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1.
The carbohydrate chains of the bronchial-mucus glycoproteins of six cystic fibrosis patients with blood group O were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high-performance liquid chromatography. Structural characterization was performed by 500-MHz 1H-NMR spectroscopy in combination with quantitative sugar analysis. The established structures range in size from tetra- up to heptasaccharides. They are all sialyl analogs of neutral oligosaccharides that were characterized previously [Lamblin G., Boersma A., Lhermitte M., Roussel P., Mutsaers J. H. G. M., Van Halbeek H. & Vliegenthart J. F. G. (1984) Eur. J. Biochem. 143, 227-236]. The NeuAc residue was found to occur either in alpha (2----3)-linkage to Gal, or in alpha (2----6)-linkage to GalNAc-ol or Gal.  相似文献   

2.
The carbohydrate chains of the respiratory-mucus glycoproteins of a patient suffering from bronchiectasis due to Kartagener's syndrome were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by consecutive straight-phase high-performance liquid chromatography (HPLC) on a silica-based alkylamine column, and reverse-phase HPLC on a silica-based octadecyl column, respectively. The structures of the oligosaccharidealditols were determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis. The 24 structures established range in size from disaccharides to heptasaccharides. Novel oligosaccharides obtained from the bronchiectasis mucus glycoproteins are: (formula; see text) 23 of the 24 monosialyl oligosaccharides characterized can be conceived of as extensions of neutral oligosaccharides purified from the bronchial mucus of this patient [Klein, A. et al. (1988) Eur. J. Biochem. 171, 631-642; Breg, J. et al. (1988) Eur. J Biochem. 171, 643-654]. The sialic acid residue was found to occur either in alpha (2----3)- or alpha (2----6)-linkage to a galactosyl residue or in alpha (2----6)-linkage to GalNAc-ol.  相似文献   

3.
The N-linked oligosaccharides, released by hydrazinolysis from the major 55-kDa family, PZP3, of porcine zona pellucida glycoproteins, were separated into neutral (28%) and acidic (72%) carbohydrate chains by anion-exchange HPLC. By competition assay, it was shown that the mixture of neutral chains has the sperm-receptor activity, while that of the acidic chains has no activity. Their carbohydrate structures were analyzed after the reducing ends were modified with 2-aminopyridine. The neutral chains were fractionated into several components by reverse-phase and normal-phase HPLC. By sequential glycosidase digestion and 500-MHz 1H-NMR spectroscopy, the structures of three major components were determined. The structures of some of the minor components were analyzed only by sequential glycosidase digestion. By these analyses, it was found that a diantennary complex-type structure with a fucose residue was predominant in the neutral chains. Furthermore, the analyses of the endo-beta-galactosidase digests of the acidic chains revealed that the partially sulfated and sialylated N-acetyllactosamines are linked to the non-reducing ends of diantennary, triantennary, and tetra-antennary complex-type neutral chains, forming heterogeneous acidic chains.  相似文献   

4.
The O-glycosidically linked carbohydrate units of ovomucin were released from serine and threonine in peptide as oligosaccharide chains by alkali treatment with and without borohydride. Two sulfated oligosaccharides were fractionated by using gel filtration and ion-exchange chromatography. The yield of sulfated oligosaccharides released by alkali treatment was higher in the presence of borohydride than in the absence of borohydride. The sulfated oligosaccharides released by alkali treatment with borohydride were as follows: an oligosaccharide composed of N-acetylgalactosaminitol, galactose, N-acetylneuraminic acid and sulfate in a molar ratio of about 1: 1: 1: 1 and another oligosaccharide in a molar ratio of about 1:1: 0.6: 0.5.  相似文献   

5.
Mucin glycopeptides were prepared from the salivary mucins of 20 healthy donors with blood group O. The carbohydrate chains of the high-molecular-weight mucins were released by alkaline borohydride treatment. Neutral and monosialylated oligosaccharide-alditols were purified by ion-exchange chromatography, gel filtration, and HPLC. The structures of the oligosaccharide-alditols were determined by high-resolution 1H-NMR spectroscopy in combination with fast atom bombardment mass spectrometry and methylation analysis. Thirty-seven oligosaccharide-alditols were characterized and illustrate the extreme diversity of the salivary mucins carbohydrate chains. This diversity might represent a mosaic of bacterial adhesion sites and be involved in the early events of the nonimmune defense of the oral cavity. Among these 37 oligosaccharide-alditols, 31 have not been previously described in human saliva and five of these are novel structures: [formula: see text]  相似文献   

6.
Human Tamm-Horsfall glycoprotein has been purified from the urine of one male. The Asn-linked carbohydrate chains were enzymically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, and separated from the remaining protein by gel-permeation chromatography on Bio-Gel P-100. Fractionation of the intact (sulfated) sialylated carbohydrate chains was achieved by a combination of three liquid-chromatographic techniques, namely, anion-exchange FPLC on Q-Sepharose, amine-adsorption HPLC on Lichrospher-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. In total, more than 150 carbohydrate-containing fractions were obtained, some of which still contained mixtures of oligosaccharides. The primary structure of 30 N-glycans, including 10 novel oligosaccharides, were determined by one- and two-dimensional 1H-NMR spectroscopy at 500 MHz or 600 MHz. The types of compounds identified range from non-fucosylated, monosialylated, diantennary to fucosylated, tetrasialylated, tetraantennary carbohydrate chains, possessing the following terminal structural elements: [formula: see text]  相似文献   

7.
Oligosaccharide chains of herpes simplex virus type 2 glycoprotein gG.2   总被引:3,自引:0,他引:3  
gG.2 glycoprotein was purified by H966 monoclonal antibodies linked to Sepharose from herpes simplex virus type 2-infected HEp-2 cells labeled with [3H] glucosamine. The glycoprotein was subjected to Pronase digestion and the glycopeptides were fractionated by Con A-Sepharose in a major fraction (88.5% of total radioactivity) unbound to the lectin gel and in a minor species which bound to the lectin as a N-linked diantennary oligosaccharide. Mild and strong acid hydrolysis of Con A-unbound and Con A-bound fractions revealed that (i) both species were highly sialylated; (ii) the Con A-unbound fraction contained mainly labeled N-acetylgalactosamine, as is the case for O-linked oligosaccharides; and (iii) the Con A-bound fraction carried the vast majority of the labeled N-acetylglucosamine present in gG.2. Three size classes of oligosaccharides were separated from mild alkaline borohydride-treated Con A-unbound glycopeptides, which accounted for about 80% of the radioactivity present in the fraction. Galactosaminitol was recovered as the major labeled product in the strong acid hydrolyzates of the oligosaccharides generated by reductive beta-elimination, indicating that they were O-glycosidically linked to the peptide backbone. Thin-layer and DEAE-Sephacel chromatography of the three O-linked oligosaccharide species indicated that disialylated tetrasaccharides and monosialylated trisaccharides were the major components, whereas neutral disaccharide was a minor component. Digestion with neuraminidase and beta-galactosidase of the O-linked oligosaccharides supported the idea that the common disaccharide core was mainly of the structure beta-galactosyl-N-acetylgalactosamine. The large occurrence of O-linked oligosaccharides differentiates this type 2-specific herpes simplex virus glycoprotein from the type-common herpesvirus glycoproteins gB, gC, and gD.  相似文献   

8.
The primary structures of the O-glycosidically linked oligosaccharides isolated from glycoproteins GP I and GP II of Fusarium sp. M7-1 were established. The oligosaccharides released by alkaline borohydride treatment from the glycoproteins were purified by Bio-Gel P-4 and HPLC. This approach resulted in one monosaccharide and seven oligosaccharides. Their primary structures were resolved mainly by NMR spectrometry in combination with methylation mass spectrometry and fast atom bombardment mass spectrometry. The following structures have been determined. [formula: see text].  相似文献   

9.
The multiple cellulase-containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O-linked mainly to a 230-kDa subunit. The oligosaccharide chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by one- and two-dimensional (correlation, homonuclear Hartmann-Hahn, rotating-frame nuclear Overhauser enhancement) 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast-atom-bombardment mass spectrometry. The following carbohydrate structures could be established: [formula: see text] The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B. cellulosolvens and of Clostridium thermocellum [Gerwig, G. J., Kamerling, J. P., Vliegenthart, J. F. G., Morag (Morgenstern), E., Lamed, R. & Bayer, E. A. (1991) Eur. J. Biochem. 196, 115-122], having 3, 5 and 6 as common elements. The furanose form of a terminal alpha-D-galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome-like entity of B. cellulosolvens.  相似文献   

10.
The structures of large O-glycosidically linked oligosaccharides derived from swine trachea mucin glycoprotein were established. Reduced oligosaccharides released by treatment with alkaline borohydride were separated by gel filtration on Bio-Gel P-6 and the neutral oligosaccharides were isolated by chromatography on DEAE-cellulose. Eight oligosaccharides (DIa to BII), ranging in length from 8 to 15 sugar units, were isolated. On the basis of carbohydrate composition and analytical data from sequential treatment with exoglycosidases and permethylation analysis, the following structures were assigned to these oligosaccharides: (Formula: see text).  相似文献   

11.
A Lewis-b-active glycosphingolipid containing a repetitive type-1 chain carbohydrate core was isolated from human colonic adenocarcinoma cell line Colo205. This glycosphingolipid was purified by HPLC and preparative high-performance thin-layer chromatography and its structure elucidated by positive-ion fast-atom-bombardment mass spectrometry with collision-induced disassociation, 1H-NMR spectroscopy and methylation analysis. The glycosphingolipid was found to be a trifucosylated derivative of this novel carbohydrate core, having the following structure: [formula; see text].  相似文献   

12.
Pooled human milk oligosaccharides were separated into neutral and several acidic oligosaccharide fractions by preparative anion-exchange chromatography (AEC) using AG 1-X2. The oligosaccharides were eluted stepwise using deionized water and three different concentrations of ammonium acetate buffer, pH 6.8. The elution order of the compounds was determined directly by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the AEC effluent without any cleanup or concentration steps. Up to a concentration of 500 mM ammonium acetate, the masses of acidic oligosaccharides could be detected by screening the fractions in an automated mode. The combination of the improved chromatographic procedure, the applied MALDI matrices, and operating parameters is suitable for the detection of neutral oligosaccharides as well as acidic oligosaccharides. The method provides high sensitivity and mass accuracy, including for the high-molecular-weight monosialylated oligosaccharides up to 2751.5 Da. The applied ionic strength of the anion-exchange eluents enables a rapid and an unambiguous composition assignment by MALDI-MS for neutral, monosialylated, and disialylated oligosaccharides from human milk. The acidic fractions have to be desalted by electrodialysis and were finally analyzed by HPAEC-PAD to get a high-resolution "fingerprint" of structures present in each fraction. From these analyses, it can be concluded that the isomeric variety of monosialylated oligosaccharides occurring in human milk is higher than estimated before.  相似文献   

13.
Sialidosis urine was fractionated by gel filtration on Bio-Gel P-6. All pooled fractions containing carbohydrates showed the presence of small amounts of GalNAc in non-reducing position, besides free N-acetyllactosamine type of oligosaccharides as major constituents. The fractions were subjected to reductive alkaline borohydride degradation, after which the major part of GalNAc was recovered as N-acetyl-D-galactosaminitol (GalNAc-ol). The GalNAc-ol-containing material was separated from the N-glycosidic oligosaccharides by a second gel-filtration step on AcA 202. Subsequently, the O-glycosidic sialyloligosaccharide-alditols were subfractionated by anion-exchange chromatography on Mono Q. Structural analysis by 500-MHz 1H-NMR spectroscopy revealed two major components in all fractions, namely: NeuAc alpha 2-3Gal beta 1-3GalNAc-ol and NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GalNAc-ol. Furthermore, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-6]GalNAc-ol was found as a minor component in some of the fractions. The presence of these carbohydrate chains in Bio-Gel fractions differing in molecular mass suggested that they are derived from glycopeptides which are heterogeneous in their peptide part.  相似文献   

14.
Two O-glycosidically linked acidic polysaccharides, AP I and AP II, were released, respectively, from glycoproteins GP I and GP II of Fusarium sp. M7-1 by alkaline borohydride treatment and purified by gel filtration chromatography. They were found to be apparently homogeneous on gel filtration chromatography and analytical ultracentrifugation. Their molecular weights were estimated to be 8.2 x 10(4) and 3.1 x 10(4), respectively. The various oligosaccharide fragments obtained from AP I and AP II by acetolysis and partial acid hydrolysis were purified by gel filtration chromatography and HPLC. Their primary structures were resolved mainly by NMR spectrometry in combination with methylation mass spectrometry. Analyses of the acetolysis and partial acid hydrolysis products and the 1H-NMR spectrum of AP I and AP II showed that they are analogues. Thus, we propose that the main parts of the acidic polysaccharides have the following structures. [formula: see text] *X, unidentified oligosaccharide chains. The numbers on the left and the numbers in parentheses outside the brackets indicate the approximate number of side chains of AP I and AP II, respectively, the saccharide sequences of which are not specified.  相似文献   

15.
Mucin glycopeptides were prepared from the respiratory mucus of a non-secretor, chronic bronchitis patient with blood group O, Le(a+b-). Oligosaccharides were released by alkaline-borohydride treatment and purified by anion-exchange chromatography, size exclusion chromatography, and HPLC on a silica-bonded alkylamine column. Structural studies employed 500-MHz 1H-NMR spectroscopy, fast-atom-bombardment mass spectrometry and methylation analysis. Twenty-four neutral oligosaccharides, ranging in size from disaccharides to hexasaccharides, were fully characterized in this study. None of the structures contained an alpha(1----2)-linked fucose residue, in keeping with the non-secretor status of the patient. Seven of the structures had fucose present in alpha(1----3)-linkage in the X-determinant, while only one oligosaccharide (compound 14b) was seen with fucose alpha(1----4)-linked in the Le(a) determinant. The following eight structures isolated from the mucins of the non-secretor patient had not been found previously in the mucins of secretor individuals: [formula: see text] This study confirms that the blood group status of an individual is reflected in the carbohydrate structures of the secreted mucins. Furthermore, it clearly illustrates the need for detailed carbohydrate structural studies of mucins from different individuals before any attempt can be made to correlate observed differences in oligosaccharide profiles to disease status.  相似文献   

16.
Sulfated N-linked carbohydrate chains in porcine thyroglobulin   总被引:3,自引:0,他引:3  
N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on Lichrosorb-NH2, and analyzed by 500-MHz 1H-NMR spectroscopy and, partially, by permethylation analysis. Of the acidic oligosaccharide-alditols, the following sulfated carbohydrate chains could be identified: NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3[(SO3Na----3)Gal beta 1----4GlcNAc beta1----2-Mana alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc-ol and NeuAc alpha 2----6Gal beta 1----4(SO3Na----)0-1 GlcNAc beta 1----2-Man alpha 1----3[NeuAc alpha 2----6Gal beta 1----4(SO3Na----6)1-0GlcNAc beta 1----2Man alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc- ol. The sulfated structural elements for porcine thyroglobulin form novel details of N-linked carbohydrate chains. They contribute to the fine structure of these oligosaccharides and are another type of expression of microheterogeneity.  相似文献   

17.
The glycosylation of human cytokeratins was investigated in cultured human keratinocytes and A431 cells by metabolic labeling with [3H]glucosamine. In the presence of tunicamycin, keratinocytes incorporated [3H]glucosamine into a vitamin A-regulated acidic 53-kDa component of the cytoskeleton which was identified as cytokeratin 13 by one- and two-dimensional immunoblotting with specific monoclonal antibodies. This cytoskeletal component was also labeled with [3H]glucosamine in A431 cells but not in KB cells, which do not express cytokeratin 13. Its labeling was resistant to tunicamycin, suggesting that [3H]glucosamine had not been incorporated into N-linked oligosaccharides. Acid hydrolysis followed by paper and ion-exchange chromatography showed that the radioactivity in electrophoretically purified cytokeratin 13 was still present as glucosamine. Radioactivity was completely removed by treatment with beta-N-acetylglucosaminidase, suggesting that it was present in terminal N-acetylglucosamine residues. The labeled carbohydrate was released by alkaline borohydride treatment and was bound by a phenylboronic acid column, indicating an O-glycosidic linkage. On Bio-Gel P-2 columns, the beta-eliminated carbohydrate co-eluted with authentic N-acetylglucosaminitol. The results indicate that cytokeratin 13 contains single residues of N-acetylglucosamine O-glycosidically linked to the polypeptide chain.  相似文献   

18.
From bronchial mucins of cystic fibrosis patients with blood group O, the carbohydrate chains were released in the form of oligosaccharide-alditols by alkaline borohydride treatment. Application of high-performance liquid chromatography directly on the pool of neutral oligosaccharides afforded 23 fractions. Twenty oligosaccharide structures were characterized by employing 500-MHz 1H-NMR spectroscopy in conjunction with sugar analysis. Thirteen among these had been revealed before to occur in human bronchial mucins [Van Halbeek, H., Dorland, L., Vliegenthart, J. F. G., Hull, W. E., Lamblin, G., Lhermitte, M., Boersma, A., and Roussel, P. (1982) Eur. J. Biochem. 127, 7-20], when paper-chromatographic fractionation of this pool of neutral oligosaccharides was employed. High-performance liquid chromatography enabled to obtain another seven pentasaccharide and hexasaccharide alditols; the largest-size representatives are: (Formula see text). Thereby, this approach afforded deeper insight into the structural heterogeneity displayed by the carbohydrate chains of bronchial mucins.  相似文献   

19.
The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.  相似文献   

20.
The total monosialylated oligosaccharide fraction from pooled human milk was isolated by gel filtration and ion-exchange chromatography. Further separation by HPLC using a mobile phase containing an ion-pairing reagent of triethylamine gave five new monosialylated oligosaccharides. Structural analysis was carried out by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz NMR spectroscopy. Combined structural data revealed the following new structures: [formula: see text]  相似文献   

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