Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In search of synthetic high affinity ligands for the mannose receptor, we synthesized a series of lysine-based oligomannosides containing two (M2L) to six (M6L5) terminal alpha-D-mannose groups that are connected with the backbone by flexible elongated spacers (16 A). The synthesized cluster mannosides were all able to displace binding of biotinylated ribonuclease B and tissue-type plasminogen activator to isolated human mannose receptor. The affinity of these cluster mannosides for the mannose receptor was continuously enhanced from 18-23 microM to 0.5-2.6 nM, with mannose valencies increasing from two to six. On average, expansion of the cluster mannoside with an additional alpha-D-mannose group resulted in a 10-fold increase in its affinity for the mannose receptor. M3L2 to M6L5 displayed negative cooperative inhibition of ligand binding to the mannose receptor, suggesting that binding of these mannosides involves multiple binding sites. The nanomolar affinity of the most potent ligand, the hexamannoside M6L5 makes it the most potent synthetic cluster mannoside for the mannose receptor yet developed. As a result of its high affinity and accessible synthesis, M6L5 not only is a powerful tool to study the mechanism of ligand binding by the mannose receptor, but it is also a promising targeting device to accomplish cell-specific delivery of genes and drugs to liver endothelial cells or macrophages in bone marrow, lungs, spleen, and atherosclerotic plaques.
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PMID:Lysine-based cluster mannosides that inhibit ligand binding to the human mannose receptor at nanomolar concentration. 891 Apr 12

The kinetic properties of UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) purified to homogeneity from lactating bovine mammary gland have been investigated. GlcNAc-phosphotransferase transferred GlcNAc 1-phosphate from UDP-GlcNAc to the synthetic acceptor alpha-methylmannoside, generating GlcNAc-1-phospho-6-mannose alpha-methyl, the structure of which was confirmed by mass spectroscopy. GlcNAc-phosphotransferase was active between pH 5.7 and 9.3, with optimal activity between pH 6.6 and 7.5. Activity was strictly dependent on Mg2+ or Mn2+. The Km for Mn2+ was 185 microM. The Km for UDP-GlcNAc was 30 microM, and that for alpha-methylmannoside was 63 mM. The enzyme was competitively inhibited by UDP-Glc, with a Ki of 733 microM. The 166-kDa subunit was identified as the catalytic subunit by photoaffinity labeling with azido-[beta-32P]UDP-Glc. Purified GlcNAc-phosphotransferase utilizes the lysosomal enzyme uteroferrin approximately 163-fold more effectively than the non-lysosomal glycoprotein ribonuclease B. Antibodies to GlcNAc-phosphotransferase blocked the transfer to cathepsin D, but not to alpha-methylmannoside, suggesting that protein-protein interactions are required for the efficient utilization of glycoprotein acceptors. These results indicate that the purified bovine GlcNAc-phosphotransferase retains the specificity for lysosomal enzymes as acceptors previously observed with crude preparations.
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PMID:Bovine UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase. II. Enzymatic characterization and identification of the catalytic subunit. 894 Jan 56

Glycans derivatized with 2-aminoacridone have been analyzed consecutively by reverse-phase high-performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MECC). The 2-aminoacridone derivatizing agent used in the present study is highly hydrophobic and is well separated from the glycan derivatives in both separation techniques, ensuring that excess reagent does not interfere with the oligosaccharide analysis. The methodology outlined uses the high resolving power of capillary electrophoresis to determine the heterogeneity of samples after collection and preconcentration by HPLC. Collected glycan samples are submitted for mass spectrometric analysis to determine molecular weight. This methodology has been applied to linear oligosaccharides derived from dextran and to N-linked mannose-rich glycans from ribonuclease B.
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PMID:A coordinated high-performance liquid chromatographic, capillary electrophoretic, and mass spectrometric approach for the analysis of oligosaccharide mixtures derivatized with 2-aminoacridone. 897 25

The 175-kDa mannose receptor is one of the receptors that mediates the clearance of tissue-type plasminogen activator (t-PA). The affinity of t-PA for the mannose receptor is much higher than the affinity of other high-mannose-type oligosaccharide-containing glycoproteins. In order to find an explanation for this high affinity, we studied the biochemical interaction of various forms of t-PA with the isolated human mannose receptor in several in vitro binding assays. t-PA showed a high affinity (Ki = 0.2 nM) for the mannose receptor and the interaction could be fully inhibited by mannan or polyclonal antibodies against the mannose receptor. The interaction was not affected by non-glycosylated t-PA. The high affinity differed slightly between t-PAs synthesized by various cell types (range Ki 0.2-0.7 nM) and between various glycoforms of t-PA. No statistically significant difference in affinity between t-PA and t-PA complexed to inhibitors was observed. In contrast to intact t-PA, a trypsin digest of t-PA had a low affinity (Ki = 0.5 microM) for the mannose receptor. Both intact and trypsin digests of the high-mannose-type oligosaccharide-containing glycoproteins ribonuclease B and ovalbumin had a low affinity (Ki 0.5-1.5 microM) for the mannose receptor. We conclude that neither protein-protein interactions, nor the complex-type oligosaccharides and the fucose residue on t-PA contribute significantly to the high-affinity binding of t-PA. We suggest that the conformation of the high-mannose-type oligosaccharide on t-PA is influenced by the protein moiety of t-PA in such a way that the oligosaccharide has a high affinity for the mannose receptor.
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PMID:Role of carbohydrate and protein in the binding of tissue-type plasminogen activator to the human mannose receptor. 949 74

Corneal proteoglycans have chondroitin/dermatan and keratan sulfate (KS) chains and belong to the leucine-rich proteoglycan gene family. Corneal KS is N-linked to Asn of an NX(S/T) site through a complex oligosaccharide linkage region. Only some sites receive KS, whereas others remain in a high mannose form. To determine whether the attachment of KS was biased toward specific sites, we isolated trypsin-digested KS-containing fragments of chick corneal proteoglycans and sequenced the peptides. Results showed that all of the peptides sequenced aligned to the deduced amino acid sequence of either chick lumican or chick keratocan at the first, third, and fourth potential N-linked sites. Sites 1 and 4 in lumican and keratocan are in a homologous location. By analogy with the structure of ribonuclease inhibitor (a Leu-rich repeat containing protein), the KS chains would extend outward on the outer face of a horseshoe-like structure. The amino acid sequences surrounding the potential N-linked sites were also compared. Sites receiving KS tend to have a higher occurrence of aromatic residues, in particular Phe, located within 3 amino acids of NX(S/T). These conserved Phe residues may have a role in the conversion of high mannose N-linked oligosaccharides to polylactosamine and/or keratan sulfate.
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PMID:Identification of the N-linked oligosaccharide sites in chick corneal lumican and keratocan that receive keratan sulfate. 954 93

Oligomers of glucose and oligosaccharides released from glycoproteins were derivatized with 2-aminobenzamide. As this fluorophore imparts no charge to the oligosaccharides, several strategies were investigated to achieve capillary electrophoresis (CE) separation of both neutral and charged derivatized glycans. Micellar electrokinetic capillary chromatography (MEKC) with the addition of anionic surfactants was evaluated as a first approach: sodium dodecyl sulfate (SDS) produced the best separation of the oligoglucose fragments, where the migration was inversely related to their degree of polymerization. To demonstrate the applicability of this method for complex carbohydrate analysis, oligosaccharide mixtures derived from ribonuclease B (RNase B) and alpha-acid glycoprotein (alpha-AGP) were analyzed. A satisfactory separation for the high-mannose structures found in RNase B could be obtained, whereas charged oligosaccharides from alpha-AGP were poorly resolved. Cyclodextrin-modified CE was chosen as the second approach: the effect of the addition of sulfobutylether-beta-cyclodextrin (SBE-beta-CD) or sulfobutylether-gamma-cyclodextrin (SBE-gamma-CD) on the electrophoretic mobilities and resolution of neutral and charged oligosaccharides was then studied. Selectivity of sialylated structures could be further improved by using anionic cyclodextrins (CDs) instead of micelles. However, this latter approach failed to baseline-resolve the different high-mannose structures of RNase B. A successful separation of the complex mixture of oligosaccharides from alphaalpha-AGP was obtained with the addition of 4% of SBE-gamma-CD and triethylamine (TEA) in a phosphate buffer, pH 6.7.
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PMID:Investigation of micelles and anionic cyclodextrins as pseudostationary phases for the capillary electrophoresis separation of oligosaccharides derivatized with 2-amino-benzamide. 984 71

Using ribonuclease B and human alpha 1-acid glycoprotein (AGP) as model glycoproteins, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with collision-induced dissociation (CID) is validated here as an effective tool for oligosaccharide sequencing. The spectra acquired for high-mannose and complex oligosaccharide structures show characteristic fragments resulting from cleavages of the glycosidic bonds and a few cross-ring cleavages. Esterification of the sialic acid residues is essential in stabilizing the acidic N-linked oligosaccharides. An important analytical feature observed in all acquired spectra is the occurrence of cleavages on the same antenna up to the branching point, as deduced from the absence of fragmentation due to the simultaneous cleavages on two or more antennas.
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PMID:Matrix-assisted laser desorption/ionization mass spectrometry of neutral and acidic oligosaccharides with collision-induced dissociation. 1020 63

The efficiencies in derivatization of reducing carbohydrates were compared by capillary electrophoresis using maltose as a model with nine monoaminobenzene derivatives by reductive amination in the presence of sodium cyanoborohydride. We found that aminobenzene derivatives substituted at the 3-position showed good reactivity with reducing carbohydrates as expected from the reaction mechanism, although the fluorescence intensities and molar absorptivities of these derivatives were not as high as those of 2- and 4-aminobenzene derivatives. The reagents, 3-aminobenzamide and 3-aminobenzoic acid, which showed the highest reactivity, were applied to the labeling of carbohydrate chains obtained from some sialic acid-containing glycoprotein samples, and also high-mannose and hybrid-type oligosaccharides. Capillary electrophoresis of these labeled carbohydrate chains in an inner surface-modified capillary with (50% phenyl)methylpolysiloxane allowed excellent separation of sialic acid-containing carbohydrate chains derived from fetuin and thyroglobulin as well as high mannose-type and hybrid-type carbohydrates derived from bovine pancreas ribonuclease B, soybean agglutinin and hen ovalbumin. The lower limit of calibration was as low as the 10(-16) mol (injected amount) with helium-cadmium laser induced detection.
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PMID:3-Aminobenzamide and 3-aminobenzoic acid, tags for capillary electrophoresis of complex carbohydrates with laser-induced fluorescent detection. 1059

A procedure for the enzymatic synthesis of neoglycoenzymes is described. The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was overexpressed in Escherichia coli as a fusion protein linked to glutathione S-transferase (GST). GST-Endo-A fusion was extracted as a soluble protein. The fusion protein was purified to homogeneity with glutathione-Sepharose 4B and showed transglycosylation activity toward high-mannose-type glycopeptides without removing the GST moiety. The GST-Endo-A immobilized on glutathione-Sepharose 4B retained its transglycosylation activity. The immobilized enzyme could transfer (Man)(6)GlcNAc en bloc to partially deglycosylated ribonuclease B without damaging its enzyme activity. The immobilized GST-Endo-A should be very useful for synthesizing active neoglycoenzymes attached with homogeneous N-linked oligosaccharides.
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PMID:Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-beta-N-acetylglucosaminidase A. 1062 87

Conglutinin is a serum lectin of the innate immune system, which binds high mannose N-glycans when these are appropriately presented on proteins. Here we use the conglutinin-ribonuclease B (RNaseB)-recognition system as a model to investigate the structural basis of selective recognition of protein-bound oligosaccharides by this carbohydrate-binding receptor. Conglutinin shows little binding to the isolated RNaseB-Man(8 )glycoform, and no binding to Man(5-6) glycoforms. In contrast, when the protein moiety is reduced and denatured we observe that conglutinin binds strongly to the isolated RNaseB-Man(8) glycoform and weakly to the Man(5-6) glycoforms. These results are in accord with observations on the binding to the N-glycans in the absence of carrier protein. NMR analyses of native RNaseB-Man(8) and -Man(5-6) glycoforms reveal that the three-dimensional structure of the protein moiety is essentially identical to that of non-glycosylated RNase (RNaseA). Thus there are no perceptible differences between the RNase protein forms that could account for differential availability of the N-glycan for conglutinin-binding. After reduction and denaturation, the NMR spectrum became typical of a non-structured polypeptide, although the conformational preferences of the N-glycosidic linkage were unchanged, and most importantly, the Man(8 )oligosaccharide retained the average conformational behavior of the free oligosaccharide irrespective of the carrier protein fold. This conformational freedom is clearly not translated into full availability of the oligosaccharide for the carbohydrate-recognition protein. We propose, therefore, that the differing bioactivity of the N-glycan is a reflection of the existence of different geometries of presentation of the carbohydrate determinant in relation to the protein surface within the glycan:carrier protein ensemble.
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PMID:Carrier protein-modulated presentation and recognition of an N-glycan: observations on the interactions of Man(8) glycoform of ribonuclease B with conglutinin. 1118 59


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