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.

A strain of human influenza A virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay.

A human strain of influenza virus (A, H1N1) was shown to bind in an unexpected way to leukocyte and other gangliosides when compared with avian virus (A, H4N6) as assayed on TLC plates. The human strain bound only to species with about 10 or more sugars, while the avian strain bound to a wide range of gangliosides including the 5-sugar gangliosides. By use of specific lectins, antibodies, and FAB and MALDI-TOF mass spectrometry an attempt was done to preliminary identify the sequences of leukocyte gangliosides recognized by the human strain. The virus binding pattern did not follow binding by VIM-2 monoclonal antibody and was not identical with binding by anti-sialyl Lewis x antibody. There was no binding by the virus of linear NeuAcalpha3- or NeuAcalpha6-containing gangliosides with up to seven monosaccharides per mol of ceramide. Active species were minor NeuAcalpha6-containing molecules with probably repeated HexHexNAc units and fucose branches. This investigation demonstrates marked distinctions in the recognition of gangliosides between avian and human influenza viruses. Our data emphasize the importance of structural factors associated with more distant parts of the binding epitope and the complexity of carbohydrate recognition by human influenza viruses.

Polyglycosylceramides recognized by Helicobacter pylori: analysis by matrix-assisted laser desorption/ionization mass spectrometry after degradation with endo-beta-galactosidase and by fast atom bombardment mass spectrometry of permethylated undegraded material.

Human erythrocyte polyglycosylceramides (PGCs) are recognized by the gastric pathogen Helicobacter pylori and are based on a successively extended and highly branched N-acetyllactosamine core linked to ceramide and substituted by fucose and sialic acid. As a step in the identification of the binding epitope we earlier characterized intact PGCs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS (Karlsson,H., Johansson,L., Miller-Podraza,H., and Karlsson,K-A. [1999] Glycobiology, 9, 765-778). In the present work, PGCs from human blood group O erythrocytes were digested with endo-ss-galactosidase (Bacterioides fragilis), an enzyme which cleaves the bond 3Galss1-4GlcNAc in linear but not branched poly-N-acetyllactosamine chains. The enzymatic digestion resulted in a mixture of neutral and sialic acid-containing glycolipids together with terminal and internal sequences of mainly neutral oligosaccharides. The products were analyzed by MALDI-TOF MS in both positive and negative ion mode which gave spectra where the ions could be assigned to structures of the neutral and acidic components, respectively. Among glycolipids found were [structure in text] where R could be H, Fuc or NeuAc. Also observed were structures as [structure in text] which indicated linear extension along both branches. Observed at higher masses were fully branched structures obtained by stepwise extension with [structure in text] where R could be H, Fuc or NeuAc. Most probably further branching may occur along both the (1-->3)- and the (1-->6)-linked branches to give a partly dendritic structure. Structures with more than one sialic acid substituted could not be observed in the MALDI spectrum. Complementary information of the terminal sequences was obtained by FAB-MS analysis of permethylated undegraded PGCs. High-temperature gas chromatography/mass spectrometry of reduced and permethylated products from enzyme hydrolysis documented that Fuc was present in a blood group O sequence, Fuc-Hex-HexN-. Fucose may be placed on short (monolactosamine) or longer branches, while sialic acid seems to be restricted to monolactosamine branches. The conclusion is that human erythrocyte PGCs display microheterogeneity within terminal and internal parts of the poly-N-acetyllactosamine chains. The first branch from the ceramide end may be located at the second or third Gal and possibly also on the first Gal. Other branches may occur on every N-acetyllactosamine unit in fully branched domains, or there may be linear extensions between branches resulting in incompletely branched structures. The extended linear sequences may be present in both 3- and 6-linked antennae. Terminal structures are based on one, two or maybe higher number of N-acetyllactosamine units.

Neu5Acalpha3Gal is part of the Helicobacter pylori binding epitope in polyglycosylceramides of human erythrocytes.

The sialic acid dependent binding by the human pathogen Helicobacter pylori to polyglycosylceramides of human erythrocytes was investigated. Polyglycosylceramides, complex glycosphingolipids with a branched N-acetyllactosamine core, were isolated from human erythrocytes, blood group O, and subfractionated after peracetylation by anion-exchange chromatography. Three subfractions were deacetylated, analysed by matrix-assisted laser desorption ionization-time of flight MS and 2D 1H NMR spectroscopy. The observed mass ranges were m/z = 3093-7622, 3968-7255 and 3459-7987 in the mass spectra of the first, second and third fractions, respectively. The observed ions agreed with the general formula Hex(x+2)HexNAcxFucyNeu5AczCer. Two-dimensional 1H total correlation spectra of the mixtures showed that the first fraction contained 3-linked sialic acid and the second and third fractions contained both 3-linked and 6-linked sialic acid. Thin-layer chromatogram binding assays using the lectins from Maackia amurensis, specific for Neu5Acalpha3Galbeta4GlcNAc, and Sambucus nigra, specific for Neu5Acalpha6Gal/GalNAc, were used to confirm this distribution. H. pylori recognized all three fractions in the binding assay, indicating that the 3-linked, rather than 6-linked, sialic acid is essential for binding.

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.

Fingerprinting of large oligosaccharides linked to ceramide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: highly heterogeneous polyglycosylceramides of human erythrocytes with receptor activity for Helicobacter pylori.

Highly microheterogeneous polyglycosylceramides (PGCs) of human erythrocytes with an average composition of about 25 monosaccharides linked to ceramide were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The human gastric pathogen Helicobacter pylori was earlier shown to bind this glycosphingolipid mixture by thin-layer chromatogram binding assay. The receptor activity was present along the whole nonresolved chromatographic interval. Mass spectra of intact PGCs were compared with corresponding spectra of oligosaccharides enzymatically released from the ceramides. Two subfractions of PGCs containing less than one and more than one sialic acid residue per molecule were used. MALDI-MS spectra were recorded in both linear and reflectron mode with the accuracies of

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.

Analysis of 3- and 6-linked sialic acids in mixtures of gangliosides using blotting to polyvinylidene difluoride membranes, binding assays, and various mass spectrometry techniques with application to recognition by Helicobacter pylori.

A convenient approach to analyze 3- and 6-linked sialic acids in mixtures of biologically active gangliosides was developed. The procedure was adapted to work on small amounts of material and included parallel tests, which allowed direct analysis of structure and activity. The initial step in the procedure was separation of a mixture of gangliosides by thin-layer chromatography (TLC) and blotting to a polyvinylidene difluoride membrane. The gangliosides were then analyzed (a) by direct desorption from the membrane and fast atom bombardment mass spectrometry (FAB MS), (b) by membrane-binding assay using the NeuAcalpha3- and NeuAcalpha6-specific lectins from Maackia amurensis and Sambucus nigra, respectively, and (c) by TLC binding assay with radiolabeled bacteria. All experiments were performed on a mixture of gangliosides from human leucocytes, which contained species with affinity for the human gastric pathogen Helicobacter pylori. The procedure was used with good results for gangliosides with up to seven sugars per ceramide. A three-sugar ganglioside was identified as GM3 with ceramides composed of sphingosine (d18:1) and 20:0, h20:0, and 24:0 fatty acids. The sequences of four bands in the five-sugar region were consistent with sialylparagloboside (NeuAcalpha3/6Galbeta4GlcNAcbeta3Galbeta4GlcbetaCer). The ceramides were composed of d18:1 and 24:1 fatty acid in the first and third bands, and d18:1 and 16:0 fatty acid in the second and fourth bands from the top. The sialic acid was shown to be 3-linked in the upper two bands and 6-linked in the lower two bands. The same distribution of sialic acid and ceramides but the sequence elongated with one N-acetyllactosamine unit was observed in the less resolved interval containing seven-sugar glycosphingolipids. The direct comparison of binding of lectins and radiolabeled bacteria showed that H. pylori recognized 3-linked sialic acid only. These results were supported by a novel technique of analysis of the sialic acid linkage position by trifluoroacetolysis and gas chromatography/MS. Direct membrane/FAB MS was ineffective for species migrating below the seven-sugar region on the TLC. In this case, the membranes were instead cut in bands and the gangliosides extracted by methanol before analysis by FAB MS.

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.

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.

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.

Recognition of glycoconjugates by Helicobacter pylori. Comparison of two sialic acid-dependent specificities based on haemagglutination and binding to human erythrocyte glycoconjugates.

Helicobacter pylori expresses separate binding characteristics depending on growth conditions, as documented by binding to human erythrocyte glycoconjugates. Cells grown in Ham's F12 liquid medium exhibited a selective sialic acid-dependent binding to polyglycosylceramides, PGCs (Miller-Podraza et al.(1996) Glycoconjugate J13:453-60). There was no binding to traditional sialylated glycoconjugates like shorter-chain gangliosides, glycophorin or fetuin. However, cells grown on Brucella agar bound both to PGCs and other sialylated glycoconjugates. Fetuin was an effective inhibitor of haemagglutination caused by agar-grown cells, but had no or a very weak inhibitory effect on haemagglutination by F12-grown bacteria. PGCs were strong inhibitors in both cases, while asialofetuin was completely ineffective. The results indicate that H. pylori is able to express two separate sialic acid-dependent specificities, one represented by binding to fetuin, as described before, and another represented by a selective binding to PGCs.

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.

Screening for the presence of polyglycosylceramides in various tissues: partial characterization of blood group-active complex glycosphingolipids of rabbit and dog small intestines.

Twenty different human and animal tissues were investigated for the presence of polyglycosylceramides. The glycolipids were isolated by peracetylation of dry tissue residues left after conventional lipid extraction, followed by extraction with chloroform and subsequent Sephadex LH-20, Sephadex LH-60 and silica gel chromatography. In most of the cases only trace amounts of complex glycolipids were found. Distinct bands of glycosphingolipids migrating on TLC plates in a region of brain gangliosides and below were observed in bovine erythrocytes, human leukocytes and human colon mucosa. Definite fractions of polyglycosylceramides were isolated from rabbit small intestine, dog small intestine, human placenta and human leukocytes. The polyglycosylceramides of dog and rabbit intestine were characterized by colorimetric analysis, methylation analysis, mass spectrometry and immunological assays. The dog material contained branched carbohydrate chains with repeated fucosylated N-acetyllactosamine units. Rabbit intestine polyglycosylceramides resembled rabbit erythrocyte polyglycosylceramides with Hex-Hex- terminal determinants but were more complex in respect of sugar composition and structure. The material isolated from dog intestine showed A, H, Le(x) and Le(y) blood group activities. Polyglycosylceramides of human erythrocytes, placenta and leukocytes showed strong binding affinity for Helicobacter pylori, while polyglycosylceramide fractions from rabbit and dog intestine were receptor-inactive for this bacterium or displayed only weak and poorly reproducible binding.

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.

Influenza viruses display high-affinity binding to human polyglycosylceramides represented on a solid-phase assay surface.

Polyglycosylceramides (PGCs), complex glycolipids containing up to 50 or more sugar residues, are recognized as the minor components of the cell-surface membranes, but a knowledge on their tissue distribution, structure, and function is limited. In this study, the binding of influenza viruses to preparations of PGCs was investigated using a TLC overlay assay and a microwell adsorption assay. The ability of PGCs to bind influenza virus was dependent on the source from which they were derived. Preparations of PGCs from human erythrocytes were found to support binding of A and B influenza virus strains at a much lower concentration than sialyl-6-paragloboside and to be somewhat better receptors for these viruses compared to the sialylglycoprotein fetuin. A high virus-binding activity of PGCs suggests that these species could potentially serve as biologically important cell-surface receptors for influenza viruses.

Recognition of glycoconjugates by Helicobacter pylori: an apparently high-affinity binding of human polyglycosylceramides, a second sialic acid-based specificity.

Helicobacter pylori has been reported to agglutinate erythrocytes and to bind to various other cells in a sialic acid-dependent way. The binding was inhibited by sialyllactose or fetuin and other sialylated glycoproteins. The specificity apparently requires bacterial growth on agar, since we found that it was lost after growth in the nutrient mixture Ham's F12. Instead, the bacteria bound with high affinity and in a sialic acid-dependent way to polyglycosylceramides of human erythrocytes, a still incompletely characterized group of complex glycolipids. Bacteria grown in F12 medium were metabolically labelled with 35S-methionine and analysed for binding to glycolipids on thin-layer chromatograms and to glycoproteins on blots after electrophoresis, with human erythrocyte glycoconjugates in focus. There was no binding to simpler gangliosides including GM3 or sialylparagloboside, or to a mixture of brain gangliosides. In contrast, polyglycosylceramides of human erythrocyte membranes bound at a pmol level. The activity was eliminated by mild acid treatment, mild periodate oxidation or sialidase hydrolysis. Erythrocyte proteins as well as a range of reference glycoproteins did not bind except band 3, which was weakly active. However, this activity was resistant to periodate oxidation. These results indicate a second and novel sialic acid-recognizing specificity which is expressed independently of the previously described specificity.

New method for the isolation of polyglycosylceramides from human erythrocyte membranes.

A new procedure was developed for the isolation of long-chain, highly polar glycosphingolipids from human erythrocytes. The membrane material left after extraction of membrane lipids with organic solvents was peracetylated in a mixture of formamide, pyridine and acetic anhydride, and the acetylated products were then extracted with chloroform. The material was fractionated and purified by means of Sephadex LH-20, Sephadex LH-60 and silica-gel chromatography. The final preparations were mixtures of highly polar glycosphingolipids containing from 7 to 31 sugar residues relative to sphingosine. GC-MS analysis of the sugar part of the isolated fractions showed the presence of branched polyglycosyl chains of N-acetyllactosaminyl type. Endo-beta-galactosidase (Bacteroides fragilis) liberated from the deacetylated material two glycosphingolipids, which were identified by fast atom bombardment-mass spectrometry as Hex-Cer and HexNAc-Hex-Hex-Cer with sphingosine and mainly 24 and 22 carbon fatty acids. Endoglycoceramidase (Rhodococcus) degraded polyglycosylceramides to free ceramides and free polysaccharides. The released sugars were fractionated by high-pH ion-exchange chromatography into fractions differing in sialic acid content. The procedure presented in this paper can be used for large and small scale preparations of complex glycosphingolipids. It proved to be especially suitable for screening for polyglycosylceramides in different tissues.