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PubMed1992

Gaucher disease: A G+1----A+1 IVS2 splice donor site mutation causing exon 2 skipping in the acid beta-glucosidase mRNA.

Gaucher disease is the most frequent lysosomal storage disease and the most prevalent Jewish genetic disease. About 30 identified missense mutations are causal to the defective activity of acid beta-glucosidase in this disease. cDNAs were characterized from a moderately affected 9-year-old Ashkenazi Jewish Gaucher disease type 1 patient whose 80-year-old, enzyme-deficient, 1226G (Asn370----Ser [N370S]) homozygous grandfather was nearly asymptomatic. Sequence analyses revealed four populations of cDNAs with either the 1226G mutation, an exact exon 2 (delta EX2) deletion, a deletion of exon 2 and the first 115 bp of exon 3 (delta EX2-3), or a completely normal sequence. About 50% of the cDNAs were the delta EX2, the delta EX2-3, and the normal cDNAs, in a ratio of 6:3:1. Specific amplification and characterization of exon 2 and 5' and 3' intronic flanking sequences from the structural gene demonstrated clones with either the normal sequence or with a G+1----A+1 transition at the exon 2/intron 2 boundary. This mutation destroyed the splice donor consensus site (U1 binding site) for mRNA processing. This transition also was present at the corresponding exon/intron boundary of the highly homologous pseudogene. This new mutation, termed "IVS2 G+1----A+1," is the first splicing mutation described in Gaucher disease and accounted for about 3.4% of the Gaucher disease alleles in the Ashkenazi Jewish population. The occurrence of this "pseudogene"-type mutation in the structural gene indicates the role of acid beta-glucosidase pseudogene and structural gene rearrangements in the pathogenesis of this disease.

Aged↗

PubMed1992

Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase II-deficient and parent mouse lymphoma cells.

Studies on N-linked oligosaccharide processing in the mouse lymphoma glucosidase II-deficient mutant cell line (PHAR2.7) as well as the parent BW5147 cells indicated that the former maintain their capacity to synthesize complex carbohydrate units through the use of the deglucosylation mechanism provided by endomannosidase. The in vivo activity of this enzyme was evident in the mutant cells from their production of substantial amounts of glucosylated mannose saccharides, predominantly Glc2Man; moreover, in the presence of 1-deoxymannojirimycin or kifunensine to prevent processing by mannosidase I, N-linked Man8GlcNAc2 was observed entirely in the form of the characteristic isomer in which the terminal mannose of the alpha 1,3-linked branch is missing (isomer A). In contrast, parent lymphoma cells, as well as HepG2 cells in the presence of 1-deoxymannojirimycin accumulated Man9GlcNAc2 as the primary deglucosylated N-linked oligosaccharide and contained only about 16% of their Man8GlcNAc2 as isomer A. In the presence of the glucosidase inhibitor castanospermine the mutant released Glc3Man instead of Glc2Man, and the parent cells converted their deglucosylation machinery to the endomannosidase route. Despite the mutant's capacity to accommodate a large traffic through this pathway no increase in the in vitro determined endomannosidase activity was evident. The exclusive utilization of endomannosidase by the mutant for the deglucosylation of its predominant N-linked Glc2Man9GlcNAc2 permitted an exploration of the in vivo site of this enzyme's action. Pulse-chase studies utilizing sucrose-D2O density gradient centrifugation indicated that the Glc2Man9GlcNAc2 to Man8GlcNAc2 conversion is a relatively late event that is temporally separated from the endoplasmic reticulum-situated processing of Glc3Man9GlcNAc2 to Glc2Man9GlcNAc2 and in contrast to the latter takes place in the Golgi compartment.

1-Deoxynojirimycin↗

PubMed2005

[Construction of flocculation selective vector and expression of beta-glucosidase gene in Saccharomyces cerevisiae].

Selective markers used in yeast vector for gene manipulation were usually drug resistance or autotrophy. Unfortunately, drug resistance selective marker requires drug sensitive host and most industrial strains were not autotrophy. In this paper, flocculation gene (FLO1) from Saccharomyces cerevisiae ABXL-1D was amplified by PCR, sequenced and cloned to construct an expression vector. The new vector was easy to manipulate and suitable for broad host of yeasts without either autotrophy or drugs. beta-glucosidase gene from Bacillus polymyxa was cloned with the vector and expressed in Saccharomyces cerevisiae. The specific activity of beta-glucosidase of the recombinant yeast cell-free extract was 3.91 u/mg protein. The residue glucose of the recombinant yeast was considerably reduced in mixed fermentation of glucose and cellobiose. It should be favorable for ethanol fermentation when utilize lignocellulosic biomass as raw material.

Bacillus↗

PubMed1992

Region-directed mutagenesis of residues surrounding the active site nucleophile in beta-glucosidase from Agrobacterium faecalis.

The active site nucleophile of the beta-glucosidase of Agrobacterium faecalis has recently been identified by the use of inhibitors. A combination of site-directed and in vitro enzymatic mutagenesis was carried out on the beta-glucosidase to probe the structure of the active site region. Forty-three point mutations were generated at 22 different residues in the region surrounding the active site nucleophile, Glu358. Only five positions were identified which affected enzyme activity indicating that only a few key residues are important to enzyme activity, thus the enzyme can tolerate a number of single residue changes and still function. The importance of Glu358 to enzymatic function has been confirmed and other residues important to enzyme structure or function have been identified.

Amino Acid Sequence↗

PubMed2005

Immobilization of alpha-glucosidase in chitosan coated polygalacturonic acid.

Crude alpha-glucosidase from Baker's yeast was immobilized in polygalacturonic acid beads and coated with chitosan. Chemical and physical characterization were performed by using p-nitrophenyl-alpha-D-glucopyranoside (pNPG) as an artificial substrate. Operation, thermal, pH, and strorage stabilities of the free and immobilized enzyme were also examined. The stabilities of immobilized enzyme were found to be better than that of the free enzyme. Furthermore, the hydrolysis rate of the chitosan coated alpha-glucosidase polygalacturonic acid beads were studied. In conclusion, the enzyme beads appear to have good characteristics and offer the prospect that this system may find application in enzyme immobilization, in addition to controlled drug release studies.

Chitosan↗

PubMed2005

The influence of methyl jasmonate (JA-Me) and B-glucosidase on induction of resistance mechanisms of strawberry against two-spotted spider mite (Tetranychus urticae Koch.).

The goal of the research was to study the influence of methyl jasmonate (JA-Me) and beta-glucosidase treatments on fecundity and preference to infestation and oviposition of two-spotted spider mite feeding on strawberry. The experiments were conducted in laboratory conditions on leaves of Aga and Kent cultivars. Leaves were treated with: a. solution of 0.1% JA-Me in 0.05% Triton X-100 (by spraying); b. beta-glucosidase dissolved in 0.1 M citrate buffer at pH 6 (by petiole); c. 0.05% solution of the Triton X-100 (by spraying); d. 0.1 M citrate buffer at pH 6 (by petiole). In the no-choice test, application of JA-Me on leaves of strawberry caused reducing of number of eggs laid during three days of the experiment. In the choice test, which was carried out for determination of non-preference mechanism of resistance, there was a statistically significant lower number of mites on leaves treated with JA-Me compared to leaves treated with other compounds as well as to non-treated leaves after 24 hours from solutions application. Moreover, at the same experiment, females of two-spotted spider mite laid the least number of eggs on leaves treated with JA-Me. Analysis conducted using liquid chromatography method, revealed increase of the level of phenolic compounds like chlorogenic acid and rutin on leaves treated with JA-Me. Thus, it appears that JA-Me may be involved in antybiosis or non-preference mechanisms of resistance of strawberry to two-spotted spider mite.

Acetates↗

PubMed2006

A specific short dextrin-hydrolyzing extracellular glucosidase from the thermophilic fungus Thermoascus aurantiacus 179-5.

The thermophilic fungus Thermoascus aurantiacus 179-5 produced large quantities of a glucosidase which preferentially hydrolyzed maltose over starch. Enzyme production was high in submerged fermentation, with a maximal activity of 30 U/ml after 336 h of fermentation. In solid-state fermentation, the activity of the enzyme was 22 U/ml at 144 h in medium containing wheat bran and 5.8 U/ml at 48 h when cassava pulp was used as the culture medium. The enzyme was specific for maltose, very slowly hydrolyzed starch, dextrins (2-7G) and the synthetic substrate (alpha-PNPG), and did not hydrolyze sucrose. These properties suggest that the enzyme is a type II alpha-glucosidase. The optimum temperature of the enzyme was 70 degrees . In addition, the enzyme was highly thermostable (100% stability for 10 h at 60 degrees and a half-life of 15 min at 80 degrees), and stable within a wide pH range.

Culture Media↗

PubMed1991

[The value of the level of alpha-1,4-glucosidase and seminal l-carnitine in patients with oligoasthenospermia].

Samples of sperm have been obtained from 95 who consulted us for infertility. In each case seminal plasma was examined for levels of alpha-1,4-glucosidase and L-carnitine. Our results have led us to fix the threshold value of 42.6 mlU per ejaculate for alpha-1,4-glucosidase and 960 nanomoles of L-carnitine below those levels that we thought occur where the origin of the oligospermia is obstructive (series 1 patients). In series 2 patients the cause of the oligospermia purely being secretory, there is normal epididymal function and therefore the excretory doubts are proven. It is not impossible to have both pathologies because we have found this in men of the intermediate groups C and D. We have found that there is a correlation between the presence of epididymal pathology and a drop in epididymal markers which can be found in severe oligospermia (which can be epididymal in origin and not testicular). Also when there is non abnormalities in the spermogram. This last situation can occur in "invisible" abnormalities of spermaturation in the epidymus.

Biomarkers↗

PubMed1991

Sequence homology between a beta-galactosidase and some beta-glucosidases.

A significant sequence homology was found between a thermostable beta-galactosidase from Sulfolobus solfataricus and two beta-glucosidases, respectively, from Caldocellum saccharolyticum and from Agrobacterium sp. These glycosidases appear to form a new protein family, since no homology could be detected with established beta-galactosidase or beta-glucosidase families.

Amino Acid Sequence↗

PubMed1990

Analyses of catalytic activity and inhibitor binding of human acid beta-glucosidase by site-directed mutagenesis. Identification of residues critical to catalysis and evidence for causality of two Ashkenazi Jewish Gaucher disease type 1 mutations.

Analyses of catalytic properties and inhibitor binding were conducted to investigate the molecular basis of active site function of human acid beta-glucosidases (EC 3.2.1.45) expressed from normal and Gaucher disease Type 1 alleles. Comparative studies were conducted with enzymes expressed from natural (spleen and fibroblasts) alleles or from mutagenized cDNAs in Spodoptera frugiperda (Sf9) cells using the baculovirus expression system. Mutant cDNAs containing Thr43 to Lys43 (beta-GlcThr43----Lys) and Asp358 to Glu358 (beta-GlcAsp358----Glu) substitutions and two cDNAs containing Ashkenazi Jewish Gaucher disease Type 1 mutations, Arg120 to Gln120 (beta-GlcArg120----Gln) and Asn370 to Ser370 (beta-GlcAsn370----Ser) were expressed and the gene products characterized by enzymatic, immunologic, and inhibitor studies. Genotypes at the acid beta-glucosidase locus in selected Gaucher disease Type 1 patients were determined by allele-specific oligonucleotide hybridization of amplified genomic DNA. Compared with normal, recombinant or natural enzymes expressed from beta-GlcAsn370----Ser alleles had about 2-5-fold decreased specific activity based on CRIM (cross-reacting immunologic material). The beta-GlcArg120----Gln cDNA expressed catalytically inactive CRIM in Sf9; consistent with the 9-fold decreased CRIM-specific activity of the natural enzyme from a beta-GlcArg120----Gln/beta-GlcAsn370----Ser genetic compound. The beta-GlcAsp358----Glu cDNA expressed catalytically inactive CRIM in Sf9 cells. The presence of natural or recombinant enzyme expressed from beta-GlcAsn370----Ser alleles was sufficient to confer 3-5-fold increased IC50 values for deoxynojirimycin, glucosylsphingosine, and N-alkyl-glucosylamine derivatives. Progress curves for inhibition by the slow-tight binding N-alkyl-glucosylamines indicated that the beta-Glc-Asn370----Ser mutation did not alter a conformational change induced by these reaction intermediate analogues. These results provide evidence that the beta-GlcArg120----Gln and beta-GlcAsn370----Ser mutations found in Gaucher disease Type 1 patient genomes are the molecular bases of the enzymatic dysfunction. In addition, the region including Arg120 and that encompassing Asp358 and Asn370 contain residues critical to active site formation or participation in the catalytic mechanism.

Amino Acid Sequence↗

PubMed1989

Partial purification and characterization of alpha-glucosidase from Rothia dentocariosa.

Disaccharidases of oral bacteria, especially alpha-glucosidase and beta-fructofuranosidase, are considered to play an important role in the induction of dental caries. Upon the examination of disaccharidases from several strains of saccharolytic oral bacteria, we found all of those bacteria to be capable of hydrolyzing the glycosidic linkage of sucrose. One species of bacteria, Rothia dentocariosa, was found to contain a single disaccharidase, alpha-glucosidase. This enzyme was partially purified by ammonium sulfate precipitation, gel filtration and ion-exchange column chromatography. The optimum pH and temperature for the enzyme activity was found to be 6.8-7.0 and 40 degrees C, respectively. The enzyme activity was strongly inhibited by Ag+, Hg2+, Cu2+, Fe2+ and Tris (Hydroxymethyl) aminomethane.

Bacteria↗

PubMed1988

The activator protein for glucosylceramide beta-glucosidase from guinea pig liver. Improved isolation method and complete amino acid sequence.

beta-Glucosidase activator (SAP-2) is a family of heat-stable, acidic glycoproteins which stimulate enzymatic hydrolysis of glucosylceramide. In this study, we improved the purification method and found that SAP-2 is highly heterogeneous. A hot water extract of frozen guinea pig liver was fractionated by ammonium sulfate sedimentation, then chromatographed with DEAE-Sephacel, Sephadex G-75, and concanavalin A-Sepharose. A fraction binding to concanavalin A-Sepharose was purified further with a C4 high performance liquid chromatography reverse phase column. This yielded several peaks, the main one of which was studied. The specific activity of the purified SAP-2 was 35 units/micrograms (1 unit produces 50% stimulation of a basal glucosidase preparation). N-terminal amino acid sequencing showed that this preparation is a mixture of polypeptides differing in the presence or absence of one or two of the end amino acids. The complete amino acid sequence of the 81 residues in SAP-2 has been determined. Comparison of the sequence of guinea pig SAP-2 with the sequence of human sphingomyelinase activator revealed 58% homology and quite similar hydropathy profiles. Both proteins possess a highly hydrophilic region around Asn-22, which is glycosylated, and 6 cysteine residues, in oxidized form, located in the same positions. Comparison with the published nucleotide sequence for the precursor form of the human activator protein for sulfatide sulfatase (SAP-1) suggested that this activator also has a possibly glycosylated Asn and 6 Cys residues at similar positions, although the remainder of the molecule is somewhat different. Examination of another region of the precursor's nucleotide sequence, assuming a few changes in the identifications, revealed the presence of the sphingomyelinase activator. It appears that two or more activators are derived from a single precursor protein. Marked homologies were seen also with a lung surfactant protein and a sulfated glycoprotein from Sertoli cells.

Amino Acid Sequence↗

PubMedDate not supplied

[Soluble and membrane-bound forms of neutral alpha-glucosidase in human urine in normal conditions and in kidney diseases].

Soluble and membrane-bound forms of neutral alpha-glucosidase, which are immunologically similar to the corresponding forms of kidney enzymes, were found in urine of healthy persons and of patients with kidney impairments. Membrane-bound enzyme was only slightly active in urine of healthy persons and constituted 3-10% of total urine activity although the ratio of membrane-bound enzyme was simultaneously elevated under pathological conditions. The increased rate of soluble enzyme secretion was responsible for distinct activation of neutral alpha-glucosidase in urine of the patients with kidney impairments.

Humans↗

PubMed1987

The precursor of acid alpha-glucosidase is synthesized as a membrane-bound enzyme.

A pulse-chase study in human skin fibroblasts showed that a 110 kDa precursor of acid alpha-glucosidase was synthesized as a membrane-bound protein, which was solubilized in vitro not by mannose 6-phosphate or 1M KCl but by Triton X-100 or trypsin. This 110 kDa precursor form bound to the membrane was detected in control fibroblasts treated with tunicamycin and in I-cell disease fibroblasts as well. The precursors in human placenta were found also in the membrane fraction. It was concluded that the newly synthesized acid alpha-glucosidase precursor is located on the surface of the membrane, and the phosphomannosyl receptor does not participate in the enzyme-membrane binding.

Cell Membrane↗

PubMed1985

Gaucher disease types 1, 2, and 3: differential mutations of the acid beta-glucosidase active site identified with conduritol B epoxide derivatives and sphingosine.

To elucidate the genetic heterogeneity in Gaucher disease, the residual beta-glucosidase in cultured fibroblasts from affected patients with each of the major phenotypes was investigated in vitro and/or in viable cells by inhibitor studies using the covalent catalytic site inhibitors, conduritol B epoxide or its bromo derivative, and the reversible cationic inhibitor, sphingosine. These studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to these inhibitors. Group A residual enzymes had normal I50 values (i.e., the concentration of inhibitor that results in 50% inhibition) for the inhibitors and normal or nearly normal t1/2 values for conduritol B epoxide. All neuronopathic (types 2 and 3) and most non-Jewish nonneuronopathic (type 1) patients had group A residual activities and, thus, could not be distinguished by these inhibitor studies. Group B residual enzymes had about four- to fivefold increased I50 values for the inhibitors and similarly increased t1/2 values for conduritol B epoxide. All Ashkenazi Jewish type 1 and only two non-Jewish type 1 patients had group B residual activities. The differences in I50 values between groups A and B also were confirmed by determining the uninhibited enzyme activity after culturing the cells in the presence of bromo-conduritol B epoxide. Group C residual activity had intermediate I50 values for the inhibitors and represented a single Afrikaner type 1 patient: this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. These inhibition studies indicated that: Gaucher disease type 1 is biochemically heterogeneous, neuronopathic and non-Jewish nonneuronopathic phenotypes cannot be reliably distinguished by these inhibitor studies, and the Ashkenazi Jewish form of Gaucher disease type 1 results from a unique mutation in a specific active site domain of acid beta-glucosidase that leads to a defective enzyme with a decreased Vmax.

Adolescent↗

PubMed1994

Purification and characterization of the endocellular beta-glucosidase of a new strain of Candida entomophila isolated from fermenting agave (Agave sp.) juice.

A yeast strain isolated in the laboratory from fermenting agave (Agave sp.) juice was studied and classified as Candida entomophila. The beta-glucosidase of this yeast was purified by ion-exchange chromatography and gel filtration. Its molecular mass estimated by gel filtration was 400 kDa. The oligomeric structure was determined following treatment of the purified enzyme with SDS. Its optimum pH was between 5 and 6, and its optimum temperature was 60 degrees C. The enzyme was active against soluble glucosides with (1-->3)-beta, (1-->4)-beta and (1-->4)-alpha linkage configuration, and it possesses (1-->6)-alpha-arabinofuranosidase activity. It is competitively inhibited by glucose and by D-gluconic acid lactone. The enzyme was constitutive and a glucosyltransferase activity is observed in the presence of ethanol. Since the glycosides present in wines and fruit juices represent a potential source of aromatic flavour, the possible use of the yeast glucosidase for the liberation of the bound aroma is discussed.

Candida↗

PubMed1993

Role of sulfhydryl groups in the function of glucosidase I from mammary gland.

Glucosidase I initiates the processing of asparagine-linked glycoproteins by excising the distal alpha 1,2-linked glucosyl residue from the Glc3Man9GlcNAc2 oligosaccharide, soon after its en bloc transfer from the lipid-linked donor to the nascent polypeptide. 1-Deoxynojirimycin, an analog of D-glucose, is a potent competitive inhibitor of the enzyme. Sulfhydryl-seeking reagents also strongly inhibit the enzyme, implying the involvement of an -SH group in its activity. To test this hypothesis, glucosidase I was purified from the rat mammary gland and its active site was loaded with 1-deoxynojirimycin, to protect such a group(s), while -SH groups on the remaining surface of the enzyme were blocked with N-ethylmaleimide or para-chloromercuriphenylsulfonic acid. Deoxynojirimycin was removed by dialysis to expose the active site -SH group(s). This group(s) was then tagged with 3-(N-maleimidopropionyl)biocytin (MPB) and detected with 125I-streptavidin on Western blots. A series of experiments is presented to show that indeed a critical -SH group(s) is located within the catalytic site of the enzyme. Additionally, the enzyme also possesses one or more sulfhydryls and disulfide bonds in its primary structure. The experimental approach outlined here should apply to identify reactive sulfhydryl groups in other catalytically active proteins.

Animals↗

PubMed1997

[Chronic respiratory failure in a case with juvenile-onset acid alpha-glucosidase deficiency; successful therapy with nasal intermittent positive pressure ventilation (NIPPV)].

A 13-year-old boy with juvenile-onset acid alpha glucosidase deficiency was reported. Proximal muscle weakness including respiratory muscles and scoliosis progressed since nine year of age. He developed nocturnal dyspnea and daytime somnolence at age 13. His arterial blood gas analysis showed hypoxemia (PO2 54.1 mmHg) and hypercapnia (PCO2 72.3 mmHg), and spirometry showed significantly decreased vital capacity (% VC 21%). He was treated with nocturnal NIPPV employing a device for delivering bilevel positive airway pressure (Bi-PAP). Nocturnal dyspnea and daytime somnolence rapidly disappeared with nocturnal ventilatory support. Daytime arterial PO2 and PCO2 improved after the therapy, namely 74.8 mmHg and 64.1 mmHg respectively. We conclude that NIPPV is a noninvasive and effective therapy for respiratory failure in patients with chronic progressive neuromuscular disorder including acid alpha glucosidase deficiency.

Adolescent↗