Specific Determination of β-Galactocerebrosidase Activity via Competitive Inhibition of β-Galactosidase

Martino, Sabata; Tiribuzi, Roberto; Tortori, Andrea; Conti, Daniele; Visigalli, Ilaria; Lattanzi, Annalisa; Biffi, Alessandra; Gritti, Angela; Orlacchio, Aldo

doi:10.1373/clinchem.2008.115873

Cited by 45 publications

(48 citation statements)

References 29 publications

(42 reference statements)

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“…ARSA activity was determined using the 4-methylumbelliferyl-sulfate substrate and calculated by subtracting the value obtained in the presence of AgNO 3 [Arylsulphatase B (ARSB) activity] from that measured in the absence of the inhibitor (ARSA + ARSB activity). Both assays were performed according to previously described protocols (64,65) that have been validated in our previous studies (15,19). …”

Section: Methodsmentioning

confidence: 99%

Therapeutic benefit of lentiviral-mediated neonatal intracerebral gene therapy in a mouse model of globoid cell leukodystrophy

Lattanzi

Salvagno

Maderna

et al. 2014

Human Molecular Genetics

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Globoid cell leukodystrophy (GLD) is an inherited lysosomal storage disease caused by β-galactocerebrosidase (GALC) deficiency. Gene therapy (GT) should provide rapid, extensive and lifetime GALC supply in central nervous system (CNS) tissues to prevent or halt irreversible neurologic progression. Here we used a lentiviral vector (LV) to transfer a functional GALC gene in the brain of Twitcher mice, a severe GLD model. A single injection of LV.GALC in the external capsule of Twitcher neonates resulted in robust transduction of neural cells with minimal and transient activation of inflammatory and immune response. Importantly, we documented a proficient transduction of proliferating and post-mitotic oligodendroglia, a relevant target cell type in GLD. GALC activity (30–50% of physiological levels) was restored in the whole CNS of treated mice as early as 8 days post-injection. The early and stable enzymatic supply ensured partial clearance of storage and reduction of psychosine levels, translating in amelioration of histopathology and enhanced lifespan. At 6 months post-injection in non-affected mice, LV genome persisted exclusively in the injected region, where transduced cells overexpressed GALC. Integration site analysis in transduced brain tissues showed no aberrant clonal expansion and preferential targeting of neural-specific genes. This study establishes neonatal LV-mediated intracerebral GT as a rapid, effective and safe therapeutic intervention to correct CNS pathology in GLD and provides a strong rationale for its application in this and similar leukodystrophies, alone or in combination with therapies targeting the somatic pathology, with the final aim of providing an effective and timely treatment of these global disorders.

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Section: Methodsmentioning

confidence: 99%

Therapeutic benefit of lentiviral-mediated neonatal intracerebral gene therapy in a mouse model of globoid cell leukodystrophy

Lattanzi

Salvagno

Maderna

et al. 2014

Human Molecular Genetics

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“…It can be speculated that during the long incubation time (18 hr) other, not substrate-specific enzymes, present in the homogenate, can be involved in the degradation of the artificial substrate. Such an enzyme may be lysosomal β-galactosidase and its undesirable action should be specifically inhibited [17]. It should be also emphasized that enzyme activities measured in cultured skin fibroblasts are relevant to higher variability than in isolated blood leukocytes of the examined individual as the culture conditions are very changeable.…”

Section: B Bmentioning

confidence: 99%

Original article Diagnostic difficulties in Krabbe disease: a report of two cases and review of literature

Sztefko

Ługowska²,

Laure‐Kamionowska³

et al. 2012

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“…To distinguish the two enzyme activities we followed the protocol described by Martino et al which employs AgNO3 as a selective competitive inhibitor of lysosomal acid β-galactosidase. [38] Preincubation of brain and kidney lysates with 10 µM of probe 3 resulted in significant decrease of GALC activity (approximately 50% of untreated values) in these tissues, while β-galactosidase activity was unaltered ( Figure 3C and D). In contrast, incubation of the lysates with 0.5 µM of inhibitor 1 completely abrogated both enzyme activities.…”

Section: Selectivity Of Galactocerebrosidase Labelingmentioning

confidence: 90%

“…For measurement of β-galactosidase and β-galactocerebrosidase activity we followed the protocol previously described by Martino et al [38] with slight alterations. Briefly, a volume of tissue lysate equivalent to 7.5 µg of protein was completed with water (6.25 µL total volume) and exposed to the indicated concentration of ABP 3 or inhibitor 1 (6.25 µL 2x solution in McIlvaine buffer pH 4.3) for 60 min at 37°C.…”

Section: Fluorogenic Substrate Assay Using Mouse Tissue Homogenatesmentioning

confidence: 99%

A Specific Activity‐Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

et al. 2017

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Galactosylceramidase (GALC) is the lysosomal β-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a β-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a Bodipy fluorophore at C6 that allows visualizing active enzyme in cells and tissues. The detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intracerebroventricular infusion of the ABP. In conclusion, the GALCspecific ABP should find broad applications in diagnosis, drug development and evaluation of therapy of Krabbe disease.

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Specific Determination of β-Galactocerebrosidase Activity via Competitive Inhibition of β-Galactosidase

Cited by 45 publications

References 29 publications

Therapeutic benefit of lentiviral-mediated neonatal intracerebral gene therapy in a mouse model of globoid cell leukodystrophy

Therapeutic benefit of lentiviral-mediated neonatal intracerebral gene therapy in a mouse model of globoid cell leukodystrophy

Original article Diagnostic difficulties in Krabbe disease: a report of two cases and review of literature

A Specific Activity‐Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

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