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PubMed1971

Antibody-mediated activation of a defective beta-D-galactosidase. II. Immunological relationship between the normal and the defective enzyme.

Two closely related protein antigens were used to study immunogenic competition. Namely, normal beta-D-galactosidase of Escherichia coli (Z) and a genetically defective beta-D-galactosidase (AMEF) which seems to differ from the normal in one amino acid substitution. A unique characteristic of this pair of antigens is that, although they are indistinguishable in precipitation and absorption tests with antibodies, the enzymatic activity of AMEF is specifically increased several-hundredfold in the presence of antibodies directed against Z. The following results show that Z and AMEF also differ in their immunogenic ability: (a) antibodies directed against Z activated AMEF; antibodies directed against AMEF did not activate, but competed specifically with activating antibodies. (b) Animals immunized with AMEF failed to produce activating antibodies when they were subsequently challenged with Z, although the presence of some cells primed to produce activating antibodies could be demonstrated by adoptive transfer. (c) Animals preimmunized with Z were stimulated in their production of activating antibodies by AMEF challenge, although not as efficiently as with Z. A model explaining these observations by competition for the immunogenic site among antigen-sensitive cells carrying cross-reacting receptors is presented.

Adoptive Transfer↗
PubMed1961

The effect of exogenous energy sources on the synthesis of beta-galactosidase in resting-cell suspensions of Escherichia coli.

Using methyl-1-thio-beta-D-galactoside as the inducer, the biosynthesis of beta-galactosidase was observed in Escherichia coli B with only endogenous sources of nitrogen and energy available. The addition of glucose, ribose, xylose, or glycerol as exogenous energy sources to nitrogen-deficient media blocked enzyme formation. Preinduction of the resting cells failed to overcome inhibition by the added energy sources. With limited quantities of glucose, ribose, xylose, or glycerol, synthesis of beta-galactosidase resumed abruptly and continued at the rate normal for cells in nitrogen-deficient media. Comparison of enzyme activities with oxygen uptake data revealed a reduction in the rate of oxygen uptake at the time enzyme synthesis resumed in media originally containing small amounts of energy sources. This change corresponded to only a fraction of the oxygen required for complete oxidation of one of the exogenous substrates. It is suggested that inhibition by these particular exogenous substrates involves metabolism to a common repressor or interference with an energy-transfer system.

Escherichia coli↗
PubMed1998

Chronic uveitis in guinea pigs infected with varicella-zoster virus expressing Escherichia coli beta-galactosidase.

There is no small animal model that replicates chickenpox and herpes zoster, which are caused by varicella-zoster virus (VZV). Therefore, to detect VZV in tissues of infected animals, the Escherichia coli beta-galactosidase gene was inserted into the viral genome. Intravitreal inoculation of guinea pigs with virus-infected cells resulted in a chronic uveitis, with mononuclear cells in the vitreous cavity of the eye of nearly all animals. Staining with X-gal demonstrated the presence of VZV in the ciliary body or iris of approximately 40% of the animals and in retinal pigmented epithelial cells in 4 animals. X-gal staining showed VZV in the eye of 1 animal 140 days after inoculation. These experiments indicate that VZV expressing beta-galactosidase is useful for detecting virus in tissues and that VZV can cause a chronic uveitis in which virus can be detected in some animals for up to 4 months.

Animals↗
PubMed2003

Ultrasound-guided percutaneous delivery of adenoviral vectors encoding the beta-galactosidase and human factor IX genes to early gestation fetal sheep in utero.

In utero gene therapy may provide treatment of genetic diseases before significant organ damage, allow permanent genetic correction by reaching stem cell populations, and provide immune tolerance against the therapeutic transgenes and vectors. We have used percutaneous ultrasound-guided injection as a minimally invasive fetal procedure. First-generation adenoviruses encoding the nuclear localizing beta-galactosidase reporter gene or the human factor IX (hFIX) gene, or colloidal carbon were delivered via the umbilical vein (UV, n = 4), heart (intracardiac [IC], n = 2), liver parenchyma (intrahepatic [HE], n = 11), peritoneal cavity (intraperitoneal [IP], n = 14), skeletal musculature ([intramuscular [IM], n = 11), or the amniotic cavity (intraamniotic [IA], n = 14) to early-gestation fetal sheep (0.3 gestation = day 33-61). Postmortem analysis was performed at 2, 9, or 28 days after injection. Although fetal survival was between 77% and 91% for IP, HE, IA, and IM routes, no fetuses survived UV or IC procedures. The hFIX levels reaching 1900 and 401 ng/ml (IP), 30 ng/ml (HE), 66.5 and 39 ng/ml (IA), and 83 and 65.5 ng/ml (IM), respectively, were determined 2 days after injection and decreased at birth to 16.5 ng/ml (IP), 7 ng/ml (HE), 4.5 ng/ml (IA), and 4 and 0 ng/ml (IM). Polymerase chain reaction (PCR) and immunohistochemistry showed broadest hFIX transgene spread and highest localised beta-galactosidase expression, respectively, after IP administration. Antibodies were observed against vector but not against hFIX.

Adenoviridae↗
PubMed2000

The beta-galactosidase gene as a marker for hematopoietic reconstitution: individual cell analysis in a murine bone marrow transplantation model.

We have used a simple, single-gene retrovirus carrying the Escherichia coli beta-galactosidase reporter gene (lacZ), termed LlacZ. This virus was found to infect immortalized myeloid and lymphoid precursor/leukemic cell lines efficiently as well as primary murine bone marrow clonogenic progenitors, without apparent modulation of growth or phenotype. Following infection of bone marrow cells, a significant proportion of progenitors--36% of lineage-negative cells with low levels of c-kit expression (lin-/c-kit(lo)) known to be enriched with pluripotent hemopoietic stem cells, and 19% of Sca1-positive cells known to be enriched with transplantable cells with lymphomyeloid-reconstituting ability--were shown to express lacZ. Use of an LlacZ-infected population of post 5-fluorouracil bone marrow cells to reconstitute lethally irradiated mice demonstrated the presence of lacZ-expressing cells in the spleen at day 12 post-transplantation with provirus detected in individual spleen colonies (CFU-S). In the long term (3-6 months following transplantation), lacZ expression was detected in hematopoietic tissues of all recipient mice. The use of two-color in situ and flow cytometry analysis combined with lineage-specific antibodies showed lacZ expression in both myeloid and lymphoid cells in spleen and bone marrow. In addition, lacZ-expressing cells were detected in secondary recipient mice injected with bone marrow cells derived from primary LlacZ recipients. Overall, these data show the efficacy of a single gene vector for stem cell transduction, the utility of beta-galactosidase as a single cell marker for stem cell transduction and reconstitution ability, and the need for protocol optimization to see high-level multilineage gene expression.

Animals↗
PubMed2003

Homogeneous assays for cellular protein degradation using beta-galactosidase complementation: NF-kappaB/IkappaB pathway signaling.

Activation of cells by the tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) cytokines results in activation of the nuclear factor-kappaB (NF-kappaB) via proteasomal degradation of an associated IkappaB molecule. To monitor cellular IkappaB, the protein was recombinantly expressed as a fusion protein with a novel enzymatic tag, ProLabel (PL). ProLabel is a small 5.5-kDa sequence from the amino-terminal amino acids of beta-galactosidase, possesses a simple ribbon structure, and can be fused to many proteins via the amino or carboxyl terminus. Expression of this construct allows quantitative detection of the recombinant protein in crude lysates by using a method based on beta-galactosidase enzyme fragment complementation (EFC). Transient transfection of IkappaB-PL in HeLa cells generated an EFC signal that was highly correlated with a western analysis of the protein construct. ProLabel expressed alone in the cells did not show any EFC activity, due to rapid proteolytic degradation, indicating a very low background signal from the protein tag. TNF-alpha and IL-1 treatment induced a concentration-dependent degradation of IkappaB-PL, with potency values similar to those reported using other methods. IkappaBM-PL (mutant of IkappaB-PL), in contrast, did not undergo degradation for concentrations up to and including 10 ng/ml TNF-alpha or IL-1, demonstrating that degradation of IkappaB-PL was specific to the NF-kappaB pathway activation. TNF-alpha and IL-1 induced maximal IkappaB-PL degradation within 30 min of induction. This was reversed by several agents that ablate this pathway, including anti-TNF-alpha antibodies and the proteasome inhibitor, MG-132. The assay was amenable to HTS systems, with good precision and reproducibility. Z' values and coefficients of variance for IkappaB-PL degradation were 0.6 and <9%, respectively.

Dose-Response Relationship, Drug↗
PubMed1994

Linear epitopes of HIV-1, presented as hybrids with Escherichia coli beta-galactosidase or synthetic peptides.

HIV-1 B cell epitopes from gp41, the T cell epitope of p34pol, and a cluster of B and T epitopes from p17gag were selected. The epitopes were presented as synthetic peptides and as either N- or C-terminal insertions into beta-galactosidase. Hybrids were efficiently expressed in E. coli and easily purified when epitopes were inserted at the beta-galactosidase C terminus. Sera from HIV-1-infected individuals reacted in peptide- and hybrid protein-based enzyme-linked immunosorbent assays (ELISAs) mostly with the immunodominant site of gp41. The second site of gp41 and also sites from p17 and p34 appeared to be immunorecessive. A few of the HIV-1-positive sera exhibited several immunorecessive reactivities. HIV-1-positive sera from the former Soviet Union and Cuba had reactivities similar to those of American, African, and west European sera. Some sera could not be evaluated as specifically HIV-1 seropositive because of their broad reactivities with a multitude of peptides and proteins, unrelated to HIV-1. Extensive tests were performed to define unspecific reactivities by absorption, blocking, and sandwich ELISAs. The application of the hybrid protein assay substantially improved the specificity of the ELISA tests. Thus, hybrid protein-based ELISAs appeared to be more suitable than peptide-based ELISAs, especially for the evaluation of immunorecessive reactivities.

Amino Acid Sequence↗
PubMed2001

Keratan sulfate disaccharide composition determined by FACE analysis of keratanase II and endo-beta-galactosidase digestion products.

Many tissues contain glycoproteins and proteoglycans, which are substituted with N-or O-linked keratan sulfate, a glycosaminoglycan in which the lactosamine (-galbeta1,4glcNAc-) disaccharide backbone is variably modified by sulfation, fucosylation, and sialylation. We report here a rapid, sensitive, and quantitative procedure for obtaining a complete disaccharide compositional analyses for keratan sulfates after FACE separation of products generated by hydrolysis of the glycosaminoglycans with B. fragillis keratanase II and E. freundii endo-beta-galactosidase. Seven digestion end products are separable in a single electrophoretic step using Monosaccharide composition gels. These are: the unsulfated disaccharide, glcNAcbeta1,3gal, the fucosylated trisaccharide, galbeta1,2[fucalpha1,3]glcNAc6S, the mono- and disulfated disaccharides, galbeta1,4glcNAc6S or gal6Sbeta1,4glcNAc6S from the chain interior, and the sialylated mono- and disulfated trisaccharides neuAalpha2,3galbeta1,4glcNAc6S or neuAalpha2,3gal6Sbeta1,4glcNAc6S from the nonreducing terminus. FACE analyses also revealed the presence of a contaminant beta-galactosidase activity in keratanase II enzyme preparations which cleaves the disaccharide, galbeta1,4glcNAc6S to its constituent monosaccharides, gal and glcNAc6S. It was particularly prominent at enzyme concentrations > 2 mU per nmole substrate glcNH(2) or after prolonged digestion times (> 12 h), and was not inhibitable by thiogalactosides or N-acetyl-lactosamine. As these monosaccharide products would not be detectable using the commonly described analytical methods for KS hydrolase products, such as (1)H-NMR and HPLC analyses, our data illustrate that the FACE procedure represents an improved approach for accurate compositional microanalyses of corneal and skeletal keratan sulfates, especially applicable to experimentation involving small amounts (1-2 microg) of this glycosaminoglycan.

Acetylglucosaminidase↗
PubMed2000

Functional alpha-fragment of beta-galactosidase can be expressed from the mobile group I intron PpLSU3 embedded in yeast pre-ribosomal RNA derived from the chromosomal rDNA locus.

PpLSU3, a mobile group I intron found in the ribo-somal RNA genes of Physarum polycephalum, encodes the I-PpoI homing endonuclease. This enzyme represents one of the rare cases in nature where a protein is expressed from an RNA polymerase I transcript. Our previous results showed that the full length intron, but not a further processed species, is the messenger for I-PpoI, implying a role of the untranslated region (UTR) in gene expression. To study the function of the 3'-UTR in expression of the endonuclease and in splicing of the intron, we replaced the I-PpoI gene in PpLSU3 with the gene for the alpha-fragment of Escherichia coli beta-galactosidase, and then integrated this chimeric intron into all the chromosomal rDNA repeats of yeast. The resulting cells synthesized functional alpha-fragment, as evidenced by a complementation assay analogous to that used in E.coli. The beta-galactosidase activity thus provides an unusual and potentially valuable readout for Pol I transcription from chromosomal rDNA. This is the first example in which a eucaryotic homing endonuclease gene has been successfully replaced by a heterologous gene. Using deletion mutagenesis and a novel randomization approach with the alpha-fragment as a reporter, we found that a small segment of the 3'-UTR dramatically influences both splicing and protein expression.

3' Untranslated Regions↗
PubMed2000

Engineering the active center of the 6-phospho-beta-galactosidase from Lactococcus lactis.

Several amino acids in the active center of the 6-phospho-beta-galactosidase from Lactococcus lactis were replaced by the corresponding residues in homologous enzymes of glycosidase family 1 with different specificities. Three mutants, W429A, K435V/Y437F and S428D/ K435V/Y437F, were constructed. W429A was found to have an improved specificity for glucosides compared with the wild-type, consistent with the theory that the amino acid at this position is relevant for the distinction between galactosides and glucosides. The k(cat)/K(m) for o-nitrophenyl-beta-D-glucose-6-phosphate is 8-fold higher than for o-nitrophenyl-beta-D-galactose-6-phosphate which is the preferred substrate of the wild-type enzyme. This suggests that new hydrogen bonds are formed in the mutant between the active site residues, presumably Gln19 or Trp421 and the C-4 hydroxyl group. The two other mutants with the exchanges in the phosphate-binding loop were tested for their ability to bind phosphorylated substrates. The triple mutant is inactive. The double mutant has a dramatically decreased ability to bind o-nitrophenyl-beta-D-galactose-6-phosphate whereas the interaction with o-nitrophenyl-beta-D-galactose is barely altered. This result shows that the 6-phospho-beta-galactosidase and the related cyanogenic beta-glucosidase from Trifolium repens have different recognition mechanisms for substrates although the structures of the active sites are highly conserved.

Amino Acid Substitution↗
PubMed1992

Mutant forms of beta-galactosidase with an altered requirement for magnesium ions.

By random approaches we have previously isolated many variants of Escherichia coli beta-galactosidase within a short contiguous tract near the N-terminus (residues 8-12 of wild-type enzyme), some of which have increased stability towards heat and denaturants. The activity of these mutants was originally analysed and quantitated in situ in activity gels without the addition of magnesium ions to the buffer system. We now show that the improved stability is only observable under such conditions of limiting magnesium ion concentrations or in the presence of appropriate concentrations of a metal chelator. In the presence of EDTA, purified preparations of one of these mutant enzymes were much more resistant to denaturants than wild-type, but this differential was completely nullified in the presence of 1 mM Mg2+. However, the stability of this mutant enzyme in EDTA was lower than that shown by it, or the wild-type enzyme, in the presence of magnesium ions. In addition, certain alterations within another N-terminal tract (residues 27-31 of wild-type) resulted in enzymes with greater dependence on Mg2+ than natural beta-galactosidase. We conclude that a small number of residue changes in a large protein can profoundly modulate the requirement for metal ion stabilization, allowing partial abrogation of this need in certain cases. Thus, some enzymes which require divalent metal ions for structural purposes only may be engineered towards metal independence.

Amino Acid Sequence↗
PubMed1991

Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences.

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.

Amino Acid Sequence↗
PubMed1999

Regulation of the alpha-galactosidase activity in Streptococcus pneumoniae: characterization of the raffinose utilization system.

A 10.2-kb gene region was identified in the Streptococcus pneumoniae genome sequence that contains eight genes involved in regulation and metabolism of raffinose. The genes rafR and rafS are transcribed as one operon, and their gene products regulate the raffinose-dependent stimulation of a divergently transcribed second promoter (P(A)) directing the expression of aga, the structural gene for alpha-galactosidase. Raffinose-mediated transcription from P(A) results in a 500-fold increase in alpha-galactosidase activity in the cell. A third promoter within the cluster is responsible for the transcription of the remaining five genes (rafE, rafF, rafG, gtfA, and rafX), whose gene products might be involved in transport and metabolism of raffinose. The presence of additional internal promoters cannot be excluded. The aga promoter P(A) is negatively regulated by the presence of sucrose in the growth medium. Consistent with catabolite repression (CR), a DNA sequence with high homology to the CRE (cis-active element) was identified upstream of the aga promoter. Sucrose-mediated CR depends on the phosphoenolpyruvate: sucrose phosphotransferase system (PTS) but is unaffected by a mutation in a gene encoding a homolog of the CRE regulatory protein CcpA.

5' Untranslated Regions↗
PubMed1994

N-terminal amino acid sequence of persimmon fruit beta-galactosidase.

beta-Galactosidase (EC 3.2.1.23) from persimmon fruit was purified 114-fold with a 15% yield using Sephadex G-100 gel filtration, CM-Sephadex ion exchange, and Sephacryl S-200 gel filtration chromatography, with subsequent electroelution from nondenaturing polyacrylamide gel electrophoresis (PAGE) gels. The estimated molecular mass of the native beta-galactosidase by Sephacryl S-200 was 118 kD. After sodium dodecyl sulfate-PAGE of the enzyme electroeluted from native gels, two subunits with estimated molecular masses of 34 and 44 kD were observed, suggesting that the native enzyme was an aggregate of several subunits. Amino acid composition and N-terminal amino acid sequences of the two major subunits were different.

Amino Acid Sequence↗
PubMed1994

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.

Amino Acid Sequence↗
PubMed2002

Crystallization and preliminary X-ray crystallographic studies of rice alpha-galactosidase.

alpha-Galactosidases catalyze the hydrolysis of galactooligosaccharides and galactopolysaccharides to alpha-galactose residues and are widely distributed in microorganisms, plants and animals. alpha-Galactosidase from rice (Oryza sativa L. ssp. japonica) was crystallized by the hanging-drop vapour-diffusion method. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 63.1, b = 71.3, c = 85.6 A, and diffract beyond 1.9 A resolution.

Crystallization↗
PubMed2003

Crystallization and preliminary X-ray crystallographic studies of alpha-galactosidase I from Mortierella vinacea.

alpha-Galactosidases catalyze the hydrolysis of a galactosyl residue from galactooligosaccharides and galactopolysaccharides. alpha-Galactosidase I from Mortierella vinacea was crystallized in two crystal forms using the hanging-drop vapour-diffusion method. Type 1 crystals belonged to space group I422, with unit-cell parameters a = b = 142.4, c = 131.5 A, and diffracted to beyond 2.1 A resolution, while type 2 crystals belonged to space group P4, with unit-cell parameters a = b = 100.9, c = 102.7 A, and diffracted to beyond 1.6 A resolution. This enzyme crystallized as a glycoprotein tetramer and the tetrameric structure was located around the crystallographic fourfold axis.

Bacterial Proteins↗
PubMed2006

Crystallization and preliminary X-ray diffraction studies of two thermostable alpha-galactosidases from glycoside hydrolase family 36.

alpha-Galactosidases from thermophilic organisms have gained interest owing to their applications in the sugar industry. The alpha-galactosidases AgaA, AgaB and AgaA A355E mutant from Geobacillus stearothermophilus have been overexpressed in Escherichia coli. Crystals of AgaB and AgaA A355E have been obtained by the vapour-diffusion method and synchrotron data have been collected to 2.0 and 2.8 A resolution, respectively. Crystals of AgaB belong to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 113.3, c = 161.6 A. Crystals of AgaA A355E belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 150.1, c = 233.2 A.

Bacillaceae↗