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)

Previously, we reported that the epitope of a 54-kDa sperm surface sialoglycoprotein on the flagellum is masked by sialic acid residues. The epitope is referred to as a hidden determinant or cryptodeterminant. This paper reports the manner in which the epitope is masked as evaluated qualitatively and quantitatively by means of SDS-PAGE/immunoblots (Western blot) and ELISA. Immunoblotting with four specific monoclonal antibodies to the 54-kDa sialoglycoprotein--T21 (IgM), MC71 (IgG1), MC81 (IgM), and MC91 (IgM)--demonstrated that not only IgM but also IgG antibody MC71 and the Fab fragment MC71 are masked. Quantitative evaluation with ELISA to compare the antibody titration curves of the masked and unmasked antigens on sialidase-treated and untreated sperm, respectively, indicated that sialidase caused the antibody-binding ability of the epitopes to increase to a different level for each antibody. There were 32-256-, 8-16-, 16-, and 2-4-fold increases in binding to T21, MC71, MC81, and MC91 antibodies, respectively. These results suggest that the antigen-masking through the cryptodeterminant does not depend upon the subtype or the molecular mass of the antibody, but upon the biochemical nature of the epitope region that is closely related to the sialic acid. The mechanism and physiological roles of the antigen-masking are discussed.
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PMID:Masking the cryptodeterminant on the 54-kilodalton mouse sperm surface antigen. 128 29

A sialidase from Bacteroides fragilis SBT3182 was purified 2,240-fold to apparent homogeneity by ammonium sulfate precipitation and sequential chromatographies on DEAE-Toyopearl 650M, Hydroxyapatite, MonoS and Superose6 columns. The N-terminal amino acid sequence of this sialidase, Ala-Asp-X-Ile-Phe-Val-Arg-Glu-Thr-Arg-Ile-Pro-, was determined. Substrate specificity of this enzyme using a variety of sialoglycoconjugates showed a 1.5- and 2.2-fold preference for sialyl alpha 2-8 linkages when compared with alpha 2-3 and alpha 2-6 bound sialic acids, respectively. The native sialidase had a molecular weight of 165kDa, as determined by Superose6 gel filtration chromatography and consisted of three subunits each of 55kDa by SDS-polyacrylamide gel electrophoresis. This enzyme had optimal activity at pH6.1 with colominic acid as substrate.
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PMID:Purification and characterization of a sialidase from Bacteroides fragilis SBT3182. 133 98

The cDNA coding for pre-peanut agglutinin (PNA) was isolated from a bacterial expression library. It codes for a polypeptide of 273 amino acids composed of a hydrophobic signal peptide of 23 amino acids and a mature protein of 250 amino acids. The sequence of the latter is identical to that of native PNA, determined very recently by conventional methods, except that it contains 14 additional amino acids at the C-terminus. Bacterial cells harboring a plasmid with the prePNA-cDNA, produced two PNA cross-reacting proteins: one migrated on SDS-PAGE identically with the native lectin (apparent mol. wt. 31 kDa); the other, at 35 kDa, was a beta-galactosidase pre-PNA fusion protein. The former protein possessed an N-terminal sequence identical to that of the mature, native PNA, suggesting that it was processed from the 35 kDa prePNA precursor. Only the 31 kDa protein was exported into the bacterial periplasmic space, and had the ability to bind to galactose-Sepharose. The isolated processed protein had the same hemagglutinating activity as the native lectin, when assayed with sialidase-treated human erythrocytes. Like the native lectin, it did not agglutinate the untreated cells, was not inhibited by N-acetylgalactosamine, and was inhibited by Gal beta 1----3GalNAc 30-times more strongly than by galactose.
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PMID:Cloning, sequence analysis and expression in Escherichia coli of the cDNA encoding a precursor of peanut agglutinin. 133 58

The relationship between cell differentiation/tumorisation and plasma membrane glycoproteins was approached using peanut agglutinin (PNA) a lectin specific for the Gal-beta(1,3)GalNAc sequence and a homologous cell system consisted of normal rat hepatocytes (HyC) and a poorly differentiated hepatoma (ZHC). This work is focused on the molecular nature of PNA receptors. PNA bound strongly to ZHC, but bound very weakly, if at all to hepatocytes. After sialidase treatment this binding was slightly enhanced in ZHC and HyC. The total number of binding sites on ZHC was 9.6 x 10(6)/cell and 1.2 x 10(7)/cell before and after sialidase treatment respectively. In contrast, this number could not be calculated on HyC, even after sialidase treatment. The PNA receptors were isolated and identified from ZHC using affinity chromatography on immobilized PNA and lectin overlay. Two bands were revealed after SDS-PAGE of PNA receptors: a major one with a relative molecular mass of 160 kDa and a minor one of 110 kDa. The latter disappeared after sialidase treatment of ZHC suggesting the possibility that these two bands could be less and more sialylated forms of the PNA receptors, respectively. In contrast no PNA receptors could be detected on HyC. These PNA receptors could be considered O-linked glycoproteins containing the Gal-beta(1,3)GalNAc disaccharide because: i) PNA carbohydrate specificity toward this disaccharide found in this glycoprotein type; ii) their carbohydrate composition with Gal and GalNAc but not man residues; iii) their sensitivity to alkaline treatment; and iv) strong inhibition of PNA binding to ZHC with the Gal-beta(1,3)GalNAc structure. The absence of PNA receptors on HyC appeared to be related to the absence of this glycoprotein containing the disaccharide but not to the change or failure of glycosylation of the polypeptide chain of PNA receptors. The relationship between the presence of PNA receptors and differentiation/tumorisation phenomena as well as the mechanism that induced the expression of these receptors are discussed.
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PMID:Identification of peanut agglutinin receptors related to the state of tumoral liver cell differentiation. 157 2

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.
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PMID:alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins. 158 69

Human lung adenocarcinoma sub-cell lines HAL-8, HAL-24 and HAL-33, showing different lung colonization potential (LCP), were established from human lung adenocarcinoma cell line KUM-LK-2 using repeated cloning with limiting dilution technique. Cell lines HAL-8 and -33 were characterized by high and low LCP, respectively, while HAL-24 did not give rise to lung colonies. The cell surface protein and carbohydrate profiles were determined by cell surface labeling (with lactoperoxidase-dependent 125I-iodination and galactose oxidase-NaB3H4, respectively) followed by SDS-gel electrophoresis. Various carbohydrate epitopes expressed at the cell surface were analysed by cytofluorometry using various monoclonal antibodies (MAbs) directed to Le(x), sialosyl-Le(x), sialosyl dimeric Le(x), T, Tn and sialosyl-Tn structures, which are often reported as being highly expressed in a variety of human cancers, particularly adenocarcinoma. Expression of sialosyl dimeric Le(x) (defined by MAb FH6) was high on HAL-8, moderate on HAL-33, and relatively low on HAL-24. In contrast, each of the three lines showed essentially equal expression (as determined by MAb reactivity) of sialosyl-Tn (defined by MAb TKH2), Le(x) (defined by MAb SH1), and Tn (defined by MAb 1E3). The cell lines showed extremely weak expression of T (defined by MAb HH8). LCP of HAL-8 and -33 was completely inhibited by sialidase treatment of cells. It is suggested that higher expression of sialosyl dimeric Le(x) (defined by MAb FH6) in HAL-8 cells may play an important role in higher potential of blood-borne lung colonization.
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PMID:Human lung adenocarcinoma cell lines with different lung colonization potential (LCP), and a correlation between expression of sialosyl dimeric Le(x) (defined by MAb FH6) and LCP. 167 53

Two mucins were isolated from bovine submandibular glands and termed major and minor on a quantitative basis. The major mucin representing over 80% of the total glycoprotein fraction contained 37% of its dry weight as protein in contrast to 62% for the minor mucin. Differences in the amino acid composition reflected the higher proportion of typically non-glycosylated peptide in the minor mucin. The molar ratio of N-acetylgalactosamine to serine plus threonine was 0.82 in major and 0.65 in minor mucins, indicating a lower degree of substitution of potential glycosylation sites in the minor mucin. Differences in the carbohydrate composition were found largely related to the sialic acids, with higher relative amounts of N-glycoloylneuraminic acid in the minor mucin. In addition, the proportion of di-O-acetylated sialic acids was higher in the major mucin. The rate of sialidase action on the two mucins could be correlated with the content of N-glycoloylneuraminic acid in each glycoprotein. There was no difference in the type of oligosaccharide found in each mucin and the differences in relative proportions reflected the monosaccharide composition for the two mucins. Gel filtration on Sepharose CL 2B showed a lower molecular weight distribution for the minor in contrast to the major mucin which was partially excluded. Density gradient centrifugation reflected this variation. SDS-PAGE demonstrated a regular banding pattern for the major mucin with a lowest subunit size of 1.8 x 10(5) Da and aggregates in excess of 10(6) Da, while the minor mucin ranged from 3.0 x 10(5) to 10(6) Da. The chemical composition of the isolated mucins was compared with previous histochemical analysis of mucin distribution in bovine submandibular glands and indicates a possible cellular location for each mucin.
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PMID:Characterization of the major and minor mucus glycoproteins from bovine submandibular gland. 184 75

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called 'protective protein', were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed
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PMID:Structure of the lysosomal neuraminidase-beta-galactosidase-carboxypeptidase multienzymic complex. 210 3

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.
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PMID:Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5. 230 8

Immunoblots of red cell membranes stained with eluates of alloanti-Aua and alloanti-Aub show that these antibodies recognize 2 membrane components from Au(a+) and Au(b+) cells, respectively. These structures, of apparent molecular weight (Mr) 79,000 and 85,000, are identical in appearance and mobility on a 10% SDS polyacrylamide gel to the Lutheran glycoproteins identified by alloanti-Lub. Like the Lutheran glycoproteins, they showed a reduction in apparent Mr of about 1,500 after sialidase treatment. Red cell membrane components immunoprecipitated by a Lutheran-related monoclonal antibody, were analysed with Lutheran and Auberger antibodies by immunoblotting. The Lutheran glycoproteins were revealed by anti-Lub in precipitates from Au(a+b-) Lu(a-b+) and Au(a-b+) Lu(a-b+) cells, by anti-Aua in precipitates from Au(a+b-) Lu(a-b+) cells and by anti-Aub in precipitates from Au(a-b+) Lu(a-b+) cells. The same components were also recognized by anti-Lua in precipitates from Lu(a+b-) cells. Thus Aua and Aub antigens appear to be carried on the same red cell membrane structures as those carrying the Lutheran determinants. These results are particularly significant in the light of the very close phenotypic association between the Auberger and Lutheran blood groups which have been shown, by one family, to be controlled by genes at separate loci.
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PMID:Evidence that the Auberger blood group antigens are located on the Lutheran glycoproteins. 231 12


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