Characterization of Novel Human β-glucocerebrosidase Antibodies for Parkinson’s Disease Research

Background: Mutations in GBA1, which encodes the lysosome enzyme β-glucocerebrosidase (also referred to as acid β-glucosidase or GCase), are the most common genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Evidence also suggests that loss of GCase activity is implicated in PD without GBA1 mutations. Consequently, therapies targeting GCase are actively being pursued as potential strategies to modify the progression of PD and related synucleinopathies. Despite this significant interest in GCase as a therapeutic target, the lack of well-characterized GCase antibodies continues to impede progress in the development of GCase-targeted therapies. Objective: This study aims to independently evaluate human GCase (hGCase) antibodies to provide recommendations for western blot, immunofluorescence, immunoprecipitation, and AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) assays. Methods: Two mouse monoclonal antibodies, hGCase-1/17 and hGCase-1/23, were raised against hGCase using imiglucerase, the recombinant enzyme developed to treat patients, as the antigen. These novel antibodies, alongside commonly used antibodies in the field, underwent evaluation in a variety of assays. Results: The characterization of hGCase-1/17 and hGCase-1/23 using genetic models including GBA1 loss-of-function human neuroglioma H4 line and neurons differentiated from human embryonic stem cells revealed their remarkable specificity and potency in immunofluorescence and immunoprecipitation assays. Furthermore, a hGCase AlphaLISA assay with excellent sensitivity, a broad dynamic range, and suitability for high throughput applications was developed using hGCase-1/17 and hGCase-1/23, which enabled a sandwich assay configuration. Conclusions: The hGCase immunofluorescence, immunoprecipitation, and AlphaLISA assays utilizing hGCase-1/17 and hGCase-1/23 will not only facilitate improved investigations of hGCase biology, but can also serve as tools to assess the distribution and effectiveness of GCase-targeted therapies for PD and related synucleinopathies.


INTRODUCTION
The lysosomal enzyme glucocerebrosidase (GCase) (EC 3.2.1.45),encoded by the gene GBA1, hydrolyzes its lipid substrate glucosylceramide (GlcCer) to ceramide and glucose.Gaucher disease (GD; OMIM #230800, 23090, and 23100) is an autosomal recessively inherited lysosomal storage disorder caused by biallelic pathologic variants in GBA1 that result in deficient GCase activity and lysosomal accumulation of lipid substrates, including GlcCer and glucosylsphingosine (GluSph) [1].In 2009, a multicenter international collaborative study conclusively established the connection between GBA1 mutations and Parkinson's disease (PD) highlighting GBA1 variants as common genetic risk factors for the development of PD [2].GBA1 mutations also elevate the risk for dementia with Lewy bodies (DLB), providing further insight into the role of GCase in PD-related synucleinopathies [3].This strong association spurred an upsurge in research on the cellular mechanism underlying the GBA1-PD link, as well as the potential of GCase as a therapeutic target for the synucleinopathies [4][5][6].With the intensifying focus on GCase, the demand for reliable GCase antibodies for various applications has surged.In 2019, Qi et al. evaluated several existing GCase antibodies for western blotting, revealing that many had limited utility [7].While both academic and commercial groups have pursued the development of new GCase antibodies, a comprehensive characterization of these newer GCase antibodies for the research community has not been available.The development of GCase antibodies recognizing external epitopes essential for immunostaining, immunoprecipitation, and enzymelinked immunosorbent assays (ELISA) has proven challenging.Here, we report the development of two mouse monoclonal antibodies, hGCase-1/17 and hGCase-1/23, raised against human GCase (hGCase) using the recombinant enzyme imiglucerase as the antigen.Characterization of these antibodies with genetic models, including GBA1 loss-of-function human neuroglioma H4 line and human neurons differentiated from human embryonic stem cell (hESCs), demonstrated their superior specificity and potency in immunofluorescence, immunoprecipitation, and AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) assays.These wellcharacterized hGCase antibodies, soon available to the scientific community through the Michael J. Fox Foundation Research Tools Program, will prove invaluable for future translational research and biomarker development in the context of GD, PD, and related synucleinopathies.

H4 cell pellet preparation
Cells were washed in PBS, dissociated with Try-pLE Express Enzyme, quenched with cell media, and spun down at 500 × g for 5 min.Cells were resuspended in PBS, transferred to a microcentrifuge tube, re-pelleted at 500 × g for 5 min, and PBS was discarded.Pellets were flash frozen in dry ice and stored at -80 • C for subsequent experiments.

Sanger sequencing
Genomic DNA was extracted from H4 cell pellets with Machery-Nagel genomic DNA extraction kit according to manufacturer instructions (cat no: 740952.250).Primers flanking exon 4 and exon 5 of GBA1 were ordered from IDT.Sequence for forward primer (P1) is aaggcaggtctcaaactcctcac (5' to 3') and for reverse primer (P2) is agaatgggcagagtgagattctgc (5' to 3').The PCR mix was prepared by combining Ampligold Taq Mastermix (cat no: 439888) without GC enhancer with 0.5 pM of each primer, 0.5 g of genomic DNA.PCR was programmed to the following conditions: 95 • C 10 min for 1 cycle; 95 • C 30 s, 57 • C 1 min, 72 • C 1 min for 40 cycles; 72 • C 7 min for 1 cycle and held at 4 • C. A single specific band corresponding to the 761 bp amplicon was observed following DNA electrophoresis.PCR purification was performed with the QIAquick PCR Purification Kit (Qiagen, cat no: 28106) according to provided protocol.Sanger sequencing was prepared according to Genewiz Sample Submission guidelines using primers P1 or P2 mixed with PCR purification product.Sequencing results were analyzed using SnapGene software.

Protein lysate preparation for western blot
150-200 L of 1% Triton-X Lysis buffer [1% Triton X-100, 10% glycerol, 150 mM NaCl, 25 mM HEPES pH 7.4, 1 mM EDTA, 1.5 mM MgCl 2 , supplemented with one EDTA-free protease inhibitor tablet per 10 mL of buffer (Pierce, cat no A32965)] was added to H4 pellets from three confluent wells of a 6-well plate.Pellets were pulse-sonicated at 420 Hz for 10 quick pulses and rested for 15-30 min on ice.Then, the cells were centrifuged for 15 min at 13000 xg at 4 • C and the supernatant containing lysate was collected in a new tube.Protein concentration was determined by Pierce BCA Protein Assay kit (Ther-moScientific, cat no: 23225) and lysates were stored in -80 • C.
Resorufin β-D-glucopyranoside GCase assay 5 × 10 4 H4 cells were washed once with PBS and lysed in 30 l GCase lysis buffer (0.05 M citric acid, 0.05 M KH2PO4, 0.05 M K2HPO4, 0.11 M KCl, 0.01 M NaCl, 0.001 M MgCl2, pH 6.0 with 0.1% (v/v) TritonX-100, supplemented with freshly added protease inhibitor).10 l of cell lysate were mixed with 10 l of 10 mM resorufin-␤-D-glucopyranoside and baseline fluorescence was measured at t 0 immediately.The build-up of fluorescent product (resorufin) was measured after incubation for 2 h at 37 • C (λ ex = 535 nm and λ em = 595 nm) indicating GCase activity.Data was normalized to total protein levels and depicted as a percentage of untreated GBA1 WT cells.
Stock solutions for analytes and internal standards were prepared at 1 mM in DMSO and kept at -20 • C. For further spiking solutions acetonitrile/water 9/1 (v/v) was used as solvent.Calibration solutions were prepared by serial dilution in acetonitrile/water 9/1 (v/v) containing 2% DMSO.The concentration ranged from C1 = 10 M to C9 = 0.0039 M.
Analysis was conducted on a LC-MS-MS system consisting of a Waters Xevo-TQ-S mass spectrometer connected to a complete Waters Acquity I-class UPLC system with a flow through needle sample manager using a mixture of acetonitrile/methanol/water 40/40/20 (v/v/v) as wash solvent.The auto-sampler temperature was set to 15 • C. The Xevo TQ-S instrument operated in positive ion electrospray mode with both quadrupoles tuned to unit mass resolution using nitrogen as nebulization-and desolvation gas.The nebulizer gas flow was set to 150 l/h and the desolvation gas flow to 800 l/h with a temperature of 500 • C. Argon was used as collision gas at a flow rate of 0.15 ml/min.Analytes and internal standards were detected by multiple reaction monitoring mode (MRM) following the transitions m/z 462.3 to 282.3 and m/z 467.3 > 287.3 at a cone voltage of 30 V and a collision energy of 18 V.
Samples were analyzed on a BEH glycan amide column (100 × 2.1 mm, 1.7 m particle size, purchased from Waters, Switzerland) with a flow rate of 0.25 ml/min and an oven temperature of 30 • C. Eluent A consisted of 100mM ammonium acetate and for eluent B acetonitrile was used.Glycospecific separation was achieved by isocratic elution with 90% B followed by a washing step with 10% B and column reconditioning.The overall analysis time was 12 min.

Sample preparation for in vitro GluSph measurements
50 l of distilled water were added to deep frozen cell pellets and samples were shaken for 30 min at 25 • C until the cells thawed.Then, 100 l methanol containing 0.005 M internal standards was added.After shaking 5 min, the supernatant was transferred to a 96-well plate.Tubes were rinsed again with 100 l of methanol and supernatant was transferred to the same plate.The samples were evaporated to dryness and reconstituted in 100 l acetonitrile/water 90/10 (v/v) containing 1% DMSO and analyzed by LC-MS/MS.Lipids were quantified using the peak area ratio analyte/internal standard ( = response).

Western blot: Conventional chemiluminescence platform
For each sample, 40 g of protein was loaded onto a 4-20% Mini-PROTEAN TGX PVDF gel (Bio-Rad Laboratories).After transfer with the Trans-Blot Turbo transfer system (Bio-Rad Laboratories), PVDF membranes (Bio-Rad Laboratories) were dried and reactivated with methanol.Methanol was rinsed off with water and TBST [1xTBS (from 10x TBS, 0.1% Tween] and the membrane was blocked for 1 h at RT with blocking solution [1×TBST, 5% (w/v) BSA (Sigma, cat no: A-7888)].5% (w/v) NFDM (Biorad, cat no: 1706404) blocking was also performed for each antibody; 5% BSA showed clearer bands and less background compared to 5% NFDM blocking for all antibodies.PVDF membranes were probed overnight at 4 • C with primary antibodies in antibody diluent [2% (w/v) BSA, 1x TBST].Primary antibodies were diluted so that each antibody had a final concentration of approximately 0.5 g/mL.PVDF membranes were washed 3 × 10 min at RT with TBST.This was followed by incubation with Goat HRP-coupled secondary anti-mouse IgG (Abcam, cat no: ab205719, 1 : 10K dilution) or anti-rabbit IgG (Abcam, cat no: ab 205718, 1 : 60K dilution) antibodies in antibody diluent.PVDF membranes were washed 3 × 10 min with TBST.The antigen-antibody complexes were detected with SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoScientific) using the ChemiDoc XRS + Imager System (Bio-Rad).Blots were exposed for 3 seconds.

Western blot: Automated capillary platform
Simple Western's Fluorescence Separation Module kit and 12-230 kDa Fluorescence Separation Capillary Cartridges (cat no: SM-W004) were used to perform automated western blotting.Samples were prepared and loaded according to manufacturer instructions.Samples were prepared in 0.1x sample buffer (10x Sample Buffer diluted in water) and Fluorescence 5x Master Mix such that 5 g of protein was loaded per lane at a concentration of 1 mg/mL.Prepared samples in master mix as well as Biotinylated Ladder were boiled at 95 • C for 10 min.All primary antibodies were diluted in the provided Antibody Diluent 2 at 1 : 50 dilution, except for R386 and GAPDH, which were diluted 1 : 1500 in Antibody Diluent 2. 10 L of diluted antibody was loaded in each lane.10 L of HRP-conjugated mouse or rabbit secondary antibody was loaded to the corresponding lanes.Finally, 15 L peroxide-luminol mix was loaded into each lane, the cartridge was centrifuged for 5 min at 1000xg, and wash buffer was added according to the user manual.The cartridge and capillaries were loaded into the Jess instrument (ProteinSimple) and the loading times used were the default for HRP chemiluminescence detection.Results were visualized and analyzed with the Compass for Simple Western program.

Immunocytochemistry
Cells were seeded in 8-well imaging chambers at a density of 30K cells/chamber and grown for 48 h with daily feeding.After 48 h, cells were washed once in PBS and fixed in 4% paraformaldehyde (20 min, RT).Fixed cells were washed in PBS three times and incubated in primary antibody ICC antibody diluent [0.05% Saponin (filtered) and 1% BSA in PBS] overnight at 4 • C. Wells were washed three times with PBS, followed by 3 × 5 min washes with PBS, then covered with aluminum foil and incubated with secondary antibody diluted in ICC antibody diluent in RT.Three PBS wash and 3 × 5 min washes in PBS were repeated, and cells were stored in PBS with NucBlue Fixed Cell ReadyProbes Reagent (DAPI) (ThermoFisher, cat no: R37606) in 4 • C until imaging.Confocal images were acquired using a Zeiss LSM 880 confocal microscope using the 63x oil objective.

AlphaLISA
One million H4 cells were washed once with PBS and lysed in 80 l GCase lysis buffer (0.05 M citric acid, 0.05 M KH2PO4, 0.05 M K2HPO4, 0.11 M KCl, 0.01 M NaCl, 0.001 M MgCl2, pH 6.0 with 0.1% (v/v) TritonX-100, supplemented with freshly added protease inhibitor).Samples were diluted 1/5 or 1/10 in 1X Immunoassay buffer (Perkin Elmer, #AL000F).A 2-fold serial dilution of imiglucerase (Genzyme) ranging from 1250 pM -2.4 pM was generated as a standard curve.10 l of samples or standards were incubated for 4 h at RT in the dark with 1 nM of biotinylated hGCase-1/23 and 20g/ml of hGCase-1/17-conjugated Acceptor beads (beads:antibody 50 : 1).Subsequently, 40 g/ml of AlphaScreen Streptavidin Donor beads (Perkin Elmer, #6760002) were added and incubated 1 h at RT in the dark.Plate was read on a Tecan Spark plate reader with ex: 680 nm and em: 520-620 nm.GCase concentration was calculated based on a sigmoidal non-linear regression of the imiglucerase standard and then normalized to total protein concentration.

Immunoprecipitation
For immunoprecipitation, the Dynabeads™ Protein A Immunoprecipitation Kit (Thermo Fisher, #10006D) was used.10 g of hGCase-1/17 or hGCase-1/23 or mouse IgG (Merck Millipore, #NI03) were coupled to 50 l of Protein A magnetic Dynabeads in 200 l Ab Binding and Washing Buffer for 30 min at RT on a rotating wheel.Subsequently, supernatant was removed, and Ab-conjugated beads were mixed with 100 g of cell lysate and incubated for 1 h at RT on a rotating wheel (20 g of cell lysate were saved for analysis).After incubation, supernatant ( = flowthrough, FT) was saved for analysis and beads were washed 3 times in 200 l Washing Buffer.For elution, beads were resuspended in 20 l of Elution Buffer mixed with 7 l of 4X LDS sample buffer (Thermo Fisher, # NP0007) and 3 l of 10X reducing agent (Thermo Fisher, # NP0004) and heated for 10 min to 70 • C. Supernatant ( = elution) was saved for analysis.Samples were analysed by SDS-PAGE loading 10 l of each fraction per lane (lysate and FT were mixed with appropriate volumes of 4X LDS sample buffer and 10X reducing agent prior to loading).Proteins were transferred to a nitrocellulose membrane using the iBlot 2 device and membranes were blocked for 1 h at RT in 5% (w/v) Blotting Grade Blocker/PBS (Biorad, #170-6404).GCase was detected using rb mAb to hGBA antibody (abcam, #128879, clone EPR5143(3)) at 1/1000 in blocking solution.After 3 × 5 min washes in PBS, membranes were incubated with goat anti-rabbit HRP-labelled antibody (Perkin Elmer, # NEF812001EA) at 1/10000 in 1 % (w/v) Blotting Grade Blocker/PBS for 1.5 h at RT. Proteins were detected using the SuperSignal West Dura Extended Duration Substrate Kit (Thermo Fisher, #34076).

Generation of GCase loss-of-function human neuroglioma H4 cells
To support further hGCase-1/17 and hGCase-1/23 characterization, a GCase loss-of-function (GBA1 -/-) human neuroglioma H4 line was generated.H4 (ATCC HTB-148) is a hypertriploid cell line with the modal chromosome number ranging from 63 to 78, with most cells having three copies of chromosome 1 and GBA1 alleles.To disrupt the multiple GBA1 alleles in the H4 cell genome, zinc-finger nuclease (ZFN)-mediated cleavage was combined with targeted integration of a gene disruption cassette into exon 4 of GBA1 alleles, following the workflow developed previously [8].This targeting approach introduced a puromycin resistance cassette into one of the GBA1 alleles (Fig. 1A).H4 cells with the integrated resistance cassette were enriched using puromycin selection, followed by single cell cloning.Established single cell clones were screened for the resistance cassette by PCR and subsequently with an in vitro GCase activity assay using fluorogenic substrate resorufin-␤-D-glucopyranoside (Res-Glc).The most promising clone underwent Sanger sequencing, confirming frameshift indels in the other two GBA1 alleles.A pair of PCR primers, P1 and P2, were designed to generate an amplicon of 761 bp, encompassing GBA1 exons 4 and 5 for Sanger sequencing.The GBA1 allele with the integrated puromycin resis-tance cassette could not produce PCR product due to the cassette's size.The Sanger sequencing chromatogram of the chosen clone displayed double peaks near the Zinc-finger nuclease targeting site (Fig. 1B).Parsing the chromatogram with Poly Peak Parser (http://yost.genetics.utah.edu/software.ph ) [13] indicated frameshift indels in the two GBA1 alleles that were targeted by ZFN, but without the resistance cassette integration (Supplementary Figure 2A).Given the disruption of all GBA1 alleles, this H4 clone is referred to as GBA1-/-, or the GCase knockout (KO) H4 line.The GBA1-/-H4 cells lacked GCase activity in the Res-Glc in vitro GCase activity assay (Fig. 1C).Corroborating the absence of GCase activity, the GBA1-/-H4 cells showed substantial GlcSph accumulation (Fig. 1D).Moreover, there were only minor changes in the GlcSph stereoisomer galactosylsphingosine (GalSph) in the GBA1-/-H4 cells, probably due to altered lysosomal homeostasis, indicating specific GBA1 targeting (Fig. 1E).
GBA1 + /+ cells using the capillary western blotting system.Hence, the three commercial hGCase antibodies were better suited for detecting endogenous hGCase in both traditional and capillary western blot.In contrast, hGCase-1/17 and hGCase-1/23 exhibited limited utility in western blot, detecting only overexpressed hGCase in HEK293-F cells (Supplementary Figure 1) but not at endogenous levels (Fig. 2).
hGCase AlphaLISA assay with hGCase-1/17 and hGCase-1/ 23The AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay) is a bead-based luminescent amplification assay developed by PerkinElmer to detect and quantify biomolecules in diverse sample types (Fig. 6A).It offers a streamlined workflow, enhanced sensitivity, expansive dynamic range, and adaptability for high-throughput applications.In the AlphaLISA assay, a standard sandwich configuration employs two distinct antibodies recognizing non-overlapping epitopes on the target molecule.One biotinylated antibody is coupled with an Alpha streptavidin-coated Donor bead, while the other antibody is directly conjugated to the AlphaLISA Acceptor bead.When the analyte is present, the Donor and Acceptor beads come together.Upon excitation with a 680 nm laser, a photosensitizer inside the Donor bead converts ambient oxygen to an excited singlet state.Singlet oxygen diffuses up to 200 nm to produce a chemiluminescent reaction in the Acceptor bead, emitting light at 615 nm proportional to the analyte amount.
The potential of hGCase-1/17 and hGCase-1/23 for the AlphaLISA assay was explored by generating a dose-response curve with imiglucerase.The AlphaL- ISA assay utilized a sandwich configuration where hGCase-1/23 was biotinylated and associated with the Alpha Donor Bead, and hGCase-1/17 was directly conjugated to the Alpha Acceptor Bead.This assay effectively quantified imiglucerase levels, exhibiting exceptional sensitivity, broad dynamic range, and substantial signal-to-background ratio (Fig. 6B).With this assay, hGCase protein per milligram of cell lysate was estimated at approximately 20 ng in GBA1 + /+ H4 cells and around 80 ng in GBA1 + /+ neurons, while GBA1-/-H4 or human neuron cell lysates showed no hGCase protein, underscoring the assay's specificity towards hGCase (Fig. 6C,  E).Imiglucerase treatment increased hGCase levels in H4 cells and human neurons, regardless of GBA1 genotype (Fig. 6D, F).This quantitative immuno-based assay has been previously employed to determine hGCase levels in PD and DLB postmortem brains [19].

DISCUSSION
This study unveils the development and characterization of hGCase-1/17 and hGCase-1/23, two mouse monoclonal antibodies with great utility for hGCase immunofluorescence, immunoprecipitation, and AlphaLISA assays.This achievement was made possible by immunizing with imiglucerase (recombinant full length hGCase) instead of peptide fragments, and by screening for selectivity at each stage of hybridoma clone selection.The resulting antibodies exhibited remarkable selectivity for hGCase and potent performance in immunofluorescence, immunoprecipitation, and AlphaLISA assays.
Numerous pathological variants in GBA1 compromise the trafficking of hGCase from the ER to lysosomes [20][21][22], and decreased levels of lysosomal GCase has been proposed to contribute to PD risk [23,24].Consequently, hGCase is the therapeutic target in various disease-modifying therapies currently under development for PD.Strategies to restore lysosomal GCase activity include gene therapy to deliver exogenous hGCase [25] and small molecule chaperones to facilitate proper folding and trafficking of hGCase mutants [5,[26][27][28][29].The development of hGCase-1/17 and hGCase-1/23 now provides essential assays for evaluating the effectiveness of these therapeutic approaches in delivering hGCase to lysosomes in future clinical trials.

Table 1
Antibodies used to detect hGCase by western blotting