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PubMed1997

Regioselectivity of the enzymatic transgalactosidation of D- and L-xylose catalysed by beta-galactosidases.

The regioselectivity of enzymatic transgalactosidation depends on the source of the beta-galactosidase used. When the galactosyl acceptor only contains secondary hydroxyl groups, e.g., D- or L-xylose, it is possible to find an enzyme that catalyses preferentially the synthesis of any of the three regioisomers 4-, 3- and 2-O-beta-D-galactopyranosyl-D-xylose (1, 2 and 3, respectively) or 4-, 3- and 2-O-beta-D-galactopyranosyl-L-xylose (4, 5 and 6, respectively). Enriched mixtures in 1, 2 or 3 were obtained using beta-galactosidases from Escherichia coli, bovine testes or Aspergillus oryzae, respectively, by transgalactosidation reaction of O-nitrophenyl-beta-D-galactopyranoside and D-xylose, and enriched mixtures in 4, 5 or 6 were obtained in a similar way using beta-galactosidases from Aspergillus oryzae, lamb small-intestine (intestinal lactase-phloridzin hydrolase) or Saccharomyces fragilis, respectively, using L-xylose as acceptor.

Animals↗
PubMed2000

Regioselective synthesis of p-nitrophenyl glycosides of beta-D-galactopyranosyl-disaccharides by transglycosylation with beta-D-galactosidases.

The beta-D-galactosidase from porcine liver induced regiospecific transglycosylation of beta-D-galactose from beta-D-Gal-OC6H4NO2-o to OH-6 of, respectively, p-nitrophenyl glycoside acceptors of Gal, GlcNAc and GalNAc to afford beta-Gal-(1-->6)-alpha-Gal-OC6H4NO2-p, beta-Gal-(1--> 6)-beta-Gal-OC6H4NO2-p, beta-Gal-(1-->6)-alpha-GalNAc-OC6H4NO2-p, beta-Gal-(1-->6)-beta-GalNAc-OC6H4NO2-p, beta-Gal-(1-->6)-alpha-GlcNAc-OC6H4NO2-p, and beta-Gal-(1-->6)-beta-GlcNAc-OC6H4NO2-p. The enzyme showed much higher transglycosylation activity for the alpha-glycoside acceptors than the corresponding beta-glycoside acceptors. The regioselectivity of the beta-D-galactosidase from Bacillus circulans ATCC 31382 greatly depended on the nature of the acceptor. When alpha-D-GalNAc-OC6H4NO2-p and alpha-D-GlcNAc-OC6H4NO2-p were used as acceptors, the enzyme showed high potency for regioselective synthesis of beta-Gal-(1-->3)-alpha-GalNAc-OC6H4NO2-p and beta-Gal-(1-->3)-alpha-GlcNAc-OC6H4NO2-p in high respective yields of 75.9 and 79.3% based on the acceptors added. However, replacement of beta-D-Gal-OC6H4NO2-p by beta-D-GalNAc-OC6H4NO2-p did change the direction of galactosylation. The enzyme formed regioselectively beta-Gal-(1-->6)-beta-Gal-OC6H4NO2-p with (beta-Gal-1-->(6-beta-Gal-1-->)n6-beta-Gal-OC6H4NO2-p, n = 1-4). No beta-(1-->3)-linked product was detected during the reaction. Use of the two readily available beta-D-galactosidases facilitates the preparation of (1-->3)- and (1-->6)-linked disaccharide glycosides of beta-D-Gal-GalNAc and beta-D-Gal-GlcNAc.

Animals↗
PubMed2002

Residual galactosylsphingosine (psychosine) beta-galactosidase activities and associated GALC mutations in late and very late onset Krabbe disease.

BACKGROUND: Krabbe disease (globoid-cell leukodystrophy; GLD) is caused by mutations in the GALC gene. Beta-galactocerebrosidase (GALC) is a specific beta-galactosidase which is defective in GLD. About 90% of GLD patients have an infantile course by fatal cerebral demyelination, but 10% have a later onset (LOGLD) of symptoms and survive for one or several decades. METHODS: Activities of GALC towards galactosylceramide (GC) and galactosylsphingosine (psychosine; PS) were determined in white blood cells and cultured fibroblasts derived from GLD patients and controls using tritium-labelled natural substrates. In the galactosylsphingosine (psychosine) beta-galactosidase (GALC-PS) assay, a thin layer chromatographic technique was used to separate enzymatically released radioactive galactose. RESULTS: Both galactosylceramide beta-galactosidase (GALC-GC) and GALC-PS activities were reduced by at least 85% of the normal in all but 2 of the 10 GLD patients studied. In particular, one 23-year-old severely demyelinated LOGLD patient was strongly deficient (11% of the normal) in GALC-GC but apparently normal for GALC-PS activity. This patient's GALC genotype was the 30-kb-deleted/502T allele combined with a wild-type allele in the 1637C background known to slightly reduce GALC-GC activity. Further, of six LOGLD patients, both of 62- and 63-year-old brothers had the deleted allele combined with an 809G>A mutated 1637C allele. The sibs had strongly reduced GALC-GC and GALC-PS activities but became clinically remarkable only in their 50s with a severe mental downhill course in one of them. CONCLUSIONS: A GALC genotype with one deleted and one polymorphic GALC activity-reducing allele can lead to enzymatic and clinical signs of LOGLD in the absence of marked GALC-PS deficiency. If an active PS hydrolysis in the fibroblasts of a LOGLD patient also reflected such hydrolysis in the brain, the psychosine hypothesis for GLD may need to be revised.

Age of Onset↗
PubMed2002

Optimisation of an enzymatic method for beta-galactosidase.

BACKGROUND: The enzyme beta-galactosidase present in the Kupffer cells of the liver has potential as a marker of liver dysfunction prior to transplantation. Spectrophotometric methods have insufficient sensitivity. METHODS: Fluorimetric methods have the required sensitivity and we have optimised such a method in a microtitre plate format to improve its utility. beta-galactosidase acts on the substrate 4-methylumbelliferyl-galactoside (MUG) to produce 4-methylumbelliferone (4-MU), detected fluorimetrically with excitation wavelength 355 nm and emission wavelength 460 nm. RESULTS: Reaction conditions in a citrate-phosphate buffer were optimised to give maximal enzyme activity: pH was optimal at 4.4 (range investigated 3.6-5.0) and substrate concentration at 3.33 mmol/l. A small specimen volume (10 microl) in 80 microl of substrate solution produced adequate fluorescent yield after an incubation period of 30 to 60 min at 37 degrees C. Reaction was terminated by addition of 200 microl of glycine-NaOH, pH 12.8. The assay is linear to 3,000 U/ml. The intra-assay coefficient of variation (CV%) at 50, 502, and 2,012 U/ml was 4.7, 3.1, and 3.4, respectively (n=10). Inter-assay CV% at 51, 496, and 1,986 U/ml was 7.0, 4.0, and 3.9, respectively (n=10). CONCLUSIONS: The assay has greater practical utility and demonstrated significant differences in the perfusate beta-galactosidase between cold-stored and warm-perfused livers in a porcine model of transplantation.

Animals↗
PubMed2000

Characterization of beta-galactosidase in leukocytes and fibroblasts of GM1 gangliosidosis heterozygotes compared to normal subjects.

OBJECTIVES: Characterization of beta-galactosidase in leukocytes and fibroblasts of heterozygotes for GM1 type I. DESIGN AND METHODS: Leukocyte and fibroblast beta-galactosidase activity was determined fluorimetrically using 4-methylumbelliferyl-beta-D-galactoside as an artificial substrate. Optimum pH, Km, Vmax and thermostability of the enzyme at 42 degrees C were determined. RESULTS: The leukocyte and fibroblast enzyme of heterozygotes have an optimum pH of 4.0 and 4.2, respectively. In normal subjects, the optimum pH was 4.2 in both cells, according to previous studies. The Km of the enzyme of heterozygotes was determined to be 0.65 mM in leukocytes and 0.59 mM in fibroblasts. The Vmax was determined in 167.21 nmol/h/mg of protein in heterozygotes leukocytes and 541.2 nmol/h/mg of protein in heterozygotes fibroblasts compared to 291.7 and 1768.1 nmol/h/mg of protein in controls leukocytes and fibroblasts, respectively. When leukocyte and fibroblast heterozygote beta-galactosidase was preincubated at 42 degrees C, after 80 min the residual activity was determined to be 25 to 30% of the initial activity. These results are similar to the control group. CONCLUSIONS: We have found significant differences between the two groups in some investigated parameters. Both fibroblasts and leukocytes showed a virtually similar level of reliability as source of enzyme for the detection of heterozygotes.

Cells, Cultured↗
PubMed1997

Generation and characterization of transgenic mice expressing a human mutant alpha-galactosidase with an R301Q substitution causing a variant form of Fabry disease.

Transgenic mice expressing a human mutant alpha-galactosidase with an R301Q substitution, which was found in a patient with a variant form of Fabry disease, were established. The mice transcribed a sufficient amount of alpha-galactosidase mRNA, but the steady-state levels of the enzyme protein were decreased in liver, kidney and heart, only residual activity being detected in these tissues. The mice will be useful for the clarification of the defective regulation of the structurally altered enzyme protein expressed by the mutant gene at the organ or individual level as well as for the evaluation of drugs that stabilize and/or activate the mutant alpha-galactosidase.

Animals↗
PubMed1997

Maturation and degradation of beta-galactosidase in the post-Golgi compartment are regulated by cathepsin B and a non-cysteine protease.

Lysosomal beta-galactosidase precursor is processed to a mature form and associated with protective protein in lysosomes. In this study we used two cysteine protease proinhibitors, E64-d for cathepsins B, S, H, and L, and CA074Me for cathepsin B. They are converted intracellularly to active forms, E-64c and CA074, respectively. Both active compounds inhibited maturation of the exogenous beta-galactosidase precursor, but E-64c did not inhibit further degradation to an inactive 50-kDa product. We concluded that cathepsin B participated exclusively in maturation of beta-galactosidase, and a non-cysteine protease was involved in further degradation and inactivation of the enzyme molecule.

Cathepsin B↗
PubMed1998

Engineering of solvent-exposed loops in Escherichia coli beta-galactosidase.

The Escherichia coli beta-galactosidase is a high molecular mass tetrameric enzyme extensively used as a molecular marker. Despite its proven utility as a partner in fusion proteins, previous attempts to generate insertional mutants rendered inactive or poorly active enzymes, hampering its further engineering for the construction of multifunctional enzymes. We have explored several solvent-exposed loops on the tetramer, namely those spanning residues 246-254, 271-287, 578-584, 770-773, and 793-806, as acceptor sites to accommodate functional protein segments on the surface of active beta-galactosidase enzymes. An RGD-containing antigenic peptide positioned in these sites interacts efficiently with specific monoclonal antibodies as well as target integrins on the surface of mammalian cells. The resulting chimeric enzymes are soluble, stable, produced in high yields and enzymatically active. Moreover, the identified insertion sites could be appropriated for the design of promising beta-galactosidase-based molecular sensors.

Amino Acid Sequence↗
PubMed1997

A flow cytometric method for the rapid detection of beta-galactosidase transfected cells: an in vitro and in vivo study.

A flow cytometric method has been developed for the rapid analysis of lacZ transduced cells. The method described is based on an indirect immunofluorescence staining procedure using a monoclonal antibody which binds specifically to beta-galactosidase from E. coli and to beta-galactosidase fusion proteins. This technique was used for the quantification in vitro as well as in vivo of beta-galactosidase expression in B16 melanoma cells. The described method is appropriate for a variety of cell types (species, lineage), is simple, quantitative, reliable, rapid and applicable to all constructs containing the lacZ selectable markers. It should prove to be very helpful (1) for the quantification of cells expressing the lacZ reporter gene and (2) for studying gene regulation, including transfection modality, promoter efficacy, enhancer activity, and other regulatory factors.

Bacterial Proteins↗
PubMed2002

Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose.

The beta-galactosidase from an extreme thermophile, Thermus thermophilus A4 (A4-beta-Gal), is thermostable and belongs to the glycoside hydrolase family 42 (GH-42). As the first known structures of a GH-42 enzyme, we determined the crystal structures of free and galactose-bound A4-beta-Gal at 1.6A and 2.2A resolution, respectively. A4-beta-Gal forms a homotrimeric structure resembling a flowerpot. Each monomer has an active site located inside a large central tunnel. The N-terminal domain of A4-beta-Gal has a TIM barrel fold, as predicted from hydrophobic cluster analysis. The putative catalytic residues of A4-beta-Gal (Glu141 and Glu312) superimpose well with the catalytic residues of Escherichia coli beta-galactosidase. The environment around the catalytic nucleophile (Glu312) is similar to that in the case of E.coli beta-galactosidase, but the recognition mechanism for a substrate is different. Trp182 of the next subunit of the trimer constitutes a part of the active-site pocket, indicating that the trimeric structure is essential for the enzyme activity. Structural comparison with other glycoside hydrolases revealed that many features of the 4/7 superfamily are conserved in the A4-beta-Gal structure. On the basis of the results of 1H NMR spectroscopy, A4-beta-Gal was determined to be a "retaining" enzyme. Interestingly, the active site was similar with those of retaining enzymes, but the overall fold of the TIM barrel domain was very similar to that of an inverting enzyme, beta-amylase.

Amino Acid Sequence↗
PubMed2001

Characterization of alpha-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides.

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. Enzymic hydrolysis of the RO is accomplished by alpha-galactosidase. While the content of RO decreases during seed germination, the activity of alpha-galactosidase increases substantially. Two alpha-galactosidases were isolated from germinating seeds by partition in an aqueous two-phase system followed by ion-exchange and affinity chromatography. One of the enzyme preparations (P1) showed a single protein with M(r) of 33 kDa, and the second (P2) had two proteins with M(r) of 31 and 33 kDa. Maximal activities against the synthetic substrate rho-nitrophenyl-alpha-D-galactopyranoside (rhoNPGal) were detected at pH 5.0-5.5 and 45-50 degrees C. Both enzymes were fairly stable at 40 degrees C, but lost most of their activities after 30 min at 50 degrees C. The K(m) values for hydrolysis of rhoNPGal by the P1 and P2 enzymes were 1.55 and 0.76 mM, respectively. The K(m) values determined for hydrolysis of raffinose and melibiose by the P2 enzyme were 5.53 and 5.34 mM, respectively and galactose was a competitive inhibitor (K(i)=0.65 mM). To different extents, both enzymes were sensitive to inhibition by galactose, melibiose, CuSO(4), and SDS. Sucrose and beta-mercaptoethanol showed discrete inhibitory effects on both enzymes.

Binding, Competitive↗
PubMed1998

Unique primary structure of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula.

The gene of the monomeric multimetal beta-galactosidase of Saccharopolyspora rectivirgula was cloned and sequenced. Although the enzyme could be assigned as a member of beta-galactosidases belonging to the glycosyl hydrolase family 2, it has unusual structural features for beta-galactosidase of this family; it contained a unique sequence which consists of approximately 200 amino acid residues with no similarity to known proteins. This 200-residue sequence exists as if it is inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme.

Amino Acid Sequence↗
PubMed1996

Region-specific central nervous system expression and axotomy-induced regulation in sympathetic neurons of a VIP-beta-galactosidase fusion gene in transgenic mice.

To assess the activity of cis-acting elements that direct human vasoactive intestinal peptide (VIP) expression in vivo, two independent transgenic mouse lines were created using a transgene comprised of 1.9 kb of 5'-flanking sequence of the human VIP gene joined to the Escherichia coli beta-galactosidase reporter gene. Transgene expression in brain was assessed using beta-galactosidase histochemistry and compared to the distribution of endogenous VIP expression. Transgene expression was observed in most central and peripheral nervous system sites in which endogenous VIP is expressed. We investigated whether the VIP-beta-galactosidase transgene was regulated in sympathetic neurons in experimental paradigms in which VIP regulation is dependent on the release of leukemia inhibitory factor (LIF). After dissociation in vitro and postganglionic axotomy in vivo there were parallel increases in endogenous VIP and transgene expression in superior cervical ganglia. These results indicate that the 1.9 kb region of 5'-flanking sequence of the human VIP gene includes genomic elements important for cell-specific expression and LIF-dependent regulation in neurons.

Animals↗
PubMed2002

Transient detection of beta-galactosidase activity in hematopoietic cells, following reinjection of retrovirally marked autologous blood progenitors in patients with breast or ovarian cancer receiving high-dose chemotherapy.

OBJECTIVE: The aim of this report is to demonstrate the feasibility and safety of genetically modifying autologous human blood CD34(+) cells in vitro, with a retroviral vector that encodes a marker gene. The fate of genetically modified cells and their progeny was followed in vivo, after reinfusion in patients treated with high-dose chemotherapy for poor-prognosis breast or ovarian carcinomas. PATIENTS AND METHODS: Six patients received genetically modified autologous peripheral blood progenitors, together with unmanipulated aphereses, following high-dose chemotherapy. CD34(+) cells were immunoselected from aphereses, and retrovirally transduced by coculture with the retroviral vector producing cell line, to express a nuclear localized version of E. coli beta-galactosidase, encoded by a defective Moloney-murine leukemia virus-derived retroviral vector. Cells were reinfused to the patients after myeloablation, without prior ex vivo selection. RESULTS: Five out of six patients showed the transient presence of low numbers of beta-galactosidase(+) cells, as detected with an immunocytochemical assay, in the peripheral blood, during the first month following infusion. One patient had beta-galactosidase(+) clonogenic progenitors in her marrow at two months after transplantation, including HPP-CFC; intriguingly, this patient had the lowest percentage of X-gal(+) cells in her graft. Patients experienced side effects that are often observed after high-dose chemotherapy. CONCLUSIONS: Feasibility and safety of genetic modification of human hematopoietic stem and progenitor cells are demonstrated by this study. Ex vivo or in vivo selection is not mandatory, even in clinical situations where transduced cells have no survival advantage over wild-type cells; however, significant improvements in gene transfer technology are needed to achieve potentially useful levels of expression in such clinical situations.

Aged↗
PubMed1998

A live non-neurovirulent herpes simplex virus vector expresses beta-galactosidase in the nervous system of the Wistar and Sprague-Dawley strain rat for a prolonged period.

We have constructed a live non-neurovirulent herpes simplex virus vector expressing beta-galactosidase under the control of the latency associated transcript promoter without inducing inflammation. Pathogenicity of the recombinant virus (betaH1) was not observed in the cutaneous, intravenous and intracerebral infection in mice. When betaH1 was inoculated at the caudate putamen of rats, beta-galactosidase activity was observed in neurons at the inoculation site and its projecting frontal cortex. Expression of beta-galactosidase was observed in the neurons of the innervating dorsal root ganglia 45 days after inoculation of betaH1 into the hind paws of the rats. Neither inflammation nor tissue destruction was observed in both neural tissues in this study. Thus this non-neurovirulent recombinant virus is a suitable vector for expressing the foreign genes in the nervous system for the prolonged period.

Animals↗
PubMed1999

Characterization of a thermostable extracellular beta-galactosidase from a thermophilic fungus Rhizomucor sp.

An extracellular beta-galactosidase from a thermophilic fungus Rhizomucor sp. has been purified to homogeneity by successive DEAE cellulose chromatography followed by gel filtration on Sephacryl S-300. The native molecular mass of the enzyme is 250,000 and it is composed of two identical subunits with molecular mass of 120,000. It is an acidic protein with a pI of 4.2. Purified beta-galactosidase is a glycoprotein and contains 8% neutral sugar. The optimum pH and temperature for enzyme activity are 4.5 and 60 degrees C, respectively. The enzyme is stable at 60 degrees C for 4 h, and has a t(1/2) of 150 min(-1) at 70 degrees C which is one of the highest reported for fungal beta-galactosidases. Substrate specificity studies indicated that the enzyme is specific for beta-linked galactose residues with a preference for p-nitrophenyl-beta-D-galactopyranoside (pNPG). The Km and Vmax values for the synthetic substrates pNPG and o-nitrophenyl-beta-D-galactopyranoside (oNPG) were 0.66 mM and 1.32 mM; and 22.4 mmol min(-1) mg(-1) and 4.45 mmol min(-1) mg(-1), respectively, while that for the natural substrate, lactose, was 50.0 mM and 12 mmol min(-1) mg(-1). The end product galactose and the substrate analogue isopropyl thiogalactopyranoside (ITPG) inhibited the enzyme with Ki of 2.6 mM and 12.0 mM, respectively. The energy of activation for the enzyme using pNPG and oNPG were 27.04 kCal and 9.04 kCal, respectively. The active site characterization studies using group-specific reagents revealed that a tryptophan and lysine residue play an important role in the catalytic activity of the enzyme.

Binding Sites↗
PubMed2003

Association of increased autophagic inclusions labeled for beta-galactosidase with fibroblastic aging.

Replicative senescence appears after a finite number of cell divisions. After proliferation has ceased, senescent cells remain viable for long periods and metabolic modifications are observed such as lipofuscin accumulation. In order to understand this phenomenon, we examined the emergence of subcellular modifications corresponding to autophagy in MRC5 normal human fibroblasts. An increase of monodansylcadaverine fluorescence, a specific marker of autophagy, in aging compared to young fibroblasts was observed (p<0.0001). The increase of autophagic vacuoles in aging fibroblasts was confirmed by electron microscopy. We compared young versus senescent fibroblasts and showed that autophagic vacuoles, already present in young cells, became larger in senescent fibroblasts with a significant relative increase of inclusion area with respect to measured cell area (p=0.0041). However, autophagy-associated-gene expression remained stable in senescent compared to young fibroblasts, suggesting that the autophagy process per se is not enhanced. In parallel, transmission electron microscopy analysis showed that beta-galactosidase activity distribution was modified by aging: beta-galactosidase (an enzyme linked to lysosome) was scattered in young fibroblasts, but clustered at the level of autophagic vacuoles in senescent fibroblasts, suggesting a predominance of autolysosomes at this stage. These results support the hypothesis that, during fibroblast aging, the increase of autophagic vacuoles, as well as that of beta-galactosidase activity, may be associated to an increase of lysosomal mass and to an accumulation of degradative autolysosomes with lipofuscin. This phenomenon could be involved in the death of senescent fibroblasts.

Autophagy↗
PubMed2003

Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted beta-galactosidase.

The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 beta-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase, which efficiently hydrolyzes lactose at 0-20 degrees C, as indicated by its specific activity of 21-67 U mg(-1) of protein (11-35% of maximum activity) in this temperature range, as well as k(cat) of 157 s(-1), and k(cat)/K(m) of 47.5 mM(-1) s(-1) at 20 degrees C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 degrees C, thus below the optimum growth temperature of the bacterium (15 degrees C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 degrees C (190 U mg(-1) of protein) and pH 6.0-8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b beta-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K(+), Na(+), Mn(2+)-40% increase, Mg(2+)-15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu(2+). Noteworthy, Ca(2+) ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 degrees C for at least 30 days without any stabilizers.

Antarctic Regions↗