Structure of carbohydrate chains of the riboflavin-binding glycoprotein of chicken egg protein. II. 1H-NMR (500 MHz) spectroscopy of the major neutral oligosaccharides

Reductive cleavage of riboflavin-binding glycoprotein from hen egg white (RF-GPw) with LiBH4/tert-BuOH followed by NaBH4/NaOH treatment gave rise to oligosaccharide alditols, fractionated by a successive HPLC on muBondapak C18 and Zorbax NH2 columns. Seven main individual oligosaccharide alditols were isolated and their structure was investigated by 1H NMR 500-MHz spectroscopy. The structure and relative content of the main oligosaccharide chains were proved to be identical in RF-GPw and ovomucoid. Structure of polypeptide chains and their molecular weight, number of glycosylation sites and their structure had little or no effect on the glycosylation pattern in both glycoproteins. HPLC of the oligosaccharide alditols from another egg white glycoprotein, ovotransferrin, also revealed its high microheterogeneity and close resemblance to those of ovomucoid and RF-GPw.     

Isolation and characteristics of carbohydrate chains of riboflavin-binding glycoprotein from the chicken egg

Isolation and characterization of oligosaccharides of riboflavin binding glycoprotein from hen white is described. Reductive cleavage of the N-glycosylamide carbohydrate-peptide bond with LiBH4/tert-BuOH followed by NaBH4-NaOH treatment gave rise to alditols, which were fractionated by means of HPLC. Twelve alditols were isolated in quantities sufficient for the monosaccharide analysis. Possibility of an ovomucoid-type oligosaccharide structure for all the alditols is discussed.     

High-performance anion exchange-chromatography of neutral milk oligosaccharides and oligosaccharide alditols derived from mucin glycoproteins.

High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH approximately 13) has been used to separate neutral oligosaccharides from human milk as well as oligosaccharide alditols isolated by alkaline borohydride degradation of O-linked glycoproteins having blood group A and H activities. Due to the diminished retention times of the alditols compared to their reducing counterparts, a very low base concentration (approximately 15 mM) was used in the fractionation of oligosaccharide alditols. The method appears to be ineffective in fractionation of monosaccharide alditols. Although the retention times generally increased with increasing oligosaccharide chain length, linkage of Fuc alpha-(1----2) to galactose and by Fuc alpha-(1----3) or Fuc alpha-(1----4) to glcNAc may decrease the retention times of both the alditols and the reducing oligosaccharides. Branching generally increased the retention times for oligosaccharide alditols. The retention times of isomers differing in the position of fucose substitution (LNF-1 vs LNF-2) differed greatly while those of the linkage isomers LNF-2 and LNF-3 were similar but distinct. Pulsed amperometric detection is sensitive at the picomole level both for these underivatized oligosaccharides and alditols. On-line desalting with an ion-exchange membrane has been found to be effective in preparative chromatography of these oligosaccharides for NMR spectroscopy and mass spectrometry.     

Positional isomers of sulfated oligosaccharides obtained from agarans and carrageenans: preparation and capillary electrophoresis separation

Partial reductive hydrolysis was used to produce oligosaccharide alditols from repetitive sulfated galactans obtained from four Rhodophyta species: kappa-carrageenan (from Kappaphycus alvarezii), theta-carrageenan (Gigartina skottsbergii-alkali-treated lambda-carrageenan), agarose 6-sulfate (Gracilaria domingensis), and pyruvylated agarose 2-sulfate (Acanthophora spicifera-alkali-treated pyruvylated agaran sulfate). Each hydrolyzate was submitted to anion-exchange and gel-filtration chromatography, and the isolated oligosaccharide alditols were identified by 1D and 2D NMR spectroscopy and by ESI mass spectrometry. The positional isomers of the sulfated oligosaccharide alditols were then completely resolved by capillary electrophoresis in a borate buffer. Attempts to correlate the availability of the hydroxyl groups for borate complexation with the relative migration of the oligosaccharides are presented.     

Structure of the xyloglucan produced by suspension-cultured tomato cells

The xyloglucan secreted by suspension-cultured tomato (Lycopersicon esculentum) cells was structurally characterized by analysis of the oligosaccharides generated by treating the polysaccharide with a xyloglucan-specific endoglucanase (XEG). These oligosaccharide subunits were chemically reduced to form the corresponding oligoglycosyl alditols, which were isolated by high-performance liquid chromatography (HPLC). Thirteen of the oligoglycosyl alditols were structurally characterized by a combination of matrix-assisted laser-desorption ionization mass spectrometry and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Nine of the oligoglycosyl alditols (GXGGol, XXGGol, GSGGol, XSGGol, LXGGol, XTGGol, LSGGol, LLGGol, and LTGGol, [see, Fry, S.C.; York, W.S., et al., Physiologia Plantarum 1993, 89, 1-3, for this nomenclature]) are derived from oligosaccharide subunits that have a cellotetraose backbone. Very small amounts of oligoglycosyl alditols (XGGol, XGGXXGGol, XXGGXGGol, and XGGXSGGol) derived from oligosaccharide subunits that have a cellotriose or celloheptaose backbone were also purified and characterized. The results demonstrate that the xyloglucan secreted by suspension-cultured tomato cells is very complex and is composed predominantly of 'XXGG-type' subunits with a cellotetraose backbone. The rigorous characterization of the oligoglycosyl alditols and assignment of their 1H and 13C NMR spectra constitute a robust data set that can be used as the basis for rapid and accurate structural profiling of xyloglucans produced by Solanaceous plant species and the characterization of enzymes involved in the synthesis, modification, and breakdown of these polysaccharides.     

Characterisation of oligosaccharides released from human-blood-group O erythrocyte glycopeptides by the endo-beta-galactosidase of Bacteroides fragilis. A study of the enzyme susceptibility of branched poly(N-acetyllactosamine) structures

Desialylated human blood group O erythrocyte glycopeptides were digested with the endo-beta-galactosidase of Bacteroides fragilis and the enzyme-released products reduced with NaBH4 and purified by Bio-Gel P-4 chromatography. Three linear and six branched oligosaccharides of poly(N-acetylllactosamine) type, which together accounted for 90% of the oligosaccharide alditols, were characterised by fast-atom-bombardment mass spectrometry and gas-liquid chromatography/mass spectrometry. Linkage and composition data were obtained for the remaining material. The salient findings were (a) the branched oligosaccharide alditols each contained the sequence: (Formula: see text) and (b) there was no evidence for the terminal branch-point sequence: (Formula: see text). Together these observations indicate that, as with erythrocyte glycolipids described previously [Scudder, P., Hanfland, P., Uemura, K. & Feizi, T. (1984) J. Biol. Chem. 259, 6586-6592], the endo-beta-galactosidase of Bacteroides fragilis cannot hydrolyse branch-point beta-galactosidic linkages on erythrocyte membrane glycopeptides.     

The structure of carbohydrate chains of riboflavin-binding glycoprotein from chicken egg protein. III. 1H-NMR spectroscopy (500 MHz) of neutral fucosylated oligosaccharides

Using LiBH4/ButOH treatment, oligosaccharides were cleaved off the hen egg white riboflavin-binding glycoprotein. HPLC led to the isolation of four fucose-containing oligosaccharide alditols, whose structure was elucidated by means of 1H NMR 500 MHz spectroscopy. The main fucosylated oligosaccharide, also present in hen ovomucoid, was found to be a biantennary carbohydrate chain of N-acetyllactosamine type.     

Separation of neutral oligosaccharide alditols from human meconium using high-pH anion-exchange chromatography

Neutral reduced oligosaccharides are in general not sufficiently retained to achieve adequate separation and reproducible chromatography using high-pH anion-exchange chromatography. We describe a method to increase the retention using two columns in series. This method has been applied to the separation of oligosaccharides purified from human meconium glycoproteins, obtained as their alditols after alkaline-borohydride release of oligosaccharides. The neutral oligosaccharide alditols were significantly retained upon two CarboPac PA-100 columns, connected in series, and eluted in 80 mM sodium hydroxide between 4 and 10 min. Three sialylated alditols studied were substantially retained and could be eluted in a gradient of 0–500 mM sodium acetate—80 mM sodium hydroxide between 10 and 45 min. The elution patterns were based on their size, charge and linkage, such that oligosaccharide alditol isomers could be separated.     

Selective Hydrolysis of the 3,6-Anhydrogalacotosidic Linkage in Red Algal Galactan: A Combination of Reductive Acid Hydrolysis and Anhydrous Mercaptolysis

Here we report a simple method for the structural analysis of red algal galactan containing 3,6-anhydrogalactose. Structural heterogeneity in the galactan was demonstrated by this method. For selective hydrolysis of 3,6-anhydrogalactosidic linkages in the galactan, conditions for reductive mild acid hydrolysis were examined by characterizing the resulting oligosaccharide alditols by anhydrous mercaptolysis. Residues other than alditols at the reducing ends, including labile 3,6-anhydrogalactose, were liberated quantitatively as diethyl dithioacetal derivatives, whereas alditols at the reducing ends were not derivatized and were liberated as alditols intact. The liberated sugars were then separated and measured quantitatively by gas-liquid chromatography. Heating of agarose in reductive hydrolysis with 0.3 M trifluoroacetic acid in the presence of an acid-stable reducing agent, 4-methyl morpholine borane, at 80 °C for 90 min and for 90 °C for 45 min was found to be optimum for the selective hydrolysis of 3,6-anhydrogalactosidic bonds, without detectable cleavage of other glycosidic bonds.     

Characterization of N-linked gluco-oligomannose type of carbohydrate chains of glycoproteins from the ovary of the starfish Asterias rubens (L.)

Glycoproteins were isolated from the ovary of the starfish Asterias rubens (L.). After delipidation, sugar analysis revealed the presence of mannose, glucose and N-acetylglucosamine in a molar ratio of 9.0:1.3:2.3. Subsequently, hydrazinolysis, re-N-acetylation, reduction and high-voltage paper electrophoresis were carried out, resulting in a mixture of neutral oligosaccharide alditols which was fractionated on Bio-Gel P-4. The alditols, investigated by 500-MHz 1H-NMR spectroscopy, turned out to be of the oligomannose type or of the gluco-oligomannose type containing 9 mannose and 1-3 glucose residues. The most abundant compounds were established to be: (Formula: see text) and (Formula: see text).     

Fish egg polysialoglycoproteins: structures of new sialooligosaccharide chains isolated from the eggs of Oncorhynchus keta (Walbaum). Fucose-containing units with oligosialyl groups

A series of acidic oligosaccharide alditols having different neutral core oligosaccharides were isolated from salmon egg polysialoglycoproteins by alkali-borohydride treatment followed by anion-exchange chromatography and Iatrobead chromatography. Their structures were determined by methylation analysis, molecular secondary ion mass spectrometry of underivatized oligosaccharides, and enzymatic desialylation. The molecular secondary ion mass spectra of intact sialooligosaccharides exhibit pronounced quasi-molecular-ion peaks, (M + H)+, (M + Na)+, (M + 2Na - H)+, and/or (M + K)+, as well as some diagnostic sequence ion peaks. Of a number of oligosaccharide alditols, the following are novel: Fuc alpha 1 leads to 3GalNAc beta l1 leads to 3Gal beta 1 leads to 4Gal beta 1 leads to 3[(leads to 8NeuGc alpha 2)n leads to 6]GalNAcol (n = 1-6). The proton nuclear magnetic resonance spectra of these oligosaccharides are also reported and discussed.     

Structural analysis of the N-linked oligosaccharides from murine glycophorin

Glycophorins, isolated from BALB/c mouse erythrocytes, were degraded under mild and strong reductive alkaline conditions and the N-linked oligosaccharides were isolated as alditols. The oligosaccharide alditols were fractionated and purified using gel filtration, concanavalin A-Sepharose affinity chromatography, and high-performance ion-exchange chromatography. Structural analysis was carried out by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. The results revealed the presence of sialylated biantennary, triantennary, and tetraantennary complex type oligosaccharides, all fucosylated at the innermost N-acetylglucosamine residue. The tri- and tetraantennary oligosaccharide-containing fractions also contained species elongated by one and/or two N-acetyllactosamine (-3Gal beta 1-4GlcNAc beta 1-) sequences. The N-linked oligosaccharides were shown to be combined only with one (the low molecular weight) of the two mouse glycophorins.     

Structure determination of the disialylated poly-(N-acetyllactosamine)-containingO-linked carbohydrate chains of equine chorionic gonadotropin

The disialylated poly-(N-acetyllactosamine)-containingO-linked oligosaccharide alditols, released by alkaline borohydride treatment of the enzymicallyN-deglycosylated β-subunit of equine chorionic chonadotropin, were purified by fast protein liquid chromatography (FPLC) on Mono Q and analysed by fast ion bombardment mass spectrometry (FAB-MS) and¹H-NMR spectroscopy. The identified oligosaccharide alditols have the following structure: $$\begin{gathered} Neu5Ac\alpha 2 - 3\left[ {Gal\beta 1 - 4GlcNAc\beta 1 - 3} \right]_{0 - 4} Gal\beta 1 - 4GlcNAc\beta 1 - 6 \hfill \\ \begin{array}{*{20}c} { \backslash } \\ { GalNAc - ol} \\ { /} \\ {Neu5Ac\alpha 2 - 3Gal\beta 1 - 3} \\ \end{array} \hfill \\ \end{gathered}$$      

Chromatography of permethylated oligosaccharide alditols

A new chromatographic method which enables the separation of permethylated oligosaccharide alditols has been developed. The method entails chromatography on precoated silica gel plates using benzene-methanol (16:1, v/v or 10:1 v/v) as developing solvent. Separations of disaccharides were obtained on the basis of glycosidic linkage and anomeric configuration; the method can accomodate oligosaccharides containing up to 15 glycose units. The combined use of thin-layer chromatography and gas-liquid chromatography provides a powerful approach for the characterization of oligosaccharides. Retention indices are given of permethylated oligosaccharide alditols on a fused-silica capillary column bonded with DB-1.     

Structural determination of the oligosaccharide side chains from a glycoprotein isolated from the mucus of the coral Acropora formosa

An extracellular mucous glycoprotein has been isolated from the hard coral Acropora formosa. The glycoprotein contains sulfated oligosaccharide side chains attached through O-glycosidic linkages to serine and threonine, the principal amino acids (77%) in the polypeptide. The oligosaccharide side chains consist of D-arabinose, D-mannose, and N-acetyl-D-glucosamine with smaller amounts of D-galactose, L-fucose, and N-acetyl-D-galactosamine, but no sialic or uronic acids. Alkaline borohydride reductive cleavage resulted in a mixture of oligosaccharide alditols. Six oligosaccharides were purified by high performance liquid chromatography. The structures of these oligosaccharides, which do not resemble those of any other glycoprotein so far examined, were determined by a combination of gas chromatography/mass spectrometry analysis of methylation products and NMR spectroscopy. All oligosaccharides contain a reducing terminal mannitol residue with N-acetylglucosamine linked to carbon 2, 4, or 6 of the mannitol. There is no evidence for linkage of N-acetylglucosamine to any other glycoses in the glycoprotein. Galactose was detected in two oligosaccharides linked to the 4-position of mannitol. Arabinose (Ara) was found in only one oligosaccharide. This was probably due to hydrolysis of the labile arabino-furanoside linkages. Evidence is presented which indicates the arabinose occurs primarily at the terminal position of oligosaccharide side chains. The structures of the oligosaccharides isolated from the glycoprotein were: (Formula: see text).     

Primary structure of the glycans from mouse serum and milk transferrins.

A 'serotransferrin-like' protein was purified from mouse milk. This serotransferrin cross-reacts immunologically with the serotransferrin isolated from mouse plasma and not with the mouse lactotransferrin (lactoferrin). Sugar analysis of the three transferrins, i.e. serotransferrin, milk 'serotransferrin-like' protein and lactotransferrin, revealed that the major difference between the glycan primary structure of mouse serotransferrin and those of mouse milk 'serotransferrin-like' protein and lactotransferrin concerns essentially the presence of one fucose residue in the last two proteins. For structural determination, the N-glycosidically linked glycans were released from the protein by a reductive cleavage of the oligosaccharide-protein linkage under strong alkaline conditions. The primary structure of the released oligosaccharide alditols was determined by methylation analysis and 400 MHz 1H-n.m.r. spectroscopy. The oligosaccharide alditols released from milk 'serotransferrin-like' protein and lactotransferrin were identical and were identified as disialylated biantennary glycans of the N-acetyl-lactosamine type with a fucose residue alpha-1,6-linked to the N-acetylglucosamine residue conjugated to the peptide chain and having the following primary structure: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta 1-4)[Fuc(alpha 1-6)]GlcNAc(beta 1-N)Asn. The serotransferrin glycan has the same primary structure but is only partially fucosylated (10-15%).     

Structural characterization of novel complex oligosaccharides accumulated in the caprine beta-mannosidosis kidney. Occurrence of tetra- and pentasaccharides containing a beta-linked mannose residue at the nonreducing terminus

Four oligosaccharide fractions were isolated and purified from the kidney of goats affected with beta-mannosidosis by repeating Bio-Gel P-2 column chromatography. The structural characterization of the purified oligosaccharide fractions (oligosaccharides A, B, C1,2, and D) included sugar composition analysis by gas chromatography, sugar sequence analysis by mass spectrometry of their permethylated alditols, and by methylation analysis as well as anomeric configuration studies by exoglycosidase digestions. Oligosaccharides A and B were the major oligosaccharides accumulating in the kidney and were elucidated as Man beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4GlcNAc, respectively (Matsuura, F., Laine, R. A., and Jones, M. Z. (1981) Arch. Biochem. Biophys. 211, 485-493). Oligosaccharide C1,2 was a mixture of two tetrasaccharides and oligosaccharide D was a pentasaccharide. The proposed structures are: oligosaccharide C1, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc; oligosaccharide C2, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc; oligosaccharide D, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc beta 1-4GlcNAc. Tetrasaccharide C1 and pentasaccharide D are heretofore undiscovered oligosaccharides. There is no precedent for these structures in glycoproteins or other glycoconjugates. One possibility which accounts for the presence of oligosaccharide C1 and D is that a bisecting N-acetylglucosamine (the beta-N-acetylglucosamine residue linked at the C-4 position of the beta-mannosyl residue of the trimannosyl core of the asparagine-linked sugar chains) is linked by a beta-mannosyl residue. Moreover, the detection of oligosaccharides containing two N-acetylglucosamine residues at the reducing terminus, together with those containing a single N-acetylglucosamine residue, is further corroboration of species-specific differences in glycoprotein catabolic pathways (Hancock, L. W., and Dawson, G. (1984) Fed. Proc. 43, 1552) or in glycoprotein structures.     

500 MHz 1H n.m.r. of oligosaccharides of N-acetyl-lactosamine-type released from human erythrocyte glycopeptides by endo-beta-galactosidase.

500 MHz 1H n.m.r. spectroscopy has been used in structural studies of three linear and five branched oligosaccharides of N-acetyl-lactosamine-type that were released from desialylated blood group O erythrocyte glycopeptides by treatment with the endo-beta-galactosidase of Bacteroides fragilis followed by reduction. The following oligosaccharide alditols were characterized: (formula; see book)     

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

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

Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens.

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