Isolation and characterization of sulphated oligosaccharides released from bovine corneal keratan sulphate by the action of endo-beta-galactosidase

A series of oligosaccharides has been isolated from the keratan sulphate peptidoglycan (3 M NaCl fraction) of bovine cornea after digestion with the endo-beta-galactosidase of Bacteroides fragilis. Structural information on the major oligosaccharides was obtained from (a) their susceptibilities to endo-beta-galactosidase before and after desulphation, (b) their elution positions on a column of Bio-Gel P-4 and retention times on a high-performance anion-exchange column and (c) negative-ion fast-atom-bombardment mass spectrometry. More than 75% of the oligosaccharides were sulphated unbranched poly(N-acetyllactosamine) sequences, (-3/4GlcNAc beta 1-3Gal beta 1-)n, and approximately 3% was the neutral disaccharide, GlcNAc beta 1-3Gal. The sulphated disaccharide, GlcNAc-SO-3 beta 1-3Gal, accounted for almost 35% of the oligosaccharide material while 40% consisted of four oligosaccharides, unbranched tetra-, hexa-, octa- and decasaccharides of poly(N-acetyllactosamine) type, having 3, 5, 7 and 9 sulphate residues respectively. Proton nuclear magnetic resonance studies at 500 MHz (Hounsell, E. F., et al. following paper in this journal) have shown that a sulphate residue is attached to the C-6 position of each N-acetylglucosamine and each internal galactose residue of these four oligosaccharides which express to varying degrees the antigenic determinants recognised by three monoclonal antibodies to keratan sulphate (Mehmet, H. et al., paper which follows the next paper in this journal).     

Occurrence of tetra- and pentasaccharides with the sialyl-Le(a) structure in human milk

The occurrence of two novel oligosaccharides in human milk was investigated. These oligosaccharides were purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 500-MHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry, indicated the structures of these compounds to be NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNAc and NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNac beta 1----3Gal. This constitutes the first evidence for the occurrence of N-acetylglucosamine or galactose as the reducing-end residue of human milk oligosaccharides. These two oligosaccharides bound MSW 113 to nearly the same extent as sialyl-Lea hexasaccharide but to another sialyl-Le(a) structure-directed monoclonal antibody, NS-19-9, only weakly.     

Characterisation by mass spectrometry and 500-MHz proton nuclear magnetic resonance spectroscopy of penta- and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins)

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500-MHz 1H NMR are described of the major penta- and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied after depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio-Gel P4 chromatography and HPLC. Eleven penta- and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J. 256, 397-401] and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features: (table; see text) Sequences in these oligosaccharides not commonly found in mucins so far studied are chain-terminating GlcNAc alpha 1-4Gal, repeating-type-I (Gal beta 1-3GlcNAc) backbones, the backbone branch GlcNAc beta 1-6(GlcNAc beta 1-3)Gal and the backbone sequence GlcNAc beta 1-6Gal beta 1- in the absence of a substituent at C3 of galactose.     

Comparative study of the oligosaccharides released from baby hamster kidney cells and their polyoma transformant by hydrazinolysis

The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.     

Proton-nuclear magnetic resonance study of peracetylated derivatives of ten oligosaccharides isolated from human milk

Proton-nuclear magnetic resonance (NMR) spectra of peracetylated derivatives of ten structurally related oligosaccharides isolated from human milk were measured for solutions in CDCl3 at 360 MHz. The following oligosaccharides were investigated: Gal beta 1 leads to 4Glc-ol (1), GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (2), Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (3), Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (4), Gal beta 1 leads to 3GlcNAc(4 comes from 1Fuc alpha) beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (5), Fuc alpha 1 leads to 2Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (6), Fuc alpha 1 leads to 2Gal beta 1 leads to 3GlcNAc(4 comes from 1Fuc alpha)beta 1 leads to 3Gal beta 1 leads to 4Glc-ol (7), Fuc alpha 1 leads to 2Gal beta 1 leads to 4Glc-ol(3 comes from 1Fuc alpha) (8), and a 1:3 mixture of Fuc alpha 1 leads to 2Gal beta 1 leads to 4Glc-ol (9) and Gal beta 1 leads to 4Glc-ol(3 comes from 1Fuc alpha) (10). Owing to the strong downfield shifts of the resonances of protons linked to acetoxylated carbons, the problems of signal overlap are less severe and the spin systems of all constituent sugar residues can be assigned fully. The sites of glycosidic linkage can be recognized by the high-field position of the signals of protons linked to those sites; for example, type 1 (Gal beta 1 leads to 3GlcNAc) and type 2(Gal beta 1 leads to 4GlcNAc) saccharide chains can be distinguished. The sequence can be established by observing a nuclear Overhauser effect involving the anomomeric and the aglyconic proton.     

Rabbit antibodies against the human milk sialyloligosaccharide alditol of LS-tetrasaccharide a (NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH)

The sialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LS-tetrasaccharide a), a minor component of human milk, is obtained in relatively large quantities from autohydrolysates of the major milk disialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc (disialyllacto-N-tetraose). Rabbits immunized with an oligosaccharide-protein conjugate prepared from keyhole limpet hemocyanin and LS-tetrasaccharide a produce antibodies directed against the corresponding oligosaccharide alditol. The anti-LS-tetrasaccharide a sera bind 3H-labeled LS-tetrasaccharide a in a direct-binding radioimmunoassay on nitrocellulose filters. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides. Strong hapten-antibody binding (Ka greater than 10(6) M-1) requires sialic acid linked alpha 2-3 to the nonreducing terminal galactose residue of reduced lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH). Specificities of antibodies prepared against keyhole limpet hemocyanin conjugates of LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) differ only slightly from rabbit antibodies prepared against the corresponding bovine serum albumin conjugates described previously [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59].     

Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine

In the accompanying paper (Bendiak et al., 1989), the separation of a series of oligosaccharides released from asparagine residues of fetuin was described. A series of NMR experiments, which included one- and two-dimensional nuclear Overhauser enhancement, two-dimensional correlation spectroscopy, and two-dimensional relayed-coherence spectroscopy, as well as permethylation analyses, established a Gal beta 1----3(NeuAc alpha 2----6)GlcNAc beta 1----4Man unit common to a series of purified structures. These oligosaccharides contained either three, four, or five glycosidically linked sialic acid residues. The NeuAc residue in alpha 2----6 linkage to GlcNAc gives rise to diagnostic chemical shift perturbations of particular proton signals in the oligosaccharides.     

Subunit-specific sulphation of oligosaccharides relating to charge-heterogeneity in porcine lutrophin isoforms.

Lutrophin (LH) consists of an array of isoforms with different charges and bioactivities. This study was undertaken to clarify specifically how oligosaccharides of alpha and beta subunits contribute to LH isoform charges. Porcine LH (pLH) was separated into four isoforms by isoelectric focusing (IEF), followed by subunit isolation. Their oligosaccharides were released by hydrazinolysis, labelled by reduction with NaB3H4, and fractionated by HPLC with a Mono Q column into five populations differing in the number of sulphate (S) and sialic acid (N) residues, designated as Neutral, N-1, S-1, S-N and S-2. Oligosaccharides were predominantly sulphated (S-1 and S-2) and infrequently sialylated (N-1 and S-N). Further analysis, including concanavalin A (Con A) affinity chromatography, desialylation, desulphation, sequential exoglycosidase digestion and methylation, clarified the structures of the acidic oligosaccharides. All were of the biantennary complex type. Their two peripheral branches were SO4-4GalNAc beta 1-4Glc-NAc and GalNAc beta 1-4GlcNAc or GlcNAc in S-1, SO4-4GalNAc beta 1-4GlcNAc and Sia alpha 2-6Gal beta 1-4GlcNAc in S-N, and (SO4-4GalNAc beta 1-4GlcNAc)2 in S-2 (where GalNAc is N-acetylgalactosamine and GlcNAc is N-acetylglucosamine). Ten percent of S-1 and of S-N had a bisecting GlcNAc residue. Sulphate residues occurred in nearly the same amount for both subunits; however, the alpha and beta subunits were sulphated differently. S-1 predominated in the alpha subunit, while S-1 and S-2 were major components in the beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Branch specificity of bovine colostrum CMP-sialic acid: Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man beta 1----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal beta 1----4GlcNAc-R.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

Characterization of oligosaccharides in milk of bearded seal (Erignathus barbatus)

Carbohydrates were extracted from milk of a bearded seal, Erignathus barbatus (Family Phocidae). Free neutral oligosaccharides were separated by gel filtration, anion-exchange chromatography and preparative thin layer chromatography, while free acidic oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows; lactose, 2'-fucosyllactose, lacto-N-fucopentaose IV, difucosyl lacto-N-neohexaose and difucosyl decasaccharide which contained a lacto-N-neohexaose unit as well as an additional Gal(beta1-4)GlcNAc(beta1-3) unit and two residues of non-reducing Fuc(alpha1-2). The acidic oligosaccharides were thought to contain an Neu5Ac(alpha2-6) residue linked to GlcNAc or a sulfate linked to Gal at OH-3. The sialyl oligosaccharides and sulfated oligosaccharides had a lacto-N-neohexaose unit and two non-reducing Fuc(alpha1-2) residues and some of them had in addition one or two Gal(beta1-4)GlcNAc(beta1-3) units. The milk oligosaccharides of the bearded seal were compared to those of the harbour seal, which had been studied previously.     

Structures of the sugar chains of mouse immunoglobulin G

The asparagine-linked sugar chains of mouse immunoglobulin G (IgG) were quantitatively liberated as radioactive oligosaccharides from the polypeptide portions by hydrazinolysis followed by N-acetylation, and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin (RCA120) affinity high-performance liquid chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Mouse IgG was shown to contain the biantennary complex type sugar chains. Eight neutral oligosaccharide structures, viz, +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1---- 4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc, were found after the sialidase treatment. The molar ratio of the sugar chains with 2,1, and 0 galactose residues was 2:5:3. The galactose residue in the monogalactosylated sugar chains was distributed on Man alpha 1----3 and Man alpha 1----6 sides in the ratio of 1:3. The oligosaccharides were almost wholly fucosylated and contained no bisecting N-acetylglucosamine which is present in human, rabbit, and bovine IgGs.     

N-Acetylneuraminic acid and N-glycolylneuraminic acid in the O-linked oligosaccharides of a tumor cell glycoprotein. Incorporation and distribution

The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma, which differ in several cell surface properties, contain a major mucin-type glycoprotein, termed ASGP-1. The sialic acid content of MAT-C1 ASGP-1 is 2-3-fold greater than MAT-B1 ASGP-1 (Sherblom, A. P., Buck, R. L., and Carraway, K. L. (1980) J. Biol. Chem. 255, 783-790). Sialic acid analysis demonstrated that, whereas MAT-C1 ASGP-1 contained approximately equal amounts of N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGl), MAT-B1 ASGP-1 was devoid of NeuGl. MAT-B1 microsomes also did not contain NeuGl. MAT-B1 cells incubated with [3H]N-acetylmannosamine did not synthesize either labeled CMP-NeuGl or free NeuGl, even though the CMP-sialic acid synthetase was active with the substrate NeuGl. Thus, MAT-B1 cells may be deficient in the enzyme N-acetylneuraminate monooxygenase. The O-linked oligosaccharides from both MAT-B1 and MAT-C1 ASGP-1 have been shown to contain a core tetrasaccharide Gal(beta 1-4)GlcNAc(beta 1-6)(Gal(beta 1-3]GalNAc in which both galactose residues may be linked to additional sugars (Hull, S. R., Laine, R. A., Kaizu, T., Rodriquez, I., and Carraway, K. L. (1984) J. Biol. Chem. 259, 4866-4877). The distribution of NeuAc and NeuGl between the two galactose termini of the core tetrasaccharide was examined for MAT-C1 ASGP-1. Oligosaccharides were released by alkaline-borohydride treatment of MAT-C1 ASGP-1 which had been labeled with [14C]glucosamine and galactose oxidase/B3H4. Following fractionation by Bio-Gel P-4, DEAE-Sephadex, and high-performance liquid chromatography, oligosaccharides were analyzed for NeuAc and NeuGl and for susceptibility to digestion with beta-galactosidase. Three disialylated oligosaccharides were identified containing 2 mol of NeuAc (5.5% recovery), 2 mol of NeuGl (4.5%), or 1 mol each of NeuAc and NeuGl (11.1%). For monosialylated oligosaccharides, NeuGl appeared preferentially associated with the Gal(beta 1-4)GlcNAc terminus (9.0%), whereas significant amounts of oligosaccharide containing NeuAc at both the Gal(beta 1-3)GalNAc (2.6%) and Gal(beta 1-4)GlcNAc (4.5%) termini were detected. Each of the major qualitative differences between MAT-B1 and MAT-C1 oligosaccharides, including the presence of NeuGl (MAT-C1), sulfate (MAT-B1), and alpha-linked galactose (MAT-B1), occurs at the Gal(beta 1-4)GlcNAc terminus.     

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.     

Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin

The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-Sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the alpha-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-Sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fuc alpha 1----2Gal beta 1----4GlcNAc and Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol II, which has a Gal beta 1----3(Fuc alpha 1----4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fuc alpha 1----2Gal beta 1----3GlcNAc and Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups showed almost no interaction with the matrix.     

Mucin synthesis. Conversion of R1-beta 1-3Gal-R2 to R1-beta 1-3(GlcNAc beta 1-6)Gal-R2 and of R1-beta 1-3GalNAc-R2 to R1-beta 1-3(GlcNAc beta 1-6)GalNAc-R2 by a beta 6-N-acetylglucosaminyltransferase in pig gastric mucosa

A UDP-GlcNAc:R1-beta 1-3Gal(NAc)-R2 [GlcNAc to Gal(NAc)] beta 6-N-acetylglucosaminyltransferase activity from pig gastric mucosa microsomes catalyzes the formation of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal-R from GlcNAc beta 1-3Gal-R where -R is -beta 1-3GalNAc-alpha-benzyl or -beta 1-3(GlcNAc beta 1-6)GalNAc-alpha-benzyl. This enzyme is therefore involved in the synthesis of the I antigenic determinant in mucin-type oligosaccharides. The enzyme also converts Gal beta 1-3Gal beta 1-4Glc to Gal beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc. The enzyme was stimulated by Triton X-100 at concentrations between 0 and 0.2% and was inhibited by Triton X-100 at 0.5%. There is no requirement for Mn2+ and the enzyme activity is reduced to 65% in the presence of 10 mM EDTA. Enzyme products were purified and identified by proton NMR, methylation analysis and beta-galactosidase digestion. Competition studies suggest that this pig gastric mucosal beta 6-GlcNAc-transferase activity is due to the same enzyme that converts Gal beta 1-3GalNAc-R to mucin core 2, Gal beta 1-3(GlcNAc beta 1-6)GalNAc-R, and GlcNAc beta 1-3GalNAc-R to mucin core 4, GlcNAc beta 1-3(GlcNAc beta 1-6)GalNAc-R. Substrate specificity studies indicate that the enzyme attaches GlcNAc to either Gal or GalNAc in beta (1-6) linkage, provided these residues are substituted in beta (1-3) linkage by either GlcNAc or Gal. The insertion of a GlcNAc beta 1-3 residue into Gal beta 1-3GalNAc-R to form GlcNAc beta 1-3Gal beta 1-3GalNAc-R prevents insertion of GlcNAc into GalNAc. These studies establish several novel pathways in mucin-type oligosaccharide biosynthesis.     

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin.

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.     

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.     

Isolation and characterization of an endo-beta-galactosidase from Bacteroides fragilis.

Six strains of Bacteroides fragilis were examined and all found to produce endo-beta-galactosidase, an enzyme that hydrolyses internal beta-galactosidic linkages of oligosaccharides belonging to the poly-N-acetyl-lactosamine series, with the common structure GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc/Glc. The enzyme was produced without the addition of an inducer such as keratan sulphate. It was purified 7000-fold from the culture supernatant and obtained with a yield 4-10-fold greater than from sources described previously. The specificity of the enzyme towards bovine corneal keratan sulphate, milk oligosaccharides and the glycolipids lacto-N-neotetraosylceramide and lacto-N-tetraosylceramide closely resembled that of the endo-beta-galactosidase isolated from Escherichia freundii. A novel observation was that both enzymes hydrolysed the type 2 sequence, Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc, at about twice the rate of the type 1 isomer, Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc. Because of the ease of purification of the enzyme and high yield in the absence of contaminating glycosidases and proteinases, Bacteroides fragilis is a valuable source of endo-beta-galactosidase for the structural analysis of carbohydrate chains.     

Structural characterization of oligosaccharides in the milk of an African elephant (Loxodonta africana africana)

The oligosaccharides present in the milk of an African elephant (Loxodonta africana africana), collected 4 days post partum, were separated by size exclusion-, anion exchange- and high-performance liquid chromatography (HPLC) before characterisation by (1)H NMR spectroscopy. Neutral and acidic oligosaccharides were identified. Neutral oligosaccharides characterised were isoglobotriose, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc and a novel oligosaccharide that has not been reported in the milk or colostrum of any other mammal: Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc. Acidic oligosaccharides that are also found in the milk of Asian elephant were Neu5Ac(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc and Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc, while Neu5Gc(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc and Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc have not been found in Asian elephant milk. The oligosaccharides characterised contained both alpha(2-3)- and alpha(2-6)-linked Neu5Ac residues. They also contain only the type II chain, as found in most non-human, eutherian mammals.