Reductive cleavage of Xaa-proline peptide bonds by mild alkaline borohydride treatment employed to release O-glycosidically linked carbohydrate units of glycoproteins

During the deglycosylation reaction of fish egg polysialoglycoproteins under the conditions of 1 M NaBH4 in 0.1 M NaOH at 37 degrees C for 48 h, a marked loss of the glycine content has been encountered, besides the serine and threonine residues to which the carbohydrate units are linked. The chemical basis behind this phenomenon has been elucidated by amino acid analysis first of the major glycopeptides (carbohydrate-(O)Thr-Gly-Pro-Ser) derived from desialylated polysialoglycoproteins and subsequently six proline-containing peptides before and after treatment under similar conditions. It has thus been established that -Xaa-Pro- sequences are remarkably susceptible to reductive cleavage under such mild aqueous conditions. In view of the finding that the reductive cleavage of insulin B-chain, which contains a single proline residue adjacent and C-terminal to a threonine residue, led to about 80% loss of the threonine residue, deglycosylation with alkaline borohydride reagents warrants a special comment. The decreased amounts of serine or threonine residues cannot be related simply to the degree of glycosylation of these residues. The above results are therefore discussed in the relation to other work.     

Colorimetric determination of N-acetylhexosamine-terminating O-glycosidically linked saccharides in mucins and glycoproteins

A sensitive colorimetric assay for detecting mucins and glycoproteins rich in O-glycosidically linked saccharides is reported. The method combines the susceptibility of N-acetylgalactosamine terminating O-glycosidically linked saccharides to beta-elimination with the Morgan-Elson reaction for N-acetylhexosamines with free reducing ends. All mucin and mucin-type glycoproteins but none of the serum-type glycoproteins tested resulted in characteristic color production. All mucins tested gave linear responses in the range 5 to 200 micrograms and the assay was also adapted to the microscale involving the use of 96-well microtiter plates. The microassay in which the volumes of samples and reagents are scaled down 2.5-fold was particularly useful in monitoring of mucins, in the presence of other glycoconjugates, in large numbers of samples obtained during fractionation procedures. Cesium chloride, cesium bromide, potassium thiocyanate, and various detergents do not interfere with the colorimetric determination. Guanidine hydrochloride, cesium trifluoroacetate, and beta-mercaptoethanol decreased color by 30 to 45%; however, the interference was not serious to prevent the use of the method for detection of mucins in their presence. The use of the method for the specific detection of mucin during fractionation by gel filtration and density gradient centrifugation of cystic fibrosis sputum samples is demonstrated.     

Structure of the O-glycosidically linked carbohydrate units of rat brain glycoproteins.

The O-glycosidically linked carbohydrate units of rat brain glycopeptides were released as reduced oligosaccharides with NaOH/NaBH4 treatment. Five oligosaccharides were isolated using gel filtration, ion-exchange chromatography and preparative thin-layer chromatography. Studies employing periodate oxidation, methylation analysis, chromium trixide oxidation and gas-liquid chromatography-mass spectrometry indicated the following structures: (I) alpha-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (II) beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (III) N-acetylneuraminyl-[beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol], (IV) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol and (V) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)[N-acetylneuraminyl-(2 leads to 6)]-N-acetylgalactosaminitol.     

Structural analysis of the O-glycosidically linked core-region oligosaccharides of human meconium glycoproteins which express oncofoetal antigens

Glycoproteins were extracted from meconium samples of group O neonates of secretor type by pronase digestion followed by precipitation in 67% aqueous ethanol and separated into Ii antigen enriched and depleted fractions by affinity chromatography. The latter fraction strongly expressed the oncofoetal antigens recognised by natural antibodies in mouse sera and the hybridoma antibody FC 10.2, and this activity was enhanced after mild acid hydrolysis to remove sialic acid and fucose residues. Oligosaccharides were released from the mild-acid-treated fraction by base-borohydride degradation and purified by gel permeation chromatography on Bio-Gel P4 and high performance liquid chromatography on octadecylsilyl and aminopropylsilyl columns. The major oligosaccharides were characterised by fast atom bombardment and electron impact mass spectrometry, combined gas-liquid chromatography/mass spectrometry and 500-MHz proton NMR spectroscopy. Their structures, in order of abundance, were: (Formula: see text).     

Structure of O-glycosidically linked oligosaccharides from glycoproteins of Trypanosoma cruzi CL-Brener strain: evidence for the presence of O-linked sialyl-oligosaccharides

Glycoproteins on the cell surface of Trypanosoma cruzi are known to play important roles in the interaction of the parasite with the host cells. We previously determined the structures of the O-glycan chains from the sialoglycoproteins (mucin-like molecules) of the G- and Y-strains and observed significant differences between them. We now report the structures of the sialylated and nonsialylated O-linked oligosaccharides isolated from the cell surface glycoproteins of the myotropic CL-Brener strain grown in the presence of fetal calf serum. The structures of the O-linked oligosaccharide alditols obtained by reductive beta-elimination of the sialoglycoprotein were determined by a combination of methylation analysis, fast atom bombardment-mass spectrometry and nuclear magnetic resonance spectroscopy. The presence of a beta-galactopyranose substituent on the N-acetylglucosamine O-4 position shows that these O-linked oligosaccharides from CL-Brener strain belong to the same family as those isolated from mucins expressed by T. cruzi Y strain, a reticulotropic strain. In addition, novel O-glycans, including alpha2-3 mono-sialylated species are described.     

Presence of an O-glycosidically linked hexasaccharide in fetuin

Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.     

Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

Histochemical demonstration of O-glycosidically linked,type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Summary Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo-and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with -N-acetylgalactosaminidase and -L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with -galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B₄ reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo--N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from nonsecretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(1-3)-N-acetyl-D-galactosamine1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.     

Histochemical demonstration of O-glycosidically linked, type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo- and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with alpha-N-acetylgalactosaminidase and alpha-L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with alpha-galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B4 reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo-alpha-N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from non-secretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(beta 1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.     

NMR studies of mannitol-terminating oligosaccharides derived by reductive alkaline hydrolysis from brain glycoproteins

Interest in the characterisation of O-mannosyl glycan structures has been stimulated following the identification of mannitol-terminating oligosaccharides among the chains released from mammalian proteins in nervous and muscle tissues, and by the discovery of a putative human O-mannosyl transferase. Several mass spectrometry methods have been applied to structure elucidation particularly when low amounts of oligosaccharide are available for analysis. However, when sufficient amounts are available, a combination of through-bond homo- and heteronuclear, and of through-space homonuclear NMR experiments permit the complete identification of these oligosaccharide sequences. We describe here the assignment of 1H and 13C NMR chemical shifts from such experiments for four mannitol-terminating oligosaccharide alditols, GlcNAcbeta-(1-->2)Manol, Galbeta-(1-->4)GlcNAcbeta-(1-->2)Manol, Galbeta-(1-->4)[Fucalpha-(1-->3)]GlcNAcbeta-(1-->2)Manol and NeuAcalpha-(2-->3)Galbeta-(1-->4)GlcNAcbeta-(1-->2)Manol, that were released from brain glycopeptides by alkaline borohydride treatment.     

Physiochemical properties of the Vi antigen before and after mild alkaline hydrolysis

Sedimentation and diffusion constants were determined for electrophoretically purified Vi antigen before and after mild alkaline hydrolysis. The molecular weight of the intact antigen was found to be 1.7 × 10⁶. Mild alkaline hydrolysis completely removed O-acetyl, reduced the molecular weight to 1.0 × 10⁵, and lowered the intrinsic viscosity from 8.0 deciliters/g to approximately 0.5 deciliter/g. Infrared spectra of the two antigen preparations were also compared.     

Structure of O-glycosidically linked oligosaccharides synthesized by the insect cell line Sf9

The O-glycosidically linked oligosaccharides on the pseudorabies virus (PRV) glycoprotein gp50 synthesized by three different cell lines were studied. The intact membrane protein (gp50) was expressed in Vero cells and in the insect cell line Sf9. In addition, a truncated, secreted form lacking the transmembrane and cytoplasmic domains (gp50T), was expressed in CHO and Sf9 cells. The protein, both in intact and truncated form, synthesized by the two mammalian cells contained only the disaccharide Gal beta 1-3GalNAc, either unsubstituted or substituted with one or two sialic acid residues. By contrast, the major O-linked structure on gp50 and gp50T synthesized by Sf9 cells was the monosaccharide GalNAc. The Sf9 cells also linked lower amounts of Gal beta 1-3GalNAc to gp50 (12%) and gp50T (26%). None of the structures synthesized by Sf9 cells contained sialic acid. Measurements of the two relevant glycosyltransferases revealed that while all three cell lines contain comparable levels of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase activity, there is a greater variation in the levels of UDP-Gal:N-acetylgalactosamine, beta 1-3 galactosyltransferase, with the Sf9 cells containing the lowest level.     

Is the alkaline cleavage of disulfide bonds in peptides an alpha-beta elimination reaction or a hydrolysis?

The cleavage of oxidized glutathione by alkali has been studied as representative of the cleavage of protein disulfides. This process is quite different when studied in 10-4N or 2-10-1N NaOH. At low alkali concentration no spectral changes are noted; at higher hydroxyl concentration the appearance of persulfide groups (followed at 335 nm), the formation of thiocyanate (arising from cold cyanolysis of persulfide groups) and the absorbance at 240 nm follow the same kinetics. The amount of half-cystine, recovered as cysteic acid after a 3-h reaction, is significantly lower than calculated. These results confirm that oxidized glutathione undergoes beta-elimination at high pH values, and that persulfide groups absorb not only at 335 nm (as already known) but also at 240 nm where, under our conditions, the contribution of other absorbing species is not very high.