Search

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel antiadhesive drugs and vaccines. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes binding to thyroglobulin, submaxillar mucin, fetuin, and asialofetuin. The activity is known to be regulated by Mga, a regulator of streptococcal virulence factors, and is carried by the surface-associated streptococcal cysteine protease, SpeB. In the present study, we focused on the high strepadhesin activity in an S. pyogenes strain (NZ131rgg) lacking SpeB expression. By extracting surface proteins from the bacteria, a new strepadhesin protein was identified, and mass spectrometric analysis and database search identified it as a putative pullulanase. The gene was cloned, and the recombinant pullulanase (PulA) exhibited pullulanase and starch hydrolyzing activity, as well as strepadhesin activity. Sequencing of the pulA gene revealed an open reading frame with 3,498 bp encoding a protein of 1,165 amino acids with a predicted molecular mass of 129 kDa. PulA exhibited properties typical for a gram-positive surface protein with a putative signal sequence and LPKTGE cell wall anchoring motif and contained the four highly conserved regions common to pullulanases. Mutant bacteria deficient in PulA expression showed diminished strepadhesin activity on bacterial dot blot assay and reduced adherence to thyroglobulin immobilized on microtiter plates. Thus, S. pyogenes strepadhesin activity is carried by a surface-bound pullulanase, which combines glycoprotein-binding and carbohydrate-degrading activities in the same molecule.

The interaction between Streptococcus pyogenes and the host cell surface is not completely understood. Characterization of the adhesion mechanisms of the bacterium to the host cell surface is needed in order to develop new vaccines and anti-adhesion drugs. The presence of glycoprotein-binding activities among streptococcal strains was investigated. An activity binding to thyroglobulin, fetuin, asialofetuin and mucin but not non-glycosylated proteins was found to be present in the majority of the S. pyogenes strains studied. Cross-inhibition experiments suggested that the glycoproteins share a common structure recognized by the bacteria. The glycoprotein-binding activity was found to be proteinaceous, tightly attached to the bacterial surface and it also mediated the adherence of bacteria to solid surfaces coated with glycoproteins. The activity was found by transposon mutagenesis and complementation to be regulated by the multiple-gene regulator Mga, which has been implicated as a regulator of S. pyogenes virulence factors.

A procedure for the analysis of the monosaccharide composition of glycoproteins and glycolipids by methanolysis and high-performance liquid chromatography with pulsed amperometric detection is described. The advantage over previous methods is the analysis of underivatized methyl glycosides of all glycoconjugate monosaccharides including sialic acid and uronic acid in a single chromatographic step at the subnanomolar level.

To study the properties of protein-bound oligosaccharides in neuronally differentiating cells, two model systems were used: murine N1E-115 and N-18 neuroblastoma cells inducible by serum starvation and rat PC12 pheochromocytoma cells inducible by nerve growth factor. Glycopeptides were prepared from cells metabolically labeled with [3H]glucosamine and analyzed by gel filtration. The properties of the high-molecular-weight glycopeptides were studied using enzymatic digestion with neuraminidase and endo-beta-galactosidase. In contrast to other cell lines analyzed, the neuroblastoma and pheochromocytoma lines contained predominantly glycopeptides completely cleavable with endo-beta-galactosidase, which indicated that they were linear-type poly-N-acetyllactosamine glycans. The proportion of these linear chains in the high-molecular-weight fraction increased during neuronal differentiation in both cell systems. The linear nature of the glycans was also correlated with positive anti-i and negative anti-I reactivity of the cells in immunofluorescence microscopy. Specific cell surface labeling for poly-N-acetyllactosamine glycans and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several glycoprotein components, some of which showed changes during neuronal differentiation. The high proportion of linear poly-N-acetyllactosamine chains in these neuronal cell lines and its increase during neuronal differentiation suggests that these glycans may be a characteristic feature of neuronal or neuronally differentiating cells.

The derivatives obtained by permethylation of unsubstituted 2-amino-2-deoxy-hexitols and of these compounds monosubstituted at C-3, C-4, or C-6, and disubstituted at C-3 and C-6, have been analysed by g.l.c.-m.s. Each derivative can be identified on the basis of retention time and mass spectrum. In methylation analysis, methanolysis gave one derivative of each hexitol, whereas a mixture of products was formed when degradation was effected by acetolysis followed by hydrolysis. An application in the analysis of amino-sugar linkages in alkali-labile O-glycosylic oligosaccharides from rat-brain glycoproteins is described.

The O-trimethylsilyl derivatives of five reference disaccharide alditols composed of a N-acetylhexosaminitol substituted at C-3, C-4 or C-6 with a hexose moiety were studied by gas chromatography mass spectrometry. The data were used in the determination of the linkage in disaccharide alditols derived from rat brain glycoproteins and gangliosides. The O-glycosidically linked carbohydrate units of brain glycoproteins contained two oligosaccharides, alpha-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol and beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, whereas only the latter was obtained from brain gangliosides after partial acid hydrolysis and reduction.

The structurally similar polysialic acid capsules of group B meningococci and Escherichia coli K1 are poor immunogens, and attempts are currently being made to improve their immunogenicity by chemical modifications. An IgG monoclonal antibody to these polysialic acid capsules was used for the study of the presence of structurally similar components in tissue glycoproteins to investigate the reasons for the poor immunogenicity and to evaluate potential dangers in active or passive immunization. By immunoblotting polysialic acid was detected outside the brain in newborn rat kidney, heart, and muscle. It appeared in immunoblots as one component and with similar mobility to the neural cell adhesion molecule N-CAM. Specificity studies of the antibody and endosialidase treatment showed that the polysialic acid glycans detected were composed of chains as long as eight sialic acid residues or more. The polysialic acid was not detected in the corresponding tissues of the adult animal. These results indicate that polysialic acid units are developmentally regulated components of both neural and extraneural tissues, and are bound to components with properties similar to a known cell-adhesion molecule. This together with the presence of low amounts of polysialic acid even in the adult brain, suggests potential hazards in vaccination trials and suggested immunotherapy of meningitis caused by group B meningococci or E. coli K1, which should be carefully assessed.

Glycoproteins of a metastasizing line of B16 mouse melanoma and a poorly metastasizing wheat germ agglutinin-resistant clone were compared. Cell surface proteins and glycoproteins were isotopically labeled by lactoperoxidase-catalyzed iodination and by NaB3H4 reduction after oxidation by periodate or galactose oxidase and subsequently analyzed by gel electrophoresis and autoradiography. Differences were observed in the relative mobilities of several major cell surface components. Binding of 125I-labeled lectins to total cellular proteins on polyacrylamide gels following electrophoresis showed that the major wheat germ agglutinin-binding components of F1 cells were altered in Wa-4 cells. Similar differences were not observed in concanavalin A-binding components. Total cellular glycopeptides were analyzed after separation into structurally distinct classes. The acidic "complex" N-glycosidic glycopeptides from the resistant cells were of lower molecular weight than those from the parent cells. No differences were observed among the mannose-rich N-glycosidic glycopeptides or the alkali-labile O-glycosidic oligosaccharides. Structural studies involving methylation analysis revealed that in the altered glycopeptides of the resistant cells the amount of neuraminic acid residues was decreased to one-half, concomitant with an increase in the amount of fucose. The lost sialic acid was bound to C-3 of galactose, whereas the increased fucose was found on C-3 of 4-substituted N-acetylglucosamine. A possible basis for the glycosylation change and its relation to the biological behavior are discussed.

Glycopeptides with complex carbohydrate structure were isolated from delipidated human erythrocyte membranes after digestion with pronase. The poly(glycosyl)peptides isolated (apparent molecular weight 4000--13000) are suggested to contain 20--70 sugar residues in an alkali-stable saccharide chain linked through N-acetylglucosamine to asparagine. The main sugar components are galactose and N-acetylglucosamine, which together account for 80% of total sugars. That the compounds isolated are glycopeptides and not glycolipids is concluded from the following findings: only trace amounts of glucose and fatty acids were present, and no long-chain (sphingosine) bases could be detected; on the other hand, the amounts of mannose and amino acids found are compatible with an N-glycosidic poly(glycosyl)peptide structure. The structure of the poly(glycosyl)peptides was studied using methylation analysis, exoglycosidase treatments, acid hydrolysis of the native as well as the N-deacetylated glycopeptides, and chromium trioxide oxidation. The studies indicate that the poly(glycosyl)peptides contain a repeating-3)galactosyl(beta1-4)N-acetylglucosaminyl(beta1-structure with branch points at the C-6 of the galactose residues. The saccharide chains are terminated in N-acetylglucosaminyl, galactosyl, N-acetylneuraminyl(alpha2-3 and 6)galactosyl and fucosyl(alpha1-2)galactosyl residues, and they also contain blood group A and B determinants.

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.

Type-specific capsular polysaccharides of group B streptococci show striking structural similarity with the terminal sugar sequences of tissue glycoconjugates. The polysaccharides have been put forward as vaccines against neonatal meningitis. A potential source of hazard in immunization of pregnant mothers may be the presence of the cross-reactive components in adult or fetal tissues. A radioactive ligand binding assay was used to test human immune sera to type Ia, II and III group B streptococcal polysaccharides for binding to tissue-derived glycopeptides showing structural similarities with the streptococcal polysaccharides. Of the 13 glycopeptides of human and rat tissues studied, representing a wide selection of structures known to occur in glycoproteins, only two showed some reactivity with the antisera. The reactivity with human small intestinal glycopeptides could be explained by the presence of natural blood group A antibodies, and was not related to the streptococcal group B antibodies. The basis of the reactivity of a high-molecular-weight glycopeptide from rat kidney with some of the sera was unknown, but was unrelated to the vaccination and clearly could not be inhibited with the streptococcal polysaccharides. Thus, no immunological cross-reactions of the tissue glycopeptides studied could be demonstrated with the group B streptococcal antisera.

In order to identify the molecule components carrying polyglycosyl chains on cell surfaces a two-step enzymatic method was developed. In the first step, the cells were incubated with endo-beta-galactosidase to selectively expose terminal N-acetylglucosamine residues of the lactosamine backbone to the chains. In the second step these residues were glycosylated by incubation with galactosyltransferase and radioactive UDP-galactose. As many as 2.5-3.0 X 10(6) residues per cell could be transferred to human erythrocytes. Negligible amounts of labeling occurred if either of the enzymes was omitted from the incubations. Of the label 80% was found in glycoproteins. In accordance with previous observations, bands 3 and 4.5 were found to be the main carriers of polyglycosyl chains. In human promyelotic HL-60 leukemia cells, a major band of apparent molecular weight of 110000-140000 was labeled. In addition, bands of lower molecular weight which appear to have escaped detection by previous methods were also labeled. The novel labeling method was found to be simple to perform, uses commercially available reagents, and leads to the efficient and highly specific labeling of cell surface molecules carrying polyglycosyl chains.

Polysialosyl chains containing alpha 2-8-linked N-acetylneuraminic acid have been suggested to modulate the biological activity of a neural cell adhesion molecule. Polysialosyl glycopeptides isolated from developing brain were incubated with a bacteriophage containing endosialidase. Sialic acid oligomers up to 7 residues long were liberated both from the glycopeptides and colominic acid. The substrate specificity of the endosialidase was studied with sialic acid oligomers of different sizes prepared from colominic acid. It was found that the endosialidase required the simultaneous presence adjacent to the site of cleavage a minimum of 3 sialic acid residues on the distal side and a minimum of 5 sialic acid residues on the proximal (reducing end) side. From the fragments liberated by the enzyme the existence of polysialic acid chains up to at least 12 residues long in the glycopeptides were concluded. This was also supported by the interaction of the glycopeptides with a meningococcal group B polysaccharide antiserum, which was found to require 10 residues or more for binding. The results indicate that the brain polysialosyl glycopeptides contain a long polysialic acid segment, which is also specifically needed for certain molecular interactions. The implications of the findings for the biological properties of the neural cell adhesion molecule are discussed.

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.

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.

Dopamine beta-hydroxylase present in the soluble matrix of bovine adrenal medullary chromaffin granules contains biantennary complex oligosaccharides and high-mannose oligosaccharides in a molar ratio of approximately 2:1. The high-mannose oligosaccharides contain an average of six mannose residues. The largest biantennary oligosaccharides (40% of the total) have two complete peripheral branches consisting of sialic acid-galactose-N-acetylglucosamine, but an equal proportion lack sialic acid on one branch and the remainder lack N-acetylglucosamine and/or galactose. Affinity chromatography on lentil lectin-agarose demonstrated that 84% of the dopamine beta-hydroxylase biantennary oligosaccharides are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. Based on carbohydrate concentration and the proportions of biantennary and high-mannose oligosaccharides, it would appear that the four dopamine beta-hydroxylase subunits of Mr congruent to 75,000 are not identical with respect to their oligosaccharide moieties. In chromaffin granule membranes, high-mannose and biantennary oligosaccharides comprise 20 and 35%, respectively, of the glycoprotein carbohydrate. Almost 40% is present in the form of large complex oligosaccharides with three or more antennas, less than 3% of which have both a core fucose residue and a 2,6-substituted alpha-linked mannose residue. Chromaffin granule membranes also contain a small proportion (approximately 6%) of O-glycosidically linked glycoprotein oligosaccharides which are predominantly monosialyl derivatives of galactosyl-N-acetylgalactosamine. The ratio of N-acetyl- to N-glycolylneuraminic acid in dopamine beta-hydroxylase and the glycoproteins of chromaffin granule membranes is approximately 1.5:1, which is within the same range as that previously found in membrane gangliosides and in the chromogranins isolated from the soluble granule matrix.

The O-glycosidically linked carbohydrate units of rat brain glycopeptides were released as reduced oligosaccharides with NaOH/NaBH4 treatment. Five oligosaccharides were isolated using gel filtration, ion-exchange chromatography and preparative thin-layer chromatography. Studies employing periodate oxidation, methylation analysis, chromium trixide oxidation and gas-liquid chromatography-mass spectrometry indicated the following structures: (I) alpha-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (II) beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (III) N-acetylneuraminyl-[beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol], (IV) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol and (V) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)[N-acetylneuraminyl-(2 leads to 6)]-N-acetylgalactosaminitol.

We have identified structures in nervous tissue glycoproteins that are novel for glycoproteins in general or enriched in nervous tissue or cells of neural origin. These include: (alpha 2-8)-linked polysialic acid units, the linear form of poly-N-acetyllactosamine glycans, the sialylated X antigen determinant NeuAc(alpha 2-3)-Gal(beta 1-4) [Fuc(alpha 1-3)]GlcNAc, a series of Man-O-Ser(Thr)-linked glycans, and the O-glycosidically linked disaccharide unit Gal(alpha 1-3)GalNAc. The polysialic and poly-N-acetyllactosamine glycans are also developmentally regulated. The polysialic acid units in the cell adhesion molecule N-CAM. The poly-N-acetyllactosamine units occur in the adhesion molecule NILE (which is immunologically similar to Ng-CAM and L1) and in some other components revealed by a cell surface-labelling method specific for these glycans. The mannose-linked glycans occur in a chondroitin sulphate proteoglycan involved in neuron-glia interactions. Other biological interactions of the carbohydrates include their serving as bacterial receptors in meningitis, their serving as models for molecular mimicry by the capsules of meningitis-causing bacteria, and the role of some structures as antigens in autoimmune conditions. At the molecular level, two types of mechanisms are suggested for the glycans in molecular interactions: they may function either as mediators of interactions by serving as specific recognition ligands, or as modulators of the interactions determined by polypeptides or other molecules.