Structures of the sugar chains of a major glycoprotein present in the egg jelly coat of a starfish, Asterias amurensis

Sugar chains of a major glycoprotein, obtained from the egg jelly coat of a starfish (Asterias amurensis), were released quantitatively as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by reduction with NaB3H4. Analysis by paper electrophoresis revealed that all of them were neutral oligosaccharides. Upon Bio-Gel P-4 column chromatography, the radioactive oligosaccharide mixture was separated into four components. Structural study of each component by sequential glycosidase digestion in combination with 1H-NMR spectroscopy revealed that the glycoprotein contains the following oligosaccharides, in which R represents either proton, Glc alpha 1----, Glc alpha 1----3Glc alpha 1----, or Glc alpha 1----2Glc alpha 1----3Glc alpha 1----. (Formula: see text)     

Carbohydrates of lysosomal enzymes secreted by Tetrahymena pyriformis

The carbohydrate structures of acid phosphatase and alpha-glucosidase secreted into culture medium by Tetrahymena pyriformis strain W were studied. Their asparagine-linked sugar chains were quantitatively liberated as radioactive oligosaccharides from their polypeptide moieties by controlled hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The approximate amounts of total sugar chains liberated from 1 mol each of acid phosphatase and alpha-glucosidase were 6 and 4 mol, respectively. Paper electrophoresis revealed that only neutral oligosaccharides were obtained from both enzymes. The oligosaccharide fraction from acid phosphatase was separated into seven components by Bio-Gel P-4 column chromatography while that from alpha-glucosidase was resolved into three components. The structures of these oligosaccharides were determined by sequential glycosidase digestion in combination with methylation analysis. The sugar chains of the two enzymes can be primarily classified as high mannose-type oligosaccharides. However, they have the following characteristic features: 1) their common core is not the usual Man5 . GlcNAc2 structure, it is Man3 . GlcNAc2; 2) some of the sugar chains of acid phosphatase have 1 approximately 3 glucose residues linked to the nonreducing terminal Man alpha 1----2 residue. The structural characteristics of the sugar moieties of the two enzymes indicate that they might be produced by the so-called "alternate pathway," in which lipid-linked Glc3 . Man5 . GlcNAc2 functions as an oligosaccharide donor.     

Liquid chromatographic assay for a glucose tetrasaccharide, a putative biomarker for the diagnosis of Pompe disease

A HPLC method associated with butyl-p-aminobenzoate derivatization has been developed for the analysis of a tetraglucose oligomer, Glcalpha1-6Glcalpha1-4Glcalpha1-4Glc, designated Glc(4), in biological fluids. This tetraglucose, normally excreted in the urine, has previously been shown to be elevated in a number of pathological conditions including Pompe disease (glycogen storage disease type II), which is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase. Concentrations of Glc(4) in both urine and plasma were established for the age ranges of <1, 1-5, 6-10, 11-20, and >20 years, both in normal individuals and in a cohort of 21 patients with enzymatically confirmed Pompe disease. The Glc(4) concentration decreased with age in both groups, but all the patients had elevated Glc(4) levels compared with age-matched controls. Electrospray tandem mass spectrometry was employed to establish the homogeneity of the HPLC peak for Glc(4) and to investigate the identity of other unusual oligosaccharides excreted in patient urine. Our results demonstrate that this method is suitable for application in clinical laboratories to help establish the diagnosis of Pompe disease.     

Structural analysis of the major urinary oligosaccharides in feline alpha-mannosidosis.

Two homologous series of urinary oligosaccharides were identified by h.p.l.c. and fast-atom-bombardment mass spectrometry in feline alpha-mannosidosis. The predominant series has the composition Man2-8GlcNAc2 and a minor series the composition Man2-9GlcNAc. The structure of the most abundant oligosaccharide, which accounts for over 80% of the urinary oligosaccharide, was shown to be alpha-D-Manp(1----3)[alpha-D-Manp-(1----6)]beta-D-Manp -(1----4)-beta-D-GlcpNA c-(1----4)-D-GlcNAc by gas chromatography and mass spectrometry. Such a structure is consistent with the incomplete catabolism of complex N-linked glycans due to a deficiency of alpha-D-mannosidase in tissue lacking an endohexosaminidase activity.     

Biosynthesis of a disialylated sequence in N-linked oligosaccharides: identification of an N-acetylglucosaminide (alpha 2----6)-sialyltransferase in Golgi apparatus from rat liver

Rat liver Golgi apparatus are shown to have a CMP-N-acetylneuraminate: N-acetylglucosaminide (alpha 2----6)-sialyltransferase which catalyzes the conversion of the human milk oligosaccharide LS-tetrasaccharide-a (NeuAc alpha 2----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----4Glc) to disialyllacto -N- tetraose containing the terminal sequence: (formula: see text) found in N-linked oligosaccharides of glycoproteins. The N-acetylglucosaminide (alpha 2----6)-sialyltransferase has a marked preference for the sequence NeuAc alpha 2----3-Gal beta 1---- 3GlcNAc as an acceptor substrate. Thus, the order of addition of the two sialic acids in the disialylated structure shown above is proposed to be first the terminal sialic acid in the NeuAc alpha 2----3Gal linkage followed by the internal sialic acid in the NeuAc alpha 2---- 6GlcNAc linkage. Sialylation in vitro of the type 1 branches (Gal beta 1---- 3GlcNAc -) of the N-linked oligosaccharides of asialo prothrombin to produce the same disialylated sequence is also demonstrated.     

Structural characterization of the cell surface lipooligosaccharides from a nontypable strain of Haemophilus influenzae.

Oligosaccharides released from the lipooligosaccharides (LOS) of Haemophilus influenzae nontypable strain 2019 by mild acid hydrolysis were fractionated by size exclusion chromatography and analyzed by liquid secondary ion mass spectrometry. The major component of the heterogeneous mixture was found to be a hexasaccharide of Mr 1366, which lost two phosphoethanolamine groups upon treatment with 48% aqueous HF. The dephosphorylated hexasaccharide was purified and shown by tandem mass spectrometry, composition analysis, methylation analysis, and two-dimensional nuclear magnetic resonance studies to be Gal beta 1----4Glc beta 1----(Hep alpha 1----2Hep alpha 1----3) 4Hep alpha 1----5anhydro-KDO, where Hep is L-glycero-D-manno-heptose and KDO is 3-deoxy-D-manno-octulosonic acid. An analogous structure containing authentic KDO was generated from LOS that had been HF-treated prior to acetic acid hydrolysis, suggesting that the reducing terminal anhydro-KDO moiety is produced as an artifact of the hydrolysis procedure by beta-elimination of a phosphate substituent from C-4 of KDO. Mass spectral analyses of O-deacylated LOS and free lipid A confirmed that, in addition to the two phosphoethanolamines on the oligosaccharide and two phosphates on the lipid A, another phosphate group exists on the KDO. This KDO does not appear to be further substituted with additional KDO residues in intact H. influenzae 2019 LOS. The terminal disaccharide epitope, Gal beta 1----4Glc beta 1----, of the hexasaccharide is also present on lactosylceramide, a precursor to human blood group antigens. It is postulated that the presence of this structure on H. influenzae LOS may represent a form of host mimicry by the pathogen.     

GalNAc beta 1----3 terminated glycosphingolipids of human erythrocytes

Nonacid glycosphingolipids with 4 to 10 sugar residues isolated from pooled erythrocytes of blood group O donors have been efficiently separated as peracetylated derivatives on silicic acid. This procedure enabled a quantitative estimate of individual compounds and also revealed several GalNAc beta 1----3 terminated structures. The structural characterization of these glycolipids with 1H-NMR spectroscopy, direct inlet mass spectrometry, gas chromatography, and gas chromatography-mass spectrometry identified the compounds as GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl sphingosine and GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1-N-acetyl phytosphingosine, GalNAc beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 ceramide, and GalNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1 ceramide.     

Structural study of the asparagine-linked oligosaccharides of lipophorin in locusts

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.     

Structure determination of five sialylated trisaccharides with core types 1, 3 or 5 isolated from bovine submaxillary mucin

In this study we have investigated the structures of five sialylated trisaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high-performance liquid chromatography. Three of the trisaccharides contained NeuAc while two contained NeuGc. One oligosaccharide contained core-type 1, two contained core-type 3 and two contained core-type 5. The structures, determined by a combination of one- and two-dimensional 1H-NMR spectroscopy at 270 MHz and methylation analysis involving gas-liquid chromatography/mass spectrometry, were as follows: A4b, GalNAc alpha(1----3) [NeuAc alpha(2----6)]GalNAcol; A4c, GlcNAc beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4d, Gal beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4e, GalNAc alpha(1----3)-[NeuGc alpha(2----6)]GalNAcol; A4f, GlcNAc beta(1----3)[NeuGc alpha (2----6)]GalNAcol. The oligosaccharides occurred in the approximate molar ratios 1.0:12.0:0.3:0.2:2.0. This is the first report of oligosaccharides containing core-type 5 and of the occurrence of oligosaccharides A4b, A4e, and A4f in bovine submaxillary mucin. 1H-NMR data for structure A4e, which is a novel structure, are presented for the first time.     

Structural and conformational analysis of sialyloligosaccharides using carbon-13 nuclear magnetic resonance spectroscopy

The analysis of the carbon-13 chemical shift data of NeuAc alpha (2----3)Gal beta (1----4)Glc and NeuAc alpha (2----3)Gla beta-(1----4)GlcNAc and their respective NeuAc alpha (2----6) isomers established distinct and different conformations of the sialic acid residue, depending on the type of anomeric linkage [alpha-(2----3) vs. alpha (2----6)]. Interactions between the NeuAc residue and the Glc or GlcNAc residue are particularly strong in the case of the alpha (2----6) isomers. Similar effects are observed for the larger oligosaccharides [II3(NeuAc)2Lac and IV6NeuAcLcOse4] and even in intact glycoproteins and polysaccharides. It is proposed that the NeuAc alpha (2----3) isomers assume an extended conformation with the sialic residue at the end (terminal) of the oligosaccharide chain or branch. The NeuAc alpha (2----6) isomers are assumed to be folded back toward the inner core sugar residues.     

Structure of phosphonoglycosphingolipid containing pyruvylated galactose in nerve fibers of Aplysia kurodai

A phosphonoglycosphingolipid, designated as FGL-IIb, was identified in nerve fibers of Aplysia kurodai by two-dimensional thin layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). FGL-IIb was isolated from the nervous system of A. kurodai by Iatrobeads column chromatography using three solvent systems. Pyruvic acid was identified by thin layer chromatography as its 2,4-dinitrophenylhydrazone and established by permethylation studies to be attached as a ketal to O-3 and O-4 of the terminal galactose of the oligosaccharide chain in FGL-IIb. By sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structure of FGL-IIb was concluded to be [3,4-O-(1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3(Fuc alpha 1----2) (2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1ceramide. Its major aliphatic components were palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. This is the first report of the occurrence of pyruvylated galactose as a constituent of animal sphingolipid.     

The carbohydrate moieties of human urinary ribonuclease UL

Ribonuclease UL purified from pooled human urine contains approximately 20.7% of neutral sugar and 7.8% of aminosugar. All sugars were quantitatively released as oligosaccharides on hydrazinolysis. The oligosaccharides were converted to tritium-labeled oligosaccharides on reduction with NaB3H4. The radioactive oligosaccharide fraction was separated into a neutral and an acidic fraction on paper electrophoresis. All oligosaccharides in the acidic fraction could be converted to neutral oligosaccharides with the release of one sialic acid residue by sialidase digestion. Both fractions were shown to be mixtures of more than fourteen oligosaccharides by gel permeation chromatography. Structural studies on these oligosaccharides involving sequential exoglycosidase digestion in combination with methylation analysis revealed that ribonuclease UL contains sialylated and non-sialylated mono, bi-, tri-, and tetraantennary complex type sugar chains with N-acetyllactosamine outer chains, and tri- and tetraantennary complex type sugar chains with various numbers of Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains. An important finding was that all sialic acid residues in the acidic oligosaccharides only occur as the Sia alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 group. Both fucosylated and non-fucosylated trimannosyl cores were found among the asparagine-linked sugar chains of ribonuclease UL.     

Purification and characterization of GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase from human neuroblastoma cells. Unusual substrate specificities of the tumor enzyme

Fucosyl residues in the alpha 1----3 linkage to N-acetylglucosamine (Fuc alpha 1----3GlcNAc) on oligosaccharides of glycoproteins and glycolipids have been detected in certain human tumors and are developmentally expressed (reviewed in Foster, C. S., and Glick, M. C. (1988) Adv. Neuroblastoma Res. 2, 421-432). In order to understand control mechanisms for the biosynthesis of these fucosylated glycoconjugates, GDP-L-Fuc-N-acetyl-beta-D-glucosaminide alpha 1----3fucosyltransferase was purified from human neuroblastoma cells, CHP 134, utilizing either the immobilized oligosaccharide or disaccharide substrates. The enzyme, extracted from CHP 134 cells, was purified by DEAE- and SP-Sephadex chromatography and then by either immobilized substrate. alpha 1----3Fucosyltransferase was obtained in approximately 10% yield and was purified 45,000-fold from the cell extract. The kinetic properties of the enzyme showed an apparent KGDP-Fuc 43 microM, KGal beta 1----4GlcNAc 0.4 mM, KGal beta 1----4Glc 8.1 mM, and KFuc alpha 1----2Gal beta 1----4Glc 1.0 mM. Polyacrylamide gel electrophoresis of the affinity-purified enzyme showed two proteins which migrated, Mr = 45,000-40,000. The enzyme differed in substrate specificity, pH optimum, response to N-ethylmaleimide and ion requirements from the enzymes purified from human milk or serum. The inability of alpha 1----3fucosyltransferase to transfer to substrates containing NeuAc alpha 2----3 or alpha 2----6Gal is in contrast to the reports for the enzyme in other human tumors. This substrate specificity correlates with the oligosaccharide residues thus far defined on glycoproteins of CHP 134 cells since NeuAc and Fuc alpha 1----3GlcNAc have yet to be detected on the same oligosaccharide antenna. However, the enzyme transfers to Fuc alpha 1----2Gal beta 1----4GlcNAc/Glc with higher activity than the unfucosylated disaccharides, although neither alpha 1----2fucosyltransferase nor Fuc alpha 1----2 residues have been detected in CHP 134 cells. The different substrate specificities of alpha 1----3fucosyltransferase isolated from human tumors and normal sources leads to the suggestion that a family of alpha 1----3fucosyltransferases may exist and that they may be differentially expressed in human tumors.     

Alkali-labile oligosaccharide units of a sialoglycoprotein from rabbit erythrocyte membranes

Two glycoproteins (apparent molecular weights 120,000 and 70,000) were extracted from rabbit erythrocyte membranes, and only one (Mr 120,000), which is a sialoglycoprotein, contained O-glycosidically linked sugar chains. Alkali-labile oligosaccharide units of the sialoglycoprotein were released as reduced oligosaccharides by NaOH-NaB3H4 treatment, and then purified by gel filtration on a Bio-Gel P-4 column followed by ion-exchange chromatography. From the results of methylation analysis, mass spectrometry and chromium trioxide oxidation, the main oligosaccharide unit was determined to be a linear trisaccharide (85% by weight), NeuNGc alpha(2----3)Gal beta(1----3)GalNAcol. In addition, small amounts of a tetrasaccharide (11% by weight) and a disaccharide (4% by weight) were found, which were determined to have the following structures, NeuNGc alpha(2----3)Gal beta(1----3)[NeuNGc alpha(2----6)] GalNAcol and Gal-GalNAcol, respectively.     

Structures of neutral oligosaccharides isolated from the respiratory mucins of a non-secretor (O, Le(a+b-)) patient suffering from chronic bronchitis.

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

Definition of the surface antigens of Mycobacterium malmoense and use in studying the etiology of a form of mycobacteriosis

Mycobacterium malmoense is the latest of a roster of atypical mycobacteria implicated in pulmonary infections. Yet it lacks recognizable phenotypic features to allow its ready identification. Some 23 clinical isolates of M. malmoense were examined for homologous seroagglutination reactions and characteristic surface antigens. One group showed concordant agglutination interreactions and an identical spectrum of glycolipids and are regarded as M. malmoense sensu stricto. The glycolipids are of the newly found, trehalose-containing lipooligosaccharide class. De-O-acylation followed by high-pressure liquid chromatography revealed one major and several minor oligosaccharides. Partial acidic cleavage to release glycosidically linked trehalose, alpha-mannosidase digestion to demonstrate the presence of a non-reducing-end mannobiose, perdeuteriomethylation, partial acid hydrolysis, reduction, and O ethylation, combined with 1H nuclear magnetic resonance and electron impact and fast-atom bombardment mass spectrometry revealed the structure of the major oligosaccharide as alpha-D-Manp-(1----3) -alpha-D-Manp-(1----[2-alpha-L-Rhap-(1--]4--3)-alpha-L-Rh ap- (1----3)-alpha-D-Glcp-(1----1)-alpha-D-Glcp, in which two of the 2-alpha-L-Rhap residues are O methylated at C-3. (Man, mannose; Rha, rhamnose; Glc, glucose; p, pyranosyl). The structures of the minor oligosaccharides were also determined; they differ at the distal nonreducing end. The dominant oligosaccharide was acylated by octanoate, 2-methyleicosanoate, and 2,4-dimethylpentacosanoate to yield the major species-specific surface antigen of M. malmoense, which we regard as the most characteristic feature of the pathogen.     

alpha-Glucosidase II-deficient cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.

The kinetics of N-linked oligosaccharide processing and the structures of the processing intermediates have been examined in normal parental BW5147 mouse lymphoma cells and the alpha-glucosidase II-deficient PHAR2.7 mutant cells. The mutant cells accumulated glucosylated intermediates but were able to deglucosylate and process about 40% of their oligosaccharides to complex-type. This processing was not due to residual alpha-glucosidase II activity since the alpha-glucosidase inhibitors 1-deoxynojirimycin (DNJ) and N-butyl-DNJ did not prevent it. Parent cells also showed alpha-glucosidase II-independent processing in the presence of DNJ and N-butyl-DNJ. Membrane preparations from both parent and mutant cells had endo alpha-mannosidase activity, that is, split Glc1,2Man9GlcNAc to Glc1,2Man plus Man8GlcNAc, indicating that this was a candidate for an alternate route to complex oligosaccharide formation in the mutant cells. A balance study in which the cellular glycoproteins, intracellular water soluble saccharides, and saccharides secreted into the medium were isolated and analyzed from [2-3H]mannose-labeled mutant cells showed that the cells formed the di- and trisaccharides Glc1Man and Glc2Man in amounts equivalent to the deglucosylated oligosaccharides found in the cellular glycoproteins. This result shows unequivocally that the alpha-glucosidase II-deficient mutant cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.     

1-deoxynojirimycin impairs oligosaccharide processing of alpha 1-proteinase inhibitor and inhibits its secretion in primary cultures of rat hepatocytes

1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat alpha 1-proteinase inhibitor. In normal hepatocytes alpha 1-proteinase inhibitor was present in the cells as a 49,000 Mr high mannose type glycoprotein with oligosaccharide side chains having the composition Man9GlcNAc and Man8GlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated alpha 1-proteinase inhibitor as a 51,000 Mr glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against alpha-glucosidase, the largest species being Glc3Man9GlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of 1-deoxynojirimycin inhibited glycosylation resulting in the formation of some alpha 1-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of alpha 1-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of alpha 1-proteinase inhibitor secreted from 1-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3H]fucose and concanavalin A-Sepharose chromatography. It was found that 1-deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alpha 1-proteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these alpha 1-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of alpha 1-proteinase inhibitor.     

Structural studies on a binding site for Dolichos biflorus agglutinin in the small intestine of the mouse

Glycoproteins which bound to Dolichos biflorus agglutinin (DBA) were isolated from the small intestine of 129/Sv mice. Among oligosaccharides released from the carbohydrate moieties of the glycoproteins by endo-beta-galactosidase, the major one with N-acetylgalactosamine at the non-reducing end was isolated by QAE-Sephadex A-25 column chromatography. The structure of the oligosaccharide was elucidated to be GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc beta 1----3Gal by compositional analysis, methylation analysis before and after mild acid hydrolysis, sequential glycosidase digestion, secondary ion mass spectrometry (SIMS), and nuclear magnetic resonance spectroscopy. The SIMS signal of m/z 1,071 was consistent with the presence of the branched sequence, GalNAc(NeuAc)GalGlcNAc, and the signal was also detected in the high-molecular-weight fraction obtained after endo-beta-galactosidase digestion. The pentasaccharide identified here has the terminal structure of ganglioside GM2, and an apparently identical one has been identified as the epitope of blood group Sda and the DBA binding site in human T-H urinary glycoprotein. Thus, the present result has extended our knowledge of the biological meaning of the oligosaccharide structure and has established that GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc is a DBA binding site in the small intestine of the mouse.     

Structures of asparagine-linked oligosaccharides of human placental fibronectin

The asparagine-linked sugar chains of fibronectin purified from human placenta were quantitatively released as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharides were fractionated by their charge on an anion-exchange column chromatography. All of the acidic oligosaccharides could be converted to neutral oligosaccharides by sialidase digestion. These oligosaccharides were then fractionated by serial affinity chromatography using immobilized lectin columns. Study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed the following information as to the structures of the sugar chains of human placental fibronectin: 1) nine sugar chains are included in one molecule; 2) all sialic acid residues are exclusively linked at the C-3 position of the galactose residues; 3) bi-, tri-, and tetraantennary complex-type oligosaccharides with the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)-GlcNac as their cores were found; 4) the bisecting N-acetylglucosamine residue and the Gal beta 1----4GlcNAc beta 1----repeating groups are included in some of the sugar chains.