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.     

Carbohydrate moieties of peanut agglutinin receptors isolated from human gastric cancer cells

Receptors for peanut agglutinin (PNA) were isolated from Kato III human gastric cancer cells by affinity chromatography on PNA agarose, and were labeled by the galactose oxidase-NaB3H4 method. Alkaline NaBH4 treatment of the labeled receptors released two small oligosaccharide alcohols, which were identified as Gal beta 1----3GalNAc-ol and Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAc-ol. Higher oligosaccharides and glycopeptides of both N- and O-linked type were also detected, but they did not appear to bear PNA binding sites. The presence of oligo-N-acetyllactosamine units in the N-linked type sugars was indicated by endo-beta-galactosidase digestion.     

Thyroid cell surface glycoproteins. Nature and disposition of carbohydrate units and evaluation of their blood group I activity

The distribution along the polypeptide of the carbohydrate units of two major calf thyroid cell surface glycoproteins, GP-1 and GP-3, was obtained from a study of their glycopeptides obtained after Pronase digestion. The GP-3 molecule (Mr = 20,000) yielded two large glycopeptides (Mr = 9,500 and 7,000) in equimolar amounts which each consisted of one N-linked (Mr = 5,400) and several small O-linked oligosaccharides accounting for a total of nine carbohydrate attachment sites in a 27-amino acid residue segment of the peptide chain. The Pronase treatment of GP-1 (Mr = 100,000) revealed the presence of a large protease-resistant fragment (Mr = 50,000) which contained 34 carbohydrate units (eight N-linked and 26 O-linked) in a segment of 105 amino acids. In addition to these densely glycosylated peptides (one glycosylation site/3 amino acid residues), small glycopeptides with polymannose saccharide units were found in the digests of both proteins. The occurrence of repeating N-acetyllactosamine sequences in the N-linked carbohydrate units of GP-1 and GP-3 was suggested by the composition and size of the oligosaccharides released by hydrazinolysis and was demonstrated by endo-beta-galactosidase treatment. The cleavage products from digestion with this enzyme were identified as NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----3Gal, Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal, Gal beta 1----4GlcNAc beta 1----3Gal, and GlcNAc beta 1----3Gal with the tetrasaccharides constituting the predominant species. The terminal alpha-D-Gal residues accounted for the binding of GP-1 and GP-3 glycopeptides to Bandeiraea simplicifolia I-agarose; concanavalin A-Sepharose affinity chromatography indicated that most of the N-linked carbohydrate units of both glycoproteins contained more than two branches. Difference in the branching on the poly-N-acetyllactosamine sequences of GP-1 and GP-3 was suggested by the finding that only the latter glycoprotein, as well as its glycopeptides, reacted with anti-blood group I antibodies; neither glycoprotein demonstrated blood group i antigenicity. Examination of cultured thyroid follicular cells revealed that both I and i determinants were present at the cell surface.     

Urinary endo-beta-galactosidase capable of depolymerizing polylactosaminoglycans

Human urine was found to contain an endo-beta-galactosidase capable of depolymerizing sulfated and non-sulfated polylactosaminoglycans. Using 0.05 M sodium phosphate buffer, pH 7.0, this enzyme was not retained by DEAE-Sephadex A-50 or concanavalin A-Sepharose. The urinary endo-beta-galactosidase liberated a disaccharide with chromatographic mobility identical to 6-O-sulfo-GlcNAc beta 1----3Gal as one of the major products from keratan sulfates isolated from whale nasal cartilage, bovine cornea, and human costal cartilage. It also liberated GlcNAc beta 1----3 Gal as one of the major oligosaccharides from erythroglycan. The oligosaccharide profiles produced from various keratan sulfates and erythroglycan by the action of urinary endo-beta-galactosidase are quite similar to those produced by Escherichia freundii endo-beta-galactosidase (Nakagawa, H., Yamada, T., Chien, J.-L., Gardas, A., Kitamikado, M., Li, S.-C., and Li, Y.-T. (1980) J. Biol. Chem. 255, 5955-5959). The presence of urinary endo-beta-galactosidase indicates the existence of a new catabolic pathway for polylactosaminoglycans. This pathway involves the cleavage of internal beta-galactosyl linkages of the glycan chain.     

Substrate specificity of endo-beta-galactosidases from Flavobacterium keratolyticus and Escherichia freundii is different from that of Pseudomonas sp

The substrate specificity of endo-beta-galactosidase of Pseudomonas sp. was found to differ from that of Flavobacterium keratolyticus or Escherichia freundii, based on the following experimental results. The endo-beta-galactosidases from these three bacteria released 6-O-sulfo-GlcNAc beta 1-3Gal as one of the major products from keratan sulfates from different sources. In addition to the sulfated disaccharide, Flavobacterium and Escherichia enzymes produced GlcNAc beta 1-3Gal, which is also an integral repeating unit of keratan sulfate, whereas the Pseudomonas enzyme did not release any non-sulfated disaccharide. Tetrasaccharides were prepared from the teleost skin keratan sulfate by digestion with Pseudomonas enzyme followed by gel filtration on Sephadex G-50 chromatography. A part of the tetrasaccharide fraction was hydrolyzed by Flavobacterium enzyme to produce 6-O-sulfo-GlcNAc beta 1-3Gal and GlcNAc beta 1-3Gal, whereas the fraction was completely resistant to retreatment with the Pseudomonas enzyme. Endo-beta-galactosidases from F. keratolyticus and E. freundii hydrolyzed the internal beta-1,4-galactosyl linkage of various neolacto-type glycosphingolipids to produce glucosylceramides. However, these glycosphingolipids were completely resistant to the Pseudomonas enzyme. These findings clearly show that the sulfation on the N-acetylglucosamine adjacent to galactose in the lactosaminoglycans is essential for expression of the Pseudomonas enzyme, but not for that of the Flavobacterium or Escherichia enzyme.     

Embryonal lactosaminoglycan. The structure of branched lactosaminoglycans with novel disialosyl (sialyl alpha 2----9 sialyl) terminals isolated from PA1 human embryonal carcinoma cells

Lactosaminoglycan glycopeptides were isolated from human PA1 embryonal carcinoma cells and their structures were elucidated. The glycopeptides were digested by Escherichia freundii endo-beta-galactosidase before and after the modifications by exoglycosidases. The core glycopeptides and oligosaccharides thus obtained and the intact glycopeptides were analyzed by methylation, fast atom bombardment-mass spectrometry, and high-performance liquid chromatography. Based on these experiments, the structures of PA1 lactosaminoglycans were found to have the following unique features. 1) Three lactosaminoglycan fractions of different molecular weights were isolated by Sephadex G-50 gel filtration. Lactosaminoglycans of the highest molecular weight (GpI) have tetra-antennary cores, those of intermediate molecular weight (GpII) have triantennary cores and those of low molecular weight (GpIII) have triantennary and tetra-antennary cores. 2) GpI is composed of 22-26 lactosaminyl units and 7-9 branched galactose residues, GpII is composed of 16-22 lactosaminyl units and 5-7 branched galactose residues, and GpIII is composed of 12-16 lactosaminyl units and 3-4 branched galactose residues. 3) Each branch is short and is composed of the Gal beta 1----4GlcNAc beta 1----6 structure. 4) Sialic acid is preferentially linked to nonreducing terminal regions and a significant amount of the novel disialosyl structure, NeuNAc alpha 2----9NeuNAc alpha 2----3/6Gal, is present at the terminals of the longer polylactosaminyl side chains. 5) These lactosaminoglycans are carried by cell surface glycoproteins of Mr = 80,000 approximately 120,000, as evidenced by lectin-agarose chromatography.     

Purification and characterization of a novel alpha-mannosidase from Aspergillus saitoi

An alpha-mannosidase differing from 1,2-alpha-mannosidase was found to occur in Aspergillus saitoi. By a series of column chromatographies the enzyme was purified up to 1,000-fold, and its properties were studied in detail. The enzyme preparation, which was practically free from other exoglycosidases, showed a pH optimum of 5.0. In contrast to 1,2-alpha-mannosidase, the enzyme was strongly activated by Ca2+ ions. p-Nitrophenyl alpha-mannopyranoside was not hydrolyzed by the enzyme. Accordingly, the substrate specificity of the new alpha-mannosidase was studied by using a variety of tritium-labeled oligosaccharides. Studies with linear oligosaccharides revealed that the enzyme cleaves the Man alpha 1----3Man linkage more than 10 times faster than the Man alpha 1----6Man and the Man alpha 1----2Man linkages. Furthermore, it cleaves the Man alpha 1----6Man linkage of the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT only after its Man alpha 1----3 residue is removed. Because of this specificity, the enzyme can be used as an effective reagent to discriminate R----Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT from its isomeric counterparts, Man alpha 1----6(R----Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, in which R represents sugars.     

A new endo-beta-galactosidase acting on the Gal beta 1-3Gal linkage of the proteoglycan linkage region.

A new type of endo-beta-galactosidase acting on the linkage region of peptidochondroitin sulfate was isolated from the mid-gut gland of the mollusk Patinopecten. The purification procedure included ammonium sulfate precipitation, Sephacryl S-200HR gel filtration, DEAE-Sephacel chromatography, and TSKgel Phenyl-5PW RP high performance liquid chromatography. The purified enzyme was free from exoglycosidases, sulfatases, and phosphatases. The specificity of the enzyme was as follows. 1) It acted on the internal galactoside linkage of sugar chains; 2) it specifically hydrolyzed the galactosylgalactose (Gal beta 1-3Gal) linkage, but not the galactosylxylose (Gal beta 1-4Xyl) linkage in the linkage region of peptidoglycans; 3) the enzyme activity was unaffected by the type of glycosaminoglycan, chondroitin sulfate, dermatan sulfate or heparan sulfate used as a substrate; 4) keratan sulfate and some oligosaccharides from glycolipid were not degraded by the enzyme. These properties of the endo-beta-galactosidase characterize it as a new endo-beta-galactosidase with unique specificity.     

Endo-beta-D-galactosidases of Bacteroides fragilis and Escherichia freundii hydrolyze linear but not branched oligosaccharide domains of glycolipids of the neolacto series

The specificities of the endo-beta-galactosidases of Bacteroides fragilis and Escherichia freundii towards linear and branched oligosaccharides of the lacto-N-glycosyl series were investigated using as substrates glycolipids containing (a) linear neolactotetra - or hexaosyl sequences, (b) branched biantennary neolactooctaosyl sequences, and (c) triantennary neolactononaor dodecaglycosyl sequences. Glycolipid and oligosaccharide hydrolysis products were identified by tlc and/or paper chromatography. The rate of hydrolysis was assessed in time course experiments in which the oligosaccharides released were quantified as 3H-labeled alditols. The salient observations were as follows. (i) With the substrates thus far tested in the present and a previous study ( Scudder , P., Uemura , K., Dolby , J., Fukuda, M.N., and Feizi , T. (1983) Biochem. J. 213, 485-494), the endo-beta-galactosidases from B. fragilis and E. freudii have indistinguishable specificities. (ii) The beta-galactosidic linkage of the branch point sequence (Formula: see text) is completely resistant to hydrolysis by these enzymes, although the unbranched sequence GlcNAc beta 1-3Gal beta 1-4GlcNAc/Glc is readily cleaved. (iii) At an optimal concentration of detergent, the endo-beta-galactosidase susceptibility of the GlcNAc beta 1-3Gal beta 1-4Glc sequence near the ceramide moiety of branched glycolipids is similar to that of the corresponding sequence in linear glycolipids.     

Glycosphingolipid carriers of carbohydrate antigens of human myeloid cells recognized by monoclonal antibodies

Six monoclonal antibodies with known specificities for the carbohydrate antigens i, X or Y, and seven anti-myeloid antibodies (determinants unknown) selected for their differing reaction patterns with human leucocytes were tested in chromatogram binding assays for reactions with myeloid cell glycolipids derived from normal human granulocytes and chronic myelogenous leukemia cells. Antigenicities were found exclusively on minor glycolipids which were barely or not at all detectable with orcinol-sulphuric acid stain. Among these, a neutral glycosphingolipid bound the anti-i antibody Den and chromatographed as the ceramide octasaccharide, Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer. Several species of neutral glycosphingolipids with six to more than ten monosaccharides were detected which carry the X antigen and others the Y antigen: Gal beta 1----4(Fuc alpha 1----3)GlcNAc and Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc, respectively. In addition, three new types of carbohydrate specificities were detected among the myeloid cell glycolipids. Two were associated with neutral glycolipids: the first, recognised by anti-myeloid antibodies VIM-1 and VIM-10, was expressed on a distinct set of glycolipids with six or more monosaccharides, and the second, recognized by VIM-8, was expressed on glycolipids with more than ten monosaccharides. The third specificity, recognised by the anti-myeloid antibody VIM-2, was expressed on slow migrating sialoglycolipids with backbone structures of the poly-N-acetyllactosamine type that are susceptible to degradation with endo-beta-galactosidase. Thus, we conclude that the i and Y antigens occur among the glycolipids of normal myeloid and chronic myelogenous leukemia cells and that a high proportion of hybridoma antibodies raised against differentiation antigens of myeloid cells are directed at carbohydrate structures.     

Isolation and fine-structure characterization of four monoclonal antibodies reactive with glycoconjugates containing terminal GlcNAc residues: application to aspects of lacto-series tumor antigen biosynthesis.

A series of murine monoclonal antibodies, each reactive with terminal GlcNAc residues expressed on glycolipids, have been isolated after immunization with the glycolipid nLc5 (GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1---- 4Glc beta 1----1Cer). The derived antibodies, designated TE-4, TE-5, TE-6, and TE-7, were tested for binding specificity with a variety of terminal GlcNAc-containing oligosaccharides expressed on glycolipids and glycoproteins. Antibody TE-4 was found to be reactive only with linear and branched terminal GlcNAc beta 1----3Gal containing structures present in lacto-series carbohydrates irrespective of core chain length. The binding specificity of TE-7 was similar except that no reactivity was observed with the short chain structure Lc3 and was weakly reactive with branched agalacto-I structures, suggesting a longer recognition epitope than for the TE-4 antibody. Antibodies TE-5 and TE-6 reacted with terminal GlcNAc beta 1----3Gal structures and as well GlcNAc beta 1----2(6)Man structures present on BSA-oligosaccharide conjugates. Weak binding was also observed with GlcNAc beta 1----6Gal structures with these antibodies. TE-5 was found to be particularly sensitive to low amounts of terminal GlcNAc-containing glycolipids in both solid phase assays and in TLC-immunostaining studies of neutral glycolipids extracted from colonic adenocarcinoma cell lines and tumors. No reactivity was observed with internal GlcNAc residues with any antibody tested. The panel of antibodies was applied to studies of binding to Triton X-100-solubilized fractions from normal mucosal and adenocarcinoma cell lines after desialylation and Smith degradation to expose terminal GlcNAc residues on glycoproteins and glycolipids. Binding of antibodies TE-4 and TE-7 was restricted to adenocarcinoma-derived cell fractions. Application of these antibodies in studies of lacto-series core chain synthesis and in immunodiagnostic procedures after initial treatments to concentrate lacto-series antigens into terminal GlcNAc-containing structures is discussed.     

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.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.     

Developmental change and genetic defect in the carbohydrate structure of band 3 glycoprotein of human erythrocyte membrane.

The chemical structure of Band 3 glycopeptide prepared from erythrocytes of normal adult (blood group OI), umbilical cord vessels (Oi), and an i adult variant who fails to develop I antigen (Oi), has been compared. Band 3 glycopeptide of cord erythrocytes gave, on permethylation analysis, predominantly 2,4,6-tri-O-methylgalactose and 3,6-di-O-methyl-2-N-methylacetamido-2-deoxyglucose, whereas the same glycopeptide of normal adult erythrocytes gave much higher amounts of 2,3,4,6-tetra-O-methylgalactose and 2,4-di-O-methylgalactose as compared with that of cord erythrocytes. Band 3 glycopeptide from i adult showed the same methylation pattern as cord-Band 3 glycopeptide. In accordance with these results, Band 3 glycopeptide of cord and i adult erythrocytes were hydrolyzed to mostly small oligosaccharides by endo-beta-galactosidase from Escherichia freundii, whereas that of normal adult produced a number of oligosaccharides with various sizes which was caused by branched structures. Based on these results and structures of released oligosaccharides, the major developmental change of carbohydrate structure in the erythrocyte membrane is the conversion of linear repeating Galbeta1 leads to 4GlcNAcbeta1 leads to 3Gal to a branched Galbeta 1 leads to 4GlcNAcbeta 1 leads to 3 (R leads to 6) Gal structure. i individual may result from the lack of the branching enzyme.     

Purification and characterization of endo-beta-galactosidase from Escherichia freundii induced by hog gastric mucin

A new procedure for inducing and purifying endo-beta-galactosidase from Escherichia freundii was described. The enzyme was found to be induced with high efficiency in culture medium containing Smith-degraded hog gastric mucin, which was prepared from a commercially available starting material. Endo-beta-galactosidase was then purified by ammonium sulfate fractionation, DEAE-Sephadex chromatography, and affinity chromatography on Sepharose conjugated with the Smith-degraded mucin. The enzyme thus purified by only three steps showed no other glycosidase or protease activities and had higher specific activity compared to the previous method. This new method has a great advantage since the gastric mucin is abundantly available and the efficiency of enzyme production was high without significant induction of exoglycosidase. The hydrolysis of oligosaccharides, glycosphingolipid, and keratansulfate was studied by using this newly purified enzyme. Kinetic data indicate that hydrolyzability of these substrates is largely affected by substrate concentration, enzyme concentration and the structure of substrates. Based on these results, the specificity of E. freundii endo-beta-galactosidase was discussed.     

Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.

The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.01 M, 0.1 M and 0.5 M-N-acetylglucosamine (GlcNAc), to yield weak (WGA-W), intermediate (WGA-I) and strong (WGA-S) affinity fractions. WGA-W and WGA-I contained 'N'- and 'O'-linked oligosaccharides bound to separate polypeptides. WGA-S consisted almost entirely of N-linked components. Our analytical work was concentrated mainly on the N-linked fractions. In these carbohydrates WGA affinity was directly proportional to molecular size but inversely related to N-acetylneuraminic acid content. The binding of the weak-affinity fraction was dependent on N-acetylneuraminic acid, but the intermediate- and strong-binding species interacted with the lectin by N-acetylneuraminic acid-independent mechanisms. N-linked glycopeptides in each WGA-binding class were almost totally degraded to monosaccharides by the concerted action of the exoglycosidases neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. Treatment with endo-beta-galactosidase caused partial depolymerization, yielding some disaccharides but also a heterogeneous population of partially degraded components. These findings suggest that WGA binds with high affinity to internal GlcNAc residues in large oligosaccharides containing repeat sequences of Gal beta(1----4)GlcNAc beta(1----3) (i.e. polylactosamine-type glycans). N-Acetylneuraminic acid is involved only in low-affinity interactions with WGA. WGA therefore displays an intricate pattern of saccharide specificities that can be profitably utilized for structural analysis of complex carbohydrates.     

Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines

Structures of O-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipitation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. O-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestion, Smith degradation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal beta 1----3GalNAcOH, NeuNAc alpha 2----6GalNAcOH, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAcOH, Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3)Gal beta 1----3)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with O-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of O-linked oligosaccharides which differ in core structures as well as in sialylation.     

Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes

Sialylated fucosyl lactosaminoglycan was isolated from human neutrophilic granulocytes and its structure was elucidated. The lactosaminoglycan glycopeptides were digested by endo-beta-galactosidase and "the core portion" and released oligosaccharides were analyzed by permethylation, fast atom bombardment mass spectrometry, and exoglycosidases. In addition, lactosaminoglycan saccharides were obtained by hydrazinolysis and the structures of fractionated sialyl oligosaccharides were analyzed by fast atom bombardment mass spectrometry and permethylation coupled with exoglycosidase treatment. The structure of one of the major components was found to be: (Formula: see text). This structure is unique in that 1) four linear polylactosaminyl side chains are attached to the core portion, 2) the side chain arising from position 4 of 2,4-linked mannose contains one or more alpha 1----3 fucosyl residues, 3) the side chain arising from position 6 of 2,6-linked mannose is terminated with NeuNAc alpha 2----3Gal(Fuc alpha 1----3)GlcNAc, sialyl Lex, and 4) the side chain arising from position 2 of 2,4-linked mannose is terminated with sialic acid through alpha 2----6 linkage.     

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.     

Biosynthesis of blood group i-active polylactosaminoglycans. Partial purification and properties of an UDP-GlcNAc:N-acetyllactosaminide beta 1----3-N-acetylglucosaminyltransferase from Novikoff tumor cell ascites fluid

An N-acetylglucosaminyltransferase has been partially purified from Novikoff tumor cell ascites fluid by affinity chromatography on concanavalin A-Sepharose. The enzyme was obtained in a highly concentrated form after lyophilization. The enzyme appeared to be highly specific for acceptor oligosaccharides and glycoproteins carrying a terminal Gal beta 1----4GlcNAc beta 1----R unit. Characterization of products formed by the enzyme in vitro by methylation analysis and 1H NMR spectroscopy revealed that the enzyme catalyzed the formation of a GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-R sequence. The enzyme therefore could be described as an UDP-GlcNAc:Gal beta 1----4GlcNAc beta-R beta 1----3-N-acetylglucosaminyltransferase. Acceptor specificity studies with oligosaccharides that form part of N-glycans revealed that the presence of a Gal beta 1----4GlcNAc beta 1----2(Gal beta 1----4GlcNAc beta 1----6)Man pentasaccharide in the acceptor structure is a requirement for optimal activity. Studies on the branch specificity of the enzyme showed that the branches of this pentasaccharide structure, when contained in tri- and tetraantennary oligosaccharides, are highly preferred over other branches for attachment of the 1st and 2nd mol of GlcNAc into the acceptor molecule. The enzyme also showed activity toward oligosaccharides related to blood group I- and i-active polylactosaminoglycans. In addition the enzyme together with calf thymus UDP-Gal:GlcNAc beta-R beta 1----4-galactosyltransferase was capable of catalyzing the synthesis of a series of oligomers of N-acetyllactosamine. Competition studies revealed that all acceptors were acted upon by a single enzyme species. It is concluded that the N-acetylglucosaminyltransferase functions in both the initiation and the elongation of polylactosaminoglycan chains of N-glycoproteins and possibly other glycoconjugates.