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)

The mean lipoamidase activity in human breast milk was found to be 0.073 nmol/min per mg (S.D. = 0.028, range = 0.020-0.123, n = 44). The mean lipoamidase activity is approximately 3-fold higher in milk than that in serum (0.023 nmol/min per mg, S.D. = 0.016, range = 0.001-0.059, n = 32). Lipoamidase was purified to 4400-fold by a four-step procedure from 330 ml of human breast milk. The purified enzyme was identified as a single band (Mr = 135,000) by sodium dodecyl sulfate/polyacrylamide electrophoresis. Analysis by Edman degradation indicated that the N-terminal amino acid was glycine. These results strongly suggest that milk lipoamidase is composed of a single polypeptide chain. The enzyme is considered to be a glycoprotein since it reacted positively to periodate-Schiff (PAS) staining. The isoelectric point of the enzyme was 4.2. After treatment of lipoamidase with sialidase, its position on isoelectric focusing gel moved from pH 4.2 to 4.6. This is strongly indicative that lipoamidase contains sialic acid residues. The optimum pH for the enzyme activity is 7.0. The Michaelis constant (KM) for lipoyl p-aminobenzoate is calculated as 25 microM. The enzyme activity was completely lost by heating 60 degrees C for 5 min. The effects of thiol-reactive agents, such as 2-mercaptoethanol (ME) and p-chloromercuribenzoate, were not significant. However, the enzyme activity was completely inhibited by 50 microM diisopropylfluorophosphate. Thus, this enzyme seemed to contain an essential serine residue in the active site.
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PMID:Isolation and characterization of human breast milk lipoamidase. 319 15

We have studied function and structure of the low density lipoprotein (LDL) receptors in a monensin-resistant (Monr-31) mutant isolated from Chinese hamster ovary (CHO) cells. To assay the ability of the receptor to bind LDL, we employed three methods, 125I-LDL binding to the cells at 4 degrees C, 125I-LDL binding to the receptor-phospholipid complex (Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1980) J. Biol. Chem. 255, 11442-11447), and ligand blotting (Daniel, T.O., Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1983) J. Biol. Chem. 258, 4606-4611). The LDL receptor number was similar in both CHO and Monr-31, but the binding affinity was reduced in the mutant. The semi-quantitative immunoblotting assay with an antibody directed against the COOH-terminal 14 amino acids and the ligand-blotting assay with LDL also showed that the relative steady-state level of the receptor in Monr-31 was comparable to that in CHO, whereas the binding capacity of the receptor in Monr-31 was lower than that in CHO. The precursor and degradation forms of the LDL receptors produced in the mutant cells were similar in size to those in the parental cells, but the apparent molecular mass of the mature receptor protein in sodium dodecyl sulfate-polyacrylamide gels was reduced about 5000 daltons in the mutant. These results suggest a structural change at the NH2-terminal LDL binding domain. Tests of the effects of tunicamycin, endo-alpha-N-acetylgalactosaminidase (O-glycanase), and sialidase (neuraminidase) on the molecular size of the mature receptors indicated that the reduced size of the receptor in the mutant cells resulted from altered oligosaccharide chain(s) linked to serine/threonine residues in the binding domain. We compared the molecular sizes and binding activity of human LDL receptors in several clones derived from CHO and Monr-31 cells which were transfected with human LDL receptor cDNA. The human LDL receptors produced in the transfected clones of Monr-31 were also smaller in molecular size and lower in binding capacity than those produced in the transfected clones of CHO. These results suggest that both structural and functional alteration of the LDL receptor of Monr-31 is not caused by a mutation in the structural gene of the LDL receptor but by altered processing or maturation of the receptor. The correlation of the decrease in molecular size and reduced binding capacity of the LDL receptor is discussed.
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PMID:Low binding capacity and altered O-linked glycosylation of low density lipoprotein receptor in a monensin-resistant mutant of Chinese hamster ovary cells. 330 76

Normal human fibroblasts have two extreme modes of existence in culture, quiescent and proliferative. The growth and division of these cells are usually well regulated by the action of various endogenously generated stimulators and inhibitors. We have speculated that an extracellular sialidase may be involved in the regulation of growth and that inhibition of this activity might decrease or abolish cell growth. To test this hypothesis, we have incubated preconfluent cultures of fibroblasts in the presence and absence of a potent sialidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. Treatment of cells with this inhibitor resulted in the inhibition of an extracellular sialidase activity for up to 24 h and had a marked growth inhibitory effect in a concentration-dependent manner. The effect of the inhibitor on cell proliferation was specific and reversible. During a chase period of 48 h after pulse labeling cells with [3H]N-acetylmannosamine and [14C]serine, there was a 15% decrease of [3H]sialic acid in the membrane-bound GM3 with 80 microM inhibitor in the medium, as compared with a 32% decrease in the controls. Our results suggest that an extracellular sialidase may participate in cell-surface modifications that accompany (or control) the changes observed when cells traverse the cell cycle, from the quiescent to the proliferative phase.
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PMID:Growth control of human foreskin fibroblasts and inhibition of extracellular sialidase activity by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. 339 28

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

Newcastle disease virus sialidase was found to exhibit strict specificity for hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage contained in glycoprotein oligosaccharides both N-linked to asparagine and O-linked to threonine or serine under conditions that left oligosaccharides containing the NeuAc alpha 2 leads to 2 leads to 6Gal and NeuAc alpha 2 leads to 6GallNAc linkages intact. This was determined, in part, by examining the viral sialidase for its ability to hydrolyze glycoprotein oligosaccharides derivatized with purified sialyltransferases to contain the [14C]NeuAc alpha 2 leads to 3Gal, [14C]NeuAc alpha 2 leads to 6GalNAc, and [14C]NeuAc alpha 2 leads to 6Gal linkages. The viral sialidase was also tested for hydrolysis of the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages on the N-linked oligosaccharides of alpha 1-acid glycoprotein. Selective hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage was shown by periodate oxidation and by 500-MHz 1H-NMR spectroscopy of native and sialidase-treated glycopeptides. The NMR spectra, together with composition data, further indicated that the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages were localized to specific branches of the major tri- and tetraantennary oligosaccharides of alpha 1-acid glycoprotein. The results indicate that the Newcastle disease virus sialidase can initiate the selective degradation of N-linked oligosaccharide branches containing the NeuAc alpha 2 leads to 3Gal linkage.
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PMID:Newcastle disease virus contains a linkage-specific glycoprotein sialidase. Application to the localization of sialic acid residues in N-linked oligosaccharides of alpha 1-acid glycoprotein. 629 Apr 80

Glycophorin A was digested with glycoprotease (Pasteurella haemolytica) and the digest was fractionated by a combination of high-pressure column chromatographies to produce the glycopeptides GPA-1 to GPA-6. Sequence analysis of the glycopeptides revealed that two serine residues (Ser-14 and Ser-15) are not glycosylated, Thr-17 and Ser-19 being glycosylated instead, in disagreement with the accepted structure. The glycopeptides thus obtained were treated with sialidase and beta-galactosidase. The Tn antigenicity, as assayed by the binding to a monoclonal anti-Tn antibody (MLS 128), was found exclusively in the glycopeptides including three (cluster I) or four (cluster II) consecutive residues of GalNAc-Ser/Thr, whereas the glycopeptide (GPA-2) containing two nonconsecutive GalNAc-Ser/Thr residues had practically no Tn antigenicity. The immunoreactivities of GPA-1 and GPA-3, containing both clusters I and II, and GPA-4, containing cluster II, were 63% (calcd. 67%), 81% (calcd. 86%), and 50% (calcd. 50%), respectively, of the immunoreactivity of GPA-5 or GPA-6, containing cluster I (the average being taken as the basis), based on the reactivity per GalNAc residue. These results indicate that clusters I and II react with the antibody to the same extent. The structure consisting of three consecutive glycosylated Ser/Thr residues may be essential for Tn antigenicity in the light of previous results for ovine submaxillary mucin.
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PMID:Epitopic structure of Tn glycophorin A for an anti-Tn antibody (MLS 128). 768 97

Lysosomal neuraminidase (sialidase) occurs in a high molecular weight complex with the glycosidase beta-galactosidase and the serine carboxypeptidase protective protein/cathepsin A (PPCA). Association of the enzyme with PPCA is crucial for its correct targeting and lysosomal activation. In man two genetically distinct storage disorders are associated with either a primary or a secondary deficiency of lysosomal neuraminidase: sialidosis and galactosialidosis. In the mouse the naturally occurring inbred strain SM/J presents with a number of phenotypic abnormalities that have been attributed to reduced neuraminidase activity. SM/J mice were originally characterized by their altered sialylation of several lysosomal glycoproteins. This defect was linked to a single gene, neu-1 , on chromosome 17, which was mapped by linkage analysis to the H-2 locus. In addition, these mice have an altered immune response that has also been coupled to a deficiency of the Neu-1 neuraminidase. Here we report the identification in SM/J mice of a single amino acid substitution (L209I) in the Neu-1 protein which is responsible for the partial deficiency of lysosomal neuraminidase. We propose that the reduced activity is caused by the enzyme's altered affinity for its substrate, rather than a change in substrate specificity or turnover rate. The mutant enzyme is correctly compartmentalized in lysosomes and maintains the ability to associate with its activating protein, PPCA. We propose that it is this mutation that is responsible for the SM/J phenotype.
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PMID:A point mutation in the neu-1 locus causes the neuraminidase defect in the SM/J mouse. 942 40

We have generated rat monoclonal antibodies (MoAbs) against cell surface antigens of the mouse endothelioma cell line bEND.3. Three antibodies (V.1A7, V.5C7, and V.7C7) were selected, all of which recognize a 75-kD antigen on bEND.3 cells and bind selectively to endothelial cells in cryostat sections of mouse tissues. A cDNA for the antigen was isolated from a bEND.3 pCDM8 expression library by using transient expression in COS-7 cells and immunoselection with the three MoAbs. This cDNA coded for a novel, type I membrane protein of 248 amino acids with an extracellular domain rich in threonine and serine residues (35%). The protein is sensitive to O-sialoglycoprotein endopeptidase, indicating that it belongs to the class of sialomucin-like proteins. Therefore, we suggest the name endomucin. Treatment of isolated endomucin by sialidase and O-glycosidase reduced the apparent molecular weight to 45 kD and abolished binding of all three antibodies, indicating that carbohydrates are directly or indirectly involved in the formation of the antibody epitopes. Immunohistological analysis of all examined mouse tissues showed that endomucin is an endothelial antigen found in venous endothelium as well as in capillaries, but not on arterial endothelium. Interestingly, high endothelial venules of peripheral and mesenteric lymph nodes as well as of Peyers's patches were negative for staining with the three MoAbs.
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PMID:Biochemical characterization and molecular cloning of a novel endothelial-specific sialomucin. 986 58

The 'small' (43 kDa) sialidase of Clostridium perfringens is inhibited by low concentrations of mercury ions. For the investigation of possible functional roles of the enzyme's four cysteine residues at the amino acid positions 2, 282, 333 and 349, they were separately altered to serine by site-directed mutagenesis. The four mutant sialidases expressed in E. coli and purified by metal chelate chromatography were markedly reduced in specific activity when compared to the wild-type enzyme but with the exception of C282S exhibited similar K(M)-values indicating an unchanged mode of substrate binding. The substrate specificity was also conserved for C2S, C282S, and C333S. Only the C349S sialidase exhibited a higher relative activity with colominic acid and the alpha2,6-linked sialic acid of sialyllactose compared to the alpha2,3-linked isomer than the other mutants. Chemical modifications with the thiol-blocking reagents N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB) and HgCl2 had little effect on the C282S sialidase, e.g., 6% inhibition by 5 mM NEM compared to reductions in activity between 65 and 90% for the wild-type and other mutant enzymes, supporting the idea that among the enzyme's cysteines, Cys-282 has the highest structural or functional significance. The results also explain the higher mercury tolerance of Salmonella typhimurium and Clostridium tertium sialidases, which have the positions equivalent to Cys-282 altered to Val and Thr, respectively, indicating that the thiol group of Cys-282, despite being situated near the active site, is not involved in catalysis.
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PMID:Effect of cysteine modifications on the activity of the 'small' Clostridium perfringens sialidase. 987 Mar 52

The human V2 vasopressin receptor contains one consensus site for N-linked glycosylation at asparagine 22 in the predicted extracellular amino terminal segment of the protein. This segment also contains clusters of serines and threonines that are potential sites for O-glycosylation. Mutagenesis of asparagine 22 to glutamine abolished N-linked glycosylation of the V2 receptor (N22Q-V2R), without altering its function or level of expression. The N22Q-V2R expressed in transfected cells migrated in denaturing acrylamide gels as two protein bands with a difference of 7000 Da. Protein labeling experiments demonstrated that the faster band could be chase to the slower one suggesting the presence of O-linked sugars. Sialidase treatment of membranes from cells expressing the N22Q-V2R or of immunoprecipitated metabolically labeled V2R accelerated the migration of the protein in acrylamide gels demonstrating the existence of O-glycosylation, the first time this type of glycosylation has been found in a G protein coupled receptor. Synthesis of metabolically labeled receptor in the presence of 1 mM phenyl-N-acetyl-alpha-D-galactosaminide, a competitive inhibitor of N-acetyl-alpha-D-galactose and N-acetylneuraminic acid transferases, also produced a receptor that migrated faster in denaturing gels. Serines and threonines present in the amino terminus were analyzed by alanine scanning mutagenesis to identify the acceptor sites. O-glycosylation was found at most serines and threonines present in the amino terminus. Because the disappearance of a site opened the availability of others to the transferases, the exact identification of the acceptor sites was not feasible. The wild type V2R expressed in HEK 293, COS, or MDCK cells underwent N- and O-linked glycosylation. The mutant V2R bearing all serine/threonine substitutions by alanine at the amino terminus yielded a receptor functionally indistinguishable from the wild type protein, whose mobility in polyacrylamide gels was no longer affected by sialidase treatment.
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PMID:O-Glycosylation of the V2 vasopressin receptor. 1036 43


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