Analysis of glucocerebrosidase activity using N-(1-[14C]hexanoyl)-d-erythro-glucosylsphingosine demonstrates a correlation between levels of residual enzyme activity and the type of Gaucher disease

Glucosylceramide, a degradation product of complex glycosphingolipids, is hydrolysed in lysosomes by glucocerebrosidase (GlcCerase). Mutations in the human GlcCerase gene cause a reduction in GlcCerase activity and accumulation of glucosylceramide, which results in the onset of Gaucher disease, the most common lysosomal storage disease. Significant clinical heterogeneity is observed in Gaucher disease, with three main types known, but no clear correlation has been reported between the different types and levels of residual GlcCerase activity. We now demonstrate that a correlation exists by using a radioactive, short-acyl chain substrate, N-(1-[14C]hexanoyl)-D-erythro-glucosylsphingosine ([14C]hexanoyl-GlcCer). This substrate rapidly transferred into biological membranes in the absence of detergent [Futerman and Pagano (1991) Biochem. J. 280, 295-302] and was hydrolyzed to N-(1-[14C]hexanoyl)-D-erythro-sphingosine ([14C]hexanoyl-Cer) both in vitro and in situ, with an acid pH optimum. A strict correlation was observed between levels of [14C]hexanoyl-GlcCer hydrolysis and Gaucher type in human skin fibroblasts. The mean residual activity measured in vitro for 3 h incubation in type 1 Gaucher fibroblasts (the mild form of the disease) was 46.3 +/- 4.6 nmol of [14C]hexanoyl-Cer formed per mg protein (n = 9), and in type 2 and 3 fibroblasts (the neuronopathic forms of the disease) was 19.6 +/- 6.5 (n = 9). A similar correlation was observed when activity was measured in situ, suggesting that the clinical severity of a lysosomal storage disease is related to levels of residual enzyme activity.

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Is there a role for scintigraphic imaging of bone manifestations in Gaucher disease?

Nuklearmedizin ◽

10.3413/nukmed-0142 ◽

2008 ◽

Vol 47 (06) ◽

pp. 239-247 ◽

Cited By ~ 8

Author(s):

S. Kohlfürst ◽

H. J. Gallowitsch ◽

E. Kresnik ◽

P. Lind ◽

A. B. Mehta ◽

...

Keyword(s):

Bone Marrow ◽

Lysosomal Storage Disease ◽

Gaucher Disease ◽

Storage Disease ◽

Lysosomal Storage ◽

Scintigraphic Imaging ◽

Imaging Methods ◽

X Ray ◽

Deficient Activity

SummaryGaucher disease is the most prevalent inherited, lysosomal storage disease and is caused by deficient activity of the enzyme β-glucocerebrosidase. Bone and bone marrow alterations are frequent in the most prevalent non-neuronopathic form of Gaucher disease. Imaging of bone manifestations in Gaucher disease is performed by a variety of imaging methods, conventional X-ray and MRI as the most frequently and most important ones. However, different modalities of scintigraphic imaging have also been used. This article gives an overview on scintigraphic imaging with respect to bone manifestations in Gaucher disease discussing the advantages and limitations of scintigraphic imaging in comparison to other imaging methods.

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Assay of β-glucosidase 2 (GBA2) activity using lithocholic acid β-3-O-glucoside substrate for cultured fibroblasts and glucosylceramide for brain tissue

Biological Chemistry ◽

10.1515/hsz-2018-0438 ◽

2019 ◽

Vol 400 (6) ◽

pp. 745-752

Author(s):

Klaus Harzer ◽

Yildiz Yildiz ◽

Stefanie Beck-Wödl

Keyword(s):

Lysosomal Storage Disease ◽

Gaucher Disease ◽

Storage Disease ◽

Lithocholic Acid ◽

Artificial Substrates ◽

Lysosomal Storage ◽

Spastic Paraplegia ◽

Cultured Fibroblasts ◽

Normal Controls

Abstract Beta (β)-glucosidase 2 (GBA2) is deficient in a form of human spastic paraplegia due to defects in GBA2 (SPG46). GBA2 was proposed as a modifier of Gaucher disease, a lysosomal storage disease resulting from deficient β-glucosidase 1; GBA1. Current GBA2 activity assays using artificial substrates incompletely model the activity encountered in vivo. We studied GBA2 activity, using lithocholic acid β-glucoside or glucosylceramide as natural β-glucosidase substrates in murine tissues or cultured patient fibroblasts with the pathologic genotypes: Gba1−/−; Gba2−/−; GBA1−/−; GBA2+/− and found expected and unexpected deviations from normal controls.

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Lipoid Proteinosis: In Vivo and in Vitro Evidence for a Lysosomal Storage Disease

Journal of Investigative Dermatology ◽

10.1111/1523-1747.ep12525454 ◽

1981 ◽

Vol 76 (2) ◽

pp. 119-125 ◽

Cited By ~ 43

Author(s):

Eugene A. Bauer ◽

Daniel J. Santa-Cruz ◽

Arthur Z. Eisen

Keyword(s):

Lysosomal Storage Disease ◽

Storage Disease ◽

Lysosomal Storage ◽

Lipoid Proteinosis

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Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants

AJP Cell Physiology ◽

10.1152/ajpcell.00426.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. C1076-C1082 ◽

Cited By ~ 78

Author(s):

Gary Hin-Fai Yam ◽

Nils Bosshard ◽

Christian Zuber ◽

Beat Steinmann ◽

Jürgen Roth

Keyword(s):

Enzyme Activity ◽

Fabry Disease ◽

Competitive Inhibitor ◽

Apparent Difference ◽

Residual Enzyme Activity ◽

Lysosomal Storage ◽

Electron Microscopic ◽

Pharmacological Chaperone ◽

Mutant Proteins

Fabry disease is a lysosomal storage disorder caused by deficiency of α-galactosidase A (α-Gal A) resulting in lysosomal accumulation of glycosphingolipid globotriosylceramide Gb3. Misfolded α-Gal A variants can have residual enzyme activity but are unstable. Their lysosomal trafficking is impaired because they are retained in the endoplasmic reticulum (ER) by quality control. Subinhibitory doses of the competitive inhibitor of α-Gal A, 1-deoxygalactonojirimycin (DGJ), stabilize mutant α-Gal A in vitro and correct the trafficking defect. We showed by immunolabeling that the chaperone-like action of DGJ significantly reduces the lysosomal Gb3 storage in human Fabry fibroblasts harboring the novel mutations T194I and V390fsX8. The specificity of the DGJ effect was proven by RNA interference. Electron microscopic morphometry demonstrated a reduction of large-size, disease-associated lysosomes and loss of characteristic multilamellar lysosomal inclusions on DGJ treatment. In addition, the pre-Golgi intermediates were decreased. However, the rough ER was not different between DGJ-treated and untreated cells. Pulse-chase experiments revealed that DGJ treatment resulted in maturation and stabilization of mutant α-Gal A. Genes involved in cell stress signaling, heat shock response, unfolded protein response, and ER-associated degradation show no apparent difference in expression between untreated and DGJ-treated fibroblasts. The DGJ treatment has no apparent cytotoxic effects. Thus our data show the usefulness of a pharmacological chaperone for correction of the lysosomal storage in Fabry fibroblasts harboring different mutations with residual enzyme activity. Pharmacological chaperones acting on misfolded, unstable mutant proteins that exhibit residual biological activity offer a convenient and cost-efficient therapeutic strategy.

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