Helicobacter pylori-binding gangliosides of human gastric adenocarcinoma.

Acidic and neutral glycosphingolipids were isolated from a human gastric adenocarcinoma, and binding of Helicobacter pylori to the isolated glycosphingolipids was assessed using the chromatogram binding assay. The isolated glycosphingolipids were characterized using fast atom bombardment mass spectrometry and by binding of antibodies and lectins. The predominating neutral glycosphingolipids were found to migrate in the di- to tetraglycosylceramide regions as revealed by anisaldehyde staining and detection with lectins. No binding of H. pylori to these compounds was obtained. The most abundant acidic glycosphingolipids, migrating as the GM3 ganglioside and sialyl-neolactotetraosylceramide, were not recognized by the bacteria. Instead, H. pylori selectively interacted with slow-migrating, low abundant gangliosides not detected by anisaldehyde staining. Binding-active gangliosides were isolated and characterized by mass spectrometry, proton nuclear magnetic resonance, and lectin binding as sialyl-neolactohexaosylceramide (NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and sialyl-neolactooctaosylceramide (NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer).

Different glycosphingolipid composition in human neutrophil subcellular compartments.

The binding of a number of carbohydrate-recognizing ligands to glycosphingolipids and polyglycosylceramides of human neutrophil subcellular fractions (plasma membranes/secretory vesicles of resting and ionomycin-stimulated cells, specific and azurophil granules) was examined using the chromatogram binding assay. Several organelle-related differences in glycosphingolipid content were observed. The most prominent difference was a decreased content of the GM3 ganglioside in plasma membranes of activated neutrophils. Gangliosides recognized by anti-VIM-2 antibodies were detected mainly in the acid fractions of azurophil and specific granules. Slow-migrating gangliosides and polyglycosylceramides with Helicobacter pylori-binding activity were found in all acid fractions. A non-acid triglycosylceramide, recognized by Gal(alpha)4Gal-binding Escherichia coli, was detected in the plasma membrane/secretory vesicles but not in the azurophil and specific granules. Although no defined roles of glycosphingolipids have yet been conclusively established with respect to neutrophil function, the fact that many of the identified glycosphingolipids are stored in granules, is in agreement with their role as receptor structures that are exposed on the neutrophil cell surface upon fusion of granules with the plasma membrane. Accordingly, we show that neutrophil granules store specific carbohydrate epitopes that are upregulated to the plasma membrane upon cell activation.

Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope.

The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcalpha3-Galbeta) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcalpha3Galbeta is preferred to NeuAcalpha3Galbeta. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.

Isolectins from Solanum tuberosum with different detailed carbohydrate binding specificities: unexpected recognition of lactosylceramide by N-acetyllactosamine-binding lectins.

Glycosphingolipid recognition by two isolectins from Solanum tuberosum was compared by the chromatogram binding assay. One lectin (PL-I) was isolated from potato tubers by affinity chromatography, and identified by MALDI-TOF mass spectrometry as a homodimer with a subunit molecular mass of 63,000. The other (PL-II) was a commercial lectin, characterized as two homodimeric isolectins with subunit molecular masses of 52,000 and 55,000, respectively. Both lectins recognized N-acetyllactosamine-containing glycosphingolipids, but the fine details of their carbohydrate binding specificities differed. PL-II preferentially bound to glycosphingolipids with N-acetyllactosamine branches, as Galbeta4GlcNAcbeta6(Galbeta4GlcNAcbeta3)Galbeta4Glcbeta1C er. PL-I also recognized this glycosphingolipid, but bound equally well to the linear glycosphingolipid Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. Neolactotetraosylceramide and the B5 pentaglycosylceramide were also bound by PL-I, while other glycosphingolipids with only one N-acetyllactosamine unit were non-binding. Surprisingly, both lectins also bound to lactosylceramide, with an absolute requirement for sphingosine and non-hydroxy fatty acids. The inhibition of binding to both lactosylceramide and N-acetyllactosamine-containing glycosphingolipids by N-acetylchitotetraose suggests that lactosylceramide is also accomodated within the N-acetylchitotetraose/N-acetyllactosamine-binding sites of the lectins. Through docking of glycosphingolipids onto a three-dimensional model of the PL-I hevein binding domain, a Galbeta4GlcNAcbeta3Galbeta4 binding epitope was defined. Furthermore, direct involvement of the ceramide in the binding of lactosylceramide was suggested.

Inhibition of nonopsonic Helicobacter pylori-induced activation of human neutrophils by sialylated oligosaccharides.

Certain strains of Helicobacter pylori have nonopsonic neutrophil-activating capacity. Some H. pylori strains and the neutrophil-activating protein of H.pylori (HPNAP) bind selectively to gangliosides of human neutrophils. To determine if there is a relationship between the neutrophil-activating capacity and the ganglioside-binding ability, a number of H. pylori strains, and HPNAP, were incubated with oligosaccharides, and the effects on the oxidative burst of subsequently challenged neutrophils was measured by chemiluminescence and flow cytometry. Both by chemiluminescence and flow cytometry a reduced response was obtained by incubation of H.pylori with sialic acid-terminated oligosaccharides, whereas lactose had no effect. The reductions obtained with different sialylated oligosaccharides varied to some extent between the H. pylori strains, but in general 3'-sialyllactosamine was the most efficient inhibitor. Challenge of neutrophils with HPNAP gave no response in the chemiluminescence assay, and a delayed moderate response with flow cytometry. Preincubation of the protein with 3'-sialyllactosamine gave a slight reduction of the response, while 3'-sialyllactose had no effect. The current results suggest that the nonopsonic H. pylori-induced activation of neutrophils occurs by lectinophagocytosis, the recognition of sialylated glycoconjugates on the neutrophil cell surface by a bacterial adhesin leads to phagocytosis and an oxidative burst with the production of reactive oxygen metabolites.

Lactobacillus johnsonii La1 shares carbohydrate-binding specificities with several enteropathogenic bacteria.

The carbohydrate-binding specificities of the probiotic lactic acid bacterium Lactobacillus johnsonii La1 (a health-beneficial bacterial strain able to be incorporated into the human intestinal microflora) were investigated in vitro. First various soluble complex carbohydrates were tested as potential inhibitors of the strain adhesion onto Caco-2 intestinal epithelial cells, and then bacterial binding to glycolipids immobilized on TLC plates was probed. Two major carbohydrate-binding specificities of Lactobacillus johnsonii La1 were identified. A first one for an Endo-H treated yeast cell wall mannoprotein carrying mainly O:-linked oligomannosides, and a second one for the gangliotri- and gangliotetra-osylceramides (asialo-GM1). Similar carbohydrate-binding specificities are known to be expressed on cell surface adhesins of several enteropathogens, enabling them to adhere to the host gut mucosa. These findings corroborate the hypothesis that selected probiotic bacterial strains could be able to compete with enteropathogens for the same carbohydrate receptors in the gut.

Common architecture of the primary galactose binding sites of Erythrina corallodendron lectin and heat-labile enterotoxin from Escherichia coli in relation to the binding of branched neolactohexaosylceramide.

The heat-labile enterotoxin from Escherichia coli (LT) is responsible for so-called traveller's diarrhea and is closely related to the cholera toxin (CT). Toxin binding to GM1 at the epithelial cell surface of the small intestine initiates the subsequent diarrheal disease. However, LT has a broader receptor specificity than CT in that it also binds to N-acetyllactosamine-terminated structures. The unrelated lectin from Erythrina corallodendron (ECorL) shares this latter binding property. The findings that both ECorL and porcine LT (pLT) bind to lactose as well as to neolactotetraosylceramide suggests a common structural theme in their respective primary binding sites. Superimposing the terminal galactose of the lactoses in the respective crystal structures of pLT and ECorL reveals striking structural similarities around the galactose despite the lack of sequence and folding homology, whereas the interactions of the penultimate GlcNAcb3 in the neolactotetraosylceramide differ. The binding of branched neolactohexaosylceramide to either protein reveals an enhanced affinity relative to neolactotetraosylceramide. The b3-linked branch is found to bind to the primary Gal binding pocket of both proteins, whereas the b6-linked branch outside this site provides additional interactions in accordance with the higher binding affinities found for this compound. While the remarkable architectural similarities of the primary galactose binding sites of pLT and ECorL point to a convergent evolution of these subsites, the distinguishing structural features determining the overall carbohydrate specificities are located in extended binding site regions. In pLT, Arg13 is thus found to play a crucial role in enhancing the affinity not only for N-acetyllactosamine-terminated structures but also for GM1 as compared to human LT (hLT) and CT. The physiological relevance of the binding of N-acetyllactosamine-containing glycoconjugates to LT and ECorL is briefly discussed.

Novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits.

The B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) are structurally and functionally related. However, the carbohydrate binding specificities of the two proteins differ. While both CTB and LTB bind to the GM1 ganglioside, LTB also binds to N-acetyllactosamine-terminated glycoconjugates. The structural basis of the differences in carbohydrate recognition has been investigated by a systematic exchange of amino acids between LTB and CTB. Thereby, a CTB/LTB hybrid with a gain-of-function mutation resulting in recognition of blood group A and B determinants was obtained. Glycosphingolipid binding assays showed a specific binding of this hybrid B-subunit, but not CTB or LTB, to slowly migrating non-acid glycosphingolipids of human and animal small intestinal epithelium. A binding-active glycosphingolipid isolated from cat intestinal epithelium was characterized by mass spectrometry and proton NMR as GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)Glc NAcbeta3Galbeta4Glc NAcbeta3Galbeta4Glcbeta1Cer. Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Galalpha3(Fucalpha2)Galbeta4(Fucalpha3)GlcNAc beta. The blood group A and B determinants bind to a novel carbohydrate binding site located at the top of the B-subunit interfaces, distinct from the GM1 binding site, as found by docking and molecular dynamics simulations.

Helicobacter pylori and neutrophils: sialic acid-dependent binding to various isolated glycoconjugates.

Helicobacter pylori has been shown to agglutinate erythrocytes in a sialic acid-dependent manner. However, very few studies have examined relevant target cells in the human stomach. Neutrophils are required for the onset of gastritis, and the inflammatory reaction may be induced on contact between bacteria and neutrophils. In the present work, glycolipids and glycoproteins were isolated from neutrophils and were studied for binding by overlay with radiolabeled bacteria on thin-layer chromatograms and on membrane blots. There was a complex pattern of binding bands. The only practical binding activity found was sialic acid dependent, since treatment of glycoconjugates with neuraminidase or mild periodate eliminated binding. As shown before for binding to erythrocytes and other glycoconjugates, bacterial cells grown on agar bound to many glycoconjugates, while growth in broth resulted in bacteria that would bind only to polyglycosylceramides, which are highly heterogeneous and branched poly-N-acetyllactosamine-containing glycolipids. Approximately seven positive bands were found for glycoproteins, and the traditional ganglioside fraction showed a complex, slow-moving interval with very strong sialic-acid-dependent binding, probably explained by Fuc substitutions on GlcNAc.

Glycosphingolipid binding specificities of Neisseria meningitidis and Haemophilus influenzae: detection, isolation, and characterization of a binding-active glycosphingolipid from human oropharyngeal epithelium.

The glycosphingolipid binding specificities of Haemophilus influenzae and Neisseria meningitidis were investigated as to the binding of radiolabeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby, similar binding profiles, for the binding of the two bacteria to lactosylceramide, isoglobotriaosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, lactotetraosylceramide, neolactotetraosylceramide, and sialylneolactohexaosylceramide, were obtained. On a closer view the binding preferences of the bacteria could be differentiated into three groups. The first specificity is recognition of lactosylceramide. The second specificity is binding to gangliotriaosylceramide and gangliotetraosylceramide, since conversion of the acetamido group of the N-acetylgalactosamine of gangliotriaosylceramide and gangliotetraosylceramide to an amine prevented the binding of the bacteria, and thus the binding to these two glycosphingolipids represents a separate specificity from lactosylceramide recognition. Preincubation of H. influenzae with neolactotetraose inhibited the binding to neolactotetraosylceramide, while the binding to lactosylceramide, gangliotetraosylceramide, or lactotetraosylceramide was unaffected. Thus, the third binding specificity is represented by neolactotetraosylceramide, and involves recognition of other neolacto series glycosphingolipids with linear N-acetyllactosamine chains, such as sialyl-neolactohexaosylceramide. The relevance of the detected binding specificities for adhesion to target cells was addressed as to the binding of the bacteria to glycosphingolipids from human granulocytes, epithelial cells of human nasopharyngeal tonsils and human plexus choroideus. Binding-active neolactotetraosylceramide was thereby detected in human granulocytes and the oropharyngeal epithelium.

Epitope dissection of receptor-active gangliosides with affinity for Helicobacter pylori and influenza virus.

Receptor-active gangliosides with affinity for Helicobacter pylori and influenza virus were chemically modified and analyzed by negative ion fast atom bombardment mass spectrometry (FAB MS) or electron ionization mass spectrometry (EI MS) after permethylation. Derivatizations included mild periodate oxidation of the sialic acid glycerol tail or conversion of the carboxyl group to primary alcohol or amides. The modified gangliosides were then tested for binding affinity using thin-layer plates overlaid with labeled microbes or microbe-derived proteins. Mild periodate oxidation, which shortens sialic acid tail without destruction of sugar cores, abolished or drastically reduced binding of H. pylori and avian influenza virus to sialyl-3-paragloboside (S-3-PG). The same effect was observed in the case of binding of the human influenza virus to receptor-active gangliosides of human leukocytes. Conversion of S-3-PG or leukocyte gangliosides to primary alcohols or amides also abolished the binding. However, mild periodate oxidation had no effect on binding of NAP (neutrophil-activating protein of H. pylori) to the active ganglioside.

The lactosylceramide binding specificity of Helicobacter pylori.

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.

Binding of N-trifluoroacetyl-derivatized natural glycosphingolipids by uropathogenic Escherichia coli and Neisseria subflava.

Several neutral glycosphingolipids were hydrogenated and subjected to trifluoroacetylation in trifluoroacetic acid/trifluoroacetic acid anhydride under conditions leading to complete exchange of the N-acetyl groups of GalNAc for N-trifluoroacetyl. The derivatized glycosphingolipids were analyzed for binding by P-fimbriated uropathogenic Escherichia coli, recognizing the globo series of glycolipids (carrying Galalpha1-4Gal). Using E. coli it was shown that a GalNCO-CF3 next to the minimum binding epitope Galalpha1-4Gal did not substantially influence the binding, as did not a trifluoro acetyl group on the ceramide. Exchange of N-acetyl of GalNAc in the receptor active gangliotetraosylceramide, Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1-1Cer, for N-trifluoroacetyl, did not change the binding of two out of the three strains tested of the bacterium Neisseria subflava. Discussion concerning the binding epitopes of the bacterial adhesins to carbohydrates is based on these results.

Carbohydrate binding specificity of the neutrophil-activating protein of Helicobacter pylori.

The possible interaction of the neutrophil-activating protein of Helicobacter pylori with target cell glycoconjugates was investigated by the binding of 125I-labeled recombinant protein to glycosphingolipids from human neutrophils in solid phase assays. Thereby, a distinct binding of the neutrophil-activating protein to four bands in the acid glycosphingolipid fraction from human neutrophils was detected, whereas no binding to the non-acid glycosphingolipids or polyglycosyl ceramides from these cells was obtained. When using glycosphingolipids not present in the cell membrane of human neutrophils, it was found that the neutrophil-activating protein also bound to sulfated glycosphingolipids as sulfatide and sulfated gangliotetraosyl ceramide. Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources suggested that in human granulocytes, the neutrophil-activating protein of H. pylori preferentially recognizes glycoconjugates with a terminally unsubstituted NeuAcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta sequence.

Induction of protective immunity after escherichia coli bladder infection in primates. Dependence of the globoside-specific P-fimbrial tip adhesin and its cognate receptor.

Clinical observations suggest that immune mechanisms affect etiology and course of recurrent cystitis. A primate infection model was used to show that primary bladder infection with a uropathogenic P-fimbriated strain (binding to globoside present in the bladder wall) protects against rechallenge with homologous as well as heterologous Escherichia coli strains for up to 5-6 mo. In contrast, mutant derivatives producing P-fimbriae either lacking the tip adhesin protein or carrying an adhesin for which no bladder receptor was present, were unable to induce protection, even though they generated bladder infections of similar duration as the wild type. Therefore, the protective effect mediated by the adhesin seemed to depend upon the presence of its cognate receptor. Since the wild strain also mediated protection against mutants that lacked the adhesin, our data suggest that the globoside-binding PapG adhesin acts as an adjuvant during infection to enhance a specific response against other bacterial antigens. In fact, the globoside-binding strain DS17, but not the mutant DS17-1, unable to bind to membrane-bound globoside, elicited a secretory IgA response to LPS in urine. These in vivo findings suggest that bacterial adhesin-ligand interactions may have signaling functions of importance for the immune response.

Binding of Helicobacter pylori to sialic acid-containing glycolipids of various origins separated on thin-layer chromatograms.

Two standard strains of Helicobacter pylori, grown on solid or in liquid medium, were studied for their binding to sialic acid-containing glycosphingolipids on thin-layer plates. NCTC 11637, but not strain 11638, bound to mixtures of gangliosides of various human and animal origins with similar binding patterns and also to polyglycosylceramides of human erythrocytes, leukocytes, and placenta. There was an apparent specificity for NeuAc alpha3Gal of the neolacto series of gangliosides, since NeuAc alpha6Gal or ganglio-series gangliosides did not bind.

Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site.

Avian influenza virus strains representing most hemagglutinin (HA) subtypes were compared with human influenza A (H1N1,H3N2) and B virus isolates, including those with no history of passaging in embryonated hen's eggs, for their ability to bind free N-acetylneuraminic acid (Neu5Ac) and sialylollgosaccharides in a competitive binding assay and to attach to gangliosides in a solid-phase adsorption assay. The avian viruses, irrespective of their HA subtype, showed a higher affinity for sialyl-3-lactose and the other Neu5Ac2-3Gal-terminated oligosaccharides and a lower affinity for sialyl-6-lactose than for free Neu5Ac, indicative of specific interactions between the HA and the 3-linked Gal and poor accommodation of 6-linked Gal in the avian receptor-binding site (RBS). Human H1 and H3 strains, by contrast, were unable to bind to 3-linked Gal, interacting instead with the asialic portion of sialyl-6-(N-acetyllactosamine). Different parts of this moiety were recognized by H3 and H1 subtype viruses (Gal and GlcNAc, respectively). Comparison of the HA amino acid sequences revealed that residues in positions. 138, 190, 194, 225, 226, and 228 are conserved in the avian RBS, while the human HAs harbor substitutions at these positions. A characteristic feature of avian viruses was their binding to Neu5Ac2-3Gal-containing gangliosides. This property of avian precursor viruses was preserved in early human H3 isolates, but was gradually lost with further circulation of the H3 HA in humans. Consequently, later human H3 isolates, as well as H1 and type B human strains, were unable to bind to short Neu5Ac2-3Gal-terminated gangliosides, an incompatibility that correlated with higher glycosylation of the HA globular head of human viruses. Our results suggest that the RBS is highly conserved among HA subtypes of avian influenza virus, while that of human viruses displays distinctive genotypic and phenotypic variability.

Structural basis for differential receptor binding of cholera and Escherichia coli heat-labile toxins: influence of heterologous amino acid substitutions in the cholera B-subunit.

The closely related B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) both bind strongly to GM1 ganglioside receptors but LTB can also bind to additional glycolipids and glycoproteins. A number of mutant CT B-subunits were generated by substituting CTB amino acids with those at the corresponding positions in LTB. These were used to investigate the influence of specific residues on receptor-binding specificity. A mutated CTB protein containing the first 25 residues of LTB in combination with LTB residues at positions 94 and 95, bound to the same extent as native LTB to both delipidized rabbit intestinal cell membranes, complex glycosphingolipids (polyglycosylceramides) and neolactotetraosylceramide, but not to non-GM1 intestinal glycosphingolipids. In contrast, when LTB amino acid substitutions in the 1-25 region were combined with those in the 75-83 region, a binding as strong as that of LTB to intestinal glycosphingolipids was observed. In addition, a mutant LTB with a single Gly-33-->Asp substitution that completely lacked affinity for both GM1 and non-GM1 glycosphingolipids could still bind to receptors in the intestinal cell membranes and to polyglycosylceramides. We conclude that the extra, non-GM1 receptors for LTB consist of both sialylated and non-sialylated glycoconjugates, and that the binding to either class of receptors is influenced by different amino acid residues within the protein.

Redefinition of the carbohydrate specificity of Erythrina corallodendron lectin based on solid-phase binding assays and molecular modeling of native and recombinant forms obtained by site-directed mutagenesis.

Binding of the N-acetyllactosamine-specific lectin from Erythrina corallodendron (ECorL) to four glycosphingolipids has been tested using the microtiter well assay. The role of several amino acids in the binding site region was studied by combining binding assays and molecular modeling for native and recombinant forms of the lectin. Seven single-point mutants at positions 106 (Y106A), 108 (Y108A, T), 218 (A218G), and 219 (Q219A, N or E) were investigated. A comparison with more than 30 known sequences of legume lectins showed that ECorL is unique in displaying a tyrosine residue or a structural equivalent at position 106. Analyses of the binding results obtained for mutants at positions 106 and 108 using molecular modeling point to complex conformational dependencies between these and several other residues around the binding site. Gln 219 was found to have a large conformational flexibility, which, paradoxically, favors the binding of N-acetyllactosamine-containing glycosphingolipids. Particularly significant is the fact that ECorL exhibits a higher affinity for Fuc alpha2Gal beta4GlcNAc beta-terminated glycosphingolipids than N-acetyllactosamine-terminated ones, in accordance with molecular modeling revealing a perfect fit of the alpha2-linked fucose in a cavity extending from the Gal beta4 binding pocket. These findings lead to a redefinition of the specificity of this lectin, where the affinity for the terminal Fuc alpha2Gal beta4GlcNAc beta trisaccharide should be considered in the first place. The possible biological significance of this specificity remains to be investigated.

Unexpected carbohydrate cross-binding by Escherichia coli heat-labile enterotoxin. Recognition of human and rabbit target cell glycoconjugates in comparison with cholera toxin.

The bacterial protein enterotoxins, cholera toxin (CT) of Vibrio cholerae and heat-labile toxin (LT) of Escherichia coli, induce diarrhea by enhancing the secretory activity of the small intestine of man and rabbit (animal model). This physiological effect is mediated by toxin binding to a glycolipid receptor, the ganglioside GM1, Gal beta 3GalNAc beta 4(NeuAc alpha 3)GAl beta 4Glc beta 1Cer. However, LT, but not CT, was recently shown by us to bind also to paragloboside, Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, identified in the target cells. By molecular modeling of this tetrasaccharide in the known binding site of LT, the saccharide-peptide interaction was shown to be limited to the terminal disaccharide (N-acetyllactosamine). This sequence is expressed in many glycoconjugates, and we have therefore assayed glycolipids and glycoproteins prepared from the target tissues. In addition to paragloboside, receptor activity for LT was detected in glycoproteins of human origin and in polyglycosylceramides of rabbit. However, CT bound only to GM1. Two variants of LT with slightly different sequences, human (hLT) and porcine (pLT), were identical in their binding to target glycoproteins and polyglycosylceramides, but different regarding paragloboside, which was positive for pLT but negative for hLT. This difference is discussed on basis of modeling, taking in view the difference at position 13, with Arg in pLT and His in hLT. Although N-acetyllactosamine is differently recognized in form of paragloboside by the two toxin variants, we speculate that this sequence in human glycoproteins and rabbit polyglycosylceramides is the basis for the common binding. Much work remains, however, to clear up up this unexpected sophistication in target recognition.