Biochemical studies on sphingolipids of Artemia franciscana: novel neutral glycosphingolipids

Neutral glycosphingolipids containing one to six sugars in their oligosaccharide chains have been isolated from cysts of the brine shrimp Artemia franciscana. The structures of these glycolipids were identified by methylation analysis, partial acid hydrolysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance spectroscopy to be Glcβ1-Cer, Manβ1-4Glcβ1-Cer, Fucα1-3Manβ1-4Glcβ1-Cer, GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GlcNAcα1-2Fucα1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CPS), and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CHS). Two glycosphingolipids, CPS and CHS, were characterized as novel structures. Because Artemia contains a certain series of glycosphingolipids (-Fucα3Manβ4GlcβCer), which differ from the core sugar sequences reported thus far, we tentatively designated the glycosphingolipids characterized as nonarthro-series ones. Furthermore, CHS exhibited a hybrid structure of arthro-series and nonarthro-series sugar chain. Two novel glycosphingolipids were characterized from the brine shrimp Artemia franciscana; one was composed of arthrotetraose and a branching fucose attached to N-acetylglucosamine residue, and the other was composed of CPS with an additional N-acetylglucosamine residue attached to the branching fucose.     

Chemical characterization of milk oligosaccharides of the polar bear, Ursus maritimus

Two trisaccharides, three tetrasaccharides, two pentasaccharides, one hexasaccharide, one heptasaccharide, one octasaccharide and one decasaccharide were isolated from polar bear milk samples by chloroform/methanol extraction, gel filtration, ion exchange chromatography and preparative thin-layer chromatography. The oligosaccharides were characterized by ¹H-NMR as follows: the saccharides from one animal: Gal(α1-3)Gal(β1-4)Glc (α3′-galactosyllactose), Fuc(α1-2)Gal(β1-4)Glc (2′-fucosyllactose), Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (B-tetrasaccharide), GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (A-tetrasaccharide), Gal(α1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(α1-3)Gal(β1-4)GlcNAc(β1-3)[Gal(α1-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc; the saccharides from another animal: α3′-galactosyllactose, Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]Glc, A-tetrasaccharide, GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]Glc (A-pentasaccharide), Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc, Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]Glc (difucosylheptasaccharide) and Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3){Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (difucosyldecasaccharide). Lactose was present only in small amounts. Some of the milk oligosaccharides of the polar bear had α-Gal epitopes similar to some oligosaccharides in milk from the Ezo brown bear and the Japanese black bear. Some milk oligosaccharides had human blood group A antigens as well as B antigens; these were different from the oligosaccharides in Ezo brown and Japanese black bears.     

Carbohydrate binding specificities and crystal structure of the cholera toxin-like B-subunit from Citrobacter freundii

Enterotoxigenic Escherichia coli and Vibrio cholerae are well known causative agents of severe diarrheal diseases. Both pathogens produce AB₅ toxins, with one enzymatically active A-subunit and a pentamer of receptor-binding B-subunits. The primary receptor for both B-subunits is the GM1 ganglioside (Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer), but the B-subunits from porcine isolates of E. coli also bind neolacto-(Galβ4GlcNAcβ-)terminated glycoconjugates and the B-subunits from human isolates of E. coli (hLTB) have affinity for blood group A type 2-(GalNAcα3(Fucα2)Galβ4GlcNAcβ-)terminated glycoconjugates.     

Biochemical studies on sphingolipids of Artemia franciscana: complex neutral glycosphingolipids

Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer, and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III³(GlcNAcα2Fucα)-At₄Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important.     

Novel Leb-like Helicobacter pylori-binding glycosphingolipid created by the expression of human alpha-1,3/4-fucosyltransferase in FVB/N mouse stomach

The "Le(b) mouse" was established as a model for investigations of the molecular events following Le(b)-mediated adhesion of Helicobacter pylori to the gastric epithelium. By the expression of a human alpha-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice, a production of Le(b) glycoproteins in gastric pit and surface mucous cells was obtained in this "Le(b) mouse," as demonstrated by binding of monoclonal anti-Le(b) antibodies. To explore the effects of the human alpha-1,3/4-fucosyltransferase on glycosphingolipid structures, neutral glycosphingolipids were isolated from stomachs of transgenic alpha-1,3/4-fucosyltransferase-expressing mice. A glycosphingolipid recognized by BabA-expressing H. pylori was isolated and characterized by mass spectrometry and proton NMR as Fuc alpha 2Gal beta 3(Fuc alpha 4)GalNAc beta 4 Gal beta 4 Glc beta 1Cer, i.e., a novel Le(b)-like glycosphingolipid on a ganglio core. In addition, two other novel glycosphingolipids were isolated from the mouse stomach epithelium that were found to be nonbinding with regard to H. pylori. The first was a pentaglycosylceramide, GalNAc beta 3 Gal alpha 3(Fuc alpha 2)Gal beta 4 Glc beta 1Cer, in which the isoglobotetrasaccharide has been combined with Fuc alpha 2 to yield an isoglobotetraosylceramide with an internal blood group B determinant. The second one was an elongated fucosyl-gangliotetraosylceramide, GalNAc beta 3(Fuc alpha 2)Gal beta 3GalNAc beta 4Gal beta 4 Glc beta 1Cer.     

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids. Blood group ABH-active substances.

High resolution nuclear magnetic resonance spectra of permethylated and permethylated-reduced (LiAlH₄) derivatives were recorded in chloroform solution for the following glycosphingolipids with known structure: lactotriaosylceramide, neolactotetraosylceramide (paragloboside), two blood group H-active pentaglycosylceramides (type 1 and type 2 saccharide chains, respectively), a B-active hexaglycosylceramide, an A-active hexaglycosylceramide, and an A-active octaglycosylceramide. Good quality and resolution allow a clear-cut diagnosis of α-anomeric protons of Fuc, Gal, and GalNAc, and in most cases of all β protons. Upon reduction there is a strong deshielding effect on H-1 of Gal of Galβ1 → 3GlcNAc but not on Gal of Galβ1 → 4GlcNAc. It is therefore possible to differentiate type 1 and type 2 chains by this method, a structural difference of importance for serological specificity. Nuclear magnetic resonance spectroscopy may therefore provide conclusive information on the anomeric structure of the immunodeterminant of blood group-active glycolipids using the same derivatives as for sequence analysis by mass spectrometry.     

The EPR spectrum of cytochrome b-563 in the cytocrhome bf complex from spinach: (1993) FEBS Letters 164, 71–74

Blood group H antigen with globo-series structure, reacting with the monoclonal antibody MBrl, was isolated and characterized from human blood group O erythrocytes. The structure was identified by methylation analysis, direct probe mass spectrometry, and ¹H-nuclear magnetic resonance spectroscopy as shown below: Fucαl → 2Galβl → 3GalNAcβl → 3Galαl → 4Galβl → 4Glcβl → 1Cer     

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.     

Chemical structures of oligosaccharides in milks of the American black bear (Ursus americanus americanus) and cheetah (Acinonyx jubatus)

The milk oligosaccharides were studied for two species of the Carnivora: the American black bear (Ursus americanus, family Ursidae, Caniformia), and the cheetah, (Acinonyx jubatus, family Felidae, Feliformia). Lactose was the most dominant saccharide in cheetah milk, while this was a minor saccharide and milk oligosaccharides predominated over lactose in American black bear milk. The structures of 8 neutral saccharides from American black bear milk were found to be Gal(β1–4)Glc (lactose), Fuc(α1–2)Gal(β1–4)Glc (2′-fucosyllactose), Gal(α1–3)Gal(β1–4)Glc (isoglobotriose), Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)Glc (B-tetrasaccharide), Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)[Fuc(α1–3)]Glc (B-pentasaccharide), Fuc(α1–2)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc (difucosyl lacto-N-neotetraose), Gal(α1–3)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc (monogalactosyl monofucosyl lacto-N-neotetraose) and Gal(α1–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (Galili pentasaccharide). Structures of 5 acidic saccharides were also identified in black bear milk: Neu5Ac(α2–3)Gal(β1–4)Glc (3′-sialyllactose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Fuc(α1–2)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyl monofucosyl lacto-N-neohexaose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Gal(α1–3)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyl monogalactosyl lacto-N-neohexaose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3){Gal(α1–3)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–6)}Gal(β1–4)Glc (monosialyl monogalactosyl monofucosyl lacto-N-neohexaose), and Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3){Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–6)}Gal(β1–4)Glc (monosialyl monogalactosyl difucosyl lacto-N-neohexaose). A notable feature of some of these milk oligosaccharides is the presence of B-antigen (Gal(α1–3)[Fuc(α1–2)]Gal), α-Gal epitope (Gal(α1–3)Gal(β1–4)Glc(NAc)) and Lewis x (Gal(β1–4)[Fuc(α1–3)]GlcNAc) structures within oligosaccharides. By comparison to American black bear milk, cheetah milk had a much smaller array of oligosaccharides. Two cheetah milks contained Gal(α1–3)Gal(β1–4)Glc (isoglobotriose), while another cheetah milk did not, but contained Gal(β1–6)Gal(β1–4)Glc (6′-galactosyllactose) and Gal(β1–3)Gal(β1–4)Glc (3′-galactosyllactose). Two cheetah milks contained Gal(β1–4)GlcNAc(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (lacto-N-neohexaose), and one cheetah milk contained Gal(β1–4)Glc-3’-O-sulfate. Neu5Ac(α2–8)Neu5Ac(α2–3)Gal(β1–4)Glc (disialyllactose) was the only sialyl oligosaccharide identified in cheetah milk. The heterogeneity of milk oligosaccharides was found between both species with respect of the presence/absence of B-antigen and Lewis x. The variety of milk oligosaccharides was much greater in the American black bear than in the cheetah. The ratio of milk oligosaccharides-to-lactose was lower in cheetah (1:1–1:2) than American black bear (21:1) which is likely a reflection of the requirement for a dietary supply of N-acetyl neuraminic acid (sialic acid), in altricial ursids compared to more precocial felids, given the role of these oligosaccharides in the synthesis of brain gangliosides and the polysialic chains on neural cell adhesion.

     

Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases

Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.     

Glycosphingolipids in insects. Chemical structures of ceramide tetra-, penta-, hexa-, and heptasaccharides from Calliphora vicina pupae (Insecta: Diptera)

Four neutal fraction glycosphingolipids, designated components 4-7, were purified from the pupae of Calliphora vicina and isolated by the use of high performance liquid chromatography. Their chemical structures were determined to be: GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer and Gal(alpha 1-3)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer; and GlcNAC(beta 1-3)Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)Cer. By the use of specific exoglycosidases, it was possible to assign anomeric configurations to all the sugar residues present. Analysis of the ceramide moiety by electron-impact mass spectrometry revealed the dominant fatty acid and sphingoid to be arachidic acid (C20:0) and tetradecasphing-4-enine, respectively.     

The ruminant parasite Haemonchus contortus expresses an α1,3-fucosyltransferase capable of synthesizing the Lewis x and sialyl Lewis x antigens

Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize α1,3-fucosylated GlcNAc and terminal β-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal β-Gal, and the glycoconjugates lack the Lewis x (Lex) antigen or other related fucose-containing antigens, such as sialylated Lex, Lea, Leb Ley, or H-type 1. Direct assays of parasite extracts demonstrate the presence of an α1,3-fucosyltransferase (α1,3FT) and β1,4-N-acetylgalactosaminyltransferase (β1,4GalNAcT), but not β1,4-galactosyltransferase. The H. contortus α1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galα1→4GlcNAcβ1→3Galβ1→4Glc to form lacto-N-fucopentaose III Galβ1→ 4[Fucα1→3]GlcNAcβ1→3Galβ1→4Glc, which contains the Lex antigen, and the acceptor lacdiNAc (LDN) GalNAcβ1→4GlcNAc to form GalNAcβ1→4[Fucα1 →3]GlcNAc. The α1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 μ M and a Vmax of 10.8 nmol-mg?1 h?1. The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The α1,3FT acts best with type-2 glycan acceptors (Galβ1→4GlcNAcβ1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus α1,3FT can synthesize the Lex antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.     

Glycosphingolipids in insects. The amphoteric moiety, N-acetylglucosamine-linked phosphoethanolamine, distinguishes a group of ceramide oligosaccharides from the pupae of Calliphora vicina (Insecta: Diptera)

A group of Calliphora vicina pupal glycolipids could be segregated from the neutral glycosphingolipids, according to their two-dimensional TLC migration properties and positive reactions toward ninhydrin and fluorescamine spray reagents. These classified zwitterionic glycolipids were isolated by silica-gel column chromatography and characterized by the presence of a N-acetyl-glucosamine-bound phosphoethanolamine residue. The structural elucidation of the oligosaccharide moieties was performed by the determination of constituent carbohydrates as alditol acetates, linkage analysis by permethylation, exoglycosidase cleavage, fast-atom-bombardment mass spectrometry and NMR spectroscopy. The dominant fatty acid and sphingoid base species of the ceramide moieties were C20:0 (arachidic acid) and C14:1 (tetradecasphing-4-enine), respectively. The chemical structures of the zwitterionic, biogenetic glycosphingolipid series were determined as: (PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1- 4)Glc beta Cer; Gal(beta 1-3)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer; Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn-6')GlcNAc(beta 1- 3)Man(beta 1-4)Glc beta Cer; GlcNAc(beta 1-3)Gal(beta 1-3)GalNAc(alpha 1-4)GalNAc(beta 1-4)(PEtn- 6')GlcNAc(beta 1-3)Man(beta 1-4)Glc beta Cer.     

The hapten structure of a developmentally regulated glycolipid antigen (SSEA-1) isolated from human erythrocytes and adenocarcinoma: a preliminary note

Glycolipid antigen reacting to the monoclonal antibody directed to the developmentally regulated antigen SSEA-1 was isolated from human erythrocytes and colonic adenocarcinoma. The antigens have the Le^x (Galβl→4[Fucα]→3]GlcNAcβl→R) or Le^y (Fucαl→2Galβl→4[Fucαl→3]GlcNAcβl→R) structure at the termini of the branched polylactosaminolipid. In addition, a novel polyfucosyl structure locating exclusively at the internal GlcNAc was detected in the tumor antigen. The antibody reacts with a simple monovalent Le^x glycolipid (Galβl→4[Fucαl→3]GlcNAcβl→3Galβl→4Glcβl→Cer) previously isolated from colonic carcinoma when presented at a high density on liposomes. The antibody therefore may react to the bivalent or multivalent Le^x or Le^y structure.     

Studies on the chemical structure of neutral glycosphingolipids in eggs of the sea hare, Aplysia juliana.

Six neutral glycosphingolipids (GL-1-GL-6) were obtained from eggs of the sea hare (Aplysia juliana) and were characterized by FABMS, 1H-NMR, partial acid hydrolysis, methylation studies and GC analysis of the component sugars, fatty acids and long-chain bases. The following structures were determined to be Glc beta 1-1Cer (89%) and Gal beta 1-1Cer (11%) for GL-1, Glc beta 1-1Cer (47%) and Gal beta 1-1Cer (53%) for GL-2 having hydroxy fatty acids in the ceramide moiety, Gal beta 1-4Glc beta 1-1Cer for GL-3, Fuc alpha 1-2Gal beta 1-4Glc beta 1-1Cer for GL-4, Gal alpha 1-2Gal beta 1-4Glc beta 1-1Cer for GL-5 and GalNAc alpha 1-3(Gal alpha 1-2)Gal beta 1-4Glc beta 1-1Cer for GL-6. The fatty acid composition of each glycosphingolipid, except for GL-2, which contained 2-hydroxypalmitic acid, consisted of mostly saturated C16-C20 acids, especially palmitic acid and stearic acid. The long-chain bases of all glycosphingolipids consisted mainly of branched nonadeca-4-sphingenine and octadeca-4-sphingenine. GL-6, which was one of the major glycosphingolipids, may be a precursor of a series of phosphonoglycosphingolipids which have been isolated from the skin of A. kurodai.     

Complete analysis of the1H- and13C-NMR spectra of four blood-group A active oligosaccharides

The fully assigned 1H and 13C-NMR spectra of four group A oligosaccharides by use of multiple-relayed, coherence-transfer chemical-shift-correlated spectroscopy (multiple-RELAY-COSY) and 1H-/13C-correlation spectroscopy are reported. These analyses were performed on the following compounds: III-A; GalNAc alpha 1-3[Fuc alpha 1-2]Gal: VI-A; GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal: VII-A-1; GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-1Glycerol: VII-A-2; GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc.     

Chemical structures of oligosaccharides in milk of the raccoon (<Emphasis Type="Italic">Procyon lotor</Emphasis>)

In this study on milk saccharides of the raccoon (Procyonidae: Carnivora), free lactose was found to be a minor constituent among a variety of neutral and acidic oligosaccharides, which predominated over lactose. The milk oligosaccharides were isolated from the carbohydrate fractions of each of four samples of raccoon milk and their chemical structures determined by ¹H-NMR and MALDI-TOF mass spectroscopies. The structures of the four neutral milk oligosaccharides were Fuc(α1–2)Gal(β1–4)Glc (2′-fucosyllactose), Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (lacto-N-fucopentaose IV), Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (fucosyl para lacto-N-neohexaose) and Fuc(α1–2)Gal(β1–4)GlcNAc(β1–3)[Fuc(α1–2)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (difucosyl lacto-N-neohexaose). No type I oligosaccharides, which contain Gal(β1–3)GlcNAc units, were detected, but type 2 saccharides, which contain Gal(β1–4)GlcNAc units were present. The monosaccharide compositions of two of the acidic oligosaccharides were [Neu5Ac]₁[Hex]₆[HexNAc]₄[deoxy Hex]₂, while those of another two were [Neu5Ac]₁[Hex]₈[HexNAc]₆[deoxy Hex]_3. These acidic oligosaccharides contained α(2–3) or α(2–6) linked Neu5Ac, non reducing α(1–2) linked Fuc, poly N-acetyllactosamine (Gal(β1–4)GlcNAc) and reducing lactose.     

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids. Lewis-active and Lewis-like substances.

High resolution nuclear magnetic resonance spectra were recorded in a chloroform solution of six Lewis-active or Lewis-like glycosphingolipids in permethylated and permethylated-reduced (LiAlH₄) form. The samples were selected to cover the presently known structural variants of α-fucose linked to galactose and N-acetylglucosamine. Fucα1 → 2Gal, Fucα1 → 3GlcNAc, and Fucα1 → 4GlcNAc gave characteristic and well-separated anomeric resonances. Furthermore, upon reduction there was a strong deshielding effect on Fucα1 → 3GlcNAc and Galβ1 → 3GlcNAc (linkage vicinal to reduced amide), which makes it possible to differentiate type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains. This improved method of nuclear magnetic resonance spectroscopy is discussed in relation to sequence analysis by mass spectrometry, two microscale structural methods using the same type of derivatives and needing no degradations before analysis.     

Evidence of Regional Differences in the Lectin Histochemistry Along the Ductus Epididymis of the Lizard, Podarcis Sicula Raf.

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)α(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Acα(2,6)Galβ(1,3)GalNAc, Neu5Acα(2,6)Galβ (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Acα(2,3)Galβ (1,4)GlcNAc, Neu5Acα(2,6)Galβ(1,3)GalNAc, Galβ (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Galβ(1,4)GlcNAc and αGalNAc, the luminal surface by αGalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Acα(2,3)Galβ (1,4)GlcNAc, Neu5Acα(2,6)Galβ (1,3)GalNAc, Neu5Acα(2,6)Galβ (1,4)GlcNAc, and internal GlcNAc, expressed terminal Galβ (1,4)GlcNAc and αGalNAc in the caput, and terminal β GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal βGalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal αGalNAc only in the corpus.     

Multivalent human blood group ABH and Lewis glycotopes are key recognition factors for a lFuc>Man binding lectin from phytopathogenic Ralstonia solanacearum

Ralstonia solanacearum lectin (RSL), that might be involved in phytopathogenicity, has been defined as lFuc?Man specific. However, the effects of polyvalency of glycotopes and mammalian structural units on binding have not been established. In this study, recognition factors of RSL were comprehensively examined with natural multivalent glycotopes and monomeric ligands using enzyme linked lectin-sorbent and inhibition assays. Among the glycans tested, RSL reacted strongly with multivalent blood group A_h (GalNAcα1–3[Fucα1–2]Gal) and H (Fucα1–2Gal) active glycotopes, followed by B_h (Galα1–3[Fucα1–2]Gal), Le^a (Galβ1–3[Fucα1–4]GlcNAc) and Le^b (Fucα1–2Galβ1–3[Fucα1–4]GlcNAc) active glycotopes. But weak or negligible binding was observed for blood group precursors having Galβ1–3/4GlcNAcβ1- (I_β/II_β) residues or Galβ1–3GalNAcα1- (T_α), GalNAcα1-Ser/Thr (Tn) bearing glycoproteins. These results indicate that the density and degree of exposure of multivalent ligands of α1–2 linked lFuc to Gal at the non-reducing end is the most critical factor for binding. An inhibition study with monomeric ligands revealed that the combining site of RSL should be of a groove type to fit trisaccharide binding with highest complementarity to blood group H trisaccharide (H_L; Fucα1–2Galβ1–4Glc). The outstandingly broad RSL saccharide-binding profile might be related to the unusually wide spectrum of plants that suffer from R. solanacearum pathogenicity and provide ideas for protective antiadhesion strategies.