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Human embryonic skin fibroblasts attach and spread on surfaces on which a fucosyltransferase purified from human milk has been immobilized. The adhesion-enhancing effect of the transferase involves specific interactions of the enzyme surface with the cell surface carbohydrate acceptors, as suggested by the following findings. About 80% of human embryonic skin fibroblasts attach and spread in 1 hr on fucosyltransferase surfaces; in contrast, bovine serum albumin, fetuin, asialofetuin, and asialotransferrin surfaces fail to enhance adhesion. The adhesion-mediating activity of the transferase is destroyed by alkylation of the sulfhydryl groups or by heating. The adhesion on fucosyltransferase surfaces is inhibited by glycoprotein, glycolipid, and oligosaccharide acceptors containing the sugar sequence galactosyl-(beta 1 leads to 4)-N-acetylglucosamine, in agreement with the substrate specificity of the enzyme. The results suggest that glycosyltransferases are able to stimulate cell adhesion in a manner similar to that proposed for lectins.

Carbohydrate units of cell-surface glycoproteins of mouse B- and T-lymphocytes, labelled in their sialic acid residues by the periodate/NaB3H4 method and in their galactose residues by the galactose oxidase/NaB3H4 method after neuraminidase treatment, have been studied. Glycopeptides were prepared from the labelled cells by Pronase digestion and fractionated by concanavalin A affinity chromatography into two fractions (A and B). Alkali-labile oligosaccharides were isolated after mild NaOH/NaBH4 treatment by gel filtration. The alkali-labile oligosaccharides were further analysed by t.l.c. To study the relative proportion of neutral mannose-rich carbohydrate units (fraction C) in lymphocyte glycoproteins, glycopeptides were also prepared from unlabelled cells and subjected to concanavalin A affinity chromatography after N-[3H]acetylation of their peptide moiety. The major alkali-labile oligosaccharide component of both cell types was identified as galactosyl-(beta 1 leads to 3)-N-acetylgalactosaminitol. T-Lymphocytes were characterized by a high proportion of this oligosaccharide and a lower proportion of alkali-stable fraction A glycopeptides, whereas the opposite was observed for B-lymphocytes. The relative proportions of the concanavalin A-binding fractions B and C were similar in both cell types. The differences observed may correlate with the different surface properties of B- and T-lymphocytes.

Streptococcus suis causes meningitis, sepsis, and other serious infections in newborn and young pigs and in adult humans. The Gal alpha 1-4Gal-binding adhesin of S. suis was purified to homogeneity by ultrasonic treatment, fractional ammonium sulfate precipitation, and preparative polyacrylamide gel electrophoresis. Pigeon ovomucoid, a glycoprotein with Gal alpha 1-4Gal terminals, was used to detect the adhesin by blotting. The purified adhesin appeared as single band of an apparent size of 18 kDa and of a pI of 6.4; no disulfide bridges were present. The amount of adhesin as revealed by pigeon ovomucoid binding correlated with the hemagglutination activity of different S. suis strains. The purified adhesin bound to latex particles induced hemagglutination which was specifically inhibited with the same inhibitors as hemagglutination by the intact bacteria, thus demonstrating that the purified protein was the Gal alpha 1-4Gal-recognizing adhesin of S. suis. Two adhesin variants (PN and PO) with differing Gal alpha 1-4Gal binding specificity had the similar electrophoretic mobilities and the same N-terminal peptide sequences, indicating that they were closely related. This represents the first isolation of an adhesin with well-defined cell surface carbohydrate binding activity from Gram-positive bacteria associated with meningitis.

In the search for the biochemical basis of the control of glycosylation of cell surface carbohydrates, revertant clones were isolated from previously characterized wheat germ agglutinin-resistant clones of B16 mouse melanoma cells by selection for resistance to Lotus tetragonolobus lectin or to ricin. Comparison of the wheat germ agglutinin-resistant clones with the parent and revertant clones indicated that this phenotype was correlated with an increased sensitivity to the Lotus lectin, a 60- to 70-fold increase in alpha 1 leads to 3 fucosyltransferase activity and a decreased sialic acid content of the N-glycosidic chains of glycoproteins. The results suggest a novel type of control mechanism for lectin resistance, an increase in a glycosyltransferase activity. The presence of alpha 1 leads to 3 bound fucose on N-acetylglucosamine residues would interfere with the addition of sialic acid by alpha 2 leads to 3 linkages to galactose residues in the carbohydrate units, and this change could explain the resistance to wheat germ agglutinin and the increased sensitivity to the Lotus lectin. A change in a regulatory gene for the fucosyltransferase as a possible primary cause for the changed phenotype is discussed.

A novel type of glycopeptides comprising approximately 10% of the total protein-bound neuraminic acid in developing rat brain have been isolated and characterized. The glycopeptides displayed unique properties including precipitation with cetylpyridinium chloride, strong binding to anion exchange column, and large apparent size in gel filtration. Structural studies including methylation analysis, as well as gel filtration experiments with native and desialylated glycopeptides, suggested that they were composed of a core similar to that of normal tri- and tetraantennary N-glycosidic glycopeptides, with outer branches of the general structure (NeuAc alpha 2-8)nNeuAc alpha 2-3Gal–. The total number of sialic acid residues varied from 8 to at least 12. Similar glycopeptides were not observed in liver or kidney of the young rat or in the brain of the adult animal. These observations suggest that the polysialosylated glycopeptides represent a class of developmentally regulated carbohydrate structures characteristic of developing brain tissue.

The erythrocyte receptors for S-fimbriated Escherichia coli, which causes sepsis and meningitis in newborn infants, were investigated. Neuraminidase and trypsin treatments of erythrocytes abolished the hemagglutination ability of the bacteria. To identify the receptor glycoproteins, we separated erythrocyte membrane proteins by gel electrophoresis, blotted them to nitrocellulose, and incubated them with 125I-labeled bacteria. The only bacterium-binding bands identified corresponded to glycophorin A dimer and monomer, and the binding was abolished by neuraminidase treatment of the blot. Radiolabeled bacteria also bound to purified glycophorin A adsorbed to polyvinyl chloride microwells, and the binding was inhibited by other sialoglycoproteins and isolated sialyloligosaccharides containing the NeuAc alpha 2-3Gal sequence. Oligosaccharides which contain the NeuAc alpha 2-3Gal beta 1-3GalNAc and NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc sequence and which are identical to the O-linked saccharides of glycophorin A were twofold more effective inhibitors of binding than were other oligosaccharides containing the NeuAc alpha 2-3Gal sequence. The replacement of sialic acid in asialoerythrocytes with a purified Gal beta 1-3GalNAc alpha 2-3 sialyltransferase, which forms the O-linked NeuAc alpha 2-3Gal beta 1-3GalNAc sequence in asialoglycophorins, restored bacterial hemagglutination. These results indicated that the major erythrocyte receptor for S-fimbriated E. coli is the NeuAc alpha 2-3Gal beta 1-3GalNAc sequence of the O-linked oligosaccharide chains of glycophorin A.

The neural cell adhesion molecule (N-CAM) of rodents comprises three distinct proteins of Mr 180,000, 140,000, and 120,000 (designated N-CAM-180, -140, and -120). They are expressed in different proportions by different tissues and cell types. but the individual contribution of each form to cell adhesion is presently unknown. Previous studies have shown that the two N-CAM species of higher relative molecular mass span the membrane whereas N-CAM-120 lacks a transmembrane domain and can be released from the cell surface by phosphatidylinositol-specific phospholipase C. In this report, we provided evidence that N-CAM-120 contained covalently bound phosphatidylinositol and studied N-CAM-120 from its biosynthesis to its membrane insertion and finally to its release from the cell surface. Evidence was presented showing that the lipid tail of N-CAM-120 contained ethanolamine as is the case for other lipid-linked molecules. The phospholipid anchor was attached to the protein during the first minutes after completion of the polypeptide chain. This process took place in the endoplasmic reticulum as judged from endoglycosidase H digestion experiments. Immediately after a 2-min pulse with [35S]methionine, we detected also a short-lived precursor that had not yet acquired the lipid tail. Pulse-chase studies established that N-CAM-120 was transported to the cell surface from which it was slowly released into the extracellular milieu. The molecules recovered in the incubation medium appeared to have lost all of their bound fatty acid but only around half of the ethanolamine. Upon fractionation of brain tissue, approximately 75% of N-CAM-120 was recovered with a membrane fraction and approximately 25% in a membrane-free supernatant. A small proportion (approximately 6%) was found to be resistant to extraction by non-ionic detergent. A major posttranslational modification of N-CAM is polysialylation. Our results showed that also N-CAM-120 was polysialylated in the young postnatal brain and released in this form from cultured cerebellar cells. The presence of N-CAM in a form that can be released from the cell surface and accumulates in the extracellular fluid suggests a novel mechanism by which N-CAM-mediated adhesion may be modulated.

A novel type of N-linked glycopeptides representing a major part of the glycans in human small intestinal epithelial cells from blood group A and O individuals were isolated by gel filtrations and affinity chromatography on concanavalin A-Sepharose and Bandeiraea simplicifolialectin I-Sepharose. Sugar composition, methylation analysis, 1H NMR spectroscopy of the underivatized glycopeptides and FAB-mass spectrometry and electron impact-mass spectrometry of the permethylated glycopeptides indicated a tri- and tetra-antennary structure containing an intersecting N-acetylglucosamine and an α (1→6)-linked fucose residue in the core unit for the majority of the glycans. In contrast to most glycopeptides of other sources, the intestinal glycopeptides were devoid of sialic acid, but contained 6–7 residues of fucose. The outer branches contained the following structures:Fuc α 1–2Gal β 1–3GleNAc β 1- (H type 1)Fuc α 1–2Gal β 1–4GleNAc β 1- (H type 2)Gal β 1–4 (Fuc α 1–3)GlcNAc β 1- (X)Fuc α 1–2Gal β 1–4(Fuc α 1–3)GleNAc β 1- (Y)GalNAc α 1–3(Fuc α 1–2)Gal β 1–3GleNAc β 1- (A type 1)GalNAc α 1–3(Fuc α 1–2)Gal β 1–4GleNAc β 1- (monofucosyl A type 2)GalNAc α 1–3(Fuc α 1–2)Gal β 1–4(Fucα1–3)GleNAc β 1- (difucosyl A type 2)GalNAc α 1–3(Fuc α 1–2)Gal β 1–4 (Fuc α 1–3)GlcNAc β 1- (trifucosyl A type 2)The blood group determinant structures were mainly of type 2, whereas glycolipids from the same cells contained mainly type 1 determinants. The polyfucosylated glycans represent a novel type of blood group active glycopeptides. The unique properties of the small intestinal glycopeptides as compared with glycopeptides of other tissue sources may be correlated with the specialized functional properties of the small intestinal epithelial cells.

A total of 378 streptococcal isolates of Lancefield groups B, C, D and G were tested for their ability to hemagglutinate untreated, sialidase-treated, and endo-beta-galactosidase-treated human erythrocytes. Of the 43 strains showing positive hemagglutination, 9 were inhibitable with neutral monosaccharides. Four strains were inhibited with galactose and N-acetylgalactosamine, whereas five were inhibited with galactose only. A third, sialic acid-specific adhesion activity was suggested for two additional strains on the basis of their agglutination of native and endo-beta-galactosidase-treated but not sialidase-treated erythrocytes. All the sugar-specific agglutination activities detected were confined to Streptococcus suis strains of group D streptococci, whereas streptococci of other groups did not exhibit these types of hemagglutination activities. The adhesins were sensitive to proteases and heat treatment, which indicates that they were proteins. The hemagglutinating isolates of S. suis originated from pig brain and lung, human brain, and the tonsils of healthy pigs. No clear correlation with a particular serotype was observed. These results demonstrate the occurrence of unique sugar-specific adherence activities in S. suis, an important pig pathogen with occasional human pathogenicity.

Sixty-three Escherichia coli strains isolated from neonatal sepsis or meningitis were studied and compared with previous data on fecal or urinary pyelonephritis-associated isolates from children. Characteristics significantly associated with neonatal infection were capsular type K1 (54%), O group 18 (27%), rough-type lipopolysaccharide together with K1 capsule (19%), and S fimbriae (29%). Within the neonatal infection group, the K1 capsule and rough lipopolysaccharide were most common among the youngest infants (0 to 21 days old) and in meningitis. Hemolysin production, P fimbriae, and X adhesions (adhesions not identifiable as type 1, P, or S) were significantly more common in the two materials from infections as compared with the fecal isolates. One large clone of 11 strains (O18:K1:H7, with both type 1 and S fimbriae) and three smaller ones (O7:K1:H1 and O6:K2:H1, both with type 1 and P fimbriae and X adhesions; and R:K1:H33 with no adhesions) were identified among the strains from neonatal infections. Only O6:K2:H1 strains were also common among the strains from pyelonephritis.

Species-specific cell reaggregation in the marine sponge Microciona prolifera is mediated by an adhesion proteoglycan. Two interactions are involved in the process: a Ca(2+)-dependent homophilic binding between proteoglycan molecules and a Ca(2+)-independent binding between the molecule and cells. Both interactions are mediated by the glycan moieties of the proteoglycan. The interaction of the proteoglycan with itself has been characterized as a carbohydrate-carbohydrate interaction of multiple low affinity sites. The monoclonal antibodies Block 1 and Block 2 raised against the purified aggregation proteoglycan and selected for inhibition of aggregation bind to these glycans. In a previous report the structure, [formula: see text] was assigned to the oligosaccharide reacting with Block 1 antibody (Spillmann, D., Hård, K., Thomas-Oates, J., Vliegenthart, J. F. G., Misevic, G., Burger, M. M., and Finne, J. (1993) J. Biol. Chem. 268, 13378-13387). By the technique of attaching the water-soluble acid-degraded fragments to a lipid carrier for immunochemical detection and by chemical, enzymatic and spectroscopic methods the structure, [formula: see text] was assigned to the oligosaccharide reacting with the aggregation-blocking monoclonal antibody Block 2. The structure, [formula: see text] was assigned to a major nonreactive oligosaccharide, which outlined the molecular requirements of antibody binding of the two aggregation-associated epitopes. These data demonstrate that two different functional sites with distinct structural characteristics and antibody reactivities are involved in the reaggregation of sponge cells, a model of carbohydrate-carbohydrate-mediated cell interactions.

The cleavage specificities of seven bacteriophage endosialidases degrading the alpha 2-8-linked polysialic acid common to bacterial polysaccharides and to the cell adhesion molecule N-CAM were investigated. The bacteriophages studied represented five different phenotypic groups by protein and DNA fragment analysis and two different morphology groups by electron microscopy. Characterization of the fragments arising from the native or chemically modified substrates of different sizes showed that cleavage specificity was influenced by enzyme concentration. At the initial phase of degradation, at concentrations ranging from 20- to 100-fold, the minimum substrate size was an oligomer of eight (in one case, nine) sialic acid units that was preferably cleaved at the same position. Under exhaustive conditions, the oligomers were degraded further, and each enzyme type had its own specificity. The similar initial cleavage of polysialic acid by endosialidases associated with phages of different properties and morphology suggests a conserved mechanism of enzyme-substrate interaction. This mechanism may be conformationally determined and related to the specific properties of polysialic acid in other molecular interactions.

Methods were developed for the polyacrylamide gel electrophoretic analysis of capsular polysaccharides of bacteria with Escherichia coli K1 as a model. Conditions were determined for the rapid and gentle extraction of the K1 polysaccharide by incubation of the bacteria in a volatile buffer and for the subsequent removal of the putative phospholipid moiety attached to the reducing end of the polysaccharide. Detection of the polysaccharides after gel electrophoresis was carried out by fluorography of samples labeled by sodium borotritiide reduction or by combined alcian blue and silver staining. The smallest components could be detected only by fluorography, owing to diffusion during staining. Components of the E. coli K1 polysialic acid capsule ranging from monomers to 80 sialic-acid-unit-containing polymers could be separated as distinct bands in a ladderlike pattern. A maximum chain length of 160 to 230 sialyl residues was estimated for the bulk of the K1 polysaccharide from the nearly linear reciprocal relationship between the logarithm of the molecular size and the distance of migration. Gel electrophoresis of capsular polysaccharides of other bacterial species revealed different electrophoretic mobilities for each polysaccharide, with a ladderlike pattern displayed by the fastest-moving components. There are many potential applications of this facile method for the determination of the sizes of molecules present in a polydisperse polysaccharide sample. When combined with the simple method for the isolation of the capsule, as in the case of the K1 capsule, it provides an efficient tool for the characterization and comparison of the capsular polysaccharides of bacteria.

Purified S fimbriae and an Escherichia coli strain carrying the recombinant plasmid pANN801-4 that encodes S fimbriae were tested for adhesion to frozen sections of human kidney. The fimbriae and the bacteria bound to the same tissue domains, and in both cases the binding was specifically inhibited by the receptor analog of S fimbria, sialyl(alpha 2-3)lactose. S fimbriae bound specifically to the epithelial elements in the kidneys; to the epithelial cells of proximal and distal tubules as well as of the collecting ducts and to the visceral and parietal glomerular epithelium. In addition, they bound to the vascular endothelium of glomeruli and of the renal interstitium. No binding to connective tissue elements was observed. The results suggest that the biological function of S fimbriae is to mediate the adhesion of E. coli to human epithelial and vascular endothelial cells.

The occurrence of phosphate-containing sialyl oligosaccharides in bovine colostrum was investigated. Two major sialyl oligosaccharide phosphates were identified, one of which was structurally similar to the previously characterized sialyl oligosaccharide 1-phosphates of human urine. The second sialyl oligosaccharide phosphate of bovine colostrum was found to be of a novel type. Structural studies including monosaccharide and phosphate analysis, glycosidase and phosphatase treatments, methylation analysis, and periodate treatment indicated the structure of this compound to be NeuAc alpha 2-6Gal beta 1-4GlcNAc-6-P. This provides the first evidence for the occurrence of N-acetylglucosamine 6-phosphate as an integral component in complex carbohydrates.

Fimbriae recognizing sialyl galactosides (S fimbriae) were purified from an Escherichia coli strain. The S fimbriae were morphologically identical to type 1 and P fimbriae of E. coli and showed a hemagglutination that was abolished when erythrocytes were treated with neuraminidase. Hemagglutination by the purified fimbriae was inhibited by orosomucoid but not by its desialylated derivative. Of the oligosaccharides tested, sialyl-(alpha 2-3)-lactose and sialyl-(alpha 2-3)-N-acetyllactosamine had the strongest inhibitory activities. It was concluded that S fimbriae have the strongest affinity for (alpha 2-3)-linked sialyl galactosides. In the enzyme-linked immunosorbent assay, the hyperimmune serum to the S fimbriae reacted strongly with the homologous antigen but not with type 1, P, or nonhemagglutinating KS71C fimbriae of E. coli. Analogously, the hyperimmune sera to the other E. coli fimbriae did not react with the purified S fimbriae. The immunoprecipitation assay showed that S fimbriae on different E. coli serotypes shared immunological cross-reactivity.

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular weight aggregation factor (MAF) and is based on two functional properties, a Ca2+-independent cell binding activity and a Ca2+-dependent factor-factor self-interaction. Monoclonal antibodies were prepared against purified MAF, and one clone was characterized which selectively inhibited the MAF-MAF association activity. Binding of the blocking antibody (Block 1) to MAF demonstrated that this epitope structure was present in 1100 copies per one MAF molecule of Mr = 2 × 10(7). Such blocking antibodies precipitated a small molecular weight protein-free glycan fraction prepared from MAF by Pronase digestion, thus indicating that the highly repetitive epitope is located in the carbohydrate portion of the molecule. Since the inhibitory activity of the Block 1 antibody could only be achieved when most of the sites were occupied by Fab fragments of this antibody, the self-association of MAF seemed to be based on the polyvalency of the carbohydrate determinants. The affinity of the protein-free glycans was very low as shown by the fact that they did not display any measurable self or MAT binding activity in their monomeric form. After cross-linking them with glutaraldehyde into polymers of the size of MAF, however, the self-interaction could be reconstituted. Thus, the MAF-MAF association activity, which is needed for cell aggregation in sponges, seems to be based on multiple low affinity carbohydrate-carbohydrate interactions, which is different from most interactions mediated by adhesion molecules characterized so far.

The chondroitin sulfate proteoglycan of rat brain was digested with Pronase, and after removal of glycosaminoglycans, the resulting glycopeptides were treated with alkaline borohydride to release O-glycosidically linked oligosaccharides. These were fractionated by ion exchange chromatography, gel filtration, and preparative thin layer chromatography, and their structural properties were studied by specific enzymatic degradations, methylation analysis, and gas-liquid chromatography-mass spectrometry of disaccharides as their trimethylsilylated and permethylated derivatives. In addition to the previously characterized N-acetyl-galactosamine-linked oligosaccharides and neutral mannitol-containing oligosaccharides [GlcNAc(beta 1-3) Manol and Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Manol] (where Fuc is fucose), we have now identified the sialylated tetrasaccharide NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc (beta 1-3)Manol, which accounts for approximately 20% of the mannitol-containing oligosaccharides. The proteoglycan also contains mannose-linked keratan sulfate chains (with a molecular size of 3,000 to 10,000 Da) composed of disaccharide repeating units consisting of Gal(beta 1-4)GlcNAc-6-O-SO4(beta 1-3), with a small proportion of branch points at C-6 of galactose residues. There is approximately one keratan sulfate chain per four chondroitin sulfate chains of 18,000-19,000 Da. After alkaline borohydride treatment of the neutral and monosialyl glycopeptide fractions, the combined decrease in mannose and N-acetylgalactosamine was very close to the observed destruction of serine + threonine and was accompanied by an equimolar increase in alanine and alpha-aminobutyric acid. One half of the mannose was destroyed by alkaline borohydride treatment of the glycopeptides and stoichiometrically converted to mannitol, while there were only small changes in the relative amounts of the other sugars and amino acids. The data demonstrate that over half of the carbohydrate-peptide linkages in the proteoglycan are of the mannosyl-O-serine/threonine type.

We report the presence of alpha(2----8)-linked polysialic acid as detected with a monoclonal antibody outside the nervous tissue in the postnatal developing rat kidney. By immunoblot analysis, the reactivity was confined to a broad band of apparent molecular mass 200-250 kDa. By immunohistochemistry, the polysialic acid units were found throughout the ureteric bud-derived collecting duct system. In developing nephrons derived from the metanephrogenic mesenchyme, polysialic acid units were only regionally and transiently expressed. Furthermore, the expression of polysialic acid units was developmentally regulated, as evidenced by their gradual disappearance concomitant with postnatal kidney maturation. These findings suggest a possible role for polysialic acid units in cell-cell contact-mediated renal differentiation processes.