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:3.1.1.53 (sialidase)
2,694 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three site-specific mutations were performed in two regions of a sialidase gene from Clostridium perfringens which are known to be conserved in bacterial sialidases. The mutant enzymes were expressed in Escherichia coli and, when measured with MU-Neu5Ac as substrate, exhibited variations in enzymatic properties compared with the wild-type enzyme. The conservative substitution of Arg 37 by Lys, located in a short conserved region upstream from the four repeated sequences common in bacterial sialidase genes, was of special interest, as KM and Vmax, as well as K(i) measured with Neu5Ac2en, were dramatically changed. These data suggest that this residue may be involved in substrate binding. In addition to its low activity, this mutant enzyme has a lower temperature optimum and is active over a more limited pH range. This mutation also prevents the binding of an antibody able to inhibit the wild-type sialidase. The other mutations, located in one of the consensus sequences, were of lower influence on enzyme activity and recognition by antibodies.
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PMID:Effects of site-specific mutations on the enzymatic properties of a sialidase from Clostridium perfringens. 149 Jan 2

1. Previous studies from our laboratory demonstrated that subcultured astroglia enhance neurite outgrowth and survival of cultured neurons from embryonic rat cerebral cortex, but suppress proliferation of neuroblasts. 2. In the present study, the mechanisms of these three effects were further investigated. 3. Dissociated neurons were seeded on poly-L-lysine-coated coverslips which were plated on subcultured astroglia, and the survival, proliferation and neurite outgrowth of the neurons were investigated. Under these conditions, survival and antimitotic effects were also observed, while neurite extension was not stimulated. 4. The results clearly indicate that neuronal survival and proliferation are regulated by soluble factors produced by astroglia. 5. We also postulated that the neurite-promoting effect of astroglia is mediated by cell-cell contact. 6. This idea was confirmed by the finding that neurite extension was enhanced when the neurons were cultured directly on heat-treated astroglia. 7. The neurite-promoting effect was found to be specific to astroglia. 8. We preliminarily characterized the astroglial surface neurite-promoting factors (ASNPFs). 9. The relationship of laminin to ASNPFs was examined by using antibody to laminin. Laminin antibody did not inhibit the ASNPF activity. 10. The effect of digestion of heat-treated astroglia with enzymes (sialidase and endo-beta-galactosidase) on the ASNPF activity was also examined. 11. These enzyme treatments did not inhibit the ASNPF activity. 12. These results suggest that enhancement of the neurite-promoting activity is not associated with the sugar moiety of ASNPFs.
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PMID:Effects of astroglia on the development of cultured neurons from embryonic rat cerebral cortex. 167 7

The optimal conditions for the assay of sialidase in cerebellar granule cells cultivated in vitro, established using [3H]GD1a and 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUB-NeuNAc) as substrates, were the following: pH optimum for both substrates, 3.9; optimal molarity of sodium acetate/acetic acid buffer, 0.05 M with [3H]GD1a and 0.1 M for MUB-NeuNAc; substrate concentration for apparent maximal activity, 0.5 mM for MUB-NeuNAc and 0.1 mM for [3H]GD1a; enzyme activity linear with time up to 30 min with MUB-NeuNAc and up to 90 min with [3H]GD1a; and enzyme activity linear with enzyme protein content up to 80 micrograms with MUB-NeuNAc and up to 20 micrograms with [3H]GD1a. The assay with [3H]GD1a required the presence of Triton X-100 in a molar ratio to GD1a of 15:1. Poly-L-lysine, which was used for plating the cells, was capable of decreasing sialidase activity against [3H]GD1a/Triton X-100 when added to the incubation mixture. However, it had no effect on the enzyme working on MUB-NeuNAc. Using no more than 20 micrograms of cellular protein, the contamination, if any, by poly-L-lysine released from the dish was below the concentration limit exhibiting inhibition. Using the above optimal conditions, sialidase activity was measured during cerebellar granule cell differentiation in culture. From day 0 to day 7-8 in culture, the enzyme activity rose from 20 to 130 nmol of product released/h/mg of protein with MUB-NeuNAc and from 1 to 100 nmol of product released/h/mg of protein with [3H]GD1a.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sialidase in cerebellar granule cells differentiating in culture. 279 13

We previously described sialidase-deficient variants of the O'Take strain of mumps virus obtained by growth under the selective pressure of the competitive sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). In this report, we describe the production of a sialidase-deficient variant of the RW strain of mumps virus using an identical selection protocol. The biologic activities of the RW variant, RW(DANA)v1, were identical to those described for O'Take-(DANA)v1 and included a lack of detectable sialidase activity, unchanged hemagglutination activity, and expression of cell-to-cell fusion in infected cell monolayers. Analysis of the structural proteins of each virus by both two-dimensional tryptic peptide mapping and monoclonal antibody binding assays suggested that limited changes occurred in the hemagglutinin-neuraminidase (HN) proteins and that only the HN proteins were altered. The complete nucleotide sequence of the RW(DANA)v1 HN was determined and compared to the HN sequence of the RW parent. Two nucleotide differences accounting for two nonconservative amino acid differences were noted; an lle to a Thr at amino acid 181 and a Gln to Lys at amino acid 261 from RW to RW(DANA)v1, respectively. By comparing the data presented here with those reported for several other paramyxoviruses, we tentatively identify amino acid 181 as a critical residue in the active site of the mumps virus sialidase enzyme.
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PMID:Identification of amino acids involved in the sialidase activity of the mumps virus hemagglutinin-neuraminadase protein. 318 97

gpL115, the surface sialoglycoprotein that is defective in lymphocytes of Wiskott-Aldrich syndrome patients has been purified from large scale cultures of the lymphoblastoid line CEM. The purification entails cell lysis and solubilization of gpL115 with the detergent Nonidet P-40, sequential affinity chromatography on lentil lectin-Sepharose, wheat germ lectin-Sepharose, and, after treatment with sialidase, on peanut lectin-Sepharose. Sepharose CL-6B gel filtration removes residual protein contaminants and transfers asialo-gpL115 from Nonidet P-40-containing to sodium dodecyl sulfate-containing buffer. The yield, 1300 micrograms of homogeneous protein/10(11) cells, represents greater than 60% recovery. The amino acid composition of gpL115 has several atypical features including low lysine content, high proline content, and very high content of hydroxyamino acids (12.5 residues of serine and 12.5 residues of threonine/100 amino acids). Total carbohydrate content of gpL115 is very high, i.e. 52% for the asialo-molecule. The major carbohydrate residues of asialo-gpL115 are galactose and N-acetylgalactosamine in approximately equimolar amounts (25 and 22 residues/100 amino acids, respectively) plus severalfold lower amounts of N-acetylglucosamine, fucose, and mannose.
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PMID:Purification and chemical composition of gpL115, the human lymphocyte surface sialoglycoprotein that is defective in Wiskott-Aldrich syndrome. 371 Oct 98

Enzymes and chemicals were used to analyse the biochemical structure of the antigenic epitope recognized by GDA-J/F3 monoclonal antibody (MoAb) in the human sperm tail fibrous sheath. Treatment of sperm dried onto slides with trypsin or dispase enzymes abolished their immunofluorescence staining with GDA-J/F3 MoAb, thus indicating the proteinaceous nature of the antigen. The proteolytic cleavage of GDA-J/F3 protein by trypsin, which also caused sperm decapitation, indicated the presence of peptide bonds involving the carboxyl groups of the basic amino acids, arginine and/or lysine. The epitope was also glycosylated as demonstrated by its sensitivity to sodium metaperiodate treatment which was dose-dependent. The GDA-J/F3 antigenic epitope lacked sialic acid since pre-treatment of spermatozoa with sialidase enzyme (neuraminidase) had no effect on their reactivity with the antibody. The lack of collagenous domains in the GDA-J/F3 antigen was demonstrated by the failure of collagenase to abrogate sperm immunostaining with the MoAb. Furthermore, type VII collagen of the skin basement membrane (BM) was previously thought of as a potential target antigen for GDA-J/F3 MoAb. This was ruled out since several monoclonal and polyclonal antibodies failed to detect the antigen in the spermatozoa using immunofluorescence and Western blotting. These data, therefore, show that the target antigen for GDA-J/F3 MoAb is a non-collagenous asialo-glycoprotein, and by inference provide the first evidence for the glycosylation of the sheath proteins as another step of post-translational modification occurring during sperm tail development.
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PMID:The target antigen for GDA-J/F3 monoclonal antibody in the human sperm tail fibrous sheath is a non-collagenous asialo-glycoprotein: implications and significance. 752 22

The interferon antagonist and growth promotor sarcolectin has affinity for negatively charged carbohydrates. Isolation of cellular binding proteins will be a step to elucidate its physiological significance. Thus, resin-immobilized sarcolectin was employed as affinity ligand for chromatographic fractionation of extract from human placenta. Elution with 0.1 M NH4OH or with 0.1 M N-acetylneuraminic acid and 1 M NaCl resulted primarily in purification of a protein of molecular mass of about 12 kDa according to gel electrophoretic analysis under denaturing conditions in the presence or absence of reductive agent and 12,470 Da by laser desorption mass spectrometry. The native molecular mass, assessed by gel filtration, is approximately 28 kDa. No evidence for detectable post-translational modification by glycosylation was provided by treatment with N-glycosidase F or sialidase and subsequent electrophoretic analysis. The N-terminal sequence of the major sarcolectin-binding protein is identical to that deduced from the cDNA sequence of a human macrophage migration inhibitory factor (MIF), starting from its third amino acid, over the determined stretch of 22 amino acids. Comparison of the calculated molecular mass of 12,221 of this factor to the experimentally determined value of 12,470 excludes any extensive modification of the protein. The sarcolectin-binding protein reduces macrophage migration at a concentration of 100 ng/ml in MIF assays. Recombinant migration inhibitory factor and purified sarcolectin-binding protein reacted equally well with anti-MIF antibody in immunoblot analysis and in assays to block binding to sarcolectin. Binding of biotinylated sarcolectin, too, is nearly identical for the two protein preparations. It is optimal in the range pH 7-9 and is markedly impaired by increasing ionic strength. Chemical modification with group-specific reagents revealed that the integrity of carboxyl groups of the sarcolectin-binding protein and of lysine/arginine groups of sarcolectin are primarily important to maintain binding capacity. In addition to contribute to the understanding of the functional significance of sarcolectin this result provides a convenient procedure to purify a lymphokine.
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PMID:The major binding protein of the interferon antagonist sarcolectin in human placenta is a macrophage migration inhibitory factor. 768 62

The mucin-type carbohydrate Tn cryptantigen (GalNAc alpha 1-O-Ser/Thr, where GalNAc is N-acetyl-D-galactosamine) is expressed in many carcinomas, in haemopoietic disorders including the Tn syndrome, and on human immunodeficiency virus (HIV) coat glycoproteins, but is not expressed on normal, differentiated cells because of the expression of a Tn-processing galactosyltransferase. Using Jurkat T leukaemic cells which express high levels of Tn antigen due to deficient Tn galactosylation, we have established the Tn antigen-mediated gene transfer and demonstrate the considerable efficiency of this approach. We used poly(L-lysine) conjugates of the monoclonal antibody 1E3 directed against the Tn antigen to deliver the luciferase and beta-galactosidase reporter genes to Jurkat cells by receptor-mediated endocytosis. Addition of unconjugated 1E3 reduced transfection efficiency in a concentration-dependent manner and incubation with free GalNAc abolished DNA transfer completely, indicating that gene delivery is indeed mediated by the Tn antigen. Pre-treatment of Jurkat cells with Vibrio cholerae sialidase, which uncovers additional Tn antigens, resulted in an improvement of gene transfection. Both human and chicken adenovirus particles attached to the DNA/polylysine complex strongly augmented transgene expression. When the beta-galactosidase (lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis, up to 60% of the cells were positive in the cytochemical stain using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a chromogenic substrate. The efficiency of the transferrin receptor-mediated DNA uptake into Jurkat cells was comparatively low, although these cells were shown to express considerable amounts of transferrin receptor. We show here that a mucin-type carbohydrate antigen mediates highly efficient DNA uptake by endocytosis into Jurkat T cells. This method represents a 50-fold improvement of Jurkat cell transfection efficiency over other physical gene transfer techniques. Specific gene delivery to primary cancer cells exhibiting Tn epitopes may especially be desirable in immunotherapy protocols.
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PMID:Carbohydrate receptor-mediated gene transfer to human T leukaemic cells. 782 4

The epithelial polyanion (podocalyxin) on the foot processes (pedicels) of podocytes plays a pivotal role in maintaining slit pore integrity and excluding proteins from the glomerular filtrate. Chromatographically purified recombinant sialidase from Vibrio cholerae, a corresponding heat-inactivated enzyme, truncated enzyme (missing the last 17 amino acids from the carboxyl terminus), and the sialidase from Salmonella typhimurium strain LT2 were inoculated intraperitoneally into mice, and the resultant renal alterations were documented by a variety of functional, morphologic, and histochemical techniques. Proteinuria and renal failure developed in a dose-dependent manner after a single inoculation of sialidase from Vibrio cholerae, but not with the corresponding heat-inactivated enzyme, truncated enzyme, or the sialidase from Salmonella typhimurium strain LT2. Biotinylated lectins of known sialyl linkage specificity demonstrated that Vibrio cholerae sialidase primarily removed alpha 2-->6-linked sialic acids from the glomerulus. Furthermore, the use of a poly-L-lysine cationic gold ultrastructural probe confirmed a transient loss of charge from the endothelium and epithelium of the glomerular filtration barrier. Loss of the epithelial polyanion was accompanied by the effacement of pedicels and the apparent formation of tight junctions between adjacent podocytes. The anionic charge returned to endothelial and epithelial sites within 2 days of sialidase inoculation, but the foot process loss remained. This animal model, in addition to providing an opportunity to study basic mechanisms of renal physiology, seems to mimic minimal change disease in children, diabetic nephropathy, and the renal effects of some bacterial infections.
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PMID:In vivo enzymatic removal of alpha 2-->6-linked sialic acid from the glomerular filtration barrier results in podocyte charge alteration and glomerular injury. 864 86

Among the epilepsies, the progressive myoclonus epilepsies (PMEs) form a heterogeneous group of rare diseases characterized by myoclonus, epilepsy, and progressive neurologic deterioration, particularly dementia and ataxia. The success of the Human Genome Project and the fact that most PMEs are inherited through a mendelian or mitochondrial mode have resulted in important advances in the definition of the molecular basis of PME. The gene defects for the most common forms of PME (Unverricht-Lundborg disease, the neuronal ceroid lipofuscinoses, Lafora disease, type I sialidosis, and myoclonus epilepsy with ragged-red fibers) have been either identified or mapped to specific chromosome sites. Unverricht-Lundborg disease has been shown to be caused by mutations in the gene that codes for cystatin B, an inhibitor of cysteine protease. The most common mutation in Unverricht-Lundborg disease is an expansion of a dodecamer repeat located in a noncoding region upstream of the transcription start site of the cystatin B gene, making it the first human disease associated with instability of a dodecamer repeat. Juvenile neuronal ceroid lipofuscinosis is caused by mutations in the CLN3 gene, a gene of unknown function that encodes a 438-amino-acid protein of possible mitochondrial location. Other forms of neuronal ceroid lipofuscinosis that occur as PME and Lafora disease have been mapped by means of linkage analysis, but the corresponding gene defects remain unknown. Sialidosis has been shown to be caused by mutations in the sialidase gene, and myoclonus epilepsy with ragged-red fibers is well known to be caused by mutations in the mitochondrial gene that codes for tRNA(Lys). How the different PME gene defects described produce the various PME phenotypes, including epileptic seizures, remains unknown. The development of animal models that bear these mutations is needed to increase our knowledge of the basic mechanisms involved in the PMEs. This knowledge should lead to the development of new and effective forms of therapy, which are especially lacking for the PMEs.
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PMID:The molecular genetic bases of the progressive myoclonus epilepsies. 1051 28


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