Synthetic antigens as immunogens: Part IV. Specificity of antisera developed to Gal beta 1-3(GlcNAc beta 1-6)GalNAc and GlcNAc beta 1-6GalNAc.

Antisera to the chemically synthesized trisaccharide, Gal beta 1-3(GlcNAc beta 1-6)GalNAc, and disaccharide, GlcNAc beta 1-6GalNAc, were developed using the diazophenyl bovine serum albumin derivatives. The binding specificity of the antisera were analyzed by enzyme immunoassays with structurally related, chemically synthesized oligosaccharides. The anti-trisaccharide antibody showed no reactivity to T antigenic structures which bear the Gal beta 1-3GalNAc moiety. The immunodominant area of the Gal beta 1-3(GlcNAc beta 1-6)GalNAc was contained in the GlcNAc beta 1-6GalNAc region of the molecule. The reactivity pattern of the anti-trisaccharide antibody, although directed to the disaccharide, did not exactly duplicate the reactivity pattern of the anti-disaccharide.     

Differences in the binding specificities of Pseudomonas aeruginosa M35 and Escherichia coli C600 for lipid-linked oligosaccharides with lactose-related core regions.

Membrane glycolipids contain the lactose sequence (galactose linked to glucose), and the oligosaccharide is variously extended such that there is a cell-type-specific repertoire. In this study, binding of Pseudomonas aeruginosa M35 to lipid-linked lactose (Gal beta 1-4Glc [structure 1]), lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc [structure 2]), lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc [structure 3]), and asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc [structure 4]) was evaluated and compared with binding of Escherichia coli C600 to these compounds. Oligosaccharides were linked to the lipid phosphatidylethanolamine dipalmitoate, and the resulting neoglycolipids were resolved on thin-layer chromatograms or coated onto plastic microtiter wells. Lipid-linked structures 1 to 4 were bound by P. aeruginosa and E. coli in the chromatogram assay, but only structure 4 was bound in the microtiter well assay. As shown previously for E. coli binding to lipid-linked structures 1 to 3, binding to lipid-linked structure 4 was not inhibited with oligosaccharide, indicating a requirement for lipid and oligosaccharide. With few exceptions, sialylation and fucosylation of structures 1 to 4 resulted in impaired or abolished binding. Comparisons of binding intensities in the chromatogram assay indicated that recognition by P. aeruginosa and recognition by E. coli are not identical. Presence of the additional disaccharide unit, as in structure 2, resulted in enhanced binding of P. aeruginosa but diminished binding of E. coli relative to lactose binding; fucosylation at galactose of lactose resulted in markedly diminished binding of P. aeruginosa only. In the microtiter well assay, binding of E. coli to asialo GM1 was much weaker than P. aeruginosa binding. The saccharide-plus-lipid-dependent adhesion may be an important factor in increased susceptibility to infection of epithelia already damaged by microbial and chemical agents; the differing strengths of adhesion to the structural variants may relate to tissue tropism.     

Synthetic Antigens as Immunogens: Part IV. Specificity of Antisera Developed to Gal β1-3(GlcNAc β1-6)GalNAc and GlcNAc βl-6GalNAc

Antisera to the chemically synthesized trisaccharide, Gal β1-3(GlcNAc β1-6)GalNAc, and disaccharide, GlcNAc β1-6GalNAc, were developed using the diazophenyl bovine serum albumin derivatives. The binding specificity of the antisera were analyzed by enzyme immunoassays with structurally related, chemically synthesized oligosaccharides. The anti-trisaccharide antibody showed no reactivity to T antigenic structures which bear the Gal pl-3GalNAc moiety. The immunodominant area of the Gral β1-3(GlcNAc β 1-6)GalNAc was contained in the GlcNAc β1-6GalNAc region of the molecule. The reactivity pattern of the anti-trisaccharide antibody, although directed to the disaccharide, did not exactly duplicate the reactivity pattern of the anti-disaccharide.     

Eight lipooligosaccharides of Neisseria meningitidis react with a monoclonal antibody which binds lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc).

Eight of 12 serologically different lipooligosaccharides (LOS) of Neisseria meningitidis bound a mouse monoclonal antibody (anti-My-28) that recognizes lacto-N-neotetraose (LNnT) (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc). Among the 12 LOS immunotypes, types 2, 3, 4, 7, 8, and 9 exhibited strong binding; types 5 and 10 were moderate; and types 1, 6, 11, and 12 were negative as measured by enzyme-linked immunosorbent assays, immunodot assays, and immunoblot assays. If an LOS showed multiple components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the antibody-reactive epitope was expressed on the larger major component, of which the molecular weight was estimated to be 4,000 for most types. The expression of the reactive epitope on the LOS was influenced by the growth medium, and the epitope could be masked by sialylation when N. meningitidis was grown in tryptic soy broth. N-Acetyllactosamine inhibited the binding of the antibody to all eight reactive LOS. The antibody binding to a representative LOS was best inhibited by LNnT and next by N-acetyllactosamine but was not inhibited by lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc). These results suggest that the LNnT sequence is present in 8 of 12 immunotype LOS. The presence of the LNnT sequence, a structure expressed by a variety of human cells, in the LOS may play a role in the virulence of N. meningitidis by enabling the organism to evade host immune defenses.     

Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc.

This study was undertaken to determine whether a binding site for Clostridium difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin. Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C. difficile. The finding that binding activity could not be destroyed by heat indicated that a carbohydrate moiety might be involved. We therefore examined erythrocytes from various animal species for binding activity since erythrocytes provide a variety of carbohydrate sequences on their cell surfaces. Only rabbit erythrocytes bound the toxin, and the cells agglutinated. A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C. difficile toxin A was used to compare binding of the toxin to hamster BBMs, rabbit erythrocytes, and BBMs from rats, which are less susceptible to the action of C. difficile toxin A than hamsters. Results of this comparison indicated the following order of toxin-binding frequency: rabbit erythrocytes greater than hamster BBMs greater than rat BBMs. Binding of toxin A to hamster BBMs at 37 degrees C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4 degrees C. A similar binding phenomenon was observed with rabbit erythrocytes. Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal alpha-linked galactose. Treatment of both membrane types with alpha-galactosidase destroyed the binding activity. The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells. Toxin A did not bind to human erythrocytes from blood group A, B, or O donors. However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes, which possess the blood group B structure Gal alpha 1-3[Fuc alpha 1-2]Gal beta 1-4GlcNAc-R, bound the toxin. This indicated that toxin A was likely binding to Gal alpha 1-3Gal beta 1-4GlcNAc, a carbohydrate sequence also found on calf thyroglobulin and rabbit erythrocytes. All of the results indicate that hamster BBMs contain a carbohydrate-binding site for toxin A that has at least a Gal alpha 1-3Gal beta 1-4GlcNAc nonreducing terminal sequence.     

The K88 fimbrial adhesin of enterotoxigenic Escherichia coli binds to beta 1-linked galactosyl residues in glycosphingolipids.

The K88 fimbrial adhesin enables certain strains of enterotoxigenic Escherichia coli to adhere to the porcine small intestine. In this study, the ability of the K88 adhesin to bind to glycosphingolipids was monitored by modified enzyme-linked immunosorbent assay and thin-layer chromatography overlay binding analysis. The binding of the K88 adhesin to glycosphingolipid-coated microtiter plates was saturable, with 50% maximal binding occurring with gangliotriaosylceramide, gangliotetraosylceramide, and lactosylceramide at 67 +/- 21, 117 +/- 21, and 73 +/- 22 pM, respectively. Thin-layer chromatography overlay binding analysis demonstrated that serotype O8:K87:K88ab:H19 E. coli bound to hydroxylated galactosylceramide, gangliotriaocylceramide, gangliotetraosylceramide, and lactosylceramide but not to globotriaosylceramide, nonhydroxylated galactosylceramide, glucosides, glucosylceramide, or a mixture of ceramides. The K88 adhesin did not bind by either assay to globoside, the Forssman glycolipid, GM1, GM2, GM3, GD1a, GD2, GD3, GQ1b, or GT1b. The binding pattern observed with the K88 adhesin suggests that beta 1-linked galactosyl residues are the minimum determinant required for binding, provided they are presented correctly. It is suggested that beta 1-linked galactose residues may form the molecular basis of both glycoprotein and glycolipid receptors for the K88 fimbrial adhesin in the porcine small intestine.     

Pseudomonas aeruginosa strains possess specific adhesins for laminin.

Pseudomonas aeruginosa is a major human pathogen known to infect tissues that have been previously damaged in some way. In wounded human respiratory tissues, P. aeruginosa cells were found attached to exposed basement membranes following epithelial denudation, suggesting that the affinity for extracellular matrix proteins may account for the bacterium's opportunistic character. By using microtiter wells coated with different P. aeruginosa strains, we demonstrated that laminin binds to both colonizing bacterial strains, isolated from asymptomatic carriers, and strains isolated from infected patients. Binding of soluble laminin to piliated P. aeruginosa PAK and to the nonpiliated isogenic mutant PAK/p--was shown to be saturable. Binding of laminin to the piliated PAK strain was not different from binding to th nonpiliated PAK/p--strain but was significantly higher than binding to the avirulent, nonpiliated PAK-N1 rpoN mutant. By transmission electron microscopy, we localized the laminin-binding sites on a loose material in the outermost layer of the bacteria. Western immunoblotting results suggested that 57- and 59-kDa nonpilus adhesins from the microbial outer membranes account for the binding of P. aeruginosa to laminin. We speculate that bacterial affinity for laminin may be of biological significance in the pathogenesis of P. aeruginosa infection of injured tissues.     

Pseudomonas aeruginosa outer membrane adhesins for human respiratory mucus glycoproteins.

The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cases of cystic fibrosis. For this purpose, microtiter plate adhesion assays have been developed and have suggested that nonpilus adhesins of P. aeruginosa are the most important ones for binding to human respiratory mucins. In order to characterize these mucin-binding adhesins, outer membrane proteins (OMP) from two adhesive strains, 1244-NP and PAK-NP, and their poorly adhesive rpoN mutants, 1244-N3 and PAK-N1, were prepared by a mild extraction with Zwittergent 3-14. Mucin-binding adhesins were detected after polyacrylamide gel electrophoresis and blotting of the OMP on nitrocellulose replicas, using human bronchial mucins labeled with 125I. The binding properties of these OMP with lactotransferrin, another glycoprotein abundant in respiratory mucus, were also studied. Radiolabeled mucins detected four bands at 48, 46, 28, and 25 kDa with strain PAK-NP. With the nonmucoid strain 1244-NP, five bands were observed at 48, 46, 42, 28, and 25 kDa. The bands at 48 and 25 kDa were also visualized by radiolabeled lactotransferrin. These bands were partially or completely displaced by nonradiolabeled respiratory mucin glycopeptides but not by tetramethylurea, suggesting that they recognized carbohydrate sites. In contrast, the poorly adhesive strains showed weakly binding bands. These results demonstrate that outer membranes from two different nonpiliated P. aeruginosa strains express multiple adhesins with an affinity for human respiratory mucins and/or lactotransferrin.     

Specific binding of Streptococcus pneumoniae to two receptor saccharide structures

Specific binding of Streptococcus pneumoniae to two proposed receptor structures was studied in a solid-phase assay. The assay was based on immunodetection of the pneumococci adhering to the receptors coated to microtiter plates. Non-specific binding owing to hydrophobic forces to uncoated wells could be abolished by treatment of the plates with a blocking buffer. Binding of the pneumococcal cells was demonstrated with the glycolipid asialo-GM1 as receptor with a previously suggested specificity for the disaccharide GalNAcβ1-4Gal. A non-capsulated mutant bound with high efficiency to this receptor. Two capsulated strains also bound well, but with lower efficiency. Binding of the non-capsulated strain was also demonstrated with lactotriaosylceramide as receptor with a suggested specificity for the disaccharide GlcNAcβl-3Gal. The binding assay enables the comparison of the adherence of different strains to purified receptor molecules.     

Further studies of the specificities of monoclonal anti-i and anti-I antibodies using chemically synthesized, linear oligosaccharides of the poly-N-acetyllactosamine series

The I- and i-antigen activities of chemically synthesized, linear oligosaccharides of the neolacto series containing one, two or three N-acetyllactosamine (Gal beta 1----4GlcNAc) units have been tested by inhibition of binding of five anti-i and eight anti-I monoclonal antibodies to radioiodinated I- and i-active glycoproteins. The inhibitory activities of the milk oligosaccharides lacto-N-neotetraose (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc) and lacto-N-tetraose (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc) have also been determined. The results clearly show that: (a) the determinants that best fit the combining sites of anti-i antibodies are at least hexasaccharides of the neolacto series, (b) linear tetra- and hexasaccharides of the neolacto series can strongly inhibit the binding of anti-I antibodies of group 2 which are known to be primarily directed at the repeating Gal beta 1----4GlcNAc beta 1----3 domains of branched neolacto sequences, (c) the beta- but not the alpha-methyl anomer of the glycoside Gal beta 1----4GlcNAc beta 1-O-Me inhibits the binding of anti-I antibodies of group 1 which recognise the branch point sequence Gal beta 1----4GlcNAc beta 1----6-, (d) the reactivity of the beta-methylglycoside is impaired if the sequence is further elongated as in Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-O-Me, and (e) lacto-N-tetraose has no inhibitory activity with any of the anti-i or anti-I antibodies tested.     

Attachment of Escherichia coli via mannose- or Gal alpha 1----4Gal beta-containing receptors to human colonic epithelial cells.

The role of bacterial adhesion for the maintenance of the large-intestinal microflora has not been established. In this study, colonic cells from the adenocarcinoma cell line HT-29 or from surgical specimens were tested for the ability to bind Escherichia coli. The E. coli strains were manipulated by transformation or by mutagenesis to express either mannose-specific type 1 fimbriae (strains 506 MS and HU742) or Gal alpha 1----4Gal beta-specific P fimbriae (506 MR and HU824). Binding to HT-29 cells was seen with strains of either receptor specificity and was inhibited by alpha-methyl mannoside or globotetraosylceramide (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc-ceramide), respectively. The Gal alpha 1----4Gal beta-specific strains interacted with a loosely surface-associated substance, which was sensitive to mechanical treatment and incubation at 37 degrees C, while the mannose-specific strains bound both directly to the cell and to the loosely associated substance. Isolated colonic epithelial cells bound the mannose-specific bacteria in high numbers, while the attachment of the Gal alpha 1----4Gal beta-specific strains depended on the elution method. Cells eluted sequentially with magnetic stirring were unable to bind the Gal alpha 1----4Gal beta-specific bacteria, while elution by a more gentle method resulted in binding of these strains to material loosely associated with the epithelial cells. Thus, the binding pattern of isolated colonic epithelial cells paralleled that of the HT-29 cell line. Conceivably, binding to mannose- and Gal alpha 1----4Gal beta-containing receptors could contribute to the maintenance of E. coli in the human large intestine.     

Chemical characterisation of six oligosaccharides in a sample of colostrum of the brown capuchin, Cebus apella (Cebidae: primates)

Carbohydrates were extracted from a sample of brown capuchin colostrum and six of the component oligosaccharides were separated and purified by gel filtration and preparative thin layer chromatography. Their structures were determined by 1H-NMR to be as follows: Gal beta 1-->4[Fuc alpha 1-->3]Glc (3-fucosyllactose) Gal beta 1-->3Gal beta 1-->4Glc (beta-3'-galactosyllactose) Gal beta 1-->6Gal beta1-->4Glc (beta-6'-galactosyllactose) Gal beta-->3[Gal beta 1-->4GlcNAc beta 1-->6]Gal beta 1-->4Glc (lacto-N-novopentaose I) Gal beta 1-->4GlcNAc beta 1-->3[Gal beta 1-->4GlcNAc beta 1-->6]Gal beta 1-->4Glc (lacto-N-neohexaose) Neu5Ac alpha 2-->3Gal beta 1-->4Glc (3'-N-acetylneuraminyllactose) Of these, all except lacto-N-novopentaose I have been previously found in human milk or colostrum.     

Pseudomonas aeruginosa exoenzyme S is an adhesion.

Exoenzyme S from Pseudomonas aeruginosa has been studied as an adhesion for glycosphingolipids and buccal cells. Binding of exoenzyme S to gangliotriosylceramide (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer), gangliotetraosylceramide (Gal beta 1-3 GalNAcT beta 1-4 Gal beta 1-4Glc beta 1-1Cer), and lactosylceramide (Gal beta 1-4Glc beta 1-1Cer) separated on thin-layer chromatograms was observed. Binding curves for exoenzyme S with dilutions of gangliotetraosylceramide immobilized on plastic plates were similar to previously reported results for the intact bacteria. Binding of exoenzyme S to sialylated counterparts of these glycosphingolipids was not seen, indicating that the addition of a sialic acid residue interferes with binding. Exoenzyme S and monoclonal antibody to exoenzyme S inhibit the binding of P. aeruginosa to buccal cells. The presence of exoenzyme S on the surface of P. aeruginosa was detected by immunogold labeling of bacteria with antibodies to exoenzyme S. Results of these studies led us to conclude that exoenzyme S is an important adhesion of P. aeruginosa.     

A monoclonal antibody defining a binary N-acetyllactosaminyl structure in lactoisooctaosylceramide (IV6Gal beta 1----4GlcNAcnLc6): a useful probe for determining differential glycosylation patterns between normal and transformed human fibroblasts

Monoclonal antibodies A5 and C6 have been reported previously to recognize developmentally regulated determinants involving N-acetyllactosamine [Fenderson B. A., O'Brien D. A., Millette C. F. and Eddy E. M. (1984) Devl Biol. 103, 117-128]. In the present study, the specificity of these antibodies was determined by solid-phase radioimmunoassay and by thin-layer chromatography immunostaining using purified glycolipid standards. Antibody A5 recognized N-acetyllactosamine (type 2 chain; Gal beta 1----4GlcNAc beta 1----3R), irrespective of branching status. In contrast antibody C6 recognized the binary N-acetyllactosamine structure carried on lactoisooctaosylceramide. Antibody C6 did not react with sialosyl or alpha-galactosyl derivatives of the isooctaosyl structure, including human G10, G8 and bovine G9. Thus, unlike other anti-I antibodies, C6 provides a specific probe for both branching status and absence of terminal chain modification. Monoclonal antibodies A5, C6 and anti-I(Ma) were used to investigate glycosylation changes associated with oncogenic transformation. In contrast to results with lectins, these antibodies preferentially labeled the major glycoproteins of SV40-transformed human embryonic lung fibroblasts, including GP80, GP180, GP200 and GP250. The results suggest that increased expression of unsubstituted polylactosamine core structure at the cell surface follows SV40-transformation.     

Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids, and some LOS are sialylated.

The lipooligosaccharides (LOS) of strains of Haemophilus ducreyi, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica contain epitopes that are antigenically and structurally similar to carbohydrates present in human glycosphingolipids. LOS from strains of Haemophilus influenzae and H. influenzae biogroup aegyptius were tested for the binding of monoclonal antibodies (MAbs) that bind to human glycosphingolipids possessing Gal beta 1-4GlcNAc (MAb 3F11) and Gal alpha 1-4Gal beta 1-4Glc (MAb anti-Pk). In solid-phase radioimmunoassays, the LOS of 18 of 19 H. influenzae type b (Hib), 8 of 19 nontypeable H. influenzae, and 10 of 20 H. influenzae biogroup aegyptius strains bound MAb anti-Pk. The LOS of 13 of 19 Hib, 10 of 16 nontypeable H. influenzae, and 2 of 18 H. influenzae biogroup aegyptius strains bound MAb 3F11. Neuraminidase treatment of the strains increased the binding of MAb 3F11 by more than twofold in 47% of the H. influenzae strains, suggesting that sialic acid occluded the LOS structure recognized by MAb 3F11. The material released from neuraminidase-treated Hib LOS was confirmed to be sialic acid by high-performance anion-exchange chromatography. A recombinant plasmid containing genes involved in Hib LOS biosynthesis directed the expression (assembly) of the 3F11 epitope in Escherichia coli. These studies demonstrate that H. influenzae and H. influenzae biogroup aegyptius express at least two LOS epitopes that are similar to those present in human glycosphingolipids. Sialic acid was present on the LOS of some H. influenzae strains and prevented the binding of MAb 3F11 to its epitope. The oligosaccharide portion of sialylated LOS may also resemble sialylated oligosaccharides present in human glycosphingolipids (gangliosides).     

Adhesion of Pseudomonas aeruginosa to respiratory mucins and expression of mucin-binding proteins are increased by limiting iron during growth.

The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cystic fibrosis. Local factors in the respiratory tract, such as osmolarity or iron concentration, might influence this colonization. In the present work, we have observed that overall levels of adhesion of two nonmucoid, nonpiliated strains of P. aeruginosa, 1244-NP and PAK-NP, to human airway mucins were higher when these strains were grown in a minimal medium of low osmolarity (M9) than when they were grown in a rich medium of higher osmolarity (tryptic soy broth [TSB]). However, increasing the NaCl concentration of M9 to increase the osmolarity did not modify the level of binding. In order to find out whether these differences were due to variations in nutrients, the influence of iron concentration was investigated: the levels of binding of the two strains increased after TSB was depleted of iron and decreased after iron was added to M9. Since the outer membranes from the two strains have been shown to contain proteins reacting with human bronchial mucins, we compared the mucin-binding proteins expressed by the two strains grown in different media. When the nonpiliated strains 1244-NP and PAK-NP were grown in the different media, previously observed mucin-binding bands were detected in the 42- to 48-kDa range but additional mucin-binding bands in the 77- to 85-kDa range were detected when these strains were grown in M9 or iron-deprived TSB. These results demonstrate that the adhesion of P. aeruginosa and the expression of mucin-binding proteins in the outer membranes of nonpiliated P. aeruginosa are affected by the iron content of the medium in which the bacteria are grown and not by the osmolarity.     

Synthetic Antigens as Immunogens: Part IV. Specificity of Antisera Developed to Gal β1-3(GlcNAc β1-6)GalNAc and GlcNAc βl-6GalNAc

Antisera to the chemically synthesized trisaccharide, Gal β1-3(GlcNAc β1-6)GalNAc, and disaccharide, GlcNAc β1-6GalNAc, were developed using the diazophenyl bovine serum albumin derivatives. The binding specificity of the antisera were analyzed by enzyme immunoassays with structurally related, chemically synthesized oligosaccharides. The anti-trisaccharide antibody showed no reactivity to T antigenic structures which bear the Gal pl-3GalNAc moiety. The immunodominant area of the Gral β1-3(GlcNAc β 1-6)GalNAc was contained in the GlcNAc β1-6GalNAc region of the molecule. The reactivity pattern of the anti-trisaccharide antibody, although directed to the disaccharide, did not exactly duplicate the reactivity pattern of the anti-disaccharide.     

Blood group specific oligosaccharides from faeces of a blood group A breast-fed infant

Four different oligosaccharides were isolated from faeces collected from a blood group A, secretor, breast-fed infant. Three of these, GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-4Glc (A-tetrasaccharide), GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-4[Fuc alpha 1-3]Glc (A-pentasaccharide) and 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc (A-heptasaccharide) have previously found in urine, whereas GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (A-hexasaccharide) is a new compound. Structures were deduced by mass spectrometry of permethylated and N-trifluoroacetylated oligosaccharide alditols. The latter gave more structural information than the corresponding N-acetyl derivatives. The four oligosaccharides were tested for blood group A activity and all were found to inhibit the binding of anti-A antibody to blood group A substance.     

Recognition factors of Ricinus communis agglutinin 1 (RCA(1))

Ricinus communis agglutinin (RCA1) is one of the most important applied lectins that has been widely used as a tool to study cell surfaces and to purify glycans. Although the carbohydrate specificity of RCA1 has been described, the information obtained was mainly focused on inhibition of simple Galbeta1-related oligosaccharides and simple clusters. Here, all possible recognition factors of RCA1 of glycan binding were examined by enzyme-linked lectinosorbent (ELLSA) and inhibition assays, using known mammalian Gal/GalNAc carbohydrate structural units and natural polyvalent glycans. Among the glycoproteins (gps) tested and expressed as 50% nanogram inhibition, the high-density polyvalent Galbeta1-4GlcNAc (II) glycotopes occurring in natural gps, such as Pneumococcus type 14 capsular polysaccharide which is composed of repeating poly II residues, resulted in 9.0 x 10(4), 1.5 x 10(5), 2.3 x 10(4) and 2.1 x 10(4)-fold higher affinities to RCA1 than the monomeric Gal, linear I/II and Tri-antennary-II (Tri-II). Of the ligands tested and expressed as nanomoles of 50% inhibition, Tri-II was the best, being about 2, 4, 25.6 and 33.3 times better inhibitor than Di-II, II, I (Galbeta1-3GlcNAc) and Gal, respectively. From the results of this study, it is concluded that: (a) Galbeta1-4GlcNAc and other Galbeta1-related oligosaccharides are essential for lectin binding and their polyvalent form in macromolecules should be the most important recognition factor for RCA1; (b) the combining site of RCA1 may be a groove type, recognizing Galbeta1-4GlcNAc (II) as the major binding site; (c) its combining size may be large enough to accommodate a tetrasaccharide of beta-anomeric Gal at the non-reducing end and most complementary to human blood group I Ma active trisaccharide (Galbeta1-4GlcNAcbeta1-6Gal) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc); (d) RCA1 has a preference for the beta-anomer of Gal oligosaccharides with a Galbeta1-4 linkage > Galbeta1-6 > or = Galbeta1-3; (e) configuration of carbon-2, -3 -4 and -6 in Gal are essential for binding; (f) hydrophobic interaction in the vicinity of the binding site useful for sugar accommodation increases affinity. These results should be helpful for understanding the functional role of RCA1 and for characterizing glycotopes of mammalian complex carbohydrates.     

Pseudomonas aeruginosa adhesins for tracheobronchial mucin.

Mucins of the tracheobronchial tree are preferential sites for adherence and colonization by Pseudomonas aeruginosa. They possess specific receptors for this organism that have amino sugars as their principal constituents. Since mucins probably reflect the receptors on the cellular surfaces, we hypothesized that the bacterial adhesins previously shown to mediate the binding of P. aeruginosa to cells would also mediate bacterial binding to mucins. We therefore tested the roles of the exopolysaccharide from mucoid strains of P. aeruginosa and pili from nonmucoid strains to see whether they are indeed the adhesins for mucins. Using a microtiter plate assay of adherence to mucins, we demonstrated that the mucoid exopolysaccharide bound to mucins and enhanced the adherence of mucoid strains to this substance. Antibodies raised against the exopolysaccharide from a single mucoid strain inhibited the adherence of all mucoid strains tested. Purified pili from nonmucoid strains inhibited the binding of nonmucoid strains but not of mucoid strains. Inhibition of adherence by antibody to pili was quite specific, antibody being able to inhibit only the binding of the homologous nonmucoid strain. These data support our previous observations with tracheal cells, confirming the similarity of the adhesins for respiratory tract cells and the mucins which cover them.