Mutations in the gene encoding cytosolic β-glucosidase in Gaucher disease

doi:10.1016/j.lab.2004.03.013

Journal of Laboratory and Clinical Medicine

Volume 144, Issue 2, August 2004, Pages 65-68

https://doi.org/10.1016/j.lab.2004.03.013 Get rights and content

Abstract

Patients with Gaucher disease have a deficiency of the lysosomal acid β-glucosidase. The phenotypes of genotypically identical patients with Gaucher disease may differ markedly. We have examined the possibility that polymorphisms in another β-glucosidase are responsible for this variability in the phenotype. Sequence analysis of the gene encoding cytosolic β-glucosidase (GBA3) from 4 chromosomes revealed the presence of 4 single-nucleotide substitutions: c.316 G →A (D106N), c.1353A→G (Y451Y), c.1368T→A (Y456X), and c.1540 to 1541AG →T in the 3′ untranslated region. We examined the DNA from 62 patients with Gaucher disease who were homozygous for the 1226A→G (N370S) mutation and from 542 control subjects from various populations for these polymorphisms. Six of the possible 16 haplotypes were found, and none was over- or underrepresented among patients with the severe Gaucher disease phenotypes compared with those from patients with mild phenotypes.

Section snippets

PCR reactions and sequencing

Primers designed to amplify the entire coding region of GBA3 are listed in Table I.

We brought 1 to 4 μL of genomic DNA, 2.5 μL 20× PCR buffer (0.67 mol/L Tris, pH8.8; 0.166 mol/L [NH₄]₂SO₄; 0.067 mol/L MgCl₂; 1.7 mg/mL bovine serum albumin), 1.25 μL of a solution containing 10 mmol/L of each deoxyribonucleoside triphosphate, 1 μL of forward primer (100 ng/μL), 1 μL of reverse primer (100 ng/μL), and 0.15 μL of Taq polymerase to a final volume of 50 μL with the addition of water. The reaction

Results

We sequenced the complete coding sequences and 350 base pairs upstream from the start of transcription of the GBA3 gene of 1 normal individual and 11 patients with Gaucher disease. Four polymorphic sites were identified: a rare c.316 G→A substitution (aspartic acid 106 asparagine); a c.1353A→G substitution that is synonymous for tyrosine 451; a c.1368T→A substitution that converts tyrosine 456 to a stop codon and a substitution of a T for c.1540–541 AG in the 3′ untranslated region. Another

Discussion

It has become apparent that a great deal of phenotypic variability exists among “homozygotes” for single-gene diseases. In some instances it has been possible to show that such heterogeneity is a result of polymorphism in interacting genes. For example, glucose-6-phosphate dehydrogenase–deficient infants in whom severe neonatal jaundice develops have usually coinherited the uridine diphosphate–glucuronosyltansferase promoter polymorphism associated with Gilbert disease.¹⁶ The presence of factor

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β-glucosidases participate (EC 3.2.1.21) in the activation of glycosylated and inactive forms of various compounds during plant response to the environmental conditions. The understanding of regulatory mechanisms for β-glucosidases in each plant species and their functionality is the purpose of various investigations. Our study reports the expression analysis of 10 β-glucosidase genes in corn seedlings after a challenge infection ith Fusarium proliferatum and Ustilago maydis compared with the gene expression analysis after mechanical damage and without damage to the seedlings under in vitro culture conditions. Likewise, the heterologous sequence comparison of the β-glucosidases from Zea mays and the characterized β-glucosidases of Oryza sativa and Arabidopsis thaliana were reported. Our results suggest a potential similitude in the functionality of this group of β-glucosidase genes in maize during the response to biotic stress and the mechanical damage that also showed possible tissue-specific regulation. Our results suggest the transcription regulation of these β-glucosidases and their possible influence in the defence mechanism against fungal infections in maize.
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There is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life.
To evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases.
Enzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~ 43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0).
A 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform.
Klotho-Related Protein KLrP: Structure and Functions
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Citation Excerpt :
The KLrP and GBA2 to GD connection have been examined by several research groups. Beutler, Beutler, and West (2004) examined whether polymorphisms in KLrP are related to GD. They found four single-nucleotide substitutions in KLrP from GD patients; however, these mutations were not related to GD phenotypes.
Klotho (KL) family proteins share one or two glycoside hydrolase (GH) motifs homologous to GH family 1. However, the biological significance of GH motifs in KL family proteins remains elusive. We describe here that KL-related protein (KLrP), which is composed of a single GH motif, is a cytosolic β-glucocerebrosidase (GCase, EC 3.2.1.145). We detected a neutral conduritol B epoxide (CBE)-insensitive glucosylceramide (GlcCer)-degrading activity in the cytosol fractions of human fibroblasts, rat brains, and zebrafish embryos. KL family proteins emerged as a potent candidate for the neutral GCase using a bioinformatics approach. Recombinant human KLrP, but not α-KL, β-KL, or KLPH, exhibited GCase activity with a neutral pH optimum in the presence of CBE. We solved the crystal structures of KLrP and a KLrP mutant (E165Q) in complex with glucose, which indicate that KLrP forms a (β/α)₈TIM barrel structure with the double-displacement mechanism of the retaining β-glycosidase. Furthermore, knockdown of endogenous KLrP in CHOP cells using small interfering RNA (siRNA) decreased the CBE-insensitive neutral GCase activity and increased the cellular levels of GlcCer, which suggests that KLrP is involved in a novel GlcCer catabolism pathway. A KLrP D106N mutant was discovered in patients with severe Gaucher disease; however, this mutation did not affect the GCase activity of KLrP.
The cytosolic β-glucosidase GBA3 does not influence type 1 Gaucher disease manifestation
2011, Blood Cells, Molecules, and Diseases
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Disease severity, as measured with a commonly employed composite score, did not correlate with the GBA3 1368A haplotype of the type 1 Gaucher disease patients examined. Our findings support earlier observations by Beutler and colleagues and substantiate their conclusion that GBA3 does not influence clinical severity of type I Gaucher disease [21]. The common 1368T→A mutation in the GBA3 gene may contribute to the wide range of cytosolic β-glucosidase activity measured in human leukocytes [20].
GBA3, also known as cytosolic β-glucosidase, is thought to hydrolyze xenobiotic glycosides in man. Deficiency of glucocerebrosidase (GBA), a β-glucosidase degrading glucosylceramide, underlies Gaucher disease. We examined GBA3, which recently was proposed to degrade glucosylceramide and influence the clinical manifestation of Gaucher disease. Recombinant GBA3 was found to hydrolyze artificial substrates such as 4-methylumbelliferyl-β-D-glucoside and C6-NBD-glucosylceramide, but hydrolysis of naturally occurring lipids like glucosylceramide and glucosylsphingosine was hardly detected. Consistent with this, inhibition of GBA3 in cultured cells using a novel inhibitor (alpha-1-C-nonyl-DIX) did not result in an additional increase in glucosylceramide as compared to GBA inhibition alone. Examination of the GBA3 gene led to the identification of a common substitution in its open reading frame (1368T→A), resulting in a truncated GBA3 protein missing the last α-helix of its (β/α)₈ barrel. Both recombinant 1368A GBA3 and 1368A enzyme from spleen of a homozygous individual were found to be inactive. Amongst non-neuronopathic (type 1) Gaucher disease patients, we subsequently identified individuals being wild-type, heterozygous, or homozygous for the GBA3 1368T→A mutation. No correlation was observed between GBA3 1368A/T haplotypes and severity of type 1 Gaucher disease manifestation. In conclusion, GBA3 does not seem to modify type 1 Gaucher disease manifestation.
Glycobiology in the cytosol: The bitter side of a sweet world
2009, Biochimica et Biophysica Acta - General Subjects
Progress in glycobiology has undergone explosive growth over the past decade with more of the researchers now realizing the importance of glycan chains in various inter- and intracellular processes. However, there is still an area of glycobiology awaiting exploration. This is especially the case for the field of “glycobiology in the cytosol” which remains rather poorly understood. Yet evidence is accumulating to demonstrate that the glycoconjugates and their recognition molecules (i.e. lectins) are often present in this subcellular compartment.
Klotho-related protein is a novel cytosolic neutral β- glycosylceramidase
2007, Journal of Biological Chemistry
Citation Excerpt :
It is not clear whether any specific disease is associated with a deficiency of KLrP. Although polymorphisms were found in the gene of KLrP (GBA3) of patients with Gaucher disease, it seems unlikely that they are related to the variable phenotype of patients with this disease (37). Interestingly, Korkotian et al. (38) reported that GlcCer could escape from lysosomes in type 2 and 3 Gaucher disease patients and might affect the Ca2+ homeostasis of neuronal cells by modulating the ryanodine receptor in the ER (38).
Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-insensitive neutral glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and rat brains, and human fibroblasts. The candidates for the enzyme were assigned to the Klotho (KL), whose family members share a β-glucosidase-like domain but whose natural substrates are unknown. Among this family, only the KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when expressed in CHOP cells, in which Myc-tagged KLrP was exclusively distributed in the cytosol. In addition, knockdown of the endogenous KLrP by small interfering RNA increased the cellular level of GlcCer. The purified recombinant KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl derivatives, but each k_cat/K_m was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6Å resolution revealed that KLrP is a (β/α)₈ TIM barrel, in which Glu¹⁶⁵ and Glu³⁷³ at the carboxyl termini of β-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. The substrate-binding cleft of the enzyme was occupied with palmitic acid and oleic acid when the recombinant protein was crystallized in a complex with glucose. GlcCer was found to fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer.

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^☆: Supported by National Institutes of Health grants DK061370 and RR00833 and by the Stein Endowment Fund.

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Original articleMutations in the gene encoding cytosolic β-glucosidase in Gaucher disease☆

Abstract

Section snippets

PCR reactions and sequencing

Results

Discussion

Blood

Adv Genet

Blood Cells Mol Dis

J Biol Chem

Arch Biochem Biophys

Biochem Biophys Res Commun

Lancet

Phenotypic manifestations of Gaucher diseaseClinical features in 48 biochemically verified Type I patients and comment on Type II patients

Original article
Mutations in the gene encoding cytosolic β-glucosidase in Gaucher disease☆