,
B.
,
Florea
,
B.I.
et al.  (
2017
)
Stabilization of glucocerebrosidase by active site occupancy
.
ACS Chem. Biol.
12
,
1830
1841
45
van Smeden
,
J.
,
Dijkhoff
,
I.M.
,
Helder
,
R.W.J.
,
Al-Khakany
,
H.
,
Boer
,
D.E.C.
,
Schreuder
,
A.
et al.  (
2017
)
In situ visualization of glucocerebrosidase in human skin tissue: zymography versus activity-based probe labeling
.
J. Lipid Res.
58
,
2299
2309
46
Jebbink
,
J.M.
,
Boot
,
R.G.
,
Keijser
,
R.
,
Moerland
,
P.D.
,
Aten
,
J.
,
Veenboer
,
G.J.M.
 et al.  (
2015
)
Increased glucocerebrosidase expression and activity in preeclamptic placenta
.
Placenta
36
,
160
169
47
Meng
,
X.-L.
,
Eto
,
Y.
,
Schiffmann
,
R.
and
Shen
,
J.-S.
(
2013
)
HIV tat domain improves cross-correction of human galactocerebrosidase in a gene- and flanking sequence-dependent manner
.
Mol. Ther. Nucleic Acids
2
,
e130
48
Siebert
,
M.
,
Westbroek
,
W.
,
Chen
,
Y.-C.
,
Moaven
,
N.
,
Li
,
Y.
,
Velayati
,
A.
et al.  (
2014
)
Identification of miRNAs that modulate glucocerebrosidase activity in Gaucher disease cells
.
RNA Biol.
11
,
1291
1300
49
Choi
,
J.H.
,
Stubblefield
,
B.
,
Cookson
,
M.R.
,
Goldin
,
E.
,
Velayati
,
A.
,
Tayebi
,
N.
et al.  (
2011
)
Aggregation of α-synuclein in brain samples from subjects with glucocerebrosidase mutations
.
Mol. Genet. Metab.
104
,
185
188
50
Velayati
,
A.
,
DePaolo
,
J.
,
Gupta
,
N.
,
Choi
,
J.H.
,
Moaven
,
N.
,
Westbroek
,
W.
et al.  (
2011
)
A mutation in SCARB2 is a modifier in Gaucher disease
.
Hum. Mutat.
32
,
1232
1238
51
Tan
,
Y.L.
,
Genereux
,
J.C.
,
Pankow
,
S.
,
Aerts
,
J.M.
,
Yates
,
J.R.
and
Kelly
,
J.W.
(
2014
)
ERdj3 is an endoplasmic reticulum degradation factor for mutant glucocerebrosidase variants linked to Gaucher's disease
.
Chem. Biol.
21
,
967
976