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)

The effect of ammonium on the glycosylation pattern of the recombinant immunoadhesin tumor necrosis factor-IgG (TNFR-IgG) produced by Chinese hamster ovary cells is elucidated in this study. TNFR-IgG is a chimeric IgG fusion protein bearing one N-linked glycosylation site in the Fc region and three complex-type N-glycans in the TNF-receptor portion of each monomer. The ammonium concentration of batch suspension cultures was adjusted with glutamine and/or NH(4)Cl. The amount of galactose (Gal) and N-acetylneuraminic acid (NANA) residues on TNFR-IgG correlated in a dose-dependent manner with the ammonium concentration under which the N-linked oligosaccharides were synthesized. As ammonium increased from 1 to 15 mM, a concomitant decrease of up to 40% was observed in terminal galactosylation and sialylation of the molecule. Cell culture supernatants contained measurable beta-galactosidase and sialidase activity, which increased throughout the culture. The beta-galactosidase, but not the sialidase, level was proportional to the ammonium concentration. No loss of N-glycans was observed in incubation studies using beta-galactosidase and sialidase containing cell culture supernatants, suggesting that the ammonium effect was biosynthetic and not degradative. Several biosynthetic mechanisms were investigated. Ammonium (a weak base) is known to affect the pH of acidic intracellular compartments (e.g., trans-Golgi) as well as intracellular nucleotide sugar pools (increases UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine). Ammonium might also affect the expression rates of beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST). To separate these mechanisms, experiments were designed using chloroquine (changes intracellular pH) and glucosamine (increases UDP-GNAc pool [sum of UDP-GlcNAc and UDP-GalNAc]). The ammonium effect on TNFR-IgG oligosaccharide structures could be mimicked only by chloroquine, another weak base. No differences in N-glycosylation were found in the product synthesized in the presence of glucosamine. No differences in beta1, 4-galactosyltransferase (beta1,4-GT) and alpha2,3-sialyltransferase (alpha2,3-ST) messenger RNA (mRNA) and enzyme levels were observed in cells cultivated in the presence or absence of 13 mM NH(4)Cl. pH titration of endogenous CHO alpha2,3-ST and beta-1,4-GT revealed a sharp optimum at pH 6.5, the reported trans-Golgi pH. Thus, at pH 7.0 to 7.2, a likely trans-Golgi pH range in the presence of 10 to 15 mM ammonium, activities for both enzymes are reduced to 50% to 60%. Consequently, ammonium seems to alter the carbohydrate biosynthesis of TNFR-IgG by a pH-mediated effect on glycosyltransferase activity.
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PMID:Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms. 1079 88

In birds, the ovum is surrounded by a glycoprotein coat known as the inner perivitelline layer (IPVL), which is analogous to the mammalian zona pellucida and, as such, is the site of initial sperm binding and induction of acrosomal exocytosis (the acrosome reaction). In this study, we demonstrate that oligosaccharides isolated from chicken-IPVL glycoproteins are capable of inducing the acrosome reaction in chicken spermatozoa. Preparations containing only O-linked glycans were unable to induce the acrosome reaction whereas N-linked oligosaccharides released from the IPVL by PNGaseF treatment could induce the acrosome reaction. Addition of galactose to terminal N-acetyglucosamine residues suppressed the acrosome reaction-inducing capacity of the oligosaccharide preparation; however, this capacity could be restored by co-incubation with beta-galactosidase. This evidence suggests that the acrosome reaction-inducing factor is probably an N-linked oligosaccharide with terminal N-acetyl-glucosamine residues.
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PMID:Induction of acrosomal exocytosis in chicken spermatozoa by inner perivitelline-derived N-linked glycans. 1107 59

Recombinant adenovirus is one of the most widely used viral vectors for gene delivery. This study was designed to evaluate the ability of chitosan, a cationic, linear polysaccharide composed of beta(1,4) linked glucosamine partly containing N-acetyl-glucosamine, to enhance the in vitro infectivity of adenovirus to mammalian cells. Wild type and a fiber-mutant replication-defective recombinant adenoviruses expressing beta-galactosidase were used. In the latter, an RGD peptide, the binding site for alpha(v)beta3 and alpha(v)beta5 integrin, was introduced in the fiber knob enabling adenovirus receptor-independent viral infection. Enhanced effect of chitosan on the infectivity of both adenoviruses was observed in Chinese hamster ovary cells that do not express the receptor for adenovirus with beta-galactosidase activity assay and x-gal staining. These data indicate the receptor-independent mechanism(s) for this enhancement effect. In addition, we found that pH of the culture medium, and molecular mass and concentration of chitosan are also critical factors. Thus, the highest effect was obtained with 0.1-1 microg/ml of chitosan with molecular mass of 19K and 40K in the culture medium of pH 6.4; on the other hand, the effect was negligible with the higher chitosan concentrations (10 microg/ml or more), lower or higher molecular mass (11K and 110K) of chitosan, or at pH of 7.4. Studies using several cell lines with variable levels of adenoviral infectivity revealed that this enhanced effect is evident in the cells with poor infectivity to adenovirus. Since chitosan is biocompatible and inexpensive, these data indicate that chitosan may be a potential candidate for a non-viral vector to safely increase adenoviral infectivity to mammalian cells, particularly those with poor susceptibility to adenoviral infection.
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PMID:Receptor-independent augmentation of adenovirus-mediated gene transfer with chitosan in vitro. 1232 78

We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc(2)) as an end product from chitin. Here we sought to identify enzymes in T. kodakaraensis that were involved in the further degradation of GlcNAc(2). Through a search of the T. kodakaraensis genome, one candidate gene identified as a putative beta-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candidate protein was homologous to the beta-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to beta-galactosidases in family 42. The purified protein from recombinant Escherichia coli clearly showed an exo-beta-D-glucosaminidase (GlcNase) activity but not beta-galactosidase activity. This GlcNase (GlmA(Tk)), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80 degrees C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides. The GlcNase activity was also detected in T. kodakaraensis cells, and the expression of GlmA(Tk) was induced by GlcNAc(2) and chitin, strongly suggesting that GlmA(Tk) is involved in chitin catabolism in T. kodakaraensis. These results suggest that T. kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc(2) from chitin is first deacetylated and successively hydrolyzed to glucosamine. This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism.
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PMID:Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. 1292 90

Coproantigen ELISA based tests for diagnosis of canine echinococcosis provide high specificity and sensitivity. However, the antigenic molecules present in faeces from infected dogs have not yet been characterised. While initial attempts to determine the molecular weights of Echinococcus granulosus coproantigens by SDS-PAGE and Western blotting with coproantigen reactive capture antibodies were equivocal, they suggested presence of a significant carbohydrate component. Periodate treatment of coproantigen positive faecal supernatants resulted in a significant reduction (53%) in ELISA activity, suggesting that carbohydrates are involved in the antigenic structure of E. granulosus coproantigens. Protease treatment of antigenic molecules resulted in an 11% reduction in absorbance in ELISA, indicating that protein components were also present which affected by enzyme activity. Lectin-binding ELISA assays indicated strong affinity of E. granulosus coproantigens to concanavalin agglutinin and Lens culinaris agglutinin, and moderate binding to wheat-germ agglutinin and peanut agglutinin. No binding was detectable to Ulex europaensis agglutinin-I, Bandeiraea simplicifolia or Dolichos biflorus agglutinin. These data indicate that E. granulosus coproantigens from infected dog faeces possibly contained alpha-D-mannose and/or alpha-D-glucose, beta-galactose and N-acetyl-beta-glucosamine residues. To verify the role of carbohydrate moieties in coproantigens, faecal samples were treated with exoglycosidase and tested in the coproantigen ELISA. Treatment with beta-galactosidase or N-acetyl-beta-glucosamine reduced ELISA activity by 44 and 30%, respectively. Incubation with a panel of other specific exoglycosidases including alpha-galactosidase as well as alpha-L-fucosidase, alpha-mannosidase, beta-mannosidase, alpha-glucosidase, beta-glucosidase, beta- fructosidase, or neuraminidase, did not alter coproantigen detection in ELISA. The results indicate that coproantigens present in faeces from E. granulosus naturally infected dogs were highly glycosylated and contain beta- galactose and N-acetyl-beta-glucosamine. The putative relationship of antigenic molecules with the tapeworm glycocalyx is discussed.
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PMID:Partial characterisation of carbohydrate-rich Echinococcus granulosus coproantigens. 1457 18

In response to an estrogen, confluent monolayers of MCF-7 cell cultures develop multi-cellular nodules, termed foci. Post-confluent development of foci occurs with physiologic levels of 17beta-estradiol and are inhibited by various anti-estrogens acting through either the estrogen or aryl hydrocarbon receptors. In the present paper we report that disruption of the terminal sugars on membrane receptors results in inhibition of foci. Treatment with 0.013-0.05 units/ml of beta-galactosidase completely inhibited the development of foci while leaving the monolayer of cells intact. Trials with alpha-mannosidase resulted in a similar but less potent inhibition of foci. Lectin-fluorescent conjugates, RCA (Ricinus communis agglutinin), and ConA (Canavalia ensiformis agglutinin) were used to identify membrane surface carbohydrates on MCF-7 cells. Binding of the RCA-fluorescent conjugate was inhibited by co-treatment with galactose or lactose. Binding of ConA-fluorescent conjugate was significantly inhibited by mannose and n-acetyl-glucosamine. This is the first report of inhibition of foci development in MCF-7 cell cultures by disruption of surface carbohydrates on membrane receptors.
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PMID:Beta-galactosidase and alpha-mannosidase inhibit formation of multicellular nodules in breast cancer cell cultures. 1501 88

A GlcNase (exo-beta-D-glucosaminidase) was purified from culture supernatant of Amycolatopsis orientalis subsp. orientalis grown in medium with chitosan. The enzyme hydrolysed the terminal GlcN (glucosamine) residues in oligomers of GlcN with transglycosylation observed at late reaction stages. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The GlcNase also behaved as an exochitosanase against high-molecular-mass chitosan with K(m) and kcat values of 0.16 mg/ml and 2832 min(-1). On the basis of partial amino acid sequences, PCR primers were designed and used to amplify a DNA fragment which then allowed the cloning of the GlcNase gene (csxA) associated with an open reading frame of 1032 residues. The GlcNase has been classified as a member of glycoside hydrolase family 2 (GH2). Sequence alignments identified a group of CsxA-related protein sequences forming a distinct GH2 subfamily. Most of them have been annotated in databases as putative beta-mannosidases. Among these, the SAV1223 protein from Streptomyces avermitilis has been purified following gene cloning and expression in a heterologous host and shown to be a GlcNase with no detectable beta-mannosidase activity. In CsxA and all relatives, a serine-aspartate doublet replaces an asparagine residue and a glutamate residue, which were strictly conserved in previously studied GH2 members with beta-galactosidase, beta-glucuronidase or beta-mannosidase activity and shown to be directly involved in various steps of the catalytic mechanism. Alignments of several other GH2 members allowed the identification of yet another putative subfamily, characterized by a novel, serine-glutamate doublet at these positions.
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PMID:Two exo-beta-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases. 1631 14

Primarily, three operons, hmsHFRS, hmsT and hmsP, are responsible for the development of a Yersinia pestis biofilm, which is essential for blockage-dependent transmission of plague from fleas to mammals. Here, using specific antibodies, a polymeric beta-1,6-N-acetyl-d-glucosamine-like polysaccharide was detected in the extracellular matrix of hmsHFRS-dependent Y. pestis biofilm. The production of this exopolysaccharide (EPS) was controlled by diguanylate cyclase HmsT and EAL domain phosphodiesterase HmsP, acting as positive and negative regulators respectively. Cellular compartmentalization of soluble segments of Hms inner membrane proteins, including the putative glycosyltransferase domain of HmsR, the diguanylate cyclase/GGDEF domain of HmsT and the phosphodiesterase/EAL domain of HmsP, was determined by a combination of topology prediction algorithms and construction of C-terminal translational fusions with beta-galactosidase and alkaline phosphatase. Multiple interactions of Hms inner membrane proteins were detected using bacterial cAMP based two-hybrid system. Biochemical analyses confirmed some of these protein-protein interactions. Our results indicate that synthesis and regulation of the Y. pestis biofilm EPS occurs in the cytoplasm by a proposed Hms enzymatic complex.
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PMID:Insights into Yersinia pestis biofilm development: topology and co-interaction of Hms inner membrane proteins involved in exopolysaccharide production. 1827 44

Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with beta-galactosidase activity (Escherichia coli LacZ), beta-glucuronidase activity (Homo sapiens GusB), and beta-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-beta-D-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural beta-1,4-D-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-beta-D-glucosaminide synthetic substrate provide insight into interactions in the +1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.
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PMID:The structural basis of substrate recognition in an exo-beta-D-glucosaminidase involved in chitosan hydrolysis. 1897 64

The Escherichia coli RNA degradosome is a protein complex that plays a critical role in the turnover of numerous RNAs. The key component of the degradosome complex is the endoribonuclease RNase E, a multidomain protein composed of an N-terminal catalytic region and a C-terminal region that organizes the other protein components of the degradosome. Previously, the RNase E inhibitors RraA and RraB were identified genetically and shown to bind to the C-terminal region of RNase E, thus affecting both the protein composition of the degradosome and the endonucleolytic activity of RNase E. In the present work, we investigated the transcriptional regulation of rraB. rraB was shown to be transcribed constitutively from its own promoter, PrraB. Transposon mutagenesis and screening for increased beta-galactosidase activity from a chromosomal PrraB-lacZ transcriptional fusion resulted in the isolation of a transposon insertion in glmS, encoding the essential enzyme glucosamine-6-phosphate synthase that catalyzes the first committed step of the uridine 5'-diphospho-N-acetyl-glucosamine (UDP-GlcNAc) pathway, which provides intermediates for peptidoglycan biogenesis. The glmS852::Tn5 allele resulted in an approximately 50% lower intracellular concentration of UDP-GlcNAc and conferred a fivefold increase in the level of rraB mRNA. This allele also mediated a twofold increase in beta-galactosidase activity from a chromosomal fusion of the 5' untranslated region of the rne gene to lacZ, suggesting that a reduction in cellular concentration of UDP-GlcNAc and the resulting increased expression of RraB might modulate the action of RNase E.
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PMID:Transcriptional regulation of the Escherichia coli gene rraB, encoding a protein inhibitor of RNase E. 1971 86


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