Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary deficiency of beta-galactosidase results in GM1 gangliosidosis and Morquio B disease. Of the more than 40 disease-causing mutations described in the Gal gene to date, about 75% are of the missense type and are scattered along the length of the gene. No single, major common mutation has been associated with GM1 gangliosidosis. However, a Trp 273 Leu mutation has been commonly found in the majority of patients with Morquio B disease defined genotypically to date. We now report three new mutations in three Morquio B patients where the Trp 273 Leu mutation is absent. Two of the mutations, C1502G (Asn 484 Lys) and A1548G (Thr 500 Ala), were found in twins (one male, one female) who display a mild form of Morquio B disease and keratan sulfate in the urine. In their fibroblasts, residual activity was 1.9% and 2.1% of controls. On Western blots, the 84-kDa precursor and the 64-kDa mature protein were barely detectable. The occurrence of a 45-kDa degradation product indicates that the mutated protein reached the lysosome but was abnormally processed. In the third case, we identified only a G1363A (Gly 438 Glu) mutation (a major deletion on the second allele has not been ruled out). This female patient too displays a very mild form of the disease with a residual activity of 5.7% of control values. In fibroblasts from this case, the 84-kDa precursor and the 45-kDa degradation product were present, while the mature 64-kDa form was barely detectable. The occurrence of these three mutations in the same area of the protein may define a domain involved in keratan sulfate degradation.
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PMID:Novel mutations (Asn 484 Lys, Thr 500 Ala, Gly 438 Glu) in Morquio B disease. 1239 80

Trp-999 is a key residue for the action of beta-galactosidases (Escherichia coli). Several site specific substitutions (Phe, Gly, Tyr, Leu) for Trp-999 were made. Each substitution caused greatly decreased affinities for substrates and inhibitors that bind in the "shallow" mode, while the affinities of inhibitors that bind in the "deep" mode were not decreased nearly as much. This shows that Trp-999 is important for binding in the shallow mode. The residue is also very important for binding glucose to galactosyl-beta-galactosidase (as a transgalactosidic acceptor). Substitution greatly diminished the affinity for glucose. Substitutions also changed the activation thermodynamics and, subsequently, the rates of the catalytic reactions. The enthalpies of activation of the glycolytic bond cleavage step (galactosylation, k(2)) became less favorable while the entropies of activation of that step became more favorable as a result of the substitutions. Differing magnitudes of these enthalpic and entropic effects with ONPG as compared to PNPG caused the k(2) values for ONPG to decrease but to increase for PNPG. The enthalpies of activation for the common hydrolytic step (degalactosylation, k(3)) increased while the entropies of activation for this step did not change much. As a result, k(3) became small and rate determining for each substituted enzyme. The substitutions caused the rate constant (k(4)) of the transgalactosidic acceptor reactions with glucose (for the formation of allolactose) to become much larger and of the same order of magnitude as the normally large rate constants for transgalactosidic acceptor reactions with small alcohols. This is probably because glucose can approach with less restriction in the absence of Trp-999. However, since glucose binds very poorly to the galactosyl-beta-galactosidases with substitutions for Trp-999, the proportion of lactose molecules converted to allolactose is small. Thus, Trp-999 is also important for ensuring that an appropriate proportion of lactose is converted to allolactose.
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PMID:Trp-999 of beta-galactosidase (Escherichia coli) is a key residue for binding, catalysis, and synthesis of allolactose, the natural lac operon inducer. 1257 95

A major obstacle in gene-therapy protocols is T-cell-mediated destruction of transgene-expressing cells. Therefore new approaches are needed to prevent rapid clearance of transduced cells. We exploited the Gly-Ala repeat (GAr) domain of the Epstein-Barr virus nuclear antigen-1, since the GAr prevents cytotoxic T-lymphocyte-epitope generation. Here we show that three different enzymes (viz. the E. coli LacZ gene encoded beta-galactosidase, firefly luciferase, and HSV1 thymidine kinase) fused with the GAr retained their function. Moreover, linking GAr with beta-galactosidase successfully prevented recognition of GAr-LacZ-expressing cells by beta-galactosidase-specific CTL. Nonetheless, vaccination with a GAr-LacZ adenovirus or with an allogeneic cell line expressing GAr-LacZ resulted in the induction of beta-gal-specific CTL. This demonstrates that the GAr domain does not inhibit cross presentation of antigens, but only affects breakdown of endogenously synthesized proteins. These data demonstrate how the GAr domain can be exploited to create immuno'stealth' genes by hiding transgene products from CTL-mediated immune attack.
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PMID:Creation of immune 'stealth' genes for gene therapy through fusion with the Gly-Ala repeat of EBNA-1. 1456 61

Low gadolinium concentrations induce rapid gigaseal formation and cell adhesion to glass and plastic (polystyrene) substrates in the slime mutant of Neurospora crassa. Cellular adhesion is independent of an integrin-mediated mechanism, because pretreatment with the oligopeptide ARG-GLY-ASP-SER (RGDS) did not inhibit it, and there was no spatial correlation between integrin and adhesions. In contrast, concanavalin A and beta-galactosidase both inhibit adhesion, suggesting that adhesion is mediated by sugar moeities at the cell surface. The adhesion sites are motile in the plasma membrane, as shown by the movement of polystyrene microspheres on the cell surface. In addition to an integrin-based adhesive system, which has already been characterized in walled hyphal cells, hyphae have evolved at least two different plasma membrane-based adhesion mechanisms. The relatively non-specific sugar-mediated adhesion caused by gadolinium may be part of the mechanism of gigaseal formation in other cells. In the absence of sugar-mediated adhesion, gadolinium increases the magnitude of the gigaseal in giant unilamellar liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and cholesterol, with or without the negatively charged phosphatidylserine. Thus, gigaseal formation involves at least two different mechanisms.
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PMID:Gadolinium effects on gigaseal formation and the adhesive properties of a fungal amoeboid cell, the slime mutant of Neurospora crassa. 1513 47

The I locus in soybean (Glycine max) corresponds to a region of chalcone synthase (CHS) gene duplications affecting seed pigmentation. We sequenced and annotated BAC clone 104J7, which harbors a dominant i(i) allele from Glycine max 'Williams 82', to gain insight into the genetic structure of this multigenic region in addition to examining its flanking regions. The 103-kb BAC encompasses a gene-rich region with 11 putatively expressed genes. In addition to six copies of CHS, these genes include: a geranylgeranyltransferase type II beta subunit (E.C.2.5.1.60), a beta-galactosidase, a putative spermine and (or) spermidine synthase (E.C.2.5.1.16), and an unknown expressed gene. Strikingly, sequencing data revealed that the 10.91-kb CHS1, CHS3, CHS4 cluster is present as a perfect inverted repeat separated by 5.87 kb. Contiguous arrangement of CHS paralogs could lead to folding into multiple secondary structures, hypothesized to induce deletions that have previously been shown to effect CHS expression. BAC104J7 also contains several gene fragments representing a cation/hydrogen exchanger, a 40S ribosomal protein, a CBL-interacting protein kinase, and the amino terminus of a subtilisin. Chimeric ESTs were identified that may represent read-through transcription from a flanking truncated gene into a CHS cluster, generating aberrant CHS RNA molecules that could play a role in CHS gene silencing.
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PMID:Features of a 103-kb gene-rich region in soybean include an inverted perfect repeat cluster of CHS genes comprising the I locus. 1549 96

The mechanisms mediating responses to glycine withdrawal in budding yeast were studied using a genome-wide profiling approach. A striking pattern of repressed expression of genes with an enrichment for those involved in one-carbon metabolism and AMP biosynthesis was revealed. Sequence analysis of the promoters for the most severely repressed genes identified a conserved sequence, TGACTC, a known binding site for the transcription factors Gcn4p and Bas1p. Loss of BAS1 abolished or significantly reduced the repression of these genes in response to glycine removal but this phenotype was much less apparent in the absence of BAS2 or GCN4. Addition of a Bas1p-LexA fusion protein to a strain with a LexAop-LacZ fusion showed a strong glycine effect both in a BAS2 and a bas2 background. A Bas1p-VP16 fusion protein activated expression in a bas1bas2 strain but no glycine effect was observed while a Bas1p-Bas2p fusion protein activated expression to a lesser extent with a slight stimulation by glycine. These results suggest that glycine affects Bas1p activation of transcription rather than DNA binding and that Bas2p is not required for this affect. Glycine withdrawal repressed many of the same genes as addition of adenine, a process known to be dependent on Bas1p. However, the glycine response is independent of adenine repression, because glycine regulation occurs normally in ade strains. We did not see any difference in the degree of stimulation by glycine in the presence or absence of adenine even in Ade+ strains. Glycine regulation was also found to be dependent on an intact SHM2 gene, which encodes cytoplasmic serine hydroxymethyltransferase. A reporter plasmid containing a DNA sequence from the GCV2 promoter which confers glycine regulation on heterologous genes was introduced into the yeast deletion set to screen for genes required for glycine regulation. A number of genes, including BAS1 were required for activation by glycine but only the SHM2 gene was required for repression in the absence of glycine. We also showed that regulation of the SHM2 promoter by glycine requires Bas1p but not Bas2p or Gcn4p using a beta-galactosidase reporter. The response of the promoter to glycine required an intact SHM2 gene but was restored in a shm2 strain by addition of formate to the medium.
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PMID:Transcriptional regulation of the one-carbon metabolism regulon in Saccharomyces cerevisiae by Bas1p. 1594 49

Exosomes are vesicles of endocytic origin secreted spontaneously by dendritic cells (DCs). We have shown previously that exosomes can transfer antigen or MHC-peptide complexes between DCs, thus potentially amplifying the immune response. We had also identified milk fat globule EGF/factor VIII (MFG-E8), also called lactadherin, as one of the major exosomal proteins. MFG-E8 has two domains: an Arg-Gly-Asp sequence that binds integrins alphavbeta3 and alphavbeta5 (expressed by human DCs and macrophages) and a phosphatidyl-serine (PS) binding sequence through which it associates to PS-containing membranes (among which exosomes). MFG-E8 is thus a good candidate molecule to address exosomes to DCs. Here, we show that MFG-E8 is expressed by immature bone-marrow-derived DCs (BMDCs) and secreted in association with exosomes in vitro. We have generated mice expressing an inactive form of MFG-E8, fused to beta-galactosidase. Analyzing these mice, we demonstrate that MFG-E8 is expressed in vivo in splenic DCs. In a mouse DC-dependent, antigen-specific, CD4 T cell-stimulation assay, exosomes produced by MFG-E8-deficient BMDCs were barely less efficient than exosomes bearing MFG-E8. We conclude that MFG-E8 is efficiently targeted to exosomes but is not essential to address exosomes to mouse BMDCs. Involvement of MFG-E8/lactadherin in exosome targeting to other DC subpopulations, or to human DCs, is still possible.
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PMID:Accumulation of MFG-E8/lactadherin on exosomes from immature dendritic cells. 1598 8

We find that peptides containing -Asn-Gly- sequences typically show approximately 70-80% degree of deamidation after standard overnight (approximately 12 h) tryptic digestion at 37 degrees C. This emphasizes the need for more detailed information about the deamidation reaction in -Asn-Gly- sequences, in which two deamidated species are produced, one containing an aspartic acid (-Asp-Gly-) residue and the other containing an isoaspartic acid (-betaAsp-Gly-) residue. For the peptide SLNGEWR (54-60 beta-galactosidase, E. coli), all three components of the reaction mixture were separated by HPLC on C18 300-A sorbent, with trifluoroacetic acid as an ion-pairing modifier. Their intensity ratios suggested the elution order -betaAsp-/-Asn-/-Asp-, which was subsequently confirmed by MALDI MS and MS/MS analysis. The kinetics of the deamidation was studied in detail for the synthetic SLNGEWR parent using RP HPLC with UV detection. The half-life of this peptide was found to be approximately 8 h under digestion conditions. Analysis of a large pool of peptide retention data shows that the -betaAsp-/-Asn-/ -Asp- retention order is normally observed under the above conditions, especially if the original -NG- sequence is surrounded by hydrophobic amino acids. However, changing chromatographic conditions to 100-A pore size sorbents, or using formic acid as a modifier, increases the retention time of -betaAsp- relative to the -Asn-/-Asp- pair, so the order can sometimes be different.
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PMID:Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: Consequences for MALDI and HPLC-MALDI analysis. 1697 Mar 46

Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 beta- glycosidase (Tca beta-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both beta-galactosidase and beta-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5 mM p-nitrophenyl beta-Dgalactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5 mM p-nitrophenyl beta-D-glucopyranoside at 75degreeC. Kinetic analysis with p-nitrophenyl beta-D-galactopyranoside revealed that the kcat value of the H119G mutant was 76.3-fold lower than that of the wild type, but the Km of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency (kcat/Km) of the mutant decreased to 0.08% to that of the wild type. The kcat value of the H119G mutant for p-nitrophenyl beta- D-glucopyranoside was 5.1-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from 90 degrees C to 80-85 degrees C). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in beta- galactosidase and beta-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.
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PMID:Mutational analysis of Thermus caldophilus GK24 beta-glycosidase: role of His119 in substrate binding and enzyme activity. 1830 73

MYB transcription factor is one of the largest families in plants, which plays an important role in regulating plant development and physiological metabolism. In this study, the expression and function of the new MYB transcription factor gene GmMYBJ6 (GenBank No. DQ902863), isolated from soybean (Glycine max L.), were characterized. The expression pattern of GmMYBJ6 in different organs was examined using Northern blotting analysis. The expression of GmMYBJ6 was detected only in the leaves. The transcriptional activation ability of GmMYBJ6 protein was confirmed by the yeast assay system and the activity of beta-galactosidase was 28.48 U/mL. The green fluorescent protein expression vector p163-GFP-GmMYBJ6 was constructed and transformed into the epidermal cells of onion via particle bombardmental method. The results of instantaneous expression showed that GmMYBJ6 proteins were localized in cell nucleus. Semi-quantitative RT-PCR analysis indicated that GmMYBJ6 improved the expression of certain flavonoid biosynthetic genes, such as PAL (Phenylalanine ammonia lyase), C4H (cinnamate-4-hydroxylase), 4CL (4-coumaroyl-CoA ligase), CHS (Chalcone synthase), CHI (Chalcone isomerase), F3H (Flavanone 3-hydroxylase), and FLS (Flavonol synthase), resulting an increase of the total flavonoid levels in positive tobacco transformants. Additionally, the increasing expression of GmMYBJ6 in soybean cultivar Zhongdou 27, induced by UV-B radiation, drought, and high-salt treatment, indicated that GmMYBJ6 was associated with response to abiotic stresses.
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PMID:[Expressing and functional analysis of GmMYBJ6 from soybean]. 1958 66


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