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PubMed2001

Characterization of oligosaccharides in milk and feces of breast-fed infants by high-performance anion-exchange chromatography.

Human milk contains a large amount of oligosaccharides, which represent its third largest solute. Nevertheless, both the metabolism and the role of these substances are still largely unknown. A previous study we conducted documented that the amount of oligosaccharides excreted in the feces varies from 6% to 13% of the 24-hour ingested oligosaccharides. The aim of this study was to characterize the pattern of oligosaccharides in the feces compared with the pattern of the ingested milk. Six term newborn infants were studied at the end of the first month of life. A 7:00 AM milk sample was obtained with an electric breast pump. Feces were collected during the day of milk sampling. Analyses of oligosaccharides were performed using high-pH anion-exchange chromatography with pulsed amperometer detection. Pure milk oligosaccharides were used as reference standards. The chromatographic profile of the oligosaccharides present in the feces and in the milk samples showed more than 40 peaks, 20 of which have been identified. The oligosaccharide profile observed in the feces was similar to the pattern of oligosaccharides present in the milk ingested. A significant difference was represented by the almost complete absence of lactose in the feces of all infants and of sialyllacto-N-tetraose a and disialyllacto-N-neotetraose in 3 samples. A substantial reduction of lacto-N-tetraose was observed in 5 samples. Our results demonstrate that the oligosaccharide profile in the feces is similar to that of the ingested milk. Approximately 40% to 50% of the total ingested oligosaccharides can be found in feces of breast-fed infants.

Anions↗
PubMed1990

Two-dimensional mapping by the high-performance liquid chromatography of oligosaccharides released from glycosphingolipids by endoglycoceramidase.

A two-dimensional sugar mapping method has been developed by which sensitive, reproducible, and simple analysis can be carried out on the structures and compositions of oligosaccharides released from glycosphingolipids by endoglycoceramidase. The oligosaccharides were labeled quantitatively with an ultraviolet-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-oligosaccharides were separated first on an amide-silica column and then on a C4-silica column by high-performance liquid chromatography. The acidic ABEE-oligosaccharides were eluted as a group at the start of the chromatography while the neutral ABEE-oligosaccharides were separated according to size and structure on an amide-silica column using an eluent without salt. The acidic oligosaccharides were separated according to size and structure when rechromatographed on the same column using an eluent containing KH2PO4. NeuAc-containing ABEE-oligosaccharides were extensively separated from the corresponding NeuGc derivatives. The ABEE-oligosaccharides separated on an amide-silica column were then chromatographed on a column of C4-silica on which lactotriose and neolacto-series oligosaccharides were clearly shown to be separated from the others. On the basis of the retention times of the individual ABEE-oligosaccharides on two separate columns, 9 neutral and 15 acidic oligosaccharides derived from glycosphingolipid standards were two-dimensionally mapped without overlapping. The gangliosides of a human chondrosarcoma tissue and glycosphingolipids of tumor tissue of FBJ virus-transformed murine osteosarcoma cells were analyzed by this method in conjunction with exoglycosidase treatment. At least 11 species of glycosphingolipids were identified in both cases.

Animals↗
PubMed1994

Structural analysis of N-linked oligosaccharides of equine chorionic gonadotropin and lutropin beta-subunits.

Equine chorionic gonadotropin (eCG) and lutropin (eLH) are composed of alpha- and beta-subunits with an identical amino acid sequence but show different biological activities. To elucidate the molecular difference between these gonadotropins, the structure of the N-linked oligosaccharides of each beta-subunit was determined. N-linked sugar chains, liberated as tritum-labeled oligosaccharides by hydrazinolysis followed by N-acetylation and reduction with NaB3H4, were neutralized by sialidase digestion and/or methanolytic desulfation. Neutralized oligosaccharides were fractionated by sequential chromatography on serial lectin affinity columns and on a Bio-Gel P-4 column. Each oligosaccharide structure was determined by sequential exoglycosidase digestion in conjunction with elution profiles on lectin columns and methylation analysis. Each beta-subunit contained a single N-glycosylation site, but a high degree of microheterogeneity was observed in the structure of its N-linked oligosaccharides. eCG beta contained mono-, bi-, tri-, and tetraantennary complex-type oligosaccharides in a ratio of 3:63:13:1. eCG beta oligosaccharides contained about 16% of the bisecting GlcNAc and about 20% of poly-N-acetyllactosamine structures. Elongation of N-acetyllactosamine units showed a preference to the Man alpha 1-->6 side rather than the Man alpha 1-->3 side. Triantennary chains had only a C-2, 4-branched structure. eLH beta contained only mono- and biantennary complex-type and hybrid-type oligosaccharides in a ratio of approximately 18:67:10. eLH beta also contained bisected structures in about 18%. Oligosaccharides derived from the sulfated fraction of eLH beta contained GalNAc residues at nonreducing termini. Oligosaccharides from the sialylated/sulfated fraction of eLH beta contained both Gal and GalNAc residues at nonreducing termini, and those GalNAc residues were preferentially distributed to the Man alpha 1-->3 side of the trimannosyl core. These results clearly indicate that eCG beta and eLH beta possess structurally distinct N-linked oligosaccharides in addition to different charge groups even though they have a protein moiety identical to each other. Our results suggest that the biological activity of these hormones might be modulated by its terminal charge groups and stem structures of carbohydrate moiety synthesized in different organs.

Amino Sugars↗
PubMed1998

Oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin.

Calnexin and calreticulin are homologous molecular chaperones of the endoplasmic reticulum. Their binding to newly synthesized glycoproteins is mediated, at least in part, by a lectin site that recognizes the early N-linked oligosaccharide processing intermediate, Glc1Man9GlcNAc2. We compared the oligosaccharide binding specificities of calnexin and calreticulin in an effort to determine the basis for reported differences in their association with various glycoproteins. Using mono-, di-, and oligosaccharides to inhibit the binding of Glc1Man9GlcNAc2 to calreticulin and to a truncated, soluble form of calnexin, we show that the entire Glc alpha 1-3Man alpha 1-2Man alpha 1-2Man structure, extending from the alpha 1-3 branch point of the oligosaccharide core, is recognized by both proteins. Furthermore, analysis of the binding of monoglucosylated oligosaccharides containing progressively fewer mannose residues suggests that for both proteins the alpha 1-6 mannose branch point of the oligosaccharide core is also essential for recognition. Consistent with their essentially identical substrate specificities, calnexin and calreticulin exhibited the same relative affinities when competing for binding to the Glc1Man9GlcNAc2 oligosaccharide. Thus, differential glycoprotein binding cannot be attributed to differences in the lectin specificities or binding affinities of calnexin and calreticulin. We also examined the effects of ATP, calcium, and disulfide reduction on the lectin properties of calnexin and calreticulin. Whereas oligosaccharide binding was only slightly enhanced for both proteins in the presence of high concentrations of a number of adenosine nucleotides, removal of bound calcium abrogated oligosaccharide binding, an effect that was largely reversible upon readdition of calcium. Disulfide reduction had no effect on oligosaccharide binding by calnexin, but binding by calreticulin was inhibited by 70%. Finally, deletion mutagenesis of calnexin and calreticulin identified a central proline-rich region characterized by two tandem repeat motifs as a segment capable of binding oligosaccharide. This segment bears no sequence homology to the carbohydrate recognition domains of other lectins.

Adenosine Triphosphate↗
PubMed1981

Glycoprotein biosynthesis in animal cells grown in suspension culture. Assembly of lipid-linked saccharides and formation of protein-bound 'high-mannose' oligosaccharides.

Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into 'high-mannose' protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound 'high-mannose' oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound 'high-mannose' oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis.

Animals↗
PubMed1987

Oligosaccharides as an intravenous energy source in postsurgical patients: utilization when infused with glucose, amino acids, and lipid emulsion.

Utilization of intravenously administered oligosaccharides was evaluated in postsurgical patients by infusing oligosaccharides simultaneously with glucose, amino acids, and lipid emulsion for 4 d postoperatively. Seven patients were infused with a nutritional regimen providing glucose, amino acids, lipid emulsion, and oligosaccharides and seven patients received a similar regimen without oligosaccharides. Patients infused with oligosaccharides received an overall mean (+/- SD) of 144 +/- 41.0 g oligosaccharides per day. The mean overall excretion of total glucose (free plus oligosaccharide-bound) was significantly greater in patients infused with oligosaccharides (65.1 +/- 33.2 g/d) than in controls (1.83 +/- 1.55 g/d). Overall oligosaccharide utilization for the 4-d period was 48.7 +/- 10.1%. Plasma oligosaccharide concentrations increased from a baseline value of 2.43 +/- 1.90 mg/dL to 58.1 +/- 42.3 mg/dL after 4 d of oligosaccharide infusion, suggesting accumulation.

Adult↗
PubMed2001

Fucosylated human milk oligosaccharides vary between individuals and over the course of lactation.

Specific human milk oligosaccharides, especially fucosylated neutral oligosaccharides, protect infants against specific microbial pathogens. To study the concentrations of individual neutral oligosaccharides during lactation, a total of 84 milk samples were obtained from 12 women at 7 time periods during weeks 1-49 postpartum. The neutral oligosaccharides from each sample were isolated, perbenzoylated, resolved, and quantified by reversed-phase high-performance liquid chromatography. The resultant oligosaccharide peaks, identified by co-elution with authentic standards and mass spectrometry, ranged in size from tri- to octasaccharides. The total concentration of oligosaccharides declined over the course of lactation; the mean concentration at 1 year was less than half that in the first few weeks postpartum. One of the 12 donors produced milk fucosyloligosaccharides that were essentially devoid of alpha1,2 linkages (but contained alpha1,3- and alpha1,4-linked fucose) until late in lactation, consistent with the nonsecretor phenotype. In milk samples from the remaining 11 donors, fucosyloligosaccharides containing alpha1,2-linked fucose were prevalent, and their profiles were distinct from those of fucosyloligosaccharides devoid of alpha1,2-linked fucose. The ratio of alpha1,2-linked oligosaccharide concentrations to oligosaccharides devoid of alpha1,2-linked fucose changed during the first year of lactation from 5:1 to 1:1. Furthermore, the absolute and the relative concentrations of individual oligosaccharides varied substantially, both between individual donors and over the course of lactation for each individual. The patterns of milk oligosaccharides among individuals suggest the existence of many genotype subpopulations. This variation in individual oligosaccharide concentrations suggests that the protective activities of human milk could also vary among individuals and during lactation.

Carbohydrate Conformation↗
PubMed1993

Characterization of the high-affinity oligosaccharide-binding site of the 205-kDa porcine large granular lymphocyte lectin, a member of the leukocyte common antigen family.

Membrane lectins of mammalian large granular lymphocytes are thought to be important receptors in their non-major-histocompatibility complex-restricted activation. A triantennary desialylated oligosaccharide has been reported as the most effective triggering structure [Pospísil M., Kubrycht J., Bezouska K., Táborský O., Novák M. & Kocourek J. (1986) Immunol. Lett. 12, 83-90] while its cell surface receptor has recently been identified in pig natural killer cells as a 205-kDa membrane lectin resembling the proteins of the leukocyte common antigen family (LCA). In this study we have prepared 4-azidophenyl (photoactivatable) and 4-hydroxyphenyl (radio-iodinatable) derivatives of triantennary oligosaccharides by a new procedure which allows the natural conformation of the N-glycosidic linkage between the oligosaccharide and the respective labeling group to be retained. We used these high-affinity ligands to investigate the oligosaccharide-combining site of the 205-kDa lectin. Photoaffinity labeling of the whole cells and solubilized proteins confirmed that a 205-kDa polypeptide constitutes the major cell-surface calcium-independent receptor for triantennary oligosaccharides in pig lymphocytes. Isolation and manual sequencing of two ligand-labeled and eleven other peptides proved that the 205-kDa lectin represents a member of the LCA family expressing exons 4 and 6 during alternative splicing and that the high-affinity binding site is localized in the N-terminal 70-kDa extracellular domain. Binding studies with radiolabeled oligosaccharides and the above carbohydrate-recognition domain subjected to various chemical and enzymatic treatments indicated that the binding of oligosaccharides might be significantly modulated by sialylated O-glycosidically linked lineage-specific carbohydrate epitopes localized within this domain. Affinity chromatography of LCA isolated by conventional methods on immobilized oligosaccharides revealed that only a fraction of these cell-surface glycoproteins expressed high-affinity binding sites for the oligosaccharide ligands. Thus, N-linked oligosaccharide moieties of cell-surface glycoproteins seem to represent possible ligands of LCA that may be important in intercellular adhesion and oligosaccharide-mediated activation of lymphocytes.

Amino Acid Sequence↗
PubMed2006

Antioxidant activity and hepatoprotective potential of agaro-oligosaccharides in vitro and in vivo.

BACKGROUND: Agaro-oligosaccharides derived from red seaweed polysaccharide have been reported to possess antioxidant activity. In order to assess the live protective effects of agar-oligosaccharides, we did both in vitro and in vivo studies based on own-made agaro-oligosaccharides, and the structural information of this oligosaccharide was also determined. METHOD: Structure of agaro-oligosaccharides prepared with acid hydrolysis on agar was confirmed by matrix-assisted ultraviolet laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and NMR. The antioxidant effect of agaro-oligosaccharides on intracellular reactive oxygen species (ROS) was assessed by 2', 7'-dichlorofluorescin diacetate. Carbon tetrachloride was used to induce liver injury, some index including SOD, GSH-Px, MDA, AST, ALT were examined to determine the hepatoprotective effect of agaro-oligosaccharides. RESULTS: Agaro-oligosaccharides we got were composed of odd polymerizations with molecular weights ranged from 500 to 2500. Results from intracellular test indicated that agaro-oligosaccharides could significantly scavenge the level of oxidants in the hepatocytes, more beneficially, also associated with the improvement of cell viability In vivo studies of the antioxidant effects on tissue peroxidative damage induced by carbon tetrachloride in rat model indicated that agaro-oligosaccharides could elevate the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and decrease the level of malondialdehyde (MDA), glutamate oxaloacetate transaminase (AST), glutamic pyruvic transaminase (ALT) significantly. At 400 mg/kg, MDA level reduced 44 % and 21 % in liver and heart, SOD and GSH-Px increased to highest in liver and serum, while ALT level decreased 22.16 % in serum. CONCLUSION: Overall, the results of the present study indicate that agaro-oligosaccharides can exert their in vitro and in vivo hepatoprotective effect through scavenging oxidative damage induced by ROS.

Agar↗
PubMed2003

Possible application of milk oligosaccharides for drug development.

By applying a finger-printing method to the analysis of human milk oligosaccharides, several oligosacchartides were found to be deleted in the milk of non-secretor or Lewis negative individual. This finding afforded a clue to elucidate the enzymatic basis of blood types in humans. Furthermore, disappearance of some major oligosaccharides led to the finding of five novel minor oligosaccharides, which were hidden under the major oligosaccharides. Later on, structures of more than seventy oligosaccharides were elucidated. These oligosaccharides are derived from eleven core oligosaccharides by sialylation and/or fucosylation. All these oligosaccharides contain lactose at their reducing termini. This evidence, together with the deletion phenomena found in the milk of two blood type individuals, suggested that the oligosaccharides are formed by the concerted action of glycosyltransferases, which are responsible for formation of the sugar chains of glycoproteins on the surface of epithelial cells constructing the mucous membrane. The elongation may start by the action of iGnT. This enzyme is responsible for the addition of a beta-N-acetylglucosamine residue to the C-3 position of the galactose moiety constructing the N-acetyllactosamine group of the sugar chains of glycoconjugates. Therefore, oligosaccharides in human milk may include many structures, starting from the N-acetyllactosamine residues in the sugar chains of various glycoproteins. Many evidences, which indicate that virulent enteric bacteria and viruses start their infection by binding to particular sugar chains of glycoconjugates on the surface of their target cells, were presented recently. Therefore, milk oligosaccharides are expected to be useful to inhibit the infection of these bacteria and viruses.

ABO Blood-Group System↗
PubMed1992

Oligosaccharide structures of mucins secreted by the human colonic cancer cell line CL.16E.

Cl.16E, a stably differentiated clonal derivative of the human colonic cancer cell line HT29, was used to investigate the structure of oligosaccharide chains of mucins in colonic cancer. Secretory mucins were purified by equilibrium density gradient centrifugation in CsCl. Oligosaccharide side chains were isolated after beta-elimination. Compositional analysis of oligosaccharide-alditols performed after purification by gel filtration on a Bio-gel P-6 column showed 1) that GalNAc residues were located exclusively at the reducing ends of the chains, and 2) that fucose was absent from the preparation. Oligosaccharide-alditols were separated by high performance liquid chromatography (HPLC) on quaternary amine packings into a minor neutral fraction representing about 6.5% by weight of released oligosaccharides and four acidic fractions. Two acidic fractions, namely FI and FII encompassing mono- and disialylated structures, respectively, and containing 78% of total oligosaccharide alditols, were separated by HPLC. Structural determinations were carried out using methylation analysis, 1H NMR spectroscopy, and fast atom bombardment-mass spectrometry. Twelve oligosaccharide structures were determined which ranged in size from 3 to 8 residues. These oligosaccharides were based on core types 1, 2, and 4. Elongation of oligosaccharide chains was terminated by addition of sialic acid in alpha 2-3 linkage to Gal beta 1-3R and to Gal beta 1-4R residues. The predominant structure was a hexasaccharide (fraction FII-4). This contrasts with normal colonic mucins whose oligosaccharides were previously found to be based on core 3 structures and carry sialic acids in alpha (2-6) linkage to Gal beta 1-3R, to Gal beta 1-4R, and to GalNAc alpha-R (Podolsky, D.K. (1985) J. Biol. Chem. 260, 8262-8271; Podolsky, D.K. (1985) J. Biol. Chem. 260, 15510-15515). Collectively our findings suggest that Cl.16E colon cancer cells are able to synthesize mucin oligosaccharides of gastric type whose elongation is truncated by premature sialylation.

Adenocarcinoma↗
PubMed1987

Lectin affinity high-performance liquid chromatography. Interactions of N-glycanase-released oligosaccharides with Ricinus communis agglutinin I and Ricinus communis agglutinin II.

The structural determinants required for interaction of oligosaccharides with Ricinus communis agglutinin I (RCAI) and Ricinus communis agglutinin II (RCAII) have been studied by lectin affinity high-performance liquid chromatography (HPLC). Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of silica-bound RCAI and RCAII. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. RCAI binds oligosaccharides bearing terminal beta 1,4-linked Gal but not those containing terminal beta 1,4-linked GalNAc. In contrast, RCAII binds structures with either terminal beta 1,4-linked Gal or beta 1,4-linked GalNAc. Both lectins display a greater affinity for structures with terminal beta 1,4-rather than beta 1,3-linked Gal, although RCAII interacts more strongly than RCAI with oligosaccharides containing terminal beta 1,3-linked Gal. Whereas terminal alpha 2,6-linked sialic acid partially inhibits oligosaccharide-RCAI interaction, terminal alpha 2,3-linked sialic acid abolishes interaction with the lectin. In contrast, alpha 2,3- and alpha 2,6-linked sialic acid equally inhibit but do not abolish oligosaccharide interaction with RCAII. RCAI and RCAII discriminate between N-acetyllactosamine-type branches arising from different core Man residues of dibranched complex-type oligosaccharides; RCAI has a preference for the branch attached to the alpha 1,3-linked core Man and RCAII has a preference for the branch attached to the alpha 1,6-linked core Man. RCAII but not RCAI interacts with certain di- and tribranched oligosaccharides devoid of either Gal or GalNAc but bearing terminal GlcNAc, indicating an important role for GlcNAc in RCAII interaction. These findings suggest that N-acetyllactosamine is the primary feature required for oligosaccharide recognition by both RCAI and RCAII but that lectin interaction is strongly modulated by other structural features. Thus, the oligosaccharide specificities of RCAI and RCAII are distinct, depending on many different structural features including terminal sugar moieties, peripheral branching pattern, and sugar linkages.

Carbohydrate Conformation↗
PubMed1985

Oligosaccharide structures of isolated human colonic mucin species.

Purified human colonic mucin contains six distinct components which may be separated by DEAE-cellulose chromatography. Past studies defined the structure of oligosaccharide side chains from the most abundant species III, IV, and V which elute at intermediate salt concentrations. In these studies the structures of oligosaccharide side chains liberated from the remaining early and late eluting species I, II, and VI were determined after isolation by sequential conventional and high performance liquid chromatography through combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Mucin species I, II, and VI contained a less varied array of discrete oligosaccharide structures than that observed in the major mucin components. Mucin species I and II contained five and 10 structures, respectively, which account for 68 and 71% of total oligosaccharide content in these fractions. The predominant oligosaccharides of mucin species I included three neutral structures: a disaccharide GlcNAc beta (1-3)GalNAc-ol, a trisaccharide Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol, and a tetrasaccharide GlcNAc beta (1-4)Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol as well as two acidic components representing the sialylated forms of two of these oligosaccharides. Mucin species II contained these same oligosaccharides as well as four additional acidic structures, notably a disaccharide Neu alpha (2-6)GalNAc-ol and a hexasaccharide Gal beta (1-4)GlcNAc beta (1-3)Gal beta (1-4)GlcNAc beta (1-3) (NeuAc alpha (2-6))-GalNAc-ol, not identified in any other mucin species. The late eluting mucin species VI contained at least five discrete neutral oligosaccharides and six major acidic structures. While the majority of these structures had been previously isolated from the earlier eluting mucin species IV and V, species VI also contained di- and trisialylated oligosaccharides not identified in other mucin species. In conjunction with earlier studies of the major mucin species III, IV, and V, these data define the range of oligosaccharide structures present in human colonic mucin. These studies demonstrate that human colonic mucin possesses species with characteristic and distinguishable combinations of oligosaccharides which reflect variations of common core structures.

Carbohydrate Sequence↗
PubMed1980

The asparagine-linked sugar chains of the glycoproteins in calf thymocyte plasma membrane. Structural studies of acidic oligosaccharides.

The acidic oligosaccharide fraction obtained by paper electrophoresis of the hydrazinolysate of the plasma membrane glycoprotein mixture of calf thymocytes was fractionated by high voltage paper electrophoresis into 15 acidic components. Three of them were mixtures of two acidic oligosaccharides, while the remaining 12 were almost pure acidic oligosaccharides. The 18 oligosaccharides have a common core hexasaccharide, Man alpha 1 yields 6(man beta 1 yields 3)Man beta 1 yields 4GlcNAc beta 1 yields 4(Fuc alpha 1 yields 6)GlcNAc. The number of outer chains ranges from two to four. Three different types of outer chains, Gal beta 1 yields 4 GlcNAc beta 1 yields, Gal beta 1 yields 3 GlcNAc beta 1 yields, and Gal beta 1 yields 4 GlcNAc beta 1 yields 3Gal beta 1 yields 4GlcNAc beta 1 yields, were found to occur in the acidic oligosaccharides in various combination. In the biantennary oligosaccharides, the two outer chains are linked only at the C-2 position of the two alpha-mannosyl residues of the core hexasaccharide. All triantennary oligosaccharides have an additional outer chain at the C-4 position of the alpha-mannosyl residue constructing the Man alpha 1 yields 6Man group of the core. Two outer chains of tetraantennary oligosaccharides are linked at the C-2 and 4 positions of one alpha-mannosyl residue and the remaining two outer chains at the C-2 and 6 positions of the other alpha-mannosyl residue of the core. An interesting observation is that C-2,6 disubstitution always occurs on the alpha-mannosyl residue of the Man alpha 1 yields 6 side in all oligosaccharides with Gal beta 1 yields 4GlcNAc beta 1 yields 3Gal beta 1 yields 4GlcNAc beta 1 yields outer chain, while in other tetraantennary oligosaccharides, C-2,6 disubstitution always occurs on the alpha-mannosyl residue of the Man alpha 1 yields 3 side. No sialylated Gal beta 1 yields 3Gal beta 1 yields 4GlcNAc beta 1 yields outer chain was found in the acidic oligosaccharides studied in this paper.

Animals↗
PubMed1993

Separation and identification of O-linked oligosaccharides derived from glycoproteins by high-pH anion-exchange chromatography.

A high-pH anion-exchange chromatography (HPAEC) system with a pulsed amperometric detector was developed for analysis of O-linked oligosaccharides in glycoproteins. Since O-linked oligosaccharides are usually released from glycoproteins as alditols by beta-elimination with reduction and they are retained less on an anion-exchange resin than their reducing counterparts, a special set of HPAEC conditions optimized for separation of sialyl O-linked oligosaccharides different from those for N-linked oligosaccharide separation had to be devised. HPAEC separation of desialylated O-linked oligosaccharides is even more difficult because of the lack of carboxylic acids or anomeric hydroxyls. Therefore, a precolumn derivatization technique was developed for these oligosaccharides. Reduced desialylated oligosaccharides were de-N-acetylated followed by N-succinylation, which endowed a negative charge at each amino sugar residue in the oligosaccharides. The desialylated oligosaccharides are derivatized in the same vial and can be easily separated on HPAEC. The new HPAEC technique presented here promises to be a valuable tool for analysis of O-linked oligosaccharides in glycoproteins.

Acetylation↗
PubMed1994

Reducing-end modification of N-linked oligosaccharides with tyrosine.

The N-linked oligosaccharides from bovine fetuin were purified using newly developed preparative purification methodology. N-linked oligosaccharides were released from tryptic glycopeptides utilizing N-glycosidase F on the 5-g scale. Selective desialylation with neuraminidase from Clostridium perfringens resulted in the formation of a mono-sialyl-oligosaccharide and asialo-oligosaccharides. The reducing ends of the oligosaccharides were converted to the glycosylamine and reacted with the N-hydroxysuccinimide ester of Boctyrosine. The tyrosinated oligosaccharides were resolved into individual peaks on RP-HPLC and then characterized by proton NMR and FAB-MS. A single asialo-triantennary, an asialo-biantennary, and a mono-sialyl-triantennary oligosaccharide were recovered in good yield. Each product contained a single Boc-Tyr residue attached to the reducing-end GlcNAc residue through a beta-glycosylamide linkage. The procedure was utilized to isolate multi-micromole quantities of oligosaccharides from gram quantities of glycoprotein, thus providing a new route to purify large quantities of N-linked oligosaccharides which contain a terminal tyrosine residue. The tyrosinated oligosaccharides are valuable glycoconjugate ligands which contain a chromophore that absorbs at 280 nm and has sufficient hydrophobicity to facilitate RP-HPLC separations. Furthermore, this group can be deprotected by removal of Boc to reveal a primary amine suitable for further derivatization and can also be radioiodinated for tracking during biological experiments.

Amidohydrolases↗
PubMed1994

Reversal of tyrosinamide-oligosaccharide derivatization by Edman degradation.

In a previous report (Tamura, T., Wadhwa, M.S., and Rice, K.G. (1994) Anal. Biochem. 216, 335-344) we described the derivatization of N-linked oligosaccharides with Boc-tyrosine resulting in the formation of tyrosinamide-oligosaccharides. Attachment of Boc-tyrosine to the reducing-end of an oligosaccharide through a glycosylamide linkage provides a hydrophobic chromophore that facilitates the purification of individual N-linked oligosaccharides derived from glycoproteins on preparative reverse-phase HPLC. In the present report we extend the utility of tyrosinamide-oligosaccharides by demonstrating two additional attributes of these glycoconjugates. Edman degradation was used to reverse both sialyl and asialyl tyrosinamide-oligosaccharide derivatives resulting in the formation of reducing oligosaccharides. These can be used as analytical standards for chromatography or can be further derivatized with other probes that react with the reducing-end of an oligosaccharide. Second, we report that the fluorescence of tyrosinamide-oligosaccharides allows their sensitive detection (5 pmol) on HPLC. These two attributes expand the versatility of tyrosinamide-oligosaccharides as glycoconjugates suitable for both analytical and biological studies.

Carbohydrate Sequence↗
PubMed1996

Oligosaccharide release from frozen and paraffin-wax-embedded archival tissues.

Altered glycosylation is a feature of many solid tissue diseases such as ulcerative colitis, Crohn's disease, cancer, and connective tissue disorders. Conventionally, oligosaccharide changes have been studied by immunohistochemical techniques. We have adapted existing techniques, developed for purified protein preparations, to allow the release of intact oligosaccharides from archival tissues, so that the oligosaccharides may be structurally characterized. In our study, sections cut from paraffin-wax blocks were dewaxed and oligosaccharides were released using hydrazine and labeled with 2-aminobenzamide. Sialylated oligosaccharides were compared by passing through a GlycoSep C divinylbenzene anion exchange resin column and neutral oligosaccharides were compared by passing through a BioGel P4 column. The oligosaccharide profiles obtained from the same fresh frozen versus archival paraffin-wax-embedded, normal liver, and tumor tissues showed remarkable similarity in terms of their sialylated and neutral structures. Matrix-assisted laser desorption ionization mass spectrometry has shown that the oligosaccharides are not affected by fixation in formalin and storage in paraffin wax. The results indicate that while the proteins themselves may be denatured, oligosaccharides are not adversely affected by fixation in formalin and storage in paraffin wax. By applying these methods, oligosaccharides from archival tissues, where the natural history of the disease has been followed, may now be liberated and structurally characterized.

Animals↗