Release Of Oligosaccharides Research Articles

Effect of alkalis on N-glycosidic linkages of glycoproteins.

Effect of alkalis on N-glycosidic linkages of glycoproteins.

Isolation of sulfited oligosaccharides from glycoproteins treated with alkaline sulfite

The oligosaccharide products resulting from treatment of mucin-type glycoproteins with alkali in the presence of the sulfite anion have been investigated. Treatment of fetuin and of tryptic glycopeptides from the human erythrocyte with this reagent resulted in the release of sulfited oligosaccharides identified as N-acetylsulfohexosamine (HexNAcSO 3), α-NeuAc-(2→6)-HexNAcSO 3, and α-NeuAc-(2→3)-Gal-(1→3 or 4)-[GlcNAc-(1→6)]-HexNAcSO 3. In addition, 2.7 moles of sialic acid were released per mole of α-NeuAc-(2→6)-HexNAcSO 3 from fetuin. The sulfohexosamine moiety is formed via unsaturated intermediates from a 3- O-substituted 2-acetamido-2-deoxy- d-galactosyl residue at the carbohydrate-peptide linkage site when this residue is not substituted at O-4 by another sugar residue. A reaction mechanism accounting for the release of the sulfited oligosaccharides from a 3- O- and 6- O-substituted hexosamine is proposed in which the oligosaccharide branch attached to O-6 is obtained as a specific fragment terminating in sulfohexosamine.

Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices.

Incubations of thyroid microsomes with radiolabeled dolichyl pyrophosphoryl oligosaccharide (Glc3Man9-GlcNAc2) under conditions optimal for the N-glycosylation of protein resulted in the release, by apparently independent enzymatic reactions, of two types of neutral glucosylated polymannose oligosaccharides which differed from each other by terminating either in an N-acetylglucosamine residue (Glc3Man9GlcNAc1) or a di-N-acetylchitobiose moiety (Glc3Man9GlcNAc2). The first mentioned oligosaccharide, which was released in a steady and slow process unaffected by the addition of EDTA, appeared to be primarily the product of endo-beta-N-acetylglucosaminidase action on newly synthesized glycoprotein and such an enzyme with a neutral pH optimum capable of hydrolyzing exogenous glycopeptides and oligosaccharides (Km = 18 microM) was found in the thyroid microsomal fraction. The Glc3Man9GlcNAc2 oligosaccharide, in contrast, appeared to originate from the oligosaccharide-lipid by a rapid hydrolysis reaction which closely paralleled the N-glycosylation step, progressing as long as oligosaccharide transfer to protein occurred and terminating when carbohydrate attachment ceased either due to limitation of lipid-saccharide donor or addition of EDTA. There was a striking similarity between oligosaccharide release and transfer to protein with lipid-linked Glc3Man9GlcNAc2 serving as a 10-fold better substrate for both reactions than lipid-linked Man9-8GlcNAc2. The coincidence of transferase and hydrolase activities suggest the possibility of the existence of one enzyme with both functions. The physiological relevance of oligosaccharide release was indicated by the formation of such molecules in thyroid slices radiolabeled with [2-3H]mannose. Large oligosaccharides predominated (12 nmol/g) and consisted of two families of components; one group terminating in N-acetylglucosamine, ranged from Glc1Man9GlcNAc1 to Man5GlcNAc1 while the other contained the di-N-acetylchitobiose sequence and included Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, and Man9GlcNAc2.

Action patterns of immobilized dextranase

Dextranase, isolated from Penicillium funiculosum and P. lilacinum, was immobilized on porous, silanized-silica beads and a phenol-formaldehyde resin. A commercial dextran of relatively low molecular weight (∼2 × 10 6) was degraded by immobilized dextranase, with the formation of reducing sugars, but with little decrease in viscosity. In contrast, soluble dextranase caused rapid loss of viscosity, but only a slight increase in reducing sugar. Native dextran of high molecular weight, from Leuconostoc mesenteroides NRRL B-512 (F), was attacked very slowly by immobilized dextranase, with the release of oligosaccharides of low molecular weight.

Purification of human midcycle cervical mucin and characterization of its oligosaccharides with respect to size, composition, and microheterogeneity.

Human cervical mucin was solubilized from the gel phase of pooled midcycle cervical mucus using 6 M guanidine hydrochloride and 10 mM dithiothreitol and was then alkylated with iodoacetamide. Mucin was then purified by gel filtration on Bio-Gel A-50m resin in buffer containing 0.1% sodium dodecyl sulfate. The purified mucin gave a single band upon electrophoresis in either 5% acrylamide or 1% agarose gels. Protein comprised 21% of the glycoprotein by weight and amino acid analysis revealed a high content of Ser and Thr. Saccharide analysis yielded approximate molar ratios of Fuc:Gal:GlcNAc:GalNAc:NeuAc = 1:2:1:1:0.5. Inorganic sulfate, 1% by weight, was detected, but mannose was absent. Reductive alkali treatment of mucin resulted in release of oligosaccharides with concomitant conversion of 77% of GalNAc to its reduced derivative N-acetylgalactosaminitol (GalNAcol) thus demonstrating O-glycosidic linkage of GalNAc to protein. Reduced oligosaccharides were purified by ion exchange chromatography on DEAE-cellulose, paper chromatography, and high resolution gel filtration on Bio-Gel P-2 resin. A total of 16 reduced oligosaccharides were identified by thin layer chromatography. These included neutral, sialylated, and sulfated oligosaccharides and they varied in size from a disaccharide to a nonasaccharide. The major neutral oligosaccharide isolated (21% of recovered GalNAcol) was a tetrasaccharide, Gal:GlcNAc:GalNAcol = 2:1:1, and the major acidic oligosaccharide isolated (11% of recovered GalNAcol) was a trisaccharide, Gal:GalNAcol:NeuAc = 1:1:1.

Release of oligosaccharides from human erythrocyte membranes of different blood-group-P phenotypes by trifluoroacetolysis.

Human erythrocyte membranes of different blood-group-P phenotypes have been degraded by trifluoroacct olysis at conditions that will effect transamidation. This degradation resulted in dissolution of the membranes with concomitant degradation of proteins. Oligosaccharide chains linked to proteins and ceramides were released as their pertrifluoroacetylated derivatives, which generally are stable towards further action of the reagent. 2-Acetamido-2-deoxy-sugar residues were converted into their corresponding 2-deoxy-2-trifluoroacetamido derivatives. The mixture of pertrifluoroacetylated oligosaccharides obtained was de-O-trifluoroacetylated and de-N-trifluoroacetylated, reduced, acetylated, permethylated and analyzed by gas-liquid chromatography/mass spectrometry. Membranes obtained from two individuals with phenotype B, p̄ (known to contain an excess of lactosyl ceramide) gave almost identical results with lactose released as the main component. From AZB, Pk1 cell membranes small amounts of lactose were obtained but the trisaccharide Gal(α1-4)Gal(β1-4)Glc was the major component found. This trisaccharide is most likely derived from the Pk antigen (trihexosyl ceramide). Membranes from a donor with the phenotype B.P1 yielded small amounts of both lactose and the Pk trisaccharide together with a larger quantity of the tetrasaccharide GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc. The tetrasaccharide represents the carbohydrate portion of the P antigen (globoside). From all three types of membranes the B-specific trisaccharide Gal(α1-3)Gal was obtained. This compound is probably derived from specific degradation of blood-group-B-active glycoproteins and/or glycolipids. In addition to the oligo-saccharides mentioned above a large number of others were observed but in lower amounts.

Cell surface modification by endo-beta-galactosidase. Change of blood group activities and release of oligosaccharides from glycoproteins and glycosphingolipids of human erythrocytes.

Cell surface modification by endo-beta-galactosidase. Change of blood group activities and release of oligosaccharides from glycoproteins and glycosphingolipids of human erythrocytes.

Release of oligosaccharides from various glycosphingolipids by endo-beta-galactosidase.

Release of oligosaccharides from various glycosphingolipids by endo-beta-galactosidase.

Release of galactosyl oligosaccharides by endo-β-N-acetylglucosaminidase D

Endo-β-N-acetylglucosaminidase D from Diplococcus pneumoniae released galactosyl oligosaccharides from IgG glycopeptides treated with β-N-acetylglucosaminidase. The structure of the major oligosaccharide was proposed to be as follows. ▪Since α-mannosidase digestion of the β-N-acetylglucosaminidase-treated glycopeptides made them again resistant to the endoglycosidase, we concluded that an unsubstituted α-mannosyl residue was required for the enzymic action.

The Release of Intact Oligosaccharides from Specific Glycoproteins by Endo-β-N-acetylglucosaminidase H

Abstract Endo-β-N-acetylglucosaminidase H, purified to homogeneity from cultural filtrates of Streptomyces griseus (Tarentino, A. L., and Maley, F. (1974) J. Biol. Chem. 249, 811–817) was tested for its capacity to hydrolyze oligosaccharides from various glycoproteins. A complete and rapid release of oligosaccharide was effected from sulfitolyzed ovalbumin, bovine pancreatic deoxyribonuclease A, and ribonuclease B, and an invertase from Saccharomyces cerevisiae. These glycoproteins all contain a neutral oligosaccharide that is attached to the protein by a glycosyl asparagine bond and has the following composition: Asn-(GlcNAc)2(Man)x(GlcNAc)y. Analysis of the hydrolysis products revealed that the N-acetylglucosamine residue of di-N-acetylchitobiose distal to the asparagine is on the reducing end of the oligosaccharide, whereas the proximal N-acetylglucosamine remains with the protein. Neither the enzymatic properties nor the circular dichroic patterns of ribonuclease B and deoxyribonuclease A were altered significantly by the removal of their respective oligosaccharides. However, the elution of ribonuclease B from IRC-50 was changed, as was the electrophoretic mobility of deoxyribonuclease. Treatment of proteins that contain both neutral and acidic oligosaccharides, such as thyroglobulin, immunoglobulin M, and porcine ribonuclease, resulted in the release of the neutral chains only. A marked increase in the release of oligosaccharides from immunoglobulin M was effected by 2-mercaptoethanol. Removal of the neutral oligosaccharides did not appear to affect the immunological properties of this immunoglobulin.