Glycolipid studies in small intestine and pancreas of alpha1,3-galactosyltransferase knockout miniature swine: alpha1,3GALT-KO animals lack alphaGAL antigens and contain novel blood group H compounds.

BACKGROUND: To avoid hyperacute rejection of xeno-organs, alpha1,3-galactosyltransferase knock-out (GalT-KO) pigs have been produced. However, Galalpha1,3Gal (Gal) determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens that might interfere with the human immune response. METHODS: Glycolipids isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig were tested for immune reactivity with antibodies on thin-layer chromatograms after separation by high-performance liquid chromatography, and selected fractions were analysed by proton NMR spectroscopy. RESULTS: Immunostaining using purified human anti-Gal Abs revealed that tissues from WT animals express large amounts of Gal-antigens whereas GalT-KO tissues lacked these antigens. Proton NMR spectroscopy on small intestine fractions revealed both linear and branched nona- and decaglycosylceramides, respectively, with terminal Gal-epitopes. In corresponding GalT-KO fractions, Gal-epitopes seemed to be replaced by terminal alpha1,2fucoses. Two novel branched blood group H compounds was found in the GalT-KO intestine. CONCLUSIONS: The structural complexity of alphaGal-terminating antigens in the WT organs is very high. Knockout of alpha1,3GalT by gene-targeting results in elimination of Gal-determinants. In addition structurally novel alpha1,2fucose-terminated blood group H compounds were identified in the GalT-KO tissue. These compounds are not expected to be recognized by the human immune system.

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).

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.

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.

Binding of cholera toxin B-subunits to derivatives of the natural ganglioside receptor, GM1.

In a previous paper we showed that the B-pentamer of cholera toxin (CT-B) binds with reduced binding strength to different C(1) derivatives of N-acetylneuraminic acid (NeuAc) of the natural receptor ganglioside, GM1. We have now extended these results to encompass two large amide derivatives, butylamide and cyclohexylmethylamide, using an assay in which the glycosphingolipids are adsorbed on hydrophobic PVDF membranes. The latter derivative showed an affinity approximately equal to that earlier found for benzylamide ( approximately 0.01 relative to native GM1) whereas the former revealed a approximately tenfold further reduction in affinity. Another derivative with a charged C(1)-amide group, aminopropylamide, was not bound by the toxin. Toxin binding to C(7) derivatives was reduced by about 50% compared with the native ganglioside. Molecular modeling of C(1) and C(7) derivatives in complex with CT-B gave a structural rationale for the observed differences in the relative affinities of the various derivatives. Loss of or altered hydrogen bond interactions involving the water molecules bridging the sialic acid to the protein was found to be the major cause for the observed drop in CT-B affinity in the smaller derivatives, while in the bulkier derivatives, hydrophobic interactions with the protein were found to partly compensate for these losses.

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.

Delineation of the epitope recognized by an antibody specific for N-glycolylneuraminic acid-containing gangliosides.

P3 is a mouse monoclonal antibody (mAb) that binds to several NeuGc-containing gangliosides. It also reacts with antigens expressed in human breast tumors (Vázquez et al. (1995) Hybridoma , 14, 551-556). In this work, the binding specificity of P3 has been characterized in more detail using a panel of glycolipids that included several disialylated gangliosides and several chemical derivatives of NeuGc-GM3. The carboxyl group and the nitrogen function of sialic acid were found to play important roles in the antibody binding, whereas the glycerol tail appears to be nonrelevant. Molecular modeling was used to analyze the binding data, including the finding that P3 selectively recognizes the internal NeuGc in GD3. For this purpose, conformational studies of GD3 were performed using molecular dynamics. It was concluded that sialic acid binds the P3 antibody through its upper face (the one on which the carboxyl group is exposed) and the C4-C5 side of the sugar ring, whereas none or very little contact between the galactose residue and the protein is evident. Conformational analysis of GD3 revealed that, despite the large flexibility of the NeuGcalpha8NeuGc linkage, the P3 binding epitope on the external sialic acid is not well exposed for any of the possible conformations this linkage can adopt, whereas the internal sialic acid presents the epitope in a proper way for several of these conformations. As a final result, a coherent picture of the epitope that fits the wide binding data was obtained.

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.

Dentin phosphoprotein sequence motifs and molecular modeling: conformational adaptations to mineral crystals.

Molecular modeling has been used to investigate structural features of oligopeptides derived from possible primary structure motifs in highly phosphorylated dentin phosphoprotein (PP-H), the predominant noncollagenous protein in dentin. It contains a large number of aspartate (Asp) and phosphoserine (Pse) residues, the latter proposedly crucial for the PP-H function as a mineral nucleator. In this work, computer fitting and subsequent structural adaptation of model peptides, built exclusively from Asp and Pse, to the known crystal structures of hydroxyapatite (HAP) and octacalcium phosphate (OCP) were performed. The results show that, when considering conformational energies of fitted single strand oligo-peptides, either crystal will serve. Within a narrow range, fitting to OCP was slightly favored, except for oligo(Pse-Pse-Asp-Asp), which showed a slightly better fit to HAP. Energy differences between crystal-adapted and non-adapted freely minimized peptides showed that oligo(Pse-Asp) docked to either HAP or OCP were the energetically most favored adaptations. Fitting of minimized triple anti-parallel beta-strands of oligo(Pse-Asp) or oligo(Pse-Pse-Asp), motifs found in published sequences of rat, mouse, and bovine PP-H, revealed that a (001) crystal face of HAP, but most likely not OCP, may be formed by these beta-sheet models. The former motif is more advantageous in this respect.

Structural features of the combining site region of Erythrina corallodendron lectin: role of tryptophan 135.

The role of Trp 135 and Tyr 108 in the combining site of Erythrina corallodendron lectin (ECorL) was investigated by physicochemical characterization of mutants obtained by site-directed mutagenesis, hemagglutination-inhibition studies, and molecular modeling, including dynamics simulations. The findings demonstrate that Trp 135 in ECorL: (1) is required for the tight binding of Ca2+ and Mn2+ to the lectin because mutation of this residue into alanine results in loss of these ions upon dialysis and concomitant reversible inactivation of the mutant; (2) contributes to the high affinity of methyl alpha-N-dansylgalactosaminide (MealphaGalNDns) to the lectin; and (3) is solely responsible for the fluorescence energy transfer between the aromatic residues of the lectin and the dansyl group in the ECorL-MealphaGalNDns complex. Docking of MealphaGalNDns into the combining site of the lectin reveals that the dansyl moiety is parallel with the indole of Trp 135, as required for efficient fluorescence energy transfer, in one of the two possible conformations that this ligand assumes in the bound state. In the W135A mutant, which still binds MealphaGalNDns strongly, the dansyl group may partially insert itself into the place formerly occupied by Trp 135, a process that from dynamics simulations does not appear to be energetically favored unless the loop containing this residue assumes an open conformation. However, a small fraction of the W135A molecules must be able to bind MealphaGalNDns in order to explain the relatively high affinity, as compared to galactose, still remaining for this ligand. A model for the molecular events leading to inactivation of the W135A mutant upon demetallization is also presented in which the cis-trans isomerization of the Ala 88-Asp 89 peptide bond, observed in high-temperature dynamics simulations, appears not to be a required step.

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.

Acidic glycosphingolipids of cock testis elucidated by mass spectrometry and NMR spectroscopy.

The main acidic glycosphingolipids (GSLs) of cock testis were identified as GalCer I3-sulfate and gangliosides GM4, GM3, GD3 and GT3. They contained N-acetylneuraminic acid as the major sialic acid, and ceramides composed mainly of sphingosine (dl8:1) and C18-24 non-hydroxy fatty acids. Appreciable amounts of hydroxy fatty acids were detected only in the GM4 preparation.

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.

Molecular mimicry in the recognition of glycosphingolipids by Gal alpha 3 Gal beta 4 GlcNAc beta-binding Clostridium difficile toxin A, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13: characterization of a binding-active human glycosphingolipid, non-identical with the animal receptor.

Glycoconjugates with terminal Gal alpha 3Gal beta 4GlcNAc beta sequences have been shown to be recognized by three carbohydrate-binding proteins; toxin A of Clostridium difficile, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13. However, the biological significance of this binding specificity in humans is unclear, since unsubstituted Gal alpha 3Gal beta 4GlcNAc beta sequences are not found in human tissues, due to suppression of the gene coding for the enzyme Gal beta 3-transferase. To explore this inconsistency, the binding of toxin A, human natural anti alpha-galactosyl IgG, and the Gal-13 monoclonal antibody to various glycosphingolipids was examined using the thin-layer chromatogram binding assay. The binding to Gal alpha 3Gal beta 4GlcNAc beta-terminated glycosphingolipids of rabbit erythrocytes was confirmed. A minor binding-active compound was also detected in the non-acid glycosphingolipid fraction of human erythrocytes. This glycosphingolipid was isolated and characterized by EI mass spectrometry, gas chromatography-EI mass spectrometry after degradation, and proton NMR spectroscopy, as GalNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, corresponding to the x2 glycosphingolipid isolated before form this source. Two additional binding-active glycosphingolipids were found. One was GalNAc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4 Glc beta 1 Cer, produced from blood group A-active GalNAc alpha 3 (Fuc alpha 2) Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1 Cer by acid-induced defucosylation. The other was GlcNAc beta 3 Gal beta 4 GlcNAc beta 3 Gal beta 4 Glc beta 1 Cer, generated from NeuGc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer by enzymatic hydrolysis. A number of other glycosphingolipid sequences, including the human Le(x), Le(y), and I blood group determinants, suggested to act as receptors for toxin A, were not recognized by the three ligands. Despite the different terminal substituents and anomerity of the binding-active glycosphingolipids, calculated minimum energy conformations demonstrated topographical similarities in the spatial orientation of the terminal trisaccharides, possibly accounting for the cross-reactivity.

Glycoconjugate receptors for P-fimbriated Escherichia coli in the mouse. An animal model of urinary tract infection.

Glycosphingolipids were isolated from kidneys, urethers, and bladders (including urethrae) of C3H/HeN mice. Binding was studied of a clinical isolate and recombinant strains of uropathogenic P-fimbriated Escherichia coli to these glycolipids. A series of receptor-active glycolipids with Gal alpha 4Gal in common, previously shown to be recognized by these bacteria, was identified by use of specific monoclonal antibodies, fast-atom bombardment and electron-impact mass spectrometry, and proton nuclear magnetic resonance spectroscopy: galabiosylceramide (Gal alpha 4Gal beta Cer), globotriaosylceramide (Gal alpha 4Gal beta 4Glc beta Cer), globoside (GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer), the Forssman glycolipid (GalNAc alpha 3GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer), Gal beta 4GlcNAc beta 6(Gal beta 3)GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer, and Gal beta 4(Fuc alpha 3)GlcNAc beta 6(Gal beta 3)GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer. The binding pattern for mouse kidney glycolipids differed from that for kidney glycolipids of man and monkey. In particular, the dominant 8-sugar glycolipid in the mouse was not detected in the primates. A second difference was found in the binding of E. coli to kidney glycoproteins on blots after electrophoresis; the mouse showed distinct receptor-active bands while human and monkey did not. These differences may be of relevance when using the mouse as a model for clinical urinary tract infection of man.

Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes.

Binding studies of various glycolipids, mainly belonging to the ganglio series, to the toxins isolated from Vibrio cholerae, Escherichia coli, and Clostridium tetani have been performed, using the microtiter well assay. By using the found binding preferences in conjunction with minimum-energy conformations obtained from molecular modeling of the various ligands, binding epitopes on the natural receptor glycolipids for the toxins have been defined. The binding preferences for the cholera toxin and the heat-labile E. coli toxin are very similar, with the ganglioside GM1 being the most efficient ligand. The tetanus toxin binds strongly to gangliosides of the G1b series, with GT1b as the most efficient ligand. It is found that the binding epitope on GM1 for the cholera and heat-labile toxins to a large extent overlaps with the epitope on GQ1b for the tetanus toxin.

Comparison of the glycolipid-binding specificities of cholera toxin and porcine Escherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine.

The binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxin were investigated by binding of 125I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.

Characterization of the specificity of binding of Moluccella laevis lectin to glycosphingolipids.

The specificity of Moluccella laevis lectin was investigated by analysing its binding to glycosphingolipids separated on thin-layer chromatograms or adsorbed on microtitre wells. The binding activity of the lectin was highest for glycosphingolipids with terminal alpha-linked N-acetylgalactosamine, both in linear structures, as the Forssman glycosphingolipid, GalNAc alpha 3GalNAc beta 3Gal alpha 4Glc beta 1Cer, and in branched structures, as glycosphingolipids with the blood group A determinant, GalNAc alpha 3(Fuc alpha 2)Gal beta. In addition, the lectin bound, though considerably more weakly, to linear glycosphingolipids with terminal alpha-linked galactose. When considering the use of the M. laevis lectin for biochemical and medical purposes this cross-reactivity may be of importance.