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At least 649 records · Page 36Linked to original sources
PubMed1996

Glycosylation in human thyroglobulin: location of the N-linked oligosaccharide units and comparison with bovine thyroglobulin.

The amino acid sequence established for human thyroglobulin (hTG) from its cDNA sequence contains 20 putative N-linked glycosylation sites. We have characterized the glycopeptides contained in a tryptic digest of hTG in order to determine which sites are actually linked to carbohydrate. In addition, the distribution of oligosaccharide type(s) at these confirmed sites of N-linked glycosylation has been examined. Glycopeptides were purified using gel permeation chromatography followed by several steps of HPLC. The purified tryptic glycopeptides were characterized by gas phase sequencing and carbohydrate analysis and located within the amino acid sequence of thyroglobulin. Each of the recovered glycopeptides contained a consensus sequence for N-linked glycosylation. Of the 20 putative N-linked glycosylation sites in the human thyroglobulin polypeptide chain, 16 were shown to be actually glycosylated in the mature protein. Eight of these confirmed glycosylation sites (at positions 57, 465, 510, 729, 797, 1696, 1754, and 2230) appear to be linked to complex-type oligosaccharide units containing fucose and galactose in addition to mannose and glucosamine. Five sites (at positions 1200, 1329, 1993, 2275, and 2562) contain high mannose type units and two sites (at positions 179 and 1345) are linked to oligosaccharide units containing galactose in addition to mannose and glucosamine but no fucose and may be either hybrid or complex structures. In addition, position 928 was found to be degenerate in oligosaccharide structure and very different oligosaccharide composition types were found associated with peptides containing the same amino acid sequence. A high probability of a beta turn which would include the glycosylated asparagine residue was predicted for the amino acid sequence found at 13 of the 16 sites. The glycosylation pattern in hTG was also compared with the data recently reported for bovine thyroglobulin (bTG) (27) and as has been recently reported for bTG, no oligosaccharides of the high mannose type were found in the N-terminal portion of hTG. Only four of the 20 putative sites the sequence of hTG, at asparagine residues 91, 477, 1849, and 2102 were not represented in the purified glycopeptide population and are presumed to escape significant glycosylation.

Amino Acid Sequence↗
PubMed2000

Conformational studies of the Man8 oligosaccharide on native ribonuclease B and on the reduced and denatured protein.

Site-specific presentation of oligosaccharides in the context of carrier proteins can influence markedly their recognition by carbohydrate-binding proteins. On RNaseB, the Man5-9 N-glycans at Asn-34 are bound by the serum lectin conglutinin when the glycoprotein is reduced and denatured, but there is no binding to the N-glycans on the native form of RNaseB. The RNaseB Man8, which is a glycoform preferentially bound by conglutinin, is the subject of the present study. The conformational behavior of the protein-linked oligosaccharide Man8 is investigated on the native and on the reduced and denatured RNaseB, using a combination of NMR and theoretical calculations. Quantitative data on the NOESY crosspeaks have been obtained, thereby allowing the comparison of mobilities of homologous linkages within the glycan chain. Oligosaccharide conformations compatible with the NMR data have been explored by molecular modeling of the free oligosaccharide, using two different force fields (AMBER and SYBYL). There are some differences between the results produced by the two force fields, the AMBER simulations providing a better agreement with the experimental data. The results indicate that both on the native and on the reduced heat-denatured glycoprotein, the RNase Man8 oligosaccharide exhibits a conformational behavior very similar to that of the free oligosaccharide. However, this conformational freedom of the N-glcyan does not amount to full availability for carbohydrate-recognition proteins and enzymes.

Magnetic Resonance Spectroscopy↗
PubMed1998

Analysis of 2-aminobenzamide-labeled oligosaccharides by high-pH anion-exchange chromatography with fluorometric detection.

To establish high-pH anion-exchange chromatography with fluorometric detection (HPAEC-FD) for the sensitive oligosaccharide analysis, the system for HPAEC with pulsed amperometric detection (PAD) was modified by placing the anion micromembrane suppressor between PAD and fluoromonitor to neutralize the alkaline eluate and connecting the flow path. N-Linked oligosaccharides were released from various glycoproteins by hydrazinolysis followed by labeling with 2-aminobenzamide, and their asialooligosaccharides were analyzed by HPAEC-FD using an isocratic elution with 0.3 M NaOH solution. The results indicated that the new system using FD offers fine separation of oligosaccharides at the subpicomole level. Coinjection of reduced dextran oligomers as internal standards which are detected by PAD permits us to express elution positions of the fluorescent oligosaccharides as glucose units, resulting in a much more reliable analysis. The system was also effectively used for structural analysis of oligosaccharides by sequential glycosidase digestion. Thus, HPAEC-FD promises to be an alternative tool for the sensitive analysis of oligosaccharides.

Carbohydrate Conformation↗
PubMed1994

Transglycosylation activity of Mucor hiemalis endo-beta-N-acetyl-glucosaminidase which transfers complex oligosaccharides to the N-acetylglucosamine moieties of peptides.

A novel endo-beta-N-acetylglucosaminidase in the culture fluid of Mucor hiemalis isolated from soil was found to have transglycosylation activity. This endo-beta-N-acetylglucosaminidase, Endo-M, could liberate the complex type of asparagine-linked oligosaccharides by hydrolysis of diacetylchitobiose linkage from glycoproteins. The treatment of Endo-M with N-acetyl-glucosamine and asialotransferrin glycopeptide having the complex type of oligosaccharides resulted in the transfer of the released oligosaccharide from the glycopeptide to N-acetyl-glucosamine. The structure of the product after transfer was deduced to be (GlcNAc)2-Man-(Gal-GlcNAc-Man)2 by a combination method of pyridylamination and high performance liquid chromatography, and mass-spectrometry. The enzyme could transfer the complex type of oligosaccharide from asialotransferrin glycopeptide to bovine ribonuclease with the high-mannose type of oligosaccharide. This will lead to the construction of neoglycoproteins containing different types of oligosaccharides.

Acetylglucosamine↗
PubMed1991

Uteroferrin contains complex and high mannose-type oligosaccharides when synthesized in vitro.

Mature uteroferrin (Uf; Mr = 35,500) is a progesterone-induced acid phosphatase secreted by the pig uterus. It contains a single, unphosphorylated, high mannose-type oligosaccharide. Endometrial explants cultured in vitro secrete Uf with a Mr of 37,000 (37k Uf) having phosphorylated high mannose oligosaccharides. In this report we demonstrate that 37k Uf contains two N-linked oligosaccharides which are a mixture of complex and high mannose-type oligosaccharides. The complex-type glycopeptides are biantennary and a portion may be fucosylated on the GlcNac of the chitobiose core proximal to the peptide. Only a portion of the high mannose-type oligosaccharides are phosphorylated. The remainder appear to be typical Man6-4GlcNac2 oligosaccharides found on mature Uf.

Acid Phosphatase↗
PubMedDate not supplied

Differential processing of Asn-linked oligosaccharides on pituitary glycoprotein hormones: implications for biologic function.

Luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone from pituitary and chorionic gonadotropin from placenta are a family of glycoproteins, each consisting of an alpha and beta subunit. Within an animal species, the alpha subunit of all four hormones contains the identical amino acid sequence, while each beta subunit is distinct and confers biologic specificity to the hormone dimer. Despite sharing common alpha subunits, these hormones bear Asn-linked oligosaccharides which differ in structure. Whereas chorionic gonadotropin contains exclusively neutral and sialylated oligosaccharides, the pituitary hormones bear neutral, sialylated, sulfated, and sialylated/sulfated structures. The sulfated oligosaccharides are unique in structure and are more prevalent on certain pituitary hormones, indicating that the synthesis of these unusual oligosaccharides is tightly regulated. The differences in oligosaccharide structures in conjunction with the highly specific endocrine responses elicited by these hormones, suggest an important functional role for the oligosaccharides, such as metabolic clearance, control of hormone response, modulation of hormone potency, and/or intracellular sorting of hormones into separate secretory granules.

Animals↗
PubMed1994

Analysis of glycosphingolipid-derived oligosaccharides by high pH anion exchange chromatography.

As an adjunct to existing thin layer and column chromatographic methods for the identification of glycolipids a method that utilizes the high pH anion chromatographic (HPAEC) analysis of the oligosaccharides released from the glycolipids by endoglycoceramidase has been developed. Using a Dionex Carbo Pak PA1 column and elution with a linear gradient of sodium acetate in 0.2 M NaOH, the elution times of eight neutral and fourteen acidic oligosaccharides derived from glycolipids were determined. Under these conditions the neutral oligosaccharides were well separated from each other but some of the acidic oligosaccharides had overlapping elution times. The ganglioside-derived oligosaccharides could be further identified by treating them with sialidase or by mild acid hydrolysis and reanalysing the products by HPAEC. The method was applied to the analysis of mixed bovine brain gangliosides. The procedure provides an additional approach for the initial identification of glycolipids by analysing the component oligosaccharides rather than the intact glycolipids.

Animals↗
PubMed1989

Structure of the major O-glycosidic oligosaccharide of monkey erythrocyte glycophorin.

Sialic acids and the major O-glycosidic oligosaccharide of glycophorin MK from monkey (Japanese monkey, Macaca fuscata) erythrocyte membranes were characterized. N-Glycolylneuraminic acid (Neu5Gc) was found as the major sialic acid, which was confirmed by gas-liquid chromatography-mass spectrometry as the trimethylsilyl methyl ester. Three O-glycosidic oligosaccharide units were obtained from a tryptic glycopeptide that contained all of the carbohydrate units in glycophorin MK by mild alkaline borohydride/borotritide treatment. Carbohydrate analyses of the oligosaccharides revealed that they were composed of Neu5Gc, galactose and N-acetylgalactosaminitol in the molar ratios of 1:1:1 (trisaccharide), 2:1:1 (tetrasaccharide) and 3:1:1 (pentasaccharide). The content of oligosaccharide units was estimated to be 1:12:5 for penta-, tetra- and trisaccharide, respectively, based on the yields, the molecular weight, and the number of oligosaccharide attachment sites in the amino-acid sequence. The tetrasaccharide was the major oligosaccharide and its structure was proposed to be Neu5Gc alpha 2-3Gal beta 1-3[Neu5Gc alpha 2-6]GalNAcol.

Animals↗
PubMed1984

On the role of oligosaccharide trimming in the maturation of Sindbis and influenza virus.

The alpha-glucosidase inhibitor bromoconduritol inhibits the formation of the N-linked, complex-type oligosaccharides of the glycoproteins from influenza viruses (fowl plague virus, influenza virus PR-8) and from sindbis virus. Viral glycoproteins produced in bromoconduritol-treated chicken-embryo and baby-hamster kidney cells are fully glycosylated, but accumulate N-linked, high-mannose oligosaccharides of the composition Glc1Manx (GlcNAc)2 (x = 7, 8, and 9). Other alpha-glucosidase inhibitors (nojirimycin, deoxynojirimycin, acarbose) were not specific inhibitors of oligosaccharide processing under the conditions used in the present investigation. In bromoconduritol-treated, sindbis virus-infected chicken-embryo and baby-hamster kidney cells, the sindbis glycoproteins are metabolically stable. Specific proteolytic cleavage of the polyprotein precursors to form E2 and E1 occurs in bromoconduritol-treated chicken-embryo cells, but cleavage of PE2 to E2 is prevented in the infected baby-hamster kidney cells. Yet, release of infectious sindbis virus particles is inhibited in both cell types indicating that the formation of complex oligosaccharides is required for a late step in virus formation. The release of virus particles from influenza virus PR-8-infected bromoconduritol-treated chicken-embryo cells is not inhibited, and virus with only high-mannose oligosaccharides is formed. In contrast, when chicken-embryo cells were infected with the influenza virus fowl plague virus, release of infectious particles was inhibited. The fowl plague virus hemagglutinin is cleaved in chicken-embryo cells, in contrast to the hemagglutinin of the PR-8 virus. However, the cleavage products HA1 and HA2 do not reach the cell surface. In addition, or as a consequence, HA1 and HA2 are proteolytically broken down, whereas uncleaved hemagglutinin of PR-8 appeared metabolically stable. These results may explain the decrease in formation of fowl plague virus particles and the lack of effect on PR-8 virus in bromoconduritol-treated cells. This work thus shows different biological roles for oligosaccharide processing.

Animals↗
PubMed1996

[Structural and functional aspects of oligosaccharides in human milk].

About a century ago, pediatricians observed that in feces of breast-fed infants, compared to those of bottle-fed infants, Bifidobacterium bifidum was the predominant microorganism. It was shown thereafter that aminosugar-containing oligosaccharides are growth factors for a specific strain of Bifidobacterium. Meanwhile, more than 130 lactose-derived oligosaccharides have been identified in human milk. Some of these oligosaccharides like Lacto-N-Tetraose and Lacto-N-Fucopentaose I and II do not occur in minute amounts but in concentrations up to 1-2 g/L. As the total amount of complex oligosaccharides is between 3-6 g/L those components have to be considered as major human milk constituents. There is striking evidence that human milk oligosaccharides are potent inhibitors of bacterial adhesion to epithelial surfaces, an initial stage of infective processes. Therefore, these oligosaccharides are considered to be soluble receptor analogues of epithelial cell surfaces participating in the non-immunological defense system of human milk-fed infants.

Bifidobacterium↗
PubMed1990

Oligosaccharides in several Philippine indigenous food legumes: determination, localization and removal.

The oligosaccharide profile of raw mature seeds of seven different legumes indigenous to the Philippines was measured in 70% ethanol extracts of the seeds by thin layer chromatography using HPTLC plates and quantified by a densitometer. Based on the results, the legumes could be ranked according to decreasing oligosaccharide content or flatulence potential as follows: Sam-samping (Clitoria ternatea) greater than hyacinth bean (Dolichos lablab) greater than sabawel (Mucuna pruriens) greater than lima bean (Phaseolus lunatus) greater than swordbean (Canavalia gladiata) greater than rice bean (Vigna umbellata) greater than jack bean (Canavalia ensiformis). Sam-samping had 4.79% total oligosaccharides and hyacinth bean or batao, 3.66%. A jack bean accession had 1.79% oligosaccharides. Simple processing methods were tested to detoxify the oligosaccharides. Soaking the batao seeds had no effect while boiling even resulted in a net 23-31% increase in the levels of raffinose, stachyose and verbascose. On the other hand, two min of dry roasting resulted in complete removal of oligosaccharides whereas germination resulted in about 30-40% decrease after 1 and 2 days, respectively.

Chromatography, Thin Layer↗
PubMed2005

Enzymic transfer of alpha-L-arabinopyranosyl residues to exogenous 1,4-linked beta-D-galacto-oligosaccharides using solubilized mung bean (Vigna radiata) hypocotyl microsomes and UDP-beta-L-arabinopyranose.

A single alpha-L-arabinopyranosyl (alpha-L-Arap) residue was shown, by a combination of chemical and spectroscopic methods, to be transferred to O-4 of the nonreducing terminal galactosyl (Gal) residue of 2-aminobenzamide (2AB)-labeled galacto-oligosaccharides when these oligosaccharides were reacted with UDP-ss-L-arabinopyranose (UDP-ss-L-Arap) in the presence of a Triton X-100-soluble extract of microsomal membranes isolated from mung bean (Vigna radiata, L. Wilezek) hypocotyls. Maximum-(1-->4)-arabinopyranosyltransferase activity was obtained at pH 6.0-6.5 and 20 degrees C in the presence of 25 mM Mn2+. The enzyme had an apparent K m of 45 microM for the 2AB-labeled galactoheptasaccharide and 330 microM for UDP-ss-L-Arap. A series of 2AB-labeled galacto-oligosaccharides with a degree of polymerization (DP) between 6 and 10 that contained a single alpha-L-Arap residue linked to the former nonreducing terminal Gal residue were generated when the 2AB-labeled galactohexasaccharide (Gal6-2AB) was reacted with UDP- ss-L-Ara p in the presence of UDP-beta-D-Galp and the solubilized microsomal fraction. The mono-arabinosylated galacto-oligosaccharides are not acceptor substrates for the galactosyltransferase activities known to be present in mung bean microsomes. These results show that mung bean hypocotyl microsomes contain an enzyme that catalyzes the transfer of Arap to the nonreducing Gal residue of galacto-oligosaccharides and suggest that the presence of a alpha-L-Arap residue on the former terminal Gal residue prevents galactosylation of galacto-oligosaccharides.

Arabinose↗
PubMed2000

Functional analysis of chimeras derived from the Sinorhizobium meliloti and Mesorhizobium loti nodC genes identifies regions controlling chitin oligosaccharide chain length.

The rhizobial nodulation gene nodC encodes an N-acetylglucosaminyltransferase that is responsible for the synthesis of chitin oligosaccharides. These oligosaccharides are precursors for the synthesis of the lipo-chitin oligosaccharides that induce cell division and differentiation during the development of nitrogen-fixing root nodules in leguminous plants. The NodC proteins of Mesorhizobium loti and Sinorizobium meliloti yield chitinpentaose and chitintetraose as their main products, respectively. In order to localize regions in these enzymes that are responsible for this difference in product chain length, a set of six chimeric enzymes, comprising different combinations of regions of the NodC proteins from these two bacteria, was expressed in Escherichia coli. The oligosaccharides produced were analyzed using thin-layer chromatography. The major conclusion from this work is that a central 91-amino acid segment does not play any obvious role in determining the difference in the chain length of the major product. Furthermore, the characteristically predominant synthesis of chitintetraose by S. meliloti NodC is mainly dependent on a C-terminal region of maximally 164 amino acids; exchange of only this C-terminal region is sufficient to completely convert the M. loti chitinpentaose synthase into an S. meliloti-like chitintetraose synthase. The N-terminal region of 170 amino acids also plays a role in restricting the length of the major product to a tetramer. However, the role of the C-terminal region is clearly dominant, since exchanging the N-terminal region has no effect on the relative amounts of chitintetraose and -pentaose produced when the C-terminal region of S. meliloti NodC is present. The length of a predicted beta-strand around residue 300 in the C-terminal region of various NodC proteins is the only structural element that seems to be related to the length of the chitin oligosaccharides produced by these enzymes; the higher the amount of chitintetraose relative to chitinpentaose, the shorter the predicted beta-strand. This element may therefore be important for the effect of the C-terminal 164 amino acids on chitin oligosaccharide chain length.

Bacterial Proteins↗
PubMed1987

Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography.

A simple, sensitive, and rapid method for the analysis of structures of N-linked carbohydrates is reported. The method involves four steps: preparation of carbohydrate chains from glycopeptides by N-oligosaccharide glycopeptidase digestion; derivatization of the reducing ends of carbohydrate chains with a fluorescent reagent, 2-aminopyridine, by using sodium cyanoborohydride; separation of oligosaccharide derivatives by reverse-phase high-performance liquid chromatography; and structural analysis of oligosaccharides by sequential exoglycosidase digestion. The elution positions of 50 standard oligosaccharide derivatives were determined by HPLC. The structure of an unknown oligosaccharide can be characterized by comparison of its elution position with those of the standard compounds. The method was applied to elucidate the structures of oligosaccharides in the myeloma IgG protein, Yot.

Amidohydrolases↗
PubMed1988

The identification of furanose and pyranose ring forms of the reducing units of oligosaccharides.

Several types of oligosaccharides with glucose, arabinose, or galactose units have been prepared by chemical degradation of oligosaccharides of known structures and by enzymatic syntheses utilizing macerans amylase or yeast galactosyltransferase and appropriate substrates and cosubstrates. The ring forms of reducing units of these oligosaccharides and of oligosaccharides composed of glucose and mannose have been identified by a combined method of analysis based on methylation, gas-liquid chromatography, and mass spectrometry. In the dimethyl sulfoxide solvent used for the methylations, the oligosaccharides with arabinose or galactose units at the reducing ends occur as arabinofuranose or galactofuranose ring forms to the extent of 55 and 65%, respectively. The remainder of these monosaccharide units are present in the pyranose ring form. Oligosaccharides with glucose or mannose units at the reducing ends occur primarily in the pyranose ring form. An interesting observation is the finding that the reducing units which carry substituents linked by alpha-glycosidic linkages occur in a higher percentage in the furanose ring forms than those which carry substituents linked by beta-glycosidic linkages.

Carbohydrate Conformation↗
PubMed1990

Parameterization of contribution of sugar units to elution volumes in reverse-phase HPLC of 2-pyridylaminated oligosaccharides.

The reverse-phase high-performance liquid chromatography elution data, expressed in glucose units, of 2-pyridylaminated oligosaccharides (PA-oligosaccharides) originated from N-glycosides of glycoproteins have been analyzed with multiple regression to obtain parameters of contribution ascribable to each monosaccharide unit. For the best results, the known PA-oligosaccharides were classified into M-series (the "high mannose" type), X-series (those containing xylose), F-series (those containing L-Fuc alpha (1,6)GlcNAc-PA), and Z-series (all others). Within each series, a variable ("unit contribution") is assigned to every sugar component in the oligosaccharide, and the elution value of a given oligosaccharide was assumed to be the sum total of the unit contributions. The unit contribution obtained from such multiple regression calculations led to a set of elution values for oligosaccharides in each series, which showed very good agreement with the observed elution values. These unit contribution values are useful in predicting the elution value when the exact structure is known, and they also aid in predicting the structure from the known elution value.

Carbohydrate Sequence↗
PubMed1991

Calculated two-dimensional sugar map of pyridylaminated oligosaccharides: elucidation of the jack bean alpha-mannosidase digestion pathway of Man9GlcNAc2.

We have developed a two-dimensional (2-D) mapping of pyridylaminated oligosaccharides as an aid to structural determination of glycoprotein-derived oligosaccharides. Using the available data of reverse-phase HPLC of pyridylamino-oligosaccharides, this was further extended to parameterization of unit contribution by each sugar component, which allows the prediction of possible structures from the elution volume. We have extended this approach to the data obtained with amide-silica HPLC column to obtain a calculated 2-D mapping technique for the oligomannose-type oligosaccharides (M-series). In this method, the elution volumes of all possible pyridylamino-oligosaccharides up to the size of Glc1Man9GlcNAc2 (50 in total) are calculated from the established UC values to construct a 2-D map. To test the validity of the calculated 2-D map, the structures of intermediate PA-oligosaccharides generated during the alpha-mannosidase (jack bean) digestion of Man9GlcNAc2 (porcine thyroglobulin) were analyzed to establish the digestion pathway. The validity of this approach is substantiated by an independent deduction of the intermediate structures based on structural relationships and the coincidence of elution volumes. Our results agree well with the recently published digestion pathway of Man5GlcNAc2 by the same enzyme and that of Man9GlcNAc2 by lysosomal alpha-mannosidase.

Carbohydrate Sequence↗
PubMed1991

Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins.

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.

Amidohydrolases↗