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PubMed2002

Analysis of O-linked reducing oligosaccharides released by an in-line flow system.

Reducing O-linked oligosaccharides from bovine submaxillary mucin, bovine fetuin, and porcine gastric mucin were recovered by nonreductive alkaline beta-elimination from an in-line flow system. Glycoproteins where attached to a solid support using hydrophobic interaction with alkali-resistant Poros reversed phase beads and a flow of alkali released the oligosaccharides. The alkali was subsequently neutralized by a continuous flow through cation exchange resin. The released oligosaccharides in the flow were trapped in a cartridge filled with graphitized carbon. Salt-free oligosaccharides could be recovered as a concentrated solution by elution with organic solvents from the cartridge. The glycosylation pattern of the released oligosaccharides was compared with the conventionally released and reduced oligosaccharides recovered from alkaline beta-elimination in the presence of borohydride. In general, the recovery from the in-line release was sometimes lower than from the reductive elimination method, but it was shown that alkaline degradation of reducing oligosaccharides was limited in this system. Liquid chromatography using graphitized carbon packing and high pH mobile phases together with negative ion electrospray mass spectrometry showed that both neutral and acidic reducing oligosaccharides could be analyzed in a single run. Reducing O-linked oligosaccharides could also be recovered in this way from human glycophorin separated by SDS-PAGE. The polyacrylamide was sufficient to retain the glycoprotein in the gel while the flow of alkali released the oligosaccharides. It was also shown that the alkaline conditions for releasing O-linked oligosaccharides from fetuin would partially release some N-linked oligosaccharides, particularly in the presence of reducing agent.

Animals↗
PubMed2001

Survival of human milk oligosaccharides in the intestine of infants.

Several human milk oligosaccharides inhibit human pathogens in vitro and in animal models. In an infant, the ability of these oligosaccharides to offer protection from enteric pathogens would require that they withstand structural modification as they pass through the alimentary canal or are absorbed and excreted in urine. We investigated the fate of human milk oligosaccharides during transit through the alimentary canal by determining the degree to which breast-fed infants' urine and fecal oligosaccharides resembled those of their mothers' milk. Oligosaccharide profiles of milk from 16 breast-feeding mothers were compared with profiles of stool and urine from their infants. Results were compared with endogenous oligosaccharide profiles obtained from the urine and feces of age-, parity-, and gender-matched formula-fed infants. In all cases, oligosaccharides were extracted, purified, reduced, and separated into acidic and neutral species; the latter were perbenzoylated and subjected to reversed-phase high-performance liquid chromatography. Structures were determined by mass spectrometry after debenzoylation. Among breast-fed infants, concentrations of oligosaccharides were higher in feces than in mothers' milk, and much higher in feces than in urine. Urinary and fecal oligosaccharides from breast-fed infants resembled those in their mothers' milk. Those from formula-fed infants did not resemble human milk oligosaccharides, were found at much lower concentrations, and probably resulted from remodeling of intestinal glycoconjugates or from intestinal bacteria. Most of the human milk oligosaccharides survived transit through the gut, and some were absorbed and then excreted into the urine intact, implying that inhibition of intestinal and urinary pathogens by human milk oligosaccharides is quite likely in breast-fed infants.

Carbohydrate Conformation↗
PubMed1988

Analyses of N-linked oligosaccharides using a two-dimensional mapping technique.

We propose a two-dimensional sugar map method for the simple, reproducible, and sensitive analysis of the structures of N-linked oligosaccharides. The structure of an unknown oligosaccharide can be characterized from its position on the map. The data base for the sugar map is prepared by the use of 113 kinds of standard oligosaccharides, 58 of whose structures have been confirmed by 1H NMR spectroscopy. The present method involves six steps, (i) preparation of oligosaccharides from glycopeptides by N-oligosaccharide glycopeptidase (almond) digestion, (ii) derivatization of the reducing ends of oligosaccharides with a fluorescent reagent, 2-amino-pyridine, by using sodium cyanoborohydride, (iii) separation of oligosaccharide derivatives by high-performance liquid chromatography with an ODS-silica column, (iv) analysis of the size of each separated oligosaccharide on an amide-silica column, (v) plotting of the elution position of a sample on the two-dimensional sugar map obtained for the standard oligosaccharides, and (vi) structural analysis of the oligosaccharides by a combination of sequential exoglycosidase digestion and the steps (iii-v). The present method was applied to the identification of the structures of oligosaccharides in hen ovalbumin. It was found that two unusual oligosaccharides that have not yet been reported exist in ovalbumin.

Animals↗
PubMed1989

Incompletely processed LH molecules synthesized by rat gonadotrophs treated with inhibitors of oligosaccharide processing.

Inhibitors of N-linked oligosaccharide processing are useful tools for studies on the biological function of the oligosaccharide structures in glycoprotein hormones. We have synthesized molecules of lutropin (LH) containing high-mannose- and hybrid-type oligosaccharides using rat gonadotroph-enriched primary cultures in the presence of castanospermine (a glucosidase I inhibitor) or swainsonine (a mannosidase II inhibitor), in order to compare the actions of these molecules with that of the hormone containing complex-type oligosaccharides in the activation of the receptor-adenylate cyclase system. Treatment of gonadotrophs with the above inhibitors caused an increase in the synthesis of highly basic LH molecules (pI 9.6-10.0), because addition of charged carbohydrate moieties to these molecules was prevented. Characterization of the oligosaccharide structure performed by enzymatic treatment (endoglycosidase H and neuraminidase) and the use of immobilized lectins (wheat germ agglutinin and Ricinus communis agglutinin-120) showed that these inhibitor-synthesized LH molecules contained high-mannose- and hybrid-type (asialo and sialylated) oligosaccharides. Their immunological properties were similar to that of complex-type oligosaccharide LH, but they had significantly higher receptor-binding ability in comparison with a sialylated complex-type oligosaccharide LH (about 12-fold) and an asialo complex-type oligosaccharide LH (about 3-fold). It was noted that the incompletely processed molecules were less potent than complex-type oligosaccharide LH in the activation of adenylate cyclase of Leydig cells, showing about 40-60% of the activity induced by the sialylated complex-type oligosaccharide molecule. The present data indicate that the inhibition of terminal processing of N-linked oligosaccharides by castanospermine and swainsonine impairs the full hormonal function of rat LH.

Alkaloids↗
PubMed1987

Comparative structural study of the N-linked oligosaccharides of human normal and pathological immunoglobulin G.

The structures of oligosaccharides of normal and pathological immunoglobulin G (IgG) are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by reverse-phase high-performance liquid chromatography. It was possible to separate 15 out of the 16 kinds of oligosaccharides that have been suggested to exist in normal human IgG. High-resolution proton nuclear magnetic resonance spectroscopy was used along with chemical methods to determine the structures of the separated oligosaccharides. It has been shown that in normal IgG a biantennary complex-type oligosaccharide with a fucose residue (formula; see text) is predominant and four kinds of oligosaccharides, which are biantennary with bisecting N-acetylglucosamine and without fucose residues, exist only in a very small quantity. The results obtained for normal IgG were compared with those obtained for three myeloma IgG proteins. It has been found that the most abundant species that exist in the pathological proteins analyzed in the present work lack one or two galactose residues at the nonreducing terminal. We show that the fractions of fucose-containing oligosaccharides are markedly decreased in the heavy-chain disease protein Per. It is of particular interest that in this paraprotein the major component is a biantennary complex-type oligosaccharide that lacks a fucose residue and an oligosaccharide with the structure (Formula: see text) exists as one of the most abundant components.

Asparagine↗
PubMed1992

Different fates of the oligosaccharide moieties of lipid intermediates.

We have previously described that the N-glycosylation process was accompanied by the release of oligosaccharide-phosphates and neutral oligosaccharides. The relationship between oligosaccharide-P-P-dolichol and its metabolic products (glycoproteins, oligosaccharide-phosphates and neutral oligosaccharides) was investigated by analysing the structure of the oligosaccharide moieties and the kinetic behaviour of the various species in pulse and pulse/chase experiments. For these studies, a glycosylation mutant of Chinese hamster ovary cells (B3F7) which does not synthesize mannosylphosphoryldolichol was utilized. Evidence was obtained for the presence of two pools of oligosaccharide-P-P-dolichol which have different fates. One pool is not glucosylated, is rapidly labelled and immediately chased by mannose, and generates the oligosaccharide-phosphate species. The second pool is glucosylated, exhibits a lag time (5-10 min) prior to being labelled, and is utilized in the glycosylation of proteins and in the production of neutral oligosaccharides. We postulate that the cleavage of non-glycosylated lipid intermediates generating oligosaccharide-phosphates represents a 'bypass' in the dolichol cycle which allows direct regeneration of dolichyl phosphate. The other metabolic fate of non-glucosylated oligosaccharide-lipids, glucosylation, results in their use as effective substrates for the glycosylation of proteins or in the generation of neutral oligosaccharides.

Animals↗
PubMed2005

Prebiotic effect of fructo-oligosaccharide supplemented term infant formula at two concentrations compared with unsupplemented formula and human milk.

BACKGROUND: Human milk components, including oligosaccharides, affect the gastrointestinal flora of infants. Previous studies in adults have demonstrated that fructo-oligosaccharides increase potentially beneficial fecal bacteria, including bifidobacteria. The purpose of this study was to determine the prebiotic effect of infant formula supplemented with fructo-oligosaccharides. METHODS: Healthy term infants 2 to 6 weeks of age were enrolled in a 5-week, prospective, randomized, crossover, single-site study with a nonrandomized human milk comparator group. Washout weeks preceded and followed a week of feeding with fructo-oligosaccharide-supplemented formula (1.5 or 3.0 g/L). Stool specimens were quantitatively cultured weekly for bacteroides, lactobacilli, bifidobacteria, clostridia and enterococci and were tested for Clostridium difficile toxin. RESULTS: Seventy-two of 87 infants completed the trial; 58 were formula fed and 14 were human milk fed. Mean counts of bifidobacteria and lactobacilli were similar in all groups at entry and no group experienced a significant change in counts with fructo-oligosaccharide supplementation. After 7 days of fructo-oligosaccharide supplementation the bifidobacteria counts were greater in the 1.5 g/L fructo-oligosaccharide formula group than in the human milk fed or 3.0 g/L fructo-oligosaccharide formula groups. Formula-fed infants had higher counts of enterococci and bacteroides before fructo-oligosaccharide supplementation, and these counts did not change after supplementation. Clostridium counts increased 7 days after supplementation in the 1.5 g/L fructo-oligosaccharide formula group (P = 0.0356). No human milk fed infants had C. difficile toxin in stools. Fructo-oligosaccharide (3.0 g/L) supplementation resulted in more frequent and significantly softer stools. CONCLUSIONS: Infant formula supplemented with 1.5 or 3.0 g/L fructo-oligosaccharides was safe but had minimal effect on fecal flora and C. difficile toxin.

Bifidobacterium↗
PubMed1980

Fate of oligosaccharide-lipid intermediates synthesized by resting rat-spleen lymphocytes.

Using conditions to avoid the utilization of labelled precursors by intracellular glycosyltransferases, experiments are described demonstrating that intact rat-spleen lymphocytes are capable of utilizing exogenous GDP-mannose and UDP-N-acetylglucosamine to synthesize dolichyl monophosphate mannose and dolichyl diphosphate oligosaccharides. Kinetic and chase experiments show that dolichyl diphosphate oligosaccharides are either utilized for the transfer of their carbohydrate moieties to protein acceptors or further degraded. Since glycosylation of proteins is limited in resting lymphocytes, the degradation pathway appears as a major event in the fate of the dolichyl diphosphate oligosaccharides synthesized in vitro. These dolichyl diphosphate oligosaccharides are degraded into phospho-oligosaccharides and oligosaccharides which are released in the medium. This enzymatic cleavage of the phosphodiester bond is inhibited by bacitracin. The phospho-oligosaccharides are susceptible to alkaline phosphatase giving neutral oligosaccharides and they are cleaved by endo-N-acetyl-beta-D-glucosaminidase H leaving N-acetylglucosamine 1-phosphate and neutral oligosaccharides. These data suggest that splitting of the phosphodiester bond of colichyl diphosphate oligosaccharides, dephosphorylation and/or endo-N-acetyl-beta-D-glucosaminidase hydrolysis of the phosphorylated oligosaccharides could represent the beginning of the catabolic pathway of dolichyl diphosphate oligosaccharides.

Acetylglucosaminidase↗
PubMed1989

Structures of O-glycosidically linked oligosaccharides isolated from human meconium glycoproteins.

The structure of the major O-glycosidically linked neutral and acidic oligosaccharides isolated from human meconium glycoproteins were established. Neutral and acidic oligosaccharides were released by alkaline borohydride treatment, purified by Biogel P-6 and fractionated by high-performance liquid chromatography. This approach resulted in 50 neutral and 30 acidic oligosaccharides. The present study reports the primary structural analysis of five neutral oligosaccharides, ten monosialylated oligosaccharides, one monosialylated monosulfated oligosaccharide and three disialylated oligosaccharides, by permethylation, fast-atom-bombardment mass spectrometry analysis and 400-MHz 1H-NMR spectroscopy. The following structure have not been described previously: (formula; see text) The intestinal glycoproteins of human meconium are characterized as high molecular mass compounds with numerous carbohydrate chains of the mucin type. These mucins are a rich source of carbohydrate structures which express multiple blood group activities and occur as membrane-associated antigens, recognized by hybridoma antibodies [1-4]. A previous study [5] described the structure of fifteen free oligosaccharides derived from catabolism of O- and N-glycans accumulating in new born meconium. In the same group [6] the research has been extended to glycoasparagines with the description of thirteen of them by high resolution 1H-NMR spectroscopy analysis. From O-glycosidically linked glycoproteins, further studies [7] established the structures of nine major monosaccharides to tetrasaccharides obtained after mild acid hydrolysis and base-borohydride degradation from meconium samples of group O secretors. These oligosaccharides were derived from meconium glycopeptides which had been depleted of I- and i-antigen activities. Recently [8], neutral and acidic oligosaccharide-alditols obtained by alkaline borohydride degradation of human meconium glycoproteins have been separated by HPLC on an anion-exchange column. The neutral fraction was further purified by normal-phase and reversed-phase chromatography while acidic fractions were fractionated only by normal-phase chromatography. Thus, we have isolated several low molecular mass oligosaccharides of different size and composition and also isomers which vary in linkage position or anomer configuration. Using methylation analysis, 1H-NMR spectroscopy and fast-atom-bombardment mass spectrometry (FAB-MS), we propose the primary structure for 19 major oligosaccharides.

Binding Sites↗
PubMed2001

Structural characterization of fucose-containing oligosaccharides by high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Eight pyridylamino (PA) derivatives of fucose-containing oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by high-performance liquid chromatography (HPLC) on normal-phase and reversed-phase columns, and by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Six out of eight PA-oligosaccharides were clearly separated by both normal- and reversed-phase HPLC at a column temperature of 40 degrees C, but two PA-oligosaccharides, lacto-N-fucopentaose II [Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-3Gal beta1-4GIcPA] and lacto-N-fucopentaose III [Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-3Gal beta1-4GIcPA], were not separated. The two unresolved PA-oligosaccharides were finally separated by reversed-phase HPLC at a column temperature of 11 degrees C. MALDI-TOF mass spectra of PA-oligosaccharides demonstrated pseudo-molecular ions as the predominant signals, therefore information about the molecular mass of each PA-oligosaccharide was easily obtained. Post-source decay (PSD) MALDI-TOF mass spectra of PA-oligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PA-oligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PA-oligosaccharides by HPLC, MALDI-TOF mass spectrometry, and PSD MALDI-TOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDI-TOF mass spectrometry or HPLC and PSD MALDI-TOF mass spectrometry is quite useful for the structural characterization of sub-pmol or pmol levels of fucose-containing oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.

Carbohydrate Sequence↗
PubMed1990

Human transferrin receptor contains O-linked oligosaccharides.

We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.

Acetylgalactosamine↗
PubMed1989

Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction.

The role of the human chorionic gonadotropin (hCG) N-linked oligosaccharides in receptor binding and signal transduction was analyzed using site-directed mutagenesis and transfection studies. hCG derivatives with alterations at individual glycosylation sites were expressed in Chinese hamster ovary cells. Receptor binding studies showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on the receptor affinity of the derivatives. Similarly, absence of the N-linked oligosaccharides from the beta subunit or a single oligosaccharide from Asn-78 of alpha had no effect on the production of cAMP or on steroidogenesis. However, the absence of carbohydrate at Asn-52 of alpha decreases both the steroidogenic and cAMP responses. Furthermore, absence of this critical oligosaccharide unit on alpha unmasks differences in the two N-linked oligosaccharides on beta; the beta Asn-13 oligosaccharide but not the beta Asn-30 oligosaccharide plays a more important role in steroidogenesis. Dimers containing deglycosylated beta subunit and an alpha subunit lacking either the Asn-52 oligosaccharide or both oligosaccharides fail to stimulate cAMP or steroid formation. Moreover, these derivatives bind to receptor and behave as competitive antagonists. The use of site-directed mutagenesis was critical in uncovering site-specific functions of the hCG N-linked oligosaccharides in signal transduction and reveals the importance of the Asn-52 oligosaccharide in this process.

Adenylyl Cyclases↗
PubMed1983

Host-dependent variation of asparagine-linked oligosaccharides at individual glycosylation sites of Sindbis virus glycoproteins.

We examined the Asn-linked oligosaccharides at individual glycosylation sites of the two envelope glycoproteins of Sindbis virus, E1 and E2. The analysis was done by separating tryptic glycopeptides by reverse phase high performance liquid chromatography and analyzing the oligosaccharides from isolated glycopeptides by gel filtration chromatography. Both E1 and E2 have two glycosylation sites each in virus grown in chick embryo fibroblasts, baby hamster kidney cells, and Chinese hamster ovary cells. Our results indicate that previously observed host-dependent variation in the oligosaccharides of Sindbis virus glycoproteins are probably attributable to differences in oligosaccharide composition at individual sites of E1 and E2 and not to differences in the frequency of glycosylation of these sites. One of the two glycosylation sites in E1 has exclusively complex type oligosaccharides regardless of the host cell type. However, the second glycosylation site in E1 has high mannose oligosaccharides in virus grown in chick embryo fibroblasts, complex oligosaccharides in virus from baby hamster kidney cells, and both complex and high mannose oligosaccharides in virus from Chinese hamster ovary cells. E2 has one glycosylation site which has primarily high mannose oligosaccharides and one site which has primarily complex type oligosaccharides. Certain sites which have predominantly complex type glycans also have a small number of two specific classes of oligosaccharides which are cleaved by endo-beta-N-acetylglucosaminidase H and may be high mannose type. Our findings are discussed with regard to regulation of oligosaccharide processing in animal cells.

Animals↗
PubMed1977

Biosynthesis of lipid-linked oligosaccharides in cotton fibers. Stimulation by lipids from pig liver.

An acidic lipid fraction isolated from pig liver (Forsee, W. T. & Elbein, A.D. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2574-2578) stimulated the incorporation of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides using a particulate enzyme fraction from maturing cotton bolls. This lipid fraction did not stimulate mannose incorporation into the mannosylphosphorylpolyprenol. The 14C-oligosaccharides, formed in the presence or absence of the pig liver "acceptor" lipid, were released from the lipid-linked oligosaccharides by mild acid hydrolysis and were isolated by paper chromatography. Both sets of 14C-oligosaccharides had similar mobilities suggesting that they were qualitatively similar, except that those formed in the presence of "acceptor" lipid had much more radioactivity. The individual oligosaccharides were purified on a calibrated column of Sephadex G-25 and were then subjected to various treatments to obtain information about their structures. The molecular weights of the larger oligosaccharides ranged from about L1210 to 1720 indicating they probably contained from 6 to 10 sugar residues. Strong acid hydrolysis of the oligosaccharide with a molecular weight of 1350, followed by reduction with NaB3H4, gave a ratio of [3H]hexitol to [3H]hexosaminitol that was compatible with the molecular weight (about 5:2). However, the hexitol fraction contained small amounts of [3H]glucitol in addition to [3H]mannitol. Thus, this oligosaccharide may contain small amounts of glucose in addition to mannose and GlcNAc. When the smaller oligosaccharides (i.e. those having 5 or 6 sugars) were treated with alpha-mannosidase, essentially of all the 14C was released as mannose, but only 30 to 50% of the radioactivity could be released from the larger oligosaccharides by this enzyme. Acetolysis of the oligosaccharide with a molecular weight of 1720 released the radioactivity as mannobiose and mannotriose suggesting that these oligosaccharides contain di- and trisaccharide branches linked to the main chain in 1 leads to 6 linkages.

Animals↗
PubMed2006

The role of O-linked and N-linked oligosaccharides on the structure-function of glycoprotein hormones: development of agonists and antagonists.

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common alpha and a hormone specific beta subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the alpha and beta subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (alpha, FSHbeta and hCGbeta) asparagine-linked (N-linked) oligosaccharides. CGbeta subunit is distinguished among the beta subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure-function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.

Drug Design↗
PubMed2005

Separation, identification, and interaction of heparin oligosaccharides with granulocyte-colony stimulating factor using capillary electrophoresis and mass spectrometry.

A capillary electrophoresis (CE) method was developed for the separation of heparin oligosaccharides compatible to study the interactions between the oligosaccharides and granulocyte-colony stimulating factor (G-CSF). Unfractionated heparin was eliminitively degraded to heparin oligosaccharides by an endolytic heparinase. The degraded smaller oligosaccharides (M(r) < 1000) were baseline-separated by CE under a 50 mM phosphate buffer (pH 9.0) in 10 min. Standard heparin disaccharides and larger oligosaccharides (1000 < M(r) < 8000) were all separated under optimized separation conditions. Compared with standard heparin disaccharides, smaller oligosaccharides contained one nonsulfated, two monosulfated, and two disulfated disaccharides, but trisulfated disaccharides were not found. The smaller oligosaccharides were also identified and molecular mass was deduced by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, interactions between G-CSF and the oligosaccharides were studied by using capillary zone electrophoresis (CZE) under the above separation conditions. It was found that larger oligosaccharides could interact with G-CSF while smaller oligosaccharides were not observed to bind to G-CSF under the experimental conditions. In conclusion, the purified heparinase could selectively degrade heparin into oligosaccharides and the interaction between G-CSF and heparin was correlated with the chain length of heparin.

Chromatography, Gel↗
PubMed2005

Electrospray tandem mass spectrometry of underivatised acetylated xylo-oligosaccharides.

Acetylated neutral (Xyl(n)Ac(m)) and acidic xylo-oligosaccharides (Xyl(n)Ac(m)MeGlcA, and Xyl(n)Ac(m)MeGlcAHex) obtained by partial acid hydrolysis of Eucalyptus globulus wood glucuronoxylans and fractionated by preparative ligand exchange/size-exclusion chromatography were identified by electrospray ionisation mass spectrometry (ESI-MS). Low molecular weight acetylated xylo-oligosaccharides were studied by ESI-tandem mass spectrometry (MS/MS). All the acetylated xylo-oligosaccharides showed an abundant ion due to the neutral loss of 60 Da (CH(3)CO(2)H) in the MS/MS spectra. The presence of diacetylated xylo-oligosaccharides was confirmed by the ions formed by loss of two molecules of acetic acid. Furthermore, characteristic [Xyl(res)Ac(2)+Na](+) and [XylAc(2)+Na](+) ions, and ions due to loss of XylAc(2), indicate that both acetyl groups are located in the same Xyl residue. On the other hand, losses of Xyl(res)Ac and XylAc are also observed as well as [Xyl(res)Ac+Na](+) and [XylAc+Na](+) , indicating the location of both acetyl groups in different Xyl residues, in some cases even in adjacent xyloses. The MS/MS spectra of triacetylated xylo-oligosaccharides were complex due to the presence of different isobaric xylo-oligosaccharides containing the acetyl groups at different locations in the xylo-oligosaccharide backbone. In the MS/MS spectra of acidic xylo-oligosaccharides, the ion at m/z 387, [Xyl(res)AcMeGlcA+Na](+), indicates that the acetyl groups are preferentially linked to Xyl substituted with MeGlcA. However, acidic xylo-oligosaccharides with the acetyl and 4-O-methylglucuronic acid groups in different Xyl residues were also identified. In neutral and in acidic xylo-oligosaccharides several possible locations of the acetyl groups were identified, namely at terminal positions. In summary, ESI-MS/MS is shown to be a powerful tool for the characterisation of acetylated patterns in complex mixtures of oligosaccharides.

Acetylation↗
PubMed1997

Milk oligosaccharide profiles by reversed-phase HPLC of their perbenzoylated derivatives.

Human milk is rich in oligosaccharides, some of which inhibit toxins and pathogens involved in diseases of infants. To investigate qualitative and quantitative individual variation of human milk oligosaccharides, a sensitive method for routine identification and quantification of intact milk oligosaccharides was developed and applied to milk samples from 50 donors. The isolated, reduced neutral oligosaccharide fractions were perbenzoylated, resolved by reversed-phase HPLC, and detected at 229 nm. This method resolves most structural isomers and does not require stringent removal of lactose. Peaks were detected at the low nanogram (pmol) level and peak areas were linear from 1 to 1000 micrograms for a standard oligosaccharide. Oligosaccharide samples equivalent to 1 microliter of human milk give optimum chromatographic separation and resolution. The method gives quantitative results comparable to those obtained with classic total sugar analyses, and has an average coefficient of variation of 13%. The 12 major peaks in human milk coeluted with authentic oligosaccharide standards ranging from tri- to octasaccharides, and their identities were confirmed by mass spectrometry. Significant individual variation exists in oligosaccharide profiles; almost 70% of samples contained 2'-fucosyllactose and lacto-N-fucopentaose I as the major oligosaccharides; for the remainder, the major oligosaccharides were 3-fucosytlactose and lacto-N-fucopentaose-II or lacto-N-fucopentaose-III. This method can be used to investigate the extent and biological significance of oligosaccharide variation in human milk.

Benzoates↗