4 methylumbelliferyl β d galactopyranoside

β-glucocerebrosidase (GCase) and β-galactocerebrosidase (GALC) activities were assayed with the fluorometric substrates 4-Methylumbelliferyl β-D-glucopyranoside (Sigma-Aldrich #M3633) and 4-Methylumbelliferyl β-D-galactopyranoside (Sigma-Aldrich #M1633), respectively. All reactions were carried out in triplicate in 96-well white Opti-Plates (PerkinElmer #6005290). For GCase activity, 1 µg homogenate protein was resuspended in 15 µL 0.1 M citric acid/0.2 M disodium phosphate (pH 5) and incubated with 30 µL of 10 mM substrate dissolved in 0.1 M citric acid/0.2 M disodium phosphate (pH 5), 0.5% sodium taurocholate, 0.25% Triton X-100 [32 (link)]. Plates were covered with sealing film, shaken, and incubated at 37 °C in the dark for 1 h. Reactions were stopped with 180 µL of ice-cold stop solution (0.2 M glycine/NaOH, pH 10.4). For GALC activity, 20 µg homogenate protein was resuspended in 25 µL citrate/phosphate buffer, pH 4.5, and incubated for 30 min with 25 µL of 1 mM substrate dissolved in 50 mM sodium citrate, pH 4.5, 125 mM NaCl, 0.5% Triton X-100. Reactions were stopped with 50 µL of ice-cold stop solution (0.5 M glycine/0.3 M NaOH, pH 10). Fluorescence was measured on a Tecan M200 Pro plate reader with excitation 360 nm and emission 446 nm. Relative activity was determined after subtraction of the substrate blank.

The HA-degrading activity of HYAL1 was analyzed by "native" and "renatured protein" zymography as detailed in Puissant et al. [26 (link)]. Signals were quantified using the ImageJ software.
The enzymatic activity of lysosomal acid hydrolases and ER marker alkaline α-glucosidase was measured with the following 4-methylumbelliferyl-coupled specific substrates (Sigma-Aldrich): 4-methylumbelliferyl-β-D-galactopyranoside (β-galactosidase), 4-methylumbelliferyl-β-D-glucuronide hydrate (β-glucuronidase), 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (β-hexosaminidase) and 4-methylumbelliferyl-α-D-glucopyranoside (alkaline α-glucosidase). The samples were incubated at 37°C with 5 mM of substrate in a 50 mM citrate buffer, pH 4.5 containing 0.05% Triton X-100, except for the alkaline α-glucosidase activity assay. In this case, the reaction was conducted using 1 mM of substrate diluted in a 0.1 M glycine-NaOH solution (pH 9) containing 0.05% Triton X-100. After 6 h of reaction, a 0.1 M glycine-NaOH solution (pH 10.3) was added to stop the enzymatic activities and the fluorescence was measured at 495 nm.

Recombinant human glucosylceramidase (R&D Systems) activity was monitored using the artificial fluorescent substrate 4-methylumbelliferyl-β-d-glucopyranoside (Sigma) using the assay condition on the enzyme product datasheet. Recombinant human galactosylceramidase (R&D Systems) activity was monitored using the artificial fluorescent substrate 4-methylumbelliferyl-β-d-galactopyranoside (Sigma) using the assay conditions described on the enzyme product datasheet.

β-Gal activity was measured in a flat-bottomed 96-well plate. Azasugar solution (3 μL), 4.29 μg/μL leukocytes homogenate 1:10 (7 μL), and substrate 4-methylumbelliferyl β-d-galactopyranoside (1.47 mM, 20 μL, Sigma–Aldrich) in acetate buffer (0.1 M, pH 4.3) containing NaCl (0.1 M) and sodium azide (0.02%) were incubated at 37 °C for 1 h. The reaction was stopped by addition of sodium carbonate (200 μL; 0.5 M, pH 10.7) containing Triton X-100 (0.0025%), and the fluorescence 4-methylumbelliferone released by β-galactosidase activity was measured in SpectraMax M2 Microplate Reader (λex = 365 nm and λem = 435 nm; Molecular Devices LLC, San Jose, CA, US). Inhibition is given with respect to the control (without azasugar). Data are mean SD (n = 3).

Hit compounds confirmed with the BRET assay were subjected to the previously developed β-gal activation assay, using commercially available β-gal protein (Sigma-Aldrich) (41 (link)). With this assay, we tested the effect of identified hit molecules on β-gal self-association. Briefly, galactosidase protein was dissolved in 5 mM sodium phosphate buffer, pH 7.4, at 60 μM concentration; the dissolved protein was then dispensed into a black ViewPlate-384 (PerkinElmer) at 20 μl/well. Next, hit compounds ranging from 180 μM down to 2.4 nM were incubated with the protein for 2.5 h on ice. The fluorogenic substrate, 4-methylumbelliferyl-β-D-galactopyranoside (Sigma-Aldrich), was added at 20 μl/well to achieve the final concentration of 1 mM (34 (link)) and then incubated for 2 h on ice, and the enzymatic activity was monitored according to fluorescence increases at 455 nm with an EnSpire multimode plate reader (PerkinElmer).

β-Gal activity was measured in a flat-bottomed 96-well plate. Compounds 10–13 and 21 solution (3 μL), 4.29 μg/μL leukocytes homogenate 1:10 (7 μL), and substrate 4-methylumbelliferyl β-d-galactopyranoside (1.47 mM, 20 μL, Sigma–Aldrich) in acetate buffer (0.1 M, pH 4.3) containing NaCl (0.1 M) and sodium azide (0.02%) were incubated at 37 °C for 1 h. The reaction was stopped by the addition of sodium carbonate (200 μL; 0.5 M, pH 10.7) containing Triton X-100 (0.0025%), and the fluorescence 4-methylumbelliferone released by β-galactosidase activity was measured in SpectraMax M2 microplate reader (λex = 365 nm, λem = 435 nm; Molecular Devices). Inhibition is given with respect to the control (without compound). Percentage β-Gal inhibition is given with respect to the control (without compound). Data are mean SD (n = 3). For compounds showing β-Gal inhibitory activity higher than 40% at 1 mM concentration, the IC₅₀ values were determined by measuring the initial hydrolysis rate with 4-methylumbelliferyl β-d-galactopyranoside (1.47 mM). Data obtained were fitted by using the appropriate equation (for more details, see the Supplementary Materials).

Mice were euthanized with an overdose of ketamine (375 mg/kg) and xylazine (37.5 mg/kg). Brain, spinal cord, liver, kidney, spleen, and abdominal fat were collected, frozen on dry ice, and stored at −80°C. The brain was divided in the cerebrum and cerebellum/brainstem. Tissues were homogenized in lysis buffer (0.2 M sodium acetate, 0.1 M sodium chloride, 0.1% Triton X-100, pH 4.3) using a TissueLyser II (QIAGEN, Germantown, MD, USA) with 5 mm stainless-steel beads at 20 Hz for 30 s for three pulses.
Lysates underwent three freeze thaws alternating between a dry ice-ethanol bath and 37°C water bath. Lysates were centrifuged at 20,000 × g for 15 min at 4°C, and the supernatant was transferred to a new microcentrifuge tube and stored at −80°C. Total protein content was measured by the QuickStart Bradford Protein Assay (Bio-Rad, Hercules, CA, USA) with serial dilutions of bovine serum albumin as the protein standard. Total βgal activity was measured in the brain (coronal block containing thalamus), liver, and kidney using 4-methylumbelliferyl-β-D-galactopyranoside (Sigma-Aldrich, St. Louis, MO, USA) as the synthetic fluorogenic substrate, specific for βgal. Total βgal activity was determined by measuring the release of 4-methylumbelliferone at excitation 360 nm and emission 460 nm and normalized to total protein concentration.