TY - JOUR
AU - Orlacchio,, Aldo
AB - Abstract Background: The determination of cellular β-galactocerebrosidase activity is an established procedure to diagnose Krabbe disease and monitor the efficacy of gene/stem cell-based therapeutic approaches aimed at restoring defective enzymatic activity in patients or disease models. Current biochemical assays for β-galactocerebrosidase show high specificity but generally require large protein amounts from scanty sources such as hematopoietic or neural stem cells. We developed a novel assay based on the hypothesis that specific measurements of β-galactocerebrosidase activity can be performed following complete inhibition of β-galactosidase activity. Methods: We performed the assay using 2–7.5 μg of sample proteins with the artificial fluorogenic substrate 4-methylumbelliferone-β-galactopyranoside (1.5 mmol/L) resuspended in 0.1/0.2 mol/L citrate/phosphate buffer, pH 4.0, and AgNO3. Reactions were incubated for 30 min at 37 °C. Fluorescence of liberated 4-methylumbelliferone was measured on a spectrofluorometer (λex 360 nm, λem 446 nm). Results: AgNO3 was a competitive inhibitor of β-galactosidase [inhibition constant (Ki) = 0.12 μmol/L] and completely inhibited β-galactosidase activity when used at a concentration of 11 μmol/L. Under this condition, the β-galactocerebrosidase activity was preserved and could be specifically and accurately measured. The assay can detect β-galactocerebrosidase activity in as little as 2 μg cell protein extract or 7.5 μg tissue. Assay validation was performed using (a) brain tissues from wild-type and twitcher mice and (b) murine GALC−/− hematopoietic stem cells and neural precursor cells transduced by GALC-lentiviral vectors. Conclusions: The procedure is straightforward, rapid, and reproducible. Within a clinical context, our method unequivocally discriminated cells from healthy subjects and Krabbe patients and is therefore suitable for diagnostic applications. Globoid cell leukodystrophy (GLD),1 or Krabbe disease, is a demyelinating disease caused by a genetic deficiency of the lysosomal enzyme β-galactocerebrosidase (GALC; EC 3.2.1.46), which hydrolyzes the terminal galactose from galactosylceramide, a major lipid component of the myelin sheet (1)(2). To date, bone marrow transplantation (3)(4) and umbilical cord blood transplants (5)(6)(7) have emerged as possible treatments for GLD. Transplantation of hematopoietic stem cells (HSCT) isolated from these sources was shown to ameliorate disease manifestations if applied early in postnatal life; however, HSCT is ineffective when performed in symptomatic patients (8)(9)(10)(11). Morbidity and mortality rates associated with an allogeneic procedure largely limit the number of GLD patients who can benefit from HSCT, thereby suggesting a requirement for innovative therapies (3)(4)(5)(6)(7)(8). In this regard, we have previously examined novel gene/cell therapy approaches based on hematopoietic stem cell and/or neural stem cell transplantation (12)(13)(14). Within a clinical context, a highly analytically sensitive assay capable of detecting GALC activity in small cell or tissue protein extracts must be considered a valuable diagnostic tool. Currently, GALC activity is measured through a variety of biochemical assays based on radiolabeled galactocerebroside or fluorogenic derivatives (15)(16)(17) or histological assay (18). Notwithstanding their specificity, these substrates require an activator and relatively large amounts of protein as the source of enzyme activity, raising major sensitivity issues in light of limited availability of human biological material. To improve the diagnostic potential of biochemical tests, we developed a novel assay for GALC activity based on the fluorogenic substrate 4-methylumbelliferyl-β-d-galactoside in the presence of AgNO3, which specifically inhibits the β-galactosidase (β-Gal; EC 3.2.1.23). Materials and Methods chemicals We purchased the fluorogenic substrate 4-methylumbelliferyl derivative 4-MU-β-d-galactoside (MUGAL) and Nonidet NP40 from Sigma Chemicals, bovine serum albumin and Bio-Rad protein assay reagent from Bio-Rad Laboratories, and Sephadex S-300 from Sigma. All other reagents were of analytical grade. patients We obtained lymphocytes from Krabbe patients and healthy donors and fibroblasts from Krabbe patients, GM1 gangliosidosis patients, and healthy donors from the “Cell line and DNA bank from patients affected by genetic diseases” (Telethon Genetic Biobank Network, Diagnosi PrePostnatale Malattie Metaboliche Laboratory, G. Gaslini Institute). Additional human fibroblast cell lines were kindly provided by V. Broccoli (SCRI-HSR, Milan, Italy) according to the guidelines on human research issued by the institution’s ethics committee. animal models The twitcher mouse colony (twi +/− C57BL6 mice; Jackson Labs) was maintained in the animal facility of the Fondazione San Raffaele del Monte Tabor, Milano, Italy. All procedures were performed according to protocols approved by an internal Animal Care and Use Committee (IACUC #325 and #314) and were reported to the Ministry of Health, as per Italian law. brain tissue preparation Adult twi mice and wild-type (wt) littermates were killed by CO2 exposure and decapitated (19). Brains were either isolated and quickly frozen in liquid nitrogen or immediately treated to obtain extracts for biochemical analyses. cell preparation We isolated murine hematopoietic stem cells (mHSCs) and neural precursor cells (mNPCs) from wt and twi mice based on reported protocols (12)(14). Human neural precursor cells (hNPCs) were derived from nonimmortalized, renewable human neural precursor cell lines isolated from 12th-postconception-week human brain tissue (Advance Bioscience Resources) and cultured according to a previously described protocol (20). Briefly, hNPCs were grown and expanded in a chemically defined, serum-free medium in the presence of basic fibroblast growth factor 2 and epidermal growth factor (10 and 20 μg/L, respectively). We routinely assessed cells for multipotency and genetic stability. Serially passaged hNPC-derived neurospheres (passage 18) were collected, washed in PBS, and processed to obtain cell lysates for use in the GALC assay. U937 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (Hyclone), penicillin, streptomycin, and 2 mmol/L l-glutamine. Cells were passaged every 3 days. cell transduction with lentiviral vector galc We transduced murine and human NPCs using optimized bidirectional lentiviral vectors (LVs) allowing coordinate transcription and expression of 2 different transgenes (21) consisting of (a) the therapeutic vector that encodes the murine GALC gene tagged with hemagglutinin (HA) peptide and green fluorescent protein (GFP) (bdLV.GALC-HA.GFP) and (b) the mock vector that encodes 2 reporter genes (i.e., GFP and the truncated form of the nerve growth factor receptor; bdLV.GFP.ΔNGFR). We transduced murine HSCs and human U937 using a monocystronic PGK.GALC LV (13). Transduction of mHSCs and NPCs (both human and murine) was performed at multiplicity of infection (MOI) 10 (1 round of 12 h). Cells untransduced and transduced with an LV encoding for human or murine GALC were harvested (7 and 20 days after transduction for HSCs and NPCs, respectively), lysed to obtain cell extracts, and tested for GALC activity. Transduction of U937 cells was performed at MOI 10 and MOI 100 (1 round of 12 h). WE collected cells for enzymatic activity detection after 5 or more passages. brain and cell extract Tissues were homogenized with an Elveheim type homogenizer in 10 mmol/L sodium phosphate buffer pH 6.0 (100 g/L) with 0.1% (vol/vol) Nonidet NP40 and then subjected to 3 rounds of sonication. After 1 h, brain lysates were centrifuged (13400g) in Eppendorf microfuges for 10 min. We used supernatants as tissue extracts for biochemical analyses. All procedures were carried out at 4 °C. Cells were harvested, washed in PBS, lysed for 1 h in 10 mmol/L sodium phosphate buffer, pH 6.0, containing 0.1% (vol/vol) Nonidet NP-40, and subjected to sonication. These steps were performed at 4 °C (22)(23). We measured protein content using the Bradford Protein Assay kit with bovine serum albumin as the reference standard (24). enzyme activity We performed the β-galactosidase assay by mixing 15 μg protein extract (50 μL) with 100 μL of 1.5 mmol/L MUGAL substrate resuspended in 0.1/0.2 mol/L citrate/phosphate buffer, pH 4.0. Reactions were incubated 30 min at 37 °C and then stopped with 0.2 mol/L Glycine/NaOH, pH 10.6. We measured fluorescence of liberated 4-methylumbelliferone using a PerkinElmer LS50B spectrofluorometer (λex 360 nm, λem 446 nm) (22)(25). One enzyme unit is 1.0 μmol/min of substrate hydrolyzed at 37 °C. β-galactosidase inhibition We evaluated the effect of metal ions on β-Gal activity in the presence of ZnCl2 (0–5 mmol/L), HgCl2 (0–10 μmol/L), FeCl3 (0–5 mmol/L), and AgNO3 (0–30 μmol/L). We conducted experiments using different amounts of protein extracts, obtained from twi mice, which were reacted with 1.5 mmol/L MUGAL substrate dissolved in 0.1–0.2 mol/L citrate/phosphate buffer, pH 4.0. Reactions were incubated 30 min at 37 °C with and without metal ions. We evaluated inhibitory effects of the salts using Lineweaver-Burk plots. We obtained Km (Michaelis constant) and Vmax (limiting velocity) values using the following concentrations of the substrate MUGAL: 0.15, 0.3, 0.6, 0.9, 1.2, 1.5, 3.0, 4.0, and 6.0 mmol/L (dissolved in 0.1–0.2 mol/L citrate/phosphate buffer, pH 4.0). To determine a Ki (inhibition constant) value for AgNO3, 7.5 μg tissue extracts from twi mice were reacted with increasing concentrations (2–15 μmol/L) of the salt. sephadex s-300 gel filtration Protein extracts from wt or twi brain (1 mg) were loaded onto a Sephadex S-300 gel filtration column (1cm2 × 40cm), previously equilibrated with 10 mmol/L sodium phosphate buffer, pH 6.0, and run in the same buffer at a flow rate of 0.1 mL/min. We evaluated molecular masses using the following standards: Dextran blue (2000 kDa); apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa). The procedure was carried out at 4 °C. Fractions (0.2 mL) were collected and analyzed for enzyme activity. optimum ph We mixed partially purified GALC (7.5 μg) with preparations of the MUGAL substrate (1.5 mmol/L in 0.1–0.2 mol/L citrate/phosphate buffer) adjusted to different pH values: 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5. Reactions were carried out with and without β-Gal inhibitor. Results were expressed as the mean of at least 5 independent experiments. optimal protein concentration and time of incubation We determined the limit of detection of the GALC assay by using variable amounts (0.5–50 μg) of protein extract. To ensure linearity of the enzymatic reaction, we carried out the experiments using different incubation times, ranging between 10 and 120 min. Results were expressed as the mean of at least 5 independent experiments. km and vmax determinations We performed kinetic analyses using the following concentrations of MUGAL substrate dissolved 0.1–0.2 mol/L citrate/phosphate buffer, pH 4.0: 0.15, 0.3, 0.6, 0.9, 1.2, 1.5, 3.0, 4.0, and 6.0 mmol/L. We performed enzymatic reactions using 7.5 μg of total protein (50 μL) from partially purified GALC mixed with 100 μL MUGAL. Reactions were incubated for 30 min at 37 °C with β-Gal inhibitor. Results were expressed as the mean of at least 5 independent experiments. statistical analysis Values were expressed as mean (SE) (n = 5). Paired t-test was performed using Graphpad Prism 4 (Graphpad Software). Results inhibition of β-gal activity The screen of several metal ions to identify a specific β-Gal inhibitor showed that FeCl3, HgCl2, and ZnCl2 were unable to discriminate between GALC and β-Gal activity: in particular, FeCl3 (0–5 mmol/L) and HgCl2 (0–10 μmol/L) strongly inhibited generalized galactosidase activity in both wt (β-Gal and GALC activities) and twi mouse brain, whereas ZnCl2 (0–5 mmol/L) caused no effect at all (data not shown). On the other hand, AgNO3 exhibited differential inhibitory activity. Within a relatively narrow range of AgNO3 concentrations (10–15 μmol/L), β-Gal activity was found to decrease from 66% to 99.5% in twi brain samples and from 66% to 91% in wt brain samples (Fig. 1A ). In particular, we observed that while an 11 μmol/L AgNO3 concentration essentially abolished β-Gal in twi brain, an 18% residual activity corresponding to that of GALC was detected in the wt tissue (Fig. 1B ). The same results were obtained when AgNO3 was preincubated with the source of enzyme activity for up to 120 min (not shown). Figure 1. Open in new tabDownload slide Effect of AgNO3 on β-galactosidase activity. The wt (▪) and twi (□) mouse brain extracts were assayed for β-galactosidase activity toward MUGAL substrate in the presence of AgNO3 within 0–30 μmol/L (A) and 10–15 μmol/L (B). One enzyme unit is 1.0 μmol/min of substrate hydrolyzed at 37 °C. Values are reported as % of specific activity (mU/mg) and representative of 5 independent experiments. Figure 1. Open in new tabDownload slide Effect of AgNO3 on β-galactosidase activity. The wt (▪) and twi (□) mouse brain extracts were assayed for β-galactosidase activity toward MUGAL substrate in the presence of AgNO3 within 0–30 μmol/L (A) and 10–15 μmol/L (B). One enzyme unit is 1.0 μmol/min of substrate hydrolyzed at 37 °C. Values are reported as % of specific activity (mU/mg) and representative of 5 independent experiments. Inhibition kinetics pointed to AgNO3 as a competitive inhibitor of β-Gal enzyme (Ki = 0.112 μmol/L). In the presence of inhibitor, the Km value for the MUGAL substrate increased from 0.363 to 4.94 mmol/L, whereas no change in the Vmax value (22.2 nmol/min) was observed. Together, these data indicated that 11 μmol/L AgNO3 may be used to specifically measure GALC activity via the MUGAL substrate without interference from β-Gal. partial purification of β-galactocerebrosidase We validated the assay by measuring enzyme activity in a partially purified GALC preparation. We separated β-Gal from GALC using gel filtration chromatography (26)(27). To this end, wt or twi brain tissue extracts were loaded onto a Sephadex S-300 gel filtration column (Fig. 2 ). Gel filtration chromatography performed with wt brain extracts showed 4 peaks of generalized galactosidase activity corresponding to the following molecular masses (kDa): 640 (peak I), 520 (peak II), 400 (peak III), and 120 (peak IV) (Fig. 2A ). Chromatography of twi brain showed 2 main peaks of β-Gal activity (peak II, 520 kDa, and peak III, 400 kDa) and a small peak of activity (peak IV, 120 kDa) susceptible to complete inhibition by 11 μmol/L AgNO3 (Fig. 2B ). In the presence of 11 μmol/L AgNO3, the enzyme activities present in peaks II, III, and IV were strongly inhibited, whereas the activity separated in peak I, corresponding to GALC, was only slightly affected (Fig. 2A ). Peaks II, III, and IV might correspond to different rearrangements of β-galactosidase subunits (28). optimization of galc assay We used partially purified GALC obtained through gel filtration chromatography and wt brain extracts to identify optimal assay conditions. We investigated the pH dependence of GALC activity within a 2.5–5.5 range using MUGAL substrate in the presence of 11 μmol/L AgNO3. We found a peak of GALC activity corresponding to pH 4.0 that fell off rapidly with more acidic or alkaline conditions (data not shown). Kinetic parameters. Km and Vmax, determined via the Lineweaver-Burk plot, were 0.47 mmol/L and 20.83 nmol/min, respectively. Limit of detection of GALC assay. We measured GALC activity using a 0.5–50 μg range of total protein obtained from wt brain tissue, mHSCs extracts, and gel filtration fractions. Within a 30-min reaction time at 37 °C, the assay was capable of detecting GALC activity as low as 2 μg total protein from either cell extracts or gel filtration fractions and 7.5 μg protein from brain extracts (Fig. 3A ). Regardless the source of activity, a linear trend was observed with up to 30 μg of sample (Fig. 3A ). Under optimal assay conditions, GALC activity in wt brain was 0.31 (0.05) mU/mg, which accounts for 18% of the generalized galactosidase activity (Fig. 3B ). Moreover, we observed that β-Gal activity, calculated by subtracting the signal obtained in the presence of AgNO3 (GALC activity) from that determined in the absence of the salt (β-Gal + GALC activity), was identical [1.40 (0.09) mU/mg] in both wt (82% of the generalized galactosidase activity) and twi brain tissues (Fig. 3B ). Experiments were performed in triplicate. These data are representative of 5 independent experiments. Figure 3. Open in new tabDownload slide Optimization of GALC assay parameters. (A), Effect of protein concentration. Different amounts of extracts from wt mHSCs (♦) and wt mouse brain extracts (×) were included in the assay mixtures. (B), GALC specific activity in twi and wt mouse brain. ▨, total galactosidase activity; ▤, β-Gal activity; ◼, GALC activity. Figure 3. Open in new tabDownload slide Optimization of GALC assay parameters. (A), Effect of protein concentration. Different amounts of extracts from wt mHSCs (♦) and wt mouse brain extracts (×) were included in the assay mixtures. (B), GALC specific activity in twi and wt mouse brain. ▨, total galactosidase activity; ▤, β-Gal activity; ◼, GALC activity. We further demonstrated specificity of detection by testing human fibroblasts isolated from GM1 gangliosidosis patients, who suffer from a lack of β-Gal activity (29). We found an identical value of specific activity (0.119 mU/mg) in either the presence or the absence of AgNO3. galc assay applications GALC activity was measured in HSCs and NPCs isolated from wt and twi mice. HSCs from wt mice showed a generalized galactosidase activity of 31 (0.15) mU/mg, of which 98% was ascribed to β-Gal [30.3 (0.12) mU/mg] and 2% to GALC [0.6 (0.05) mU/mg] activity (Fig. 4A ). Figure 4. Open in new tabDownload slide GALC activity in cells transduced with LVGALC. GALC activity was measured in HSCs (A) and NPCs (B) isolated from wt and twi mice. GALC activity is restored in homozygous, defective HSCs (A) and NPCs (B) transduced with LV GALC to levels measured in wt mHSCs (A) and mNPCs (B). (C), GALC activity measured in hNPCs. The increase in GALC activity is monitored in hNPCs transduced with bdLV.GALC-HA.GFP. (D), GALC activity assayed in untransduced, mock-transduced, and transduced U937 cells by using different PGK.GALC MOIs. The increase in GALC activity correlates with the vector titer employed for the transduction. As expected, β-Gal activity was unchanged. ▨, total galactosidase activity; ▤, β-Gal activity; ◼, GALC activity. UT, untransduced cells; Mock, cells transduced with bdLV.GFP.ΔNGFR vector; T, cells transduced with bdLV.GALC-HA.GFP vector. Figure 4. Open in new tabDownload slide GALC activity in cells transduced with LVGALC. GALC activity was measured in HSCs (A) and NPCs (B) isolated from wt and twi mice. GALC activity is restored in homozygous, defective HSCs (A) and NPCs (B) transduced with LV GALC to levels measured in wt mHSCs (A) and mNPCs (B). (C), GALC activity measured in hNPCs. The increase in GALC activity is monitored in hNPCs transduced with bdLV.GALC-HA.GFP. (D), GALC activity assayed in untransduced, mock-transduced, and transduced U937 cells by using different PGK.GALC MOIs. The increase in GALC activity correlates with the vector titer employed for the transduction. As expected, β-Gal activity was unchanged. ▨, total galactosidase activity; ▤, β-Gal activity; ◼, GALC activity. UT, untransduced cells; Mock, cells transduced with bdLV.GFP.ΔNGFR vector; T, cells transduced with bdLV.GALC-HA.GFP vector. NPCs from wt mice showed generalized galactosidase activity of 9.5 (0.15) mU/mg, of which β-Gal and GALC activities represented 93% [8.75 (0.12)] and 7% [0.65 (0.08) mU/mg], respectively. Instead, GALC activity was not detected in both HSCs and NPCs from twi mice (Fig. 4A , B). The assay promptly detected a rise in GALC activity after transduction of HSCs and NPCs with lentiviral GALC vectors, showing enzyme activity levels comparable or higher to those measured in wt mHSCs [0.6 (0.05) mU/mg] (Fig. 4A ) and mNPCs [2.33 (0.12) mU/mg] (Fig. 4B ). Further, in light of their potential application as therapeutics, we tested hNPCs after transduction with the lentiviral GALC vectors (Fig. 4C ). Compared with the activities seen in mock-transduced hNPCs [0.411 (0.15) mU/mg], we found that the activity of GALC increased up to 4-fold [1.65 (0.3) mU/mg] (Fig. 4C ). Finally, we used the monocytic U937 cell line to determine the correlation between enzyme activity and MOI of the therapeutic vector PGK (21). Untransduced and mock-transduced U937 cells showed a generalized galactosidase activity of 8.25 (0.1) mU/mg, of which GALC activity accounted for 0.22 (0.05) mU/mg. In bdLV.GALC-HA.GFP-transduced cells, generalized galactosidase activity was higher, owing to a specific increase in GALC activity [3.2 (0.25) mU/mg] that was found to correlate with the vector titer used for the transduction (Fig. 4D ). As expected, β-Gal activity was unchanged (Fig. 4D ). To assess the value of the GALC assay in actual clinical diagnosis, we tested GALC activity on human fibroblasts and lymphocytes isolated from patients affected by Krabbe disease as well as from healthy donors (Table 1 ). The results were consistent with the range of values previously reported in the literature (17)(26)(30) or routinely generated through conventional methods in laboratory facilities specializing in prenatal diagnostics (G. Gaslini Institute, Diagnosi PrePostnatale Malattie Metaboliche Laboratori, Genova, Italy (31)) (Table 1 ). In particular, GALC specific activity was 4.32 (0.41) mU/mg and 0.14 (0.025) mU/mg in healthy controls and Krabbe fibroblasts, respectively. These values compared well with those determined in control and Krabbe lymphocytes [5.24 (0.34) mU/mg and 0.884 (0.09) mU/mg, respectively] (Table 1 ). Table 1. GALC activity in cells from Krabbe disease patients. Specimens . Substrates . . . . MUGAL + AgNO3 (nmol/h mg) . HMGal (nmol/h mg)1 . [3H]Gal-Cer (nmol/h mg)2 . Fibroblasts  Control 4.32 ± 0.41 3.49 ± 0.953 1.95 ± 0.464  Krabbe disease 0.14 ± 0.025 0.14 ± 0.0735 0.06 ± 0.0345 Lymphocytes  Control 5.25 ± 0.34 5.40 ± 0.753 2.73 ± 0.61  Krabbe disease 0.88 ± 0.095 0.99 ± 0.1115 0.68 ± 0.1015 Specimens . Substrates . . . . MUGAL + AgNO3 (nmol/h mg) . HMGal (nmol/h mg)1 . [3H]Gal-Cer (nmol/h mg)2 . Fibroblasts  Control 4.32 ± 0.41 3.49 ± 0.953 1.95 ± 0.464  Krabbe disease 0.14 ± 0.025 0.14 ± 0.0735 0.06 ± 0.0345 Lymphocytes  Control 5.25 ± 0.34 5.40 ± 0.753 2.73 ± 0.61  Krabbe disease 0.88 ± 0.095 0.99 ± 0.1115 0.68 ± 0.1015 1 Wiederschain et al. (17). 2 Raghavan and Krussel (15). 3 Wiederschain et al. (17) and Wiederschain et al. (26). 4 Wiederschain et al. (17) and Harzer et al. (30). 5 P < 0.01 vs control. Open in new tab Table 1. GALC activity in cells from Krabbe disease patients. Specimens . Substrates . . . . MUGAL + AgNO3 (nmol/h mg) . HMGal (nmol/h mg)1 . [3H]Gal-Cer (nmol/h mg)2 . Fibroblasts  Control 4.32 ± 0.41 3.49 ± 0.953 1.95 ± 0.464  Krabbe disease 0.14 ± 0.025 0.14 ± 0.0735 0.06 ± 0.0345 Lymphocytes  Control 5.25 ± 0.34 5.40 ± 0.753 2.73 ± 0.61  Krabbe disease 0.88 ± 0.095 0.99 ± 0.1115 0.68 ± 0.1015 Specimens . Substrates . . . . MUGAL + AgNO3 (nmol/h mg) . HMGal (nmol/h mg)1 . [3H]Gal-Cer (nmol/h mg)2 . Fibroblasts  Control 4.32 ± 0.41 3.49 ± 0.953 1.95 ± 0.464  Krabbe disease 0.14 ± 0.025 0.14 ± 0.0735 0.06 ± 0.0345 Lymphocytes  Control 5.25 ± 0.34 5.40 ± 0.753 2.73 ± 0.61  Krabbe disease 0.88 ± 0.095 0.99 ± 0.1115 0.68 ± 0.1015 1 Wiederschain et al. (17). 2 Raghavan and Krussel (15). 3 Wiederschain et al. (17) and Wiederschain et al. (26). 4 Wiederschain et al. (17) and Harzer et al. (30). 5 P < 0.01 vs control. Open in new tab Discussion Currently, GALC is evaluated in conventional assays that use a radiolabeled galactocerebroside or fluorescent-tagged derivatives as substrates (15)(16)(17). Despite being specific, these methods exhibit poor limits of detection and require relatively large amounts of protein as the source of enzyme activity. When used to test GALC activity in fibroblasts and lymphocytes of Krabbe disease patients, our assay produced activities that were comparable to values routinely reported in current diagnostic tests, but we used approximately 10% of the protein. The procedure emerges as the only GALC assay that can be implemented and completed within 2 h (Table 2 ). Figure 2. Open in new tabDownload slide Sephadex S-300 gel filtration. Identical amounts of proteins from wt (A) and twi (B) mouse brain extracts were run through a Sephadex S-300 gel filtration column. Fractions (0.2 mL) were assayed for enzyme activity using MUGAL substrate in the presence (○) and absence (•) of 11 μmol/L AgNO3. Figure 2. Open in new tabDownload slide Sephadex S-300 gel filtration. Identical amounts of proteins from wt (A) and twi (B) mouse brain extracts were run through a Sephadex S-300 gel filtration column. Fractions (0.2 mL) were assayed for enzyme activity using MUGAL substrate in the presence (○) and absence (•) of 11 μmol/L AgNO3. Table 2. Main characteristics of different GALC assay methods. Substrates . Execution time . Sample protein (μg) . MUGAL + AgNO3 90 min 2–7.5 HMGal1 180 min 20–30 [3H]Gal-Cer2 2 days 20–50 Substrates . Execution time . Sample protein (μg) . MUGAL + AgNO3 90 min 2–7.5 HMGal1 180 min 20–30 [3H]Gal-Cer2 2 days 20–50 1 Wiederschain et al. (17). 2 Raghavan and Krussel (15). Open in new tab Table 2. Main characteristics of different GALC assay methods. Substrates . Execution time . Sample protein (μg) . MUGAL + AgNO3 90 min 2–7.5 HMGal1 180 min 20–30 [3H]Gal-Cer2 2 days 20–50 Substrates . Execution time . Sample protein (μg) . MUGAL + AgNO3 90 min 2–7.5 HMGal1 180 min 20–30 [3H]Gal-Cer2 2 days 20–50 1 Wiederschain et al. (17). 2 Raghavan and Krussel (15). Open in new tab GALC and β-galactosidase share a lysosomal localization and have similar isoelectric points and the same optimal pH and substrate specificities. Both enzymes hydrolyze the O-glycosyl bond from terminal, nonreducing β-d- galactose residues in β-d-galactosides (www.brenda.org) and show activity, although under largely different kinetics, in the presence of natural substrates such as lactosyl-[N-stearoyl]ceramide, lactosyl-[N-lignoceroyl]ceramide, galactosyl-N-galactosyl-glucosylceramide, galactosyl-N-acetylgalactosaminyl-galactosyl-glucosylceramide, and many others (25)(27)(32). We hypothesized that specific measurement of GALC activity could be achieved through complete inhibition of β-Gal activity. To develop the assay, we used cells and tissue extracts from wt mice that express both β-Gal and GALC, and twi mice, a reliable animal model for GLD that specifically lacks GALC activity (2). AgNO3 was found to discriminate between β-Gal and GALC activities. Further analyses showed that AgNO3 is a potent, competitive inhibitor of β-Gal with a Ki of 0.112 μmol/L, and that a concentration of 11 μmol/L was sufficient to fully inhibit β-Gal activity and highlight GALC activity. The assay specificity was assessed using partially purified enzyme obtained through an S300 gel filtration chromatography that revealed a peak of activity (peak I) from brain extracts of wt mice that was not present in twi mice. Peak I was not inhibited by AgNO3 and corresponded to 18% of generalized galactosidase activity. These results are in agreement with the observations of Tanaka and Suzuki (27), who performed GALC assays using a radiolabeled, natural galactocerebroside substrate. Compared with other established assays that require at least 20–30 μg of proteins and longer times of incubation (16)(17), this assay emerges as rapid and sensitive. Moreover it is also specific, as demonstrated by absence of inhibitory effect on GALC in fibroblasts from GM1 gangliosidosis patients that lack β-galactosidase activity (29). In light of the potential use of engineered hematopoietic and/or neural stem cells for the treatment of GLD (12)(13)(14)(33), the assay’s performance was validated in stem cell types of murine (mHSCs and mNPCs) and human (hNPCs) origin. Enzyme activity was also measured in stem cells from twi mice transduced with a lentiviral GALC vector, suggesting that the assay can be used to monitor quality and results of viral-mediated gene therapy approaches. Based on these findings, we conclude that the assay method is ideal for performing rapid testing for the diagnosis of Krabbe disease. Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures of Potential Conflicts of Interest:Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: A. Orlacchio, S. Martino, R. Tiribuzi, A. Tortori, and D. Conti: Ministero della Salute RF-UMB-2006-339457, Italy; Fondazione Cassa Risparmio PG 2007.0149.02, and Consorzio INBB. A. Gritti, Italian Telethon Foundation grant TGT06B02 and Italian Ministry of Health, ex-art 56L289/2002. A. Biffi, Italian Telethon Foundation grant TGT06B01 and Italian Ministry of Health, ex-art 56L289/2002. A. Lattanzi, Italian Telethon Foundation grant TGT06B02. I. Visigalli, Italian Telethon Foundation grant TGT06B01. D. Conti, Italian Ministry of Health, ex-art 56L289/2002. Expert Testimony: None declared. Role of Sponsor: The funding organizations played no role in thedesign of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript. Acknowledgments: We thank Alessandro Datti for suggestions and critical reading of the manuscript. We thank M. Filocamo, from the “Cell line and DNA bank from patients affected by Genetic diseases”— Telethon Genetic Biobank Network (project no. GTB07001A), G. 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TI - Specific Determination of β-Galactocerebrosidase Activity via Competitive Inhibition of β-Galactosidase
JF - Clinical Chemistry
DO - 10.1373/clinchem.2008.115873
DA - 2009-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/specific-determination-of-galactocerebrosidase-activity-via-c0X7VaFA2f
SP - 541
VL - 55
IS - 3
DP - DeepDyve
ER -