Leb-active glycolipid in human plasma: Measurement by radioimmunoassay

A radioimmunoassay that measures Le^b-active glycolipids in human plasma has been developed using antiserum from a goat immunized with a Le^b blood group hapten, lacto-N-difucohexaose I, conjugated to polylysine. Binding by the antiserum of lacto-N-difucohexaose I conjugated to ¹²⁵I-labeled bovine serum albumin is specifically inhibited by Le^b-active ceramide hexasaccharide. Plasma levels of the glycolipid are quantitated by comparing the inhibitory activity of plasma with that of the purified Le^b-active glycolipid. Plasma samples from 35 blood group O Le(a ? b +) individuals contain Le^b-active ceramide hexasaccharide at an average concentration of 0.9 μg/ml (range: 0.2 to 2.5 μg/ml); no Le^b-active glycolipid (less than 0.02 μg/ml) could be detected in plasma from blood group O Le(a + b?) or O Le(a? b?) individuals. Plasma from A₁ Le(a ? b+) individuals contains less Le^b-active glycolipid than plasma from A₂ Le(a? b+) individuals: its level in 19 samples of A, Le(a? b+) plasma averages 0.2 μg/ml (range: 0.1 to 0.45 μg/ml), and its level in 9 samples of A₂ Le(a? b+) plasma averages 1.1 μg/ml (range 0.8 to 1.3 μg/ml). About one-third of the total Le^b-active glycolipid in whole blood is associated with erythrocytes and the rest is found in plasma.     

Immunochemistry of the Lewis-blood-group system: Partial characterization of Lea-, Leb-, and H-type 1 (LedH)-blood-group active glycosphingolipids from human plasma

Four different H-type 1 (Le^dH) blood-group-active glycosphingolipids (Le^dH-I–IV) have been isolated from the plasma of blood-group O Le(a?b?) secretors. The agglutination of O Le(a?b?) erythrocytes from secretors by 50 μl of 4 hemagglutinating units of caprine anti-Le^dH (anti-H-type 1) serum was inhibited by 0.02 μg of each of all four glycolipids. No Le^a or Le^b activities or reaction against Ulex europaeus lectin could be found. Le^dH-I, -II, -III, and -IV at 0.05, 0.01, 0.01, and 0.02 μg each are sufficient for incubation in order to convert 9 × 10⁷ O Le(a?b?) erythrocytes from nonsecretors into H-type 1 (Le^dH)-positive cells. Structural analysis of the H-type 1 glycolipids was performed in comparison to that of Le^a- and Le^b-blood-group-active glycolipids from human plasma isolated previously: Gas chromatography of peracetylated alditols revealed sugar composition. Combined gas chromatography-mass spectrometry established the glycosidic linkages. Together with the results obtained by direct inlet mass spectrometry of permethylated glycosphingolipids and by 360-MHz ¹H nuclear magnetic resonance spectroscopy (Egge, H., and Hanfland, P., 1981, Arch. Biochem. Biophys., 210, 396–404; Dabrowski, J., Hanfland, P., Egge, H., and Dabrowski, U., 1981, Arch. Biochem. Biophys., 210, 405–411) the complete structures of the oligosaccharide chains of the Le^a-, Le^b-, and H-type 1-active glycolipids were established: Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the Le^a antigens; Fucα1 → 2Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the Le^b antigens; and Fucα1 → 2Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the H-type 1 (Le^dH) glycolipids. The diverse antigens of the same blood-group specificity obviously differ from one another in their lipid residue. In addition, plasmatic neolactotetraosylceramide could be identified, differing from that of human erythrocytes by a slower migration behavior in thin-layer chromatography.     

Enzymatic synthesis of blood-group Lewis-specific glycolipids.

A blood-group Lewis precursor glycolipid was isolated from the plasma of a Lewis-negative individual [Le(a--b--)] and treated with fucosyltransferases from human gastric mucosa and GDP-fucose. Subsequently the glycolipid was adsorbed onto Le(a--b--) erythrocytes and the presence of blood-group Lewis antigens was assessed by passive hemagglutination with anti-Lewis sera. It was shown that the precursor glycolipid was enzymatically transformed to blood-group Lewis a (Lea) and Lewis b (Leb) specific glycolipids. Leb-glycolipid was also synthesized by fucosylation of an isolated Lea-glycolipid. Moreover Le(a--b--) erythrocytes were shown to develop Lea and Leb activities when subjected to enzymatic fucosylation, thus showing that Lewis-negative cells carry blood-group Lewis precursor glycolipid on the surface of their membrane. Le(a + b--) erthrocytes, upon enzymatic fucosylation, acquired Leb activity.     

Mass spectrometric analysis of permethylated glycosphingolipids II. Comparative studies on different blood-group active and related erythrocyte membrane glycosphingolipids

Three isomeric ceramide tetrasaccharides — P blood-group active globoside, lacto-N-neotetraosyl ceramide as ABH blood-group precursor, both isolated from human erythrocytes and “asiologanglioside” from human brain as reference standard — and two ceramide pentasaccharides — H blood-group active glycosphingolipid, obtained from blood-group B active ceramide hexasaccharide of human B erythrocytes after α-galactosidase treatment and ceramide pentasaccharide from rabbit erythrocytes with B-like blood-group activity — were investigated by mass spectrometry after permethylation. The carbohydrate moiety exhibits differences not only concerning the sugar sequence but also with regard to the position of some glycosidie linkages: Oligosaccharides containing N-acetylhexosamine substituted at position 4 produce spectra that are distinctly different from those containing C-3 substituted N-acetylhexosamines, thus allowing the differentiation between type 1 and type 2 carbohydrate chains. Moreover, oligosaccharide ions with a hexose at the cleavage site exhibit a fragmentation pattern different from those with a N-acetylhexosamine at the “reducing terminal”. The intensity ratio between parent ion and parent ion — 32 mass units is Q ? 3 in the first case, whereas in the latter case Q is <1. The Q-values are given for 14 oligosaccharide ions. Differences in the composition of the ceramide residues can also be deduced from the mass spectra.     

Expression of Lewis histo-blood group glycolipids in the plasma of individuals of Le(a+b+) and partial secretor phenotypes

Red cell Lewis antigens are carried by glycosphingolipids passively absorbed from plasma. Plasma was collected from a spectrum of individuals with normal and unusual Lewis/secretor phenotypes in order to investigate the glycolipid basis for the unusual phenotypes. Samples were obtained from: a Le(a+b–) ABH nonsecretor who secreted Lewis substances; a Le(a+b–) partial secretor; Le(a+b+) partial secretors; Le(a+b+) secretors; and a full range of normal Lewis/secretor phenotypes as controls. The Le(a+b+) samples represented Polynesian, Asian and Réunion Island ethnic backgrounds. Nonacid glycolipids were prepared, separated by thin-layer chromatography, and then immunostained with potent monoclonal antibodies of known specificity. Despite different serological profiles of the Le(a+b–) and Le(a+b+) Polynesian samples, their plasma glycolipid expressions were very similar, with both Le^a and Le^b co-expressed. The copresence of Le^a and Le^b in Le(a+b+) samples is in marked contrast to Caucasians with normal Lewis phenotypes, who have predominantly either Le^a or Le^b. These results suggest that there is a range of the secretor transferases in different individuals, possibly due to different penetrance or to several weak variants. We also show that Lewis epitopes on longer and/or more complex core chains appear to be predominant in the Polynesian Le(a+b+) samples. The formation of these extended glycolipids is compatible with the concept that in the presence of reduced secretor fucosyltransferase activity, increased elongation of the precursor chain occurs, which supports the postulate that fucosylation of the precursor prevents or at least markedly reduces chain elongation.Abbreviations CBA chromatogram binding assay - TLC thin-layer chromatography     

Diffusion-controlled reaction kinetics of the binding of carbon monoxide to the heme undecapeptide of cytochrome c (microperoxidase 11) in high viscosity solvents

Glycosphingolipids from human plasma with Le^a, Le^b, and H-type 1 (Le^dH) Lewis-blood-group activity have been analyzed after permethylation by electron impact mass spectrometry using an indirectly heated direct insertion probe. The spectra obtained are compared with that of permethylated neo-lactotetraosyl ceramide (Gl-3) from human plasma. The fragmentation patterns presented show clearly, that Le^a and H-type 1 glycosphingolipids are ceramide pentasaccharides while Le^b is a ceramide hexasaccharide. All Lewis-blood-group-active compounds investigated produced ions specific for type 1 carbohydrate chains. It is therefore concluded, that all compounds are derivatives of lacto-N-tetraose. The obtained spectra support the following sequences: Hexose-1→3-hexosamine[4←1-deoxyhexose]-hexose-hexose ceramide for the Le^a derivatives; deoxyhexose-hexose-1→3-hexosamine4←1-deoxyhexose]-hexose-hexose ceramide for the Le^b derivatives; and deoxyhexose-hexose-1→3-hexosamine-hexose-hexose ceramide for all H-type 1 (Le^dH) derivatives. In the case of the H-type 1 glycosphingolipids four subfractions were analyzed separately. While all four fractions contained the same carbohydrate sequence, significant differences were observed in the ceramide residues. Specific fragmentation patterns indicate the presence of sphingosine, icosasphingosine, and 4-hydroxysphinganine besides normal, unsaturated, and hydroxylated fatty acids in all Lewis-blood-group-active glycolipids.     

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.     

Immunochemistry of the Lewis-blood-group system: Proton nuclear magnetic resonance study of plasmatic Lewis-blood-group-active glycosphingolipids and related substances

The Le^a-, Le^b-, and H-type 1 (Le^dH)-blood-group-active glycosphingolipids, as well as H-I-type 2 glycolipid, lactotetraosyl ceramide, and neo-lactotetraosyl ceramide were examined by ¹H nuclear magnetic resonance at 360 MHz in dimethyl-d₆ sulfoxide as solvent. The resonances of almost all protons of the sugar rings were assigned with the aid of spin decoupling and nuclear Overhauser difference spectroscopy. The latter technique was also applied to establish the sequences and sites of glycosidic linkage. This information, combined with the chemical shift-structure correlations established in our previous work, led to an independent identification of those six glycolipids. Type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains can be distinguished by this approach. Some deviations from additivity in chemical shifts, calculated for oligosaccharides from the data on their constituent sugar residues, furnished information on the conformational changes in crowded glycolipid molecules.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Identification of Leb antigens in meconium of a phenotypically a Le(a+b−) non-secretor individual

Blood group active fucolipids of human meconium have been shown to correlate to the ABH and Lewis blood groups and to the secretor status of the corresponding children. Using a monoclonal anti-Le^b antibody and an antibody to chromatogram binding assayy the presence of Le^b antigens in meconium of a phenotypically A Le(a⁺b^?) non-secretor indivual is here demonstrated. Phenotype was determined on cord blood and saliva obtained 2 years after birth.     

Evidence for erythrocyte membrane glycoproteins being carriers of blood-group P1 determinants

The contribution of different membrane constituents to the bloodgroup P₁ activity of human erythrocytes was investigated. Pronase digestion of native red cell stroma or partition between butanol and water had no serologically detectable effect, whereas pronase-treatment of previously butanol-extracted membranes liberated virtually all blood-group P₁ determinants from the ghosts. On Laemmli gels, all P₁ activity was found in the band 4.5 region. Thus it is concluded that, in addition to the well-documented P₁ glycolipid, also membrane glycoproteins are carriers of blood-group P₁ determinants.     

Immunochemical studies on the specificity of soybean agglutinin

The specificity of the purified soybean agglutinin has been studied immunochemically by quantitative precipitin and quantitative precipitin inhibition assays. The lectin is precipitated by human A and Le^a blood-group substance, by the products of the second, third, fourth, and fifth stages of periodate oxidation of a human H blood-group substance (JS), and by precursor blood-group substances, as well as by a pig-submaxillary mucin having blood-group A activity, by partially hydrolyzed blood-group B substances (Pl fraction), and by group C streptococcal polysaccharide. The activity is attributable to terminal α-linked 2-acetamido-2-deoxy-d-galactopyranosyl or to α- or β-d-galactopyranosyl residues. The lectin did not precipitate with human blood-group H substances, with the product of the first stage of periodate oxidation (JS), with streptococcal group A polysaccharide, or with pig-submaxillary mucin devoid of blood-group A activity, and is poorly precipitated by blood-group B substances. Inhibition of precipitation with various monosaccharides indicated that the lectin is strongly specific for 2-acetamido-2-deoxy-d-galactose and for its oligosaccharides, and to a lesser extent for d-galactose and its oligosaccharides; the α-glycosides of both sugars were slightly more reactive than the β-glycosides of 2-acetamido-2-deoxy-d-galactose, and both α- and β-glycosides were more active than the free monosaccharides. Aromatic α- and β-glycosides of 2-acetamido-2-deoxy-d-galactose and d-galactose were better inhibitors than the corresponding methyl or ethyl compounds. The blood-group A trisaccharide α-d-GalNAcp-(1→3)-β-d-Galp-(1→3)-d-GlcNAc was more active than the disaccharide lectins by the use of precipitation with polysaccharides, as well as inhibition reactions, is essential to the understanding of their reactivity with cell-surface receptors.     

Immunochemical and immunohistological expression of Lewis histo-blood group antigens in small intestine including individuals of the Le(a+b+) and Le(a−b−) nonsecretor phenotypes

Histological samples and total non-acid glycosphingolipids were prepared from small intestine of human cadavers with the Le(a+b+) and Le(a–b–) nonsecretor phenotypes and contrasted with the more common Lewis phenotypes. Glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with monoclonal antibodies reactive to Lewis epitopes. Paraffin-embedded small intestine sections were also fluorescently immunostained with anti-Lewis antibodies. Unlike the common Lewis positive phenotypes, we were immunochemically able to demonstrate the copresence of large amounts of Le^a and Le^b glycolipids in the Le(a+b+) sample. In addition we demonstrated increased formation of extended Lewis structures in this phenotype. By immunohistochemistry Le^a, Le^b and type 1 precursor chain epitopes could be demonstrated in the brush border. These results show that the expression of the Le(a+b+) phenotype at the erythrocyte phenotyping level parallels the small intestinal expression of this phenotype, and the patterns of Lewis antigen expressions are unique to this phenotype. By immunohistochemistry and immunochemistry we also demonstrated the presence of trace amounts of Lewis active glycoconjugates in the small intestine of the Le(a–b–) nonsecretor and Le(a+b–) samples. In the Le(a–b–) nonsecretor Le^a and Le^b activity was absent and type 1 precursor was present in brush border, while Le^b activity was immunohistologically demonstrated in the Golgi apparatus of the deep glands. Trace amounts of both Le^a and Le^b glycolipids were identified in this sample. In parallel trace Le^b activity could also be detected in the glycolipids of the Le(a+b–) sample and could be immunohistologically demonstrated to be fully expressed in occasional cells in the deep glands of the small intestine, a pattern quite dissimilar to that of the Le(a–b–) nonsecretor. The results in this paper show that the expression of Lewis glycoconjugates in the small intestine parallel the expression of Lewis erythrocyte phenotypes. However, inappropriate Lewis activity is also seen in individuals of other phenotypes and the mechanisms by which these Lewis antigens are made appears to be different for different phenotypes.Abbreviations FITC fluorescein isothiocyanate - HPLC high-performance liquid chromatography - NeuAc N-acetyl-d-neuraminic acid - RBC red blood cell - TLC thin-layer chromatography - TRITC tetramethyl rhodamine isothiocyanate     

Structural and immunochemical identification of Leb glycolipids in the plasma of a group O Le(a-b-) secretor

Total non-acid glycosphingolipids were isolated from the plasma of a healthy red blood cell group O Le(a-b-) salivary ABH secretor individual. Glycolipids were fractionated by HPLC and combined into eight fractions based on chromatographic and immunoreactive properties. These glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with antibodies reactive to the type 1 precursor (Le^c), H type 1 (Le^d), Le^a and Le^b epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and LSIMS) and proton NMR spectroscopy. Expected blood group glycolipids, such as H type 1, (Fuc1-2Gal1-3GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified. Inconsistent with the red cell phenotype and for the first time, small quantities of Le^b blood group glycolipids (Fuc1-2Gal1-3(Fuc1-4)GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified in the plasma of a Lewis-negative individual. These findings confirm recent immunological evidence suggesting the production of small amounts of Lewis antigens by Lewis negative individuals. Abbreviations: HPLC, high performance liquid chromatography; TLC, (high performance) thin layer chromatography; EI-MS, electron impact ionisation mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; NMR, nuclear magnetic resonance spectroscopy. The sugar types are abbreviated to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose (fucose). The ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids; LCB, long chain base.     

The common structure in fucosyllactosaminolipids accumulating in human adenocarcinomas,and its possible absence in normal tissue

Two major glycolipids accumulating in a human primary liver adenocarcinoma, but absent in normal liver, were characterized as lacto-N-fucopentaosyl(III)ceramide and difucosyllacto-N-$\text{nor}$-hexaosylceramide, (Galβ1→4[Fucα1→3]GlcNAcβ1→3Galβ1→4[Fucα1→3]GlcNAcβ1→3Galβ1→4Glcβ1→1Cer), a new type of glycolipid with Le^x-determinant. Comparison of glycolipids bearing Le^x-determinant in various cases of human colonic adenocarcinoma, in adjacent normal mucosa tissue, and in erythrocytes reveals a possibility that glycolipids accumulating in human adenocarcinoma, but not in normal tissue, have a common structural unit as identified below:

     

Interaction of the Laburnum anagyroides lectin with fucoantigens

We studied interaction of the lectin from the bark of Golden Rain shrub (Laburnum anagyroides, LABA) with a number of basic fucose-containing carbohydrate antigens by changes in its tryptophan fluorescence. The strongest LABA binding was observed for the trisaccharide H of type 6 [α-L-Fucp-(1-2)-β-D-Galp-(1-4)-D-Glc, K _a = 4.2 × 10³ M^?1]. The following antigens were bound with a weaker affinity: H-disaccharide α-L-Fucp-(1-2)-D-Gal, a glucoanalogue of tetrasaccharide Le^y α-L-Fucp-(1-2)-β-D-Galp-(1-4)-[α-L-Fucp-(1-3)]-D-Glc, and 6-fucosyl-N-acetylglucosamine, a fragment of core of the N-glycans family (K _a 1.1?1.7 × 10³ M^?1). The lowest binding was observed for L-fucose (K _a = 2.7 × 10² M^?1) and trisaccharide Le^a, (β-Galp-(1-3)-[α-L-Fucp-(1-4)]-GlcNAc (K _a = 6.4 × 10² M^?1). The Le^d, Le^a, and Le^x pentasaccharides and Le^b hexasaccharide were not bound to LABA.     

A galactose specific agglutinin, a blood-group H active polysaccharide, and a trypsin inhibitor in albumin glands and eggs of Arianta arbustorum (Helicidae).

Eggs and albumin glands of the land snail Arianta arbustorum contain a powerful agglutinin which reacts specially with rabbit erythrocytes. The agglutination can be inhibited completely by di-, tri-, and oligosaccharides with α-glycosidically (1 → 6) bound galactose residues. β-Linked sugars do not inhibit the agglutinin. The agglutinin activity is not dependent on Ca²⁺ ions. Eggs and albumin glands also contain a blood-group active polysaccharide which, unlike the polysaccharide from the albumin gland of Helix pomatia (Baldo, B. A., and Uhlenbruck, G. 1973. Cross-reactive human blood group H-active polysaccharide from Helix pomatia. I. Detection with catfish anti-H and eel sera. Immunology, 25, 1–13) does not react with anti-H_eel, but does react with the agglutinins of Evonymus europaeus and Laburnum alpinum. The Arianta polysaccharide has been purified and shown to be galactogen. Finally, the occurrrence of a strong trypsin inhibitor has been demonstrated in the extracts of eggs and albumin glands. The inhibitor has been separated by column chromatography. The precipitation lines of both substances have been identified in the immunoelectrophoretogram of the extracts of albumin glands and eggs.     

Differentiation of isomeric Lewis blood groups by positive ion electrospray tandem mass spectrometry

Lewis histo-blood group antigens are one of the major classes of biologically active oligosaccharides. In this work, underivatized Lewis blood groups were studied by electrospray tandem mass spectrometry (ESI-MSⁿ) in the positive mode with three different mass analyzers: Q-TOF (quadrupole time-of-flight), QqQ (triple quadrupole), and LIT (linear ion trap). It was observed that, under collision-induced fragmentations, type 1 Lewis antigens (Le^a and Le^b) could be distinguished from type 2 (Le^x and Le^y) on the basis of specific fragmentations of the GlcNAc unit. Whereas O-4-linked sugars of the GlcNAc are lost as residues, the O-3-linked sugars undergo fragmentation both as sugar units and as sugar residues (unit −18 Da). Type 2 Lewis antigens also showed a characteristic cross-ring cleavage ^0,2A₂ of the GlcNAc. As a result, the product ions at m/z 388 and 305, characteristic of Le^x, and m/z 372, characteristic of Le^a, are proposed to distinguish the trisaccharide isomers Le^x/Le^a. Also, the product ions at m/z 534 and 305, characteristic of Le^y, and m/z 372, characteristic of Le^b, are proposed to distinguish the tetrasaccharide isomers Le^b/Le^y. These diagnostic fragment ions were further applied in the identification of Lewis type 2 antigens (Le^x and Le^y) in the lipopolysaccharide of the human gastric pathogen, Helicobacter pylori.     

Immunochemistry of the Lewis blood-group system: isolation and structures of Lewis-c active and related glycosphingolipids from the plasma of blood-group O Le(a-b-) nonsecretors

Five different glycosphingolipid fractions (GL-3, 285 micrograms; GL-5, 1090 micrograms; GL-6, 615 micrograms; GL-7, 555 micrograms; and GL-8, 155 micrograms) have been isolated from 25 liters of plasma of O Le(a-b-) nonsecretors by means of ethanol extraction, several steps of Folch distribution, and reversed-phase, silicic acid, and ion-exchange column chromatography of native or peracetylated substances. Final purification, accomplished by preparative silica gel high-performance thin-layer chromatography, led to chromatographic homogeneity of GL-3 and GL-6. In the hemagglutination inhibition as well as quantitative passive hemagglutination techniques two of these substances (GL-3, GL-5) exhibited distinct, and the other three (GL-6-GL-8) very strong, Lec blood-group activities when tested against two different Lec antisera of human or goat origin. The fragments' structures were elucidated by fast atom bombardment and electron impact mass spectrometry of permethylated derivatives in order to determine molecular weight, sugar sequence, position of branching points, and type of oligosaccharide chains, as well as fatty acid and sphingosine patterns of the ceramide residue. Combined gas-liquid chromatography and mass spectrometry of partially methylated alditol acetates identified sugar composition and glycosidic linkages. Thus, the following structures could be established: (formula; see text) In contrast to the structurally homogeneous GL-3, minor amounts of 4-O-substituted GlcNAc pointed to a small contamination of GL-6 by branched type 2 ceramide nonasaccharide analogs. Glycolipids containing hepta- or nonasaccharides as in GL-3 or GL-6 could also be identified in fractions GL-5 (ceramide heptasaccharide) and GL-7 and GL-8 (ceramide nonasaccharide). These latter fractions revealed, however, distinct heterogeneity due to the presence of a small amount of either a type 2 analog of GL-3 (GL-5) or linear, mainly type 2, ceramide hexa- (GL-5, GL-7) or octasaccharides (GL-8). In addition to previous immunochemical communications the presented Lec active structures of GL-3 and GL-6 provide evidence that 3-fucosyl-N-acetyllactosamine in combination with a type 1 based oligosaccharide sequence and a 3,6-galactosyl branching point are essential parts of the Lec antigenic determinant (as marked in the formula of GL-6).     

Carbohydrate phenotyping of human and animal milk glycoproteins

Breast-milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. The plethora of carbohydrate epitopes in breast-milk is known to differ between species, with human milk expressing the most complex one. We have investigated the expression of protein-bound carbohydrate epitopes in milk from man, cow, goat, sheep, pig, horse, dromedary and rabbit. Proteins were separated by SDS-PAGE and the presence of carbohydrate epitopes on milk proteins were analysed by Western blotting using different lectins and carbohydrate-specific antibodies. We show that ABH, Lewis (Le)^x, sialyl-Le^x, Le^a, sialyl-Le^a and Le^b carbohydrate epitopes are expressed mainly on man, pig and horse milk proteins. The blood group precursor structure H type 1 is expressed in all species investigated, while only pig, dromedary and rabbit milk proteins carry H type 2 epitopes. These epitopes are receptors for Helicobacter pylori (Le^b and sialyl-Le^x), enteropathogenic (H type 1, Le^a and Le^x) and enterotoxic Escherichia coli (heat-stable toxin; H type 1 and 2), and Campylobacter jejuni (H type 2). Thus, milk from these animals or their genetically modified descendants could have a therapeutic effect by inhibiting pathogen colonization and infection. Published in 2005.