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Glycosphingolipids and the central regulation of metabolism Sugar analogues as research tools Herrera Moro Chao, D.

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Citation for published version (APA): Herrera Moro Chao, D. (2017). Glycosphingolipids and the central regulation of metabolism: Sugar analogues as research tools.

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Download date:28 Sep 2021 Chapter 3 - - In preparation J.M.F.G. β- ing of β- and ing of β-glucosidases Application of fluorescent of fluorescent Application

β-glucopyranosyl-configured β-glucopyranosyl-configured

rea B.I., Willems L.I., Beenakker T., Li K.Y., Jiang J., Scheij S., van Scheij S., van Jiang J., K.Y., Li T., B.I., Willems L.I., Beenakker rea

neous differential fluorescent label fluorescent neous differential - simulta for cyclophellitol-aziridines Roomen C.P.A.A., Ottenhoff R., Boot R.G., Overkleeft H.S., Aerts Overkleeft R.G., R., Boot Ottenhoff C.P.A.A., Roomen Artola M., Oussoren S.V., Flo S.V., M., Oussoren Artola D., Moro A.R.A.,Marques Herrera Application of fluorescent β-glucopyrano- syl-configured cyclophellitol-aziridines for si- multaneous differential fluorescent labeling of β-glucosidases and β- galactosidases.

André R. A. Marques1, Daniela Herrera Moro1, Marta Artola2, Saskia V. Oussoren3, Bogdan I. Florea2, Lianne I. Willems2, Thomas Beenakker2, Kah-Yee Li2, Jianbing Jiang2, Saskia Scheij1, Cindy P. A. A. van Roomen1, Roelof Ottenhoff1, Rolf G. Boot3, Hermen S. Overkleeft2, Johannes M. F. G. Aerts1,3

1Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands 2Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, 2333 CC, Leiden, The Netherlands 3Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, 2333 CC, Leiden, The Netherlands *Corresponding authors: H.S. Overkleeft ([email protected]) and J.M.F.G. Aerts ([email protected]) Einsteinweg 55, 2333 CC Leiden, The Netherlands

ABSTRACT The natural product, cyclophellitol, modified at C6 with a BODIPY fluorophore specifical- ly binds to the catalytic nucleophile of lysosomal β- (GBA) in an ac- tivity-based manner. Likewise, fluorescent β-galactopyranosyl-configured cyclophellitol labels specifically lysosomal β- (GALC). In contrast, cyclophellitol aziridines equipped at the aziridine-nitrogen with a fluorophore, are in-class broad-spec- trum retaining β-glucosidase activity-based probes (ABPs). Here we document that the latter ABPs also labels with high affinity the retaining β-galactosidase GALC. With- cy clophellitol aziridine ABPs active GALC is detectedin vitro in mouse tissue homogenates and in vivo in cells and brain of mice upon intracerebroventricular administration of the probes. At elevated concentrations cyclophellitol aziridines also label lysosomal acid β-galactosidase (GLB1) as confirmed via chemical proteomics analysis with a biotinylat- ed analogue of the probe. We exploited the broad specificity of cyclophellitol aziridine to differentially visualize GBA1, GBA2, GALC and GLB1 by sequential labeling. Pre-incu- bations with cyclophellitol and high affinity GBA2 inhibitors can assist in the selective labeling of the of choice. In conclusion, cyclophellitol-aziridine derived ABPs can be employed for the simultaneous visualization of a broad spectrum of human retaining β-glycosidases.

100 INTRODUCTION The design and application of activity-based probes (ABPs) targeting human glycosidases that are implicated in inherited lysosomal storage diseases holds great promise (1–5). They offer valuable research tools to study the life cycle of targeted glycosidases in in- tact cells and organisms. Such ABPs can also be used in diagnosis of lysosomal storage disorders (LSDs) by visualizing the amount of residual active enzyme molecules in intact cells. Finally, they can be employed to study the efficacy of therapeutic interventions for LSDs. We have earlier designed different classes of ABPs for human retaining β- glu- cosidases. Taking into account that the natural product cyclophellitol is a specific and potent suicide inhibitor of lysosomal β-glucosylceramidase (, GBA) (6), we designed cyclophellitol derivatives modified at C6 (glucopyranose numbering) with a fluorescent BODIPY (compound 2 and 3, Figure 1) (7). ABPs 2 and 3 were found 3 to covalently bind, with high specificity and in an activity-based manner, to the catalytic nucleophile residue E340 of GBA (7, 8). In situ labeling of active GBA in intact cells with ABPs 2 or 3 is feasible, and allows the visualization of the intralysosomal location of the enzyme. Intravenous infusion of mice with these ABPs results in specific labeling of GBA in most tissue, but not in the brain and eye (9). Moreover, intracerebroventricular ad- ministration of ABPs2 and 3 to mice allows visualization of active GBA in brain with high spatial resolution (10). GBA is deficient in patients suffering from Gaucher disease (GD) and a reduction in the amount of active enzyme can be detected upon incubation of cultured GD fibroblasts and subsequent labeled protein analysis by gel electrophoresis and quantitative fluorescence scanning (7). The approach for GBA was then successful- ly adapted to target the lysosomal enzyme β-galactosylceramidase (galactocerebrosi- dase, GALC). A β- galactopyranosyl-configured cyclophellitol analogue modified at C6 with a BODIPY (compound 6) was synthesized and found to specifically label GALC (11) (Marques et al. submitted for publication). The latter enzyme is deficient in Krabbe dis- ease, a LSD with devastating neuropathology (12). Next, in order to generate a broad spectrum ABP, we designed N-substituted cyclophellitol aziridines (compounds 7 and 8) (9, 13) in which the fluorophore is situated roughly at the position also occupied by the aglycon in β-glucosidase substrates. Indeed ABP 8 was found to covalently and irre- versibly react with all known human retaining β-glucosidases, namely, GBA, GBA2, GBA3 and -phloridzin (9). The cytosolic enzyme GBA3 has broad substrate specificity, and is able to hydrolyze artificial β-glucosyl and β-galactosyl substrates with about equal efficiency (14). Using low nanomolar concentrations of compounds7 and 8, we were able to specifically visualize the retaining β-glucosidases GBA, GBA2, GBA3 and LPH in cell and tissue context. Next, Hoornet al. used biotinylated β-glucopyranosyl-con- figured cyclophellitol-aziridine (compound 9) in an affinity purification experiment with an Arabidopsis thaliana lysate. Besides retaining β-glucosidases, several β-xylosidases, β-glucuronidases and β-galactosidases were also identified. A concentration of 2 μM was used in the affinity purification experiment, which suggested to us that at higher concentrations also β-galactosidases may react with β-glucopyranosyl- configured -cy clophellitol-aziridine. There are two human lysosomal β-galactosidases, GALC and acid β- galactosidase (GLB1). GALC is deficient in Krabbe disease and GLB1 in GM1 ganglio- sidosis, two neuronopathic lysosomal storage disorders. With this in mind we re-exam- ined the target specificity of β-glucopyranosyl-configured cyclophellitol-aziridine ABPs (7, 8 and 9). We found that GALC is labeled at high nanomolar ABP concentration and GLB1 at micromolar concentration. The findings sparked the development of a single assay to simultaneously visualize β-glucosidases and β-galactosidases in biological sam- ples. The experimental evidence for this is here described and discussed. 101 O O O R R

HO OH HO OH F N OH OH F B O N 1: R = N3 5: R = H N 2: R = BODIPY red 6: R = BODIPY red N N : = reen 2 R BODIPY g BODIPY red 4: R = Cy5

F N B O R F N N 7 OH N N N R HO NH BODIPY green HO OH HO OH OH

7: R = BODIPY red 10 O 8: R = BODIPY reen g N 9: R = Biotin N H N N N Cy5 HO

HO HO NH N O N O HO 7 HO Cl S H OH OH HN N N N H 5 11 12 NH O N N O Biotin

Figure 1. Structure of compounds 1-12 used in study. Compound 1: β-glucopyranose az- ido-cyclophellitol; compound 2: β-glucopyranose cyclophellitol BODIPY red; compound 3: β-glucopyranose cyclophellitol BODIPY green; compound 4: β-glucopyranose cyclophellitol Cy5 blue; compound 5: β-galactopyranose cyclophellitol; compound 6: β-galactopyranose cyclophellitol BODIPY red; compound 7: β-glucopyranose cyclophellitol-aziridine BODIPY red; compound 8: β-glucopyranose cyclophellitol-aziridine BODIPY green; compound 9: biotinylated β- glucopyranose cyclophellitol-aziridine; compound 10: ABB166; compound 11: α-1-C-nonyl-DIX (anDIX); compound 12: N-[5’-adamantane-1’-yl-methoxy]-pen- tyl-1-deoxynojirimycin. MATERIALS AND METHODS Synthesis of compounds

The synthesis of cyclophellitol derivatives 1-3 (7), β-galactopyranose-configured -cy clophellitol derivatives5 -6 (11), β-glucopyranose-configured cyclophellitol aziridines7 -9 (9, 13), competitive GALC inhibitor ABB166 10 (15), GBA3 inhibitor anDIX 11 (16) and GBA2 inhibitor AMP-DNM 12 (17) was reported previously. β-Glucopyranose cyclophelli- tol Cy5 4 was synthesized by copper–catalyzed click chemistry of azidocyclophellitol 1 with Cy5 alkyne. β-Glucopyranose cyclophellitol Cy5 4: Azidocyclophellitol 1 (24.2 mg, 0.12 mmol) and the desired Cy5- alkyne (84 mg, 0.15 mmol) were dissolved in BuOH/ toluene/H2O (6 mL, 1:1:1, v/v/v). CuSO4 (0.024 mL, 1 M in H2O) and sodium ascorbate (0.024 mL, 1 M in H2O) were added and the reaction mixture was heated at 80 ºC for 18 h. Then, the solution was diluted with CH2Cl2, washed with H2O, dried over MgSO4 and concentrated under reduced pressure. The crude was purified by silica gel column chro- matography (CH2Cl2 to CH2Cl2/MeOH 9:1), subsequently purified by semipreparative 102 reversed-phase HPLC (linear gradient: 45 to 48% B in A, 12 min, solutions used A: 50mM NH4HCO3 in H2O, B: MeCN) and lyophilized to yield cyclophellitol 4 as a blue powder (33.5 mg, 44 µmol, 37%). 1H NMR (400 MHz, CD3OD): δ 8.25 (t, J = 13.1 Hz, 2H), 7.91 (d, J = 3.3 Hz, 1H), 7.49 (d, J = 7.4 Hz, 2H), 7.44 – 7.39 (m, 2H), 7.31 – 7.24 (m, 4H), 6.63 (t, J = 12.4 Hz, 1H), 6.29 (d, J = 13.8 Hz, 2H), 4.81 (dd, J = 13.9, 3.8 Hz, 1H), 4.61 (dd, J = 13.9, 8.6 Hz, 1H), 4.42 (s, 2H), 4.09 (t, J = 7.4 Hz, 2H), 3.63 (s, 3H), 3.60 (d, J = 8.0 Hz, 1H), 3.24 – 3.20 (m, 1H), 3.12 (t, J = 14.9 Hz, 1H), 3.02 – 3.00 (m, 1H), 2.41 – 2.35 (m, 1H), 2.25 (t, J = 7.3 Hz, 2H), 1.91 (s, 6H), 1.85 – 1.78 (m, 2H), 1.72 (s, 12H), 1.50 – 1.43 (m, 2H) ppm; 13C NMR (101 MHz, CD3OD): δ 175.76, 175.36, 174.58, 155.52, 155.45, 146.23, 144.24, 143.54, 142.63, 142.53, 129.77, 129.71, 126.66, 126.24, 126.19, 125.20, 123.40, 123.27, 112.03, 111.84, 104.46, 104.27, 78.23, 72.50, 68.62, 57.56, 55.46, 50.83, 50.53, 50.50, 44.75, 44.63, 36.47, 35.60, 31.56, 28.12, 27.95, 27.28, 26.39 ppm; HRMS: calculated for + 3 C42H53N6O5 [M] 721,4072, found: 721.4070. Animals

Twitcher mice (twi/twi), a natural model of Krabbe disease resulting from a mutation in the GALC , along with wild-type (wt) littermates were generated by crossing hetero- zygous (+/twi) mice in-house. The heterozygous C57BL/6J B6.CE-Galctwi/J mice (stock number 000845) were obtained from The Jackson Laboratory (Bar Harbor, USA). Mouse pups were genotyped as previously described (18). Mice (± 3 weeks old) received the rodent AM-II diet (Arie Blok Diervoeders, Woerden, The Netherlands). Animals were housed, and animal experiments were conducted, according to approved protocols by the Institutional Animal Welfare Committee of the Academic Medical Centre Amsterdam in the Netherlands. Twelve week-old wt C57Bl/6 mice were used for intracerebroventric- ular (i.c.v.) administration of ABP8 . The animals were kept in individual cages at constant temperature (23 °C+/- 2 °C) and a 12/12h light/dark cycle. They were exposed to ad libitum food and water before and after the experimental procedures.

Recombinant murine galactocerebrosidase (GALC) was expressed in human embryonic kidney 293 (HEK293) cells as previously described (12). The mouse enzyme is 83% homol- ogous to human GALC (12). The culture medium containing the secreted recombinant protein was used directly for fluorogenic substrate assays and labeling assays. Recombi- nant β-glucocerebrosidase (Cerezyme) was obtained from Genzyme. Cells

Human embryonic kidney 293 (HEK293) cells (ATCC CRL 1573) were cultured in DMEM with high glucose (Gibco) supplemented with 10% FBS (Bodinco) and 100 units/mL peni- cillin/streptomycin (Gibco). HEK293 cells with and without stable over-expression of mu- rine GALC were generated earlier (12). In vitro labeling of cell lysates

Cell lysates were prepared in 25 mM potassium phosphate buffer pH 6.5 supplemented with 0.1% (v/v) Triton X-100 and protease inhibitors (KPi buffer) by vortexing thrice (10 sec, followed by 1 min pause on ice), and protein concentration was determined (BCA kit, Pierce). In general, a volume of lysate equivalent to 20 µg of protein was labeled 103 for 1 h at 37 °C with 1 µM of ABP 8 dissolved in McIlvaine buffer (citrate-phosphate) 150 mM pH 4.3. The samples were then resolved on 10% SDS-PAGE and analyzed as described below. In situ labeling of cells

HEK293 with and without stable over-expression of murine GALC were cultured 6-well plates pre-coated with poly-L-lysine. Cells were incubated with 100 nM ABP 3, 1 µM ABP 6 or 100 nM ABP 8 in the medium for 2 h. Next the cells were washed with PBS twice and the cell lysate was prepared by scrapping the cells in KPi buffer. Molecular cloning

The design of cloning primers for mutation introduction was based on NCBI reference sequences NM_020944.2 (GBA2) and NM_020973.3 (GBA3). Full-length cDNA sequenc- es were cloned into pcDNA3.1 in frame with the myc/His vector (Invitrogen). In vitro labeling assay using mouse tissue homogenates

Animals were first anesthetized with a dose of Hypnorm (0.315 mg/mL phenyl citrate and 10 mg/mL fluanisone) and Dormicum (5 mg/mL midazolam). The given dose was 80 µL/10 g body weight. Animals were sacrificed by cervical dislocation. Tissues were collected, snap frozen in liquid N2 and stored at -80 °C. Later, homogenates from the frozen material were made in KPi buffer. For labeling experiments, a volume of tissue homogenate equivalent to 50 µg of protein was adjusted to 10 µL with water and incu- bated for 30 min at 37 °C with 1 µM of the given ABP. The samples were then resolved on 7.5% or 10% SDS-PAGE and analyzed as described below. For competition experiments the indicated concentrations of ABB166 (10) and anDIX (11) dissolved in water were pre-incubated with the tissue lysate for 30 min on ice before addition of ABP9 (final con- centration 1 μM) and subsequent incubation at 37 °C for 10 min. For the simultaneous labeling of β-glucosidases and β-galactosidases a volume of brain and kidney homoge- nate equivalent to 50 μg protein was first incubated in the indicated concentration of non- tagged probe or inhibitor: 0.125 μM of 1, 12.5 nM of 12, 62.5 nM of 5, 1.25 mM of 10 or 0.125 μM of 11. Afterwards the samples were incubated for one hour at 37 °C with 0.2 μM ABP 4 and 2 μM ABP 6 in McIlvaine buffer pH 4.6, followed by one hour incubation at 37 °C with 2 μM ABP 8. SDS-PAGE analysis and fluorescence scanning Protein samples (recombinant enzyme, cell and tissue homogenate) were denatured by adding 5x Laemmli sample buffer containing 2-mercaptoethanol (1/4th of sample volume) and boiling for 4 min at 100 °C. The samples were then run on a 7.5% or 10% SDS-PAGE gel and wet slab gels were scanned for fluorescence using the Typhoon Vari- able Mode Imager (Amersham Biosciences, Piscataway, NJ, USA), using λex 488 nm and λem 520 nm (band pass 40) for green fluorescent ABPs 3 and 8, λex 532 nm and λem 610 nm (band pass 30) for red fluorescent ABPs2 , 6 and 7, and λex 633 nm and λem 670 nm (band pass 30) for blue Cy5 fluorescent ABP4 . As loading control the gels were stained with Coomassie Brilliant Blue (CBB) and de-stained with milliQ water.

104 Proteomics

One kidney from a 5 week old wt mouse was homogenized in McIlvaine buffer 150 mM, pH 5.5 supplemented with protease inhibitors. From the homogenate 100 µL (containing 3.5 mg protein) were incubated for 2 h at 37°C with 100 µL of 30 µM ABP 9 in McIlvaine buffer 150 mM pH 5.5. The analysis was performed as previously reported (19). Peptides were desalted on stage tips (20) and analyzed with a trap-elute system on C18 reversed phase nano LC with a 45 min 10-60% ACN/0.1% formic acid gradient, hyphenated to a Thermo LTQ-orbitrap mass spectrometer using a top 3 data dependent protocol at 60.000 resolution, m/z range 300-2000, 1000 msec fill time in the Orbitrap, for MS/MS fragmentation 35 units of CID energy, 120 msec max fill time, AGC 50 e3 and a threshold of 750 counts. Ions of z=2+ and higher were selected to be fragmented twice within 10 3 sec prior to exclusion for 150 sec. Peak lists were extracted and searched against the mouse (decoy) database, with carbamido-methylation of cysteine as fixed and oxidation of methionine as variable modifications, 20 ppm peptide tolerance, trypsin as protease and 2 missed cleavages allowed using a Mascot (matrix science) search engine. Fluorogenic substrate assay of recombinant GALC Culture medium containing recombinant GALC was diluted 2/1 (v/v) with McIlvaine buf- fer pH 4.3 (10 µL total volume) and exposed to the indicated concentrations of com- pounds 8-9 (10 μL 2x solution in H2O) for 30 min at 37 °C, before addition of 100 µL substrate mix (0.23 mg/mL 4-methylumbelliferyl-β-D- galactopyranoside in McIlvaine pH 4.3/H2O 1/1 (v/v) with 0.2 M NaCl and 0.1% BSA). After incubation at 37 °C for 30 min, the reaction was quenched with 2.5 mL 0.3 M NaOH-glycine, pH 10.6 and fluorescence was measured with a fluorimeter LS55 (Perkin Elmer) using λex 366 nm and λem 445 nm. All samples were corrected for background fluorescence (sample without enzyme) and residual enzyme activity was calculated as compared to a control sample incubated in the same manner but without inhibitors. Displayed values represent mean values from triplicate experiments and error bars indicate standard deviation (SD). Graphpad Prism 5 software was used to determine apparent IC50 values. ABP i.c.v. administration

ABP intracerebroventricular (i.c.v.) administration was performed as previously de- scribed (10) with slight alterations. Briefly, stainless steel guide cannulas were implanted in the lateral ventricle using the following stereotaxic coordinates: AP 0.3, L +1.0 and V -2.2. After a recovery period of 7 days, a needle connected to a tube was introduced in the guide cannula and a solution of conduritol B-epoxide (1 mM in PBS) was admin- istered with a rate of 0.1 μL/min for 10 minutes. After 4 hours of recovery the animals received i.c.v. a solution of ABP8 (10 nM in PBS) at the same rate as above. After 2 hours, the animals were sacrificed by CO2 euthanasia and transcardially perfused with 250 mL of 0.9% of saline solution. Brains were isolated and immediately frozen for further bio- chemical and histological analysis. Analysis of brain sections Brains were cut in 30 μm slices with a cryostat and attached to SuperFrost slides (Ther- mo Scientific, Waltham, USA). All following steps were performed in the dark to protect the fluorescence of the ABP. Slides were extensively washed in 0.01M TBS to remove 105 non-specific fluorescence and were covered in DAPI (Vector Lab, Burlingame, USA) mounting media. Immunohistochemistry

For immunohistological analysis, brain slices were extensively washed in TBS, and in- cubated overnight in TBS with 0.5% (v/v) Triton X-100, 0.025% (v/v) gelatin and rab- bit-raised primary antibodies against LAMP1 (Millipore, MA, USA) and GALC (12887-1-AP, Proteintech, Chicago, IL, USA) in a concentration of 1:1000. After the primary antibody incubation, slides were incubated for 2 h with the appropriate secondary antibodies (1:1000) conjugated with fluorescent dyes; donkey anti rabbit Alexa 488/Alexa 594 (Life Technologies, Paisley, UK). The slides were then rinsed three times with TBS, mounted and covered with DAPI to be observed in a confocal laser scanning microscope (Leica SP5). Images were made using an excitation wavelength of 488 nM for Alexa 488 and ABP 8, and 561 nM for Alexa 594. For capturing of pictures a 63x objective was used. In the case of single cell images a further zoom of 3 times was used. RESULTS AND DISCUSSION Homogenates of a series of mouse tissues (liver, spleen, kidney and brain) were prepared and probed with compounds 2, 6 and 7 at different conditions (Figure 2). As reference, recombinant GALC, GBA, GBA2 and GBA3 were used. For labeling, homogenates were incubated with each of the compounds at the pH optimum for labeling GALC, GBA, GBA2 and GBA3: pH 4.3, pH 5.2 in the presence of 0.2% (w/v) taurocholate and 0.1% (v/v) Tri- ton X-100 (+/+), pH 5.8 and pH 6.0 (McIlvaine buffer), respectively. ABPs2 and 6 labeled at all conditions specifically GBA and GALC, respectively (Figure 2). In contrast to this, derivate 7 labeled several proteins including a protein with apparent molecular mass of approximately ~50 kDa. The identity of labeled ~50 kDa protein in tissue homogenates is unclear since GBA3 and GALC migrate closely in gel electrophoresis. These two enzymes markedly differ however in pH optimum (GALC, optimal activity at pH 4.3; GBA3 optimal activity at pH 6.0). The noted prominent labeling of ~50 kDa protein in brain and kidney at pH 4.3 pointed to the possibility that GALC could be targeted by ABP 7.

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Figure 2. Probing of homogenates of mouse tissues with ABPs at different conditions. Mouse brain (A), spleen (B), kidney (C) and liver (D) homogenates were incubated for 1 h at 37°C with 1 µM of ABP 2, 6 or 7 in McIlvaine buffer 150 mM at different pH values: pH 4.3, pH 5.2 +/+ (0.2% (w/v) taurocholate and 0.1% (v/v) Triton X-100), pH 5.8 and pH 6.0. The lane “ladder” contains ABP-labeled recombinantly overexpressed GALC, GBA2 and GBA3, and rGBA (Cerezyme) as reference. Slab gels were fluorescently scanned in the green (520 nm, not shown separately) and red (610 nm) channels. An “overlay” of the two channels is shown. Then, gels were stained with Coomassie Brilliant Blue (CBB) as loading control. Pre-GALC: 80-kDa precursor; GALC*: unknown GALC intermediate found in GALC overexpressing HEK cells; GALC: 50-kDa processed form.

107 To further establish whether β-glucopyranose aziridine probes bind to GALC in brain homogenates, these were labeled at pH 4.3 with compound 8 in the presence and absence of inhibitors of GBA3 (anDIX, compound 11) (16) and GALC (ABB116, compound 10) (15) (Figure 1). Labeling of the ~50 kDa protein was out competed by GALC compet- itive inhibitor 10 at millimolar concentrations, but not by GBA3 inhibitor 11, suggesting that this protein represents active GALC (Figure 3A).

Figure 3. Labeling of GALC by ABP 8. A) Brain homogenate was labeled at pH 4.3 in ab- sence and presence of different concentrations of GBA3 inhibitor anDIX (11) or GALC in- hibitor ABB116 (10). Samples were pre-incubated 30 min on ice with the inhibitors and then labeled for 10 min at 37 °C with 1 µM ABP 8 in the presence of the inhibitors. The lane “ladder” contains ABP-labeled recombinantly overexpressed GALC, GBA2 and GBA3, and rGBA (Cerezyme) as reference. Slab gels were fluorescently scanned in the green (520 nm, ABP 8) and red (610 nm, not shown separately) channels. An “overlay” of the two channels is shown. Afterwards, gels were stained with Coomassie Brilliant Blue (CBB) as loading control. Pre-GALC: 80-kDa precursor; GALC*: unknown GALC; GALC: 50-kDa pro- cessed form. B) Kidney and brain homogenates were labeled at pH 5.0 with 0.5 µM of ABP 8. Labeled proteins were visualized by fluorescence scanning after 10% SDS-PAGE (50 µg protein applied per lane). The lane “ladder” contains ABP-labeled recombinantly overex- pressed GALC, GBA2 and GBA3, and rGBA (Cerezyme) as reference. Slab gels were fluo- rescently scanned in the green (520 nm, ABP 8) and red (610 nm, not shown separately) channels. An “overlay” of the two channels is shown. Afterwards, gels were stained with Coomassie Brilliant Blue (CBB) as loading control. Pre-GALC: 80-kDa precursor; GALC*: un- known GALC; GALC: 50-kDa processed form. C) Inhibition was determined by measuring enzyme activity of GALC following pre-incubation for 30 min with compound 8 and 9 at pH 4.3. 108 Next, we incubated brain homogenates of wild-type (wt) and GALC-deficient Twitcher mice with compound 8 (0.5 µM) at pH 5.0 to allow the labeling of GBA and GBA2 as well as GALC. Gel electrophoresis and fluorescence scanning, revealed that in wt and heterozygous brain samples a ~50 kDa labeled protein is present. We also ob- served less labeled protein in the brain of heterozygous mice compared to wt and the complete absence in the brain of homozygous animals, suggesting that in this organ the labeled protein is GALC (Figure 3B). We subsequently studied the inactivation of recombinant GALC by ABP 8 and biotinylated ABP 9 by measuring loss of enzymatic activity towards 4-methylumbel- liferyl-β-D-galactopyranoside (Figure 3C). We observed that compounds 8 and 9 ir- reversibly inhibit recombinant murine GALC with high affinity yielding half-maximal inhibitory concentrations (IC50) of 1.73 µM and 4.03 µM, respectively. The IC50 of the 3 two probes for GBA are known to be in the low nanomolar range, namely 1.15 nM for ABP 8 and 0.98 nM for ABP 9 (9). ABPs 2, 3 and 4, and cyclophellitol did not inhibit at all GALC (data not shown). To further study the labeling of GALC by ABPs 7 and 8, we employed cultured HEK cells that do not express GBA3 and overexpress GALC. We therefore labeled cell lysates with compounds 3, 6 and 8 (Figure 4A). The cyclophellitol-based ABP 3 only la- beled 58-66 kDa GBA isoforms, the β- galactopyranosyl-configured cyclophellitol-epox- ide ABP 6 only labeled precursor, intermediate and mature GALC, whilst the β-glucopy- ranosyl-configured cyclophellitol-aziridine ABP8 labeled both enzymes as well as GBA2. Since the fluorophore-tagged ABPs are known to be cell-permeable, we studied in situ labeling of enzymes in intact cells. Figure 4B demonstrates the specific labeling of GBA by cyclophellitol ABP 3 in both cell-types; that of GALC in overexpressing cells by β-galactopyranosyl cyclophellitol-epoxide ABP 6 and that of GBA, GBA2 and GALC by cyclophellitol-aziridine ABP 8. As shown in Figure 4B, exposure of cells for 2 hours to low concentrations of compound8 (100 nM) results in potent labeling.

109 Figure 4. In vitro and in situ labeling HEK293 cells overexpressing GALC with ABPs. Cells (in situ) and cell homogenates (in vitro) were incubated with compounds 3 (100 nM), 6 (1 µM) and 8 (100 nM for in situ and 1 µM for in vitro). Samples (20 µg of protein) were sub- jected to 10% SDS-PAGE separation and fluorescence scanning. The lane “ladder” contains ABP-labeled recombinantly overexpressed GALC, GBA2 and GBA3, and rGBA (Cerezyme) as reference. Slab gels were stained with Coomassie Brilliant Blue (CBB) as loading control. Next, we studied the labeling of glycosidase in mouse brain following i.c.v. ad- ministration of cyclophellitol-aziridine 8. It has been previously described that i.c.v. ad- ministration of ABP2 in the brain results in a characteristically lysosomal fluorescent pat- tern, corresponding to labeled GBA molecules (10). Mice were sequentially infused with conduritol B-epoxide (CBE) and green cyclophellitol-aziridine ABP 8 (Figure 5). In the cer- ebella of wt animals a considerable lysosomal signal could still be observed even though GBA labeling was (almost completely) prevented by the pre-infusion with CBE (10). The non-lysosomal fraction of the signal can be ascribed to labeling of cytosolic GBA2 by ABP 8 (10). To exclude interference by GBA2, the same experiment was conducted in GBA2 KO mice (Figure 5). Again overlapping labeling of was apparent. This suggests that the other enzyme labeled by cyclophellitol-aziridine ABP is located in lysosomes, as is the case for GALC. Immunostaining with GALC antibody showed a significant overlap between the lysosomal signal observed in both wt as well as GBA2 KO cerebella.

110 3

Figure 5. Labeling of lysosomes in mouse brain after i.c.v. infusion of ABP 8 in animals lacking GBA2 and prior blocking of GBA with CBE. Wt (A) and GBA2 KO (B) mice were infused with CBE followed by ABP 8 (10 nM). Collected brain were sliced and examined by fluorescence microscopy. Depicted is cerebellum. Panels (from left to right): overlay, DAPI nuclear staining; ABP 8 labeling, GALC immunohistochemistry. To further establish the specificity of the labeling with cyclophellitol aziridines, attention was focused to specific lysosomal glycosidases. The lysosomal acid β-galac- tosidase (GLB1), deficient in patients with GM1 gangliosidosis, was inhibited by com- pound 8 at a concentration of 10 μM. In addition, we checked reactivity of compound 8 with recombinant human α-glucosidase or human α-galactosidase A. No significant inhibition of their enzymatic activity by ABP8 was noted (Table 2). At this concentration ABP 8 also fully inhibited lysosomal acid β-galactosidase (GLB1) indicating that at high concentrations the ABP has yet another target.

111 Table 2 – Inhibition of glycosidases by compound 5. Inhibition percentage of lysosomal enzymes following pre-incubation with ABP 8 (10 µM). GBL1 α-Gluc α-Gal GALC GBA >99 ~10 ~5 ~90 >99 We decided to analyze the inhibition of GLB1 by ABP 8 in more detail. Using mouse kidney homogenates, we determined that IC50 of ABP 8 for the enzyme is ap- proximately 3 μM. Surprisingly, the biotinylated probe 9 displays much higher affinity for this lysosomal enzyme, with an IC50 of 0.258 μM. We then explored the possibility of employing ABP 8 to differentially label GLB1. We used ABP 2 and ABP 6 to block the labeling of GBA and GALC, respectively, in control fibroblast lysates. The remaining en- zymes were then labeled with 2.5 μM of ABP 8 (Figure 6). We observed a protein with a molecular weight similar to GBA (~60 kDa) that is labeled when GBA and GALC are pre-blocked with the respective red BODIPY probes. Pre-incubation with galactopyrano- syl-configured cyclophellitol-epoxide 5 (without a fluorophore), known to inhibit GALC and GLB1, prevented labeling of the 60 kDa band. We conclude that this band is most likely ABP 8 labeled GLB1, in agreement with the known molecular weight of the pro- cessed enzyme, 64 kDa (21).

Figure 6. High affinity inhibition of acid β-galactosidase GLB1 by compounds 8 and 9. Inhibition was determined by measuring enzyme activity of GLB1 following pre-incubation for 30 min with compound 8 and 9 at pH 4.3. (B) Incubation of lysate of fibroblasts of healthy individual (50 μg protein) with ABP 8 preceded by pre-incubation with ABP2 , ABP 6 and/or inhibitor 5. To directly investigate labeling of GALC and GLB1 by cyclophellitol aziridines, we incubated mouse kidney homogenates with biotinylated compound9 . Next, labeled proteins were enriched by streptavidin bead affinity purification and their identity was determined by tryptic digestion and LC- MS/MS analysis. Among the enriched proteins, 112 the β-glucosidase GBA as well as the β-galactosidases GALC and GLB1 were identified (Supplemental Table 1). GBA2 peptides were not identified most likely due to the low abundance of the enzyme in this tissue. The broad specificity of cyclophellitol aziridines towards β-glucosidases and β-galactosidases at high concentrations invited the development of a procedure for the simultaneous visualization of the two families of enzymes. Using the cyclophellitol ABPs 4 and 6 with different fluorescent tags we can distinctively label GBA and GALC with blue and red, respectively. Cyclophellitol aziridine ABP 8 can next be employed to tar- get GBA2 and GLB1, marking them with a green fluorophore. The use of non-tagged cyclophellitol-epoxide (1) and specific inhibitors (5, 10, 11 and 12) further allows the selective labeling of the desired enzymes. This procedure can be used for the selective ABP-profiling of up to six enzymes in a single biological sample. 3

Figure 7. Sequential labeling of β-glucosidases and β-galactosidases in mouse brain and kidney homogenates. Mouse brain (A) and kidney (B) homogenates (50 μg protein) were incubated with red ABP 6 (2.5 μM) labeling GALC, blue ABP 4 (0.2 μM) labeling GBA and green ABP 8 (2.0 μM) labeling GBA2 and GLB1. Pre- incubation with non-tagged ABP epoxide (1) and specific inhibitors 5( , 10, 11 and 12) allows the selective labeling of the various enzymes.

CONCLUSIONS

Our present investigation renders conclusive evidence for labeling of the β-galactocere- brosidase GALC and at higher concentrations acid β-galactosidase GLB1 by β-glucopyra- nosyl-configured cyclophellitol- aziridines. Thus, cyclophellitol aziridines target various β-glucosidases and, at higher concentration, also two β-galactosidases. The probes are otherwise specific: no binding, or reversible inhibition, of human α-glucosidase and α-ga- lactosidases was observed. The broad targeting of β-glucosidases and β- galactosidase by cyclophellitol aziridine ABPs can be exploited in various ways. By using low nanomolar final concentrations, retaining β-glucosidases can be specifically and efficiently labeled. Labeling of specific enzymes at choice can also be accomplished by sequential exposure 113 of cell and tissue homogenates to β-glucopyranosyl- and β-galactopyranosyl-configured cyclophellitol-epoxides prior to β-glucopyranosyl-configured cyclophellitol aziridines. In- stalling different fluorophores in each of these ABPs allows specific fluorescent visualiza- tion of GBA, GBA2, GALC and GLB1 each, assisting future investigations on the interplay between glycosylceramidases during pathological conditions like Gaucher disease, Krab- be disease, GM1 gangliosidosis and GBA2 deficiency. ACKNOWLEDGEMENTS The gift of GALC overexpressing HEK293 cells by Prof. T.M. Cox is kindly acknowledged. The European Research Council (ERC AdG CHEMBIOSPHING) and the Netherlands Orga- nization for Scientific research (NWO-CW) are acknowledged for financial support. REFERENCES

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