Structural characterization of glycolipids of rat small intestine having one to eight hexoses in a linear sequence

Eight different fractions containing glycolipids with 1 to 8 hexoses in a linear sequence were isolated from rat small intestine. The structure of the major components was established by mass spectrometry and proton nuclear magnetic resonance spectroscopy of the permethylated and permethylated-reduced (LiAlH₄) derivatives and by gas-liquid chromatography of degradation products of the native and permethylated or permethylated-reduced glycolipids. The major compounds were glucosylceramide, lactosylceramide, globotriaosylceramide, and a novel tetrahexosylceramide with the structure Gal α 1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer. In addition four minor compounds having five to eight hexoses were identified with the probable structures Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Galα1 → 3Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer, and Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer. In the pentahexosylceramide fraction a novel fucolipid was also present having the probable structure Fucα1 → 2Galα1 → 3Galα1 → 4Galβ1 → 4Galβ1 → 1Cer. The lipophilic part of the glycolipids was composed of trihydroxy 18:0 and dihydroxy 18:1 long-chain bases in combination with nonhydroxy and hydroxy 16:0–24:0 fatty acids. Glycolipid studies of isolated mucosal epithelial cells and the nonepithelial intestinal residue revealed a specific cell distribution of these hexosyl compounds. The two major components, glucosylceramide and globotriaosylceramide, were mainly located in the epithelial cells together with small amounts of lactosylceramide and tetrahexosylceramide. The epithelial cells practically lacked however the penta- to octahexosylceramides. The nonepithelial residue contained all hexosyl compounds. The fucolipid was exclusively present in the epithelial cells.     

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.     

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.     

Redefinition of the Carbohydrate Binding Specificity of Helicobacter pylori BabA Adhesin

Certain Helicobacter pylori strains adhere to the human gastric epithelium using the blood group antigen-binding adhesin (BabA). All BabA-expressing H. pylori strains bind to the blood group O determinants on type 1 core chains, i.e. to the Lewis b antigen (Fucα2Galβ3(Fucα4)GlcNAc; Le(b)) and the H type 1 determinant (Fucα2Galβ3GlcNAc). Recently, BabA strains have been categorized into those recognizing only Le(b) and H type 1 determinants (designated specialist strains) and those that also bind to A and B type 1 determinants (designated generalist strains). Here, the structural requirements for carbohydrate recognition by generalist and specialist BabA were further explored by binding of these types of strains to a panel of different glycosphingolipids. Three glycosphingolipids recognized by both specialist and generalist BabA were isolated from the small intestine of a blood group O pig and characterized by mass spectrometry and proton NMR as H type 1 pentaglycosylceramide (Fucα2Galβ3GlcNAcβ3Galβ4Glcβ1Cer), Globo H hexaglycosylceramide (Fucα2Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), and a mixture of three complex glycosphingolipids (Fucα2Galβ4GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, Fucα2Galβ3GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer, and Fucα2Galβ4(Fucα3)GlcNAcβ6(Fucα2Galβ3GlcNAcβ3)Galβ3GlcNAcβ3Galβ4Glcβ1Cer). In addition to the binding of both strains to the Globo H hexaglycosylceramide, i.e. a blood group O determinant on a type 4 core chain, the generalist strain bound to the Globo A heptaglycosylceramide (GalNAcα3(Fucα2)Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer), i.e. a blood group A determinant on a type 4 core chain. The binding of BabA to the two sets of isoreceptors is due to conformational similarities of the terminal disaccharides of H type 1 and Globo H and of the terminal trisaccharides of A type 1 and Globo A.     

Effects of Divalent Cations on the Glycolipids from Cultured Mouse Neuroblastoma Cells

Abstract: The influence of divalent cations on glycosphingolipid metabolism was examined in the NB41A mouse neuroblastoma clonal cell line. HPLC methods were utilized to quantitate the effects on neutral glycolipids and monosialogangliosides. NB41A cells were shown to contain G_M3, G_M2, G_M1, G_D3, and G_D1a by HPLC and TLC. The neutral glycosphingolipids consisted of glucosylceramide (GlcCer), lactosylceramide (LacCer), GaINAc(β1→4) Gal(β1→4)Glc(β1→1)Cer (GgOse₃Cer), and GaINAc(β1→3)Gal(α1→4) Gal-(β1→4)Glc(β1→1)Cer (GbOse₃Cer) according to their HPLC behavior. Cells grown in the presence of 1.85 mm -EGTA showed a two- to threefold increase in G_M3 whereas other glycosphingolipids were only slightly affected. When cells were grown in the presence of 1.45 mm -EGTA plus 0.4 mm -EDTA a similar increase in G_M3 was observed but this change was now accompanied by decreases in G_M2, G_M1 GgOse₃Cer, and GbOse₄Cer. The EGTA-EDTA effects were reversed when growth was in the presence of Ca²⁺ sufficient to bind all chelator. Mn²⁺ replacement reversed the chelator effects differentially; G_M2 and G_M1 levels were the most sensitive to increases in Mn²⁺ concentration; GgOse₃Cer and GbOse₄Cer were also sensitive, whereas G_M3 was the least affected. These results suggest calcium serves an important regulatory role on G_M3 levels and that manganese concentration may regulate the levels of galactosamine-containing glycolipids in mouse NB41A neuroblastoma cells.     

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.     

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     

Receptors for glucocorticoids in lung tissue

Gas chromatography-mass spectrometric identification of partially methylated aminosugars has been developed: (a) various kinds of O-methylated 2-deoxy-2-(N-methyl)-acetamidohexitols were prepared from partially O-(1-methoxy)-ethylated 2-deoxy-2-acetamidohexoses, and their gas chromatography-mass spectrometric patterns were determined; (b) permethylated glycolipids gave a satisfactory yield of 2-deoxy-2-N-methyl-amidohexoses by acetolysis with 0.5 n sulfuric acid in 95% acetic acid, followed by aqueous hydrolysis; (c) the resulting partially methylated aminosugars and neutral sugars were analyzed after borohydride reduction and acetylation according to the procedure of Lindberg and associates (Björndal, Lindberg and Svennson, 1967; Björndal, Hellerqvist, Lindberg and Svensson, 1970).This method was successfully applied to analysis of aminosugar linkages in blood group B-active ceramide pentasaccharide from rabbit erythrocytes and in Forssman antigen of equine spleen. The structure of blood group B-active glycolipid of rabbit erythrocyte was found to be Galα1 → 3Galβ1 → 4G1cNAcβ1 → 3Ga11 → 4Glc → Cer, and that of Forssman antigen to be GaNAcα1 → 3GalNAcβ1 → 3Galα1 → 4Ga11 → 4Glc → Cer.     

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.     

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.     

ON THE STRUCTURE OF A NEW, FUCOSE CONTAINING GANGLIOSIDE FROM PIG CEREBELLUM

A new ganglioside, provisionally named GLIVa, was isolated in pure form from pig cerebellum. Ganglioside GLIVa is a disialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal, β 1 → 3 GalNAc, β 1 → 4 Gal, β 1 → 4 Glc, β 1 → Cer. Fucose is α-glycosidically linked to the 2-position of external galactose and one N-acetylneuraminic acid is linked to the other one by an α, 2 → 8 linkage. Thus the total structure of ganglioside GLIVa is the following: Fuc, α 1 → 2 Gal, β 1 → 3 GalNAc, β 1 → 4 (NeuAc, α 2 48 NeuAc, α 2 → 3) Gal, β 1 → 4 Glc, β 1 → Ceramide. According to the IUPAC-IUB Commission on Biochemical Nomenclature is indicated as II³α(NeuAc)₂ IV²αFuc-GgOse₄Cer.     

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.     

Preparation and characterization of antibody to galactosyl(α1 → 4)galactosyl(β1 → 4)glucosylceramide

Antiserum against galactosyl(α1 → 4)galactosyl(β1 → 4)glucosylceramide (globotriaosylceramide, Gb₃) was raised in rabbits by the administration of four weekly intramuscular injections of 1.5 mg of the purified glycolipid along with bovine serum albumin and Freund's complete adjuvant. AntiGb₃ activity was quantitated initially by immunoprecipitation employing Gb₃ mixed with 100-fold excess of lecithin and cholesterol (1 : 1 or 1 : 2, by wt.) as antigen. Subsequently, complement fixation tests done with antigen preparations containing Gb₃/lecithin/cholesterol (1 : 6 : 20, by wt.) showed antiGb₃ titres of up to 1 : 8192. Fractionation of the antiserum by BioGel A5m chromatography indicated the antibody was an IgM immunoglobulin. The partially purified antibody stimulated complement-dependent release of glucose from glucose-containing liposomes prepared with sphingomyelin/cholesterol/dicetylphosphate/Gb₃ (molar ratio, 100 : 75 : 11 : 1). The antibody crossreacted with the trisaccharide, Gal(α1 → 4)Gal(β1 → 4)Glc, but not with galactosylceramide, lactosylceramide, GM1 ganglioside, globotetraosylceramide, digalactosyldiglyceride or a number of low molecular weight saccharides.     

Structural studies of the carbohydrate moiety of human antithrombin III

Human antithrombin III contains four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively released as radioactive oligosaccharides from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB³H₄ reduction. All of the oligosaccharides, thus obtained, contain N-acetylneuraminic acid. A same neutral nonaitol was released from all acidic oligosaccharides by sialidase treatment. By combination of the sequential exoglycosidase digestion and methylation analysis, their structures were elucidated as NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6-(NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc, Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6(NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manαl → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc, and NeuAcα2 → 6Galβ1 → 4GlcNAcβ1 → 2Manα1 → 6(Galβ1 → 4GlcNAcβ1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc.     

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.     

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:

     

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.     

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.     

Chemical characterization of a blood group H type pentaglycosylceramide of human small intestine

A blood group H type pentaglycosylceramide was isolated in relatively large amounts from human adult small intestine (52mg from one individual) and human meconium (fetal origin). The structure was made likely by mass spectrometry and NMR spectroscopy of non-degraded permethylated and permethylated-LiAlH₄-reduced glycolipid and by degradaton to be Fucα1 → 2GAlβ 1 → 3GlcNAcβ 1 → 3GAlβ 1 → 4Glcβ 1 → l Cer. The ceramide was composed mainly of phytosphingosine and 2-hydroxy 16–24 carbon fatty acids. This novel type 1 chain species (Galβ 1 → 3GlcNAc) was not accompanied by the type 2 chain isomer (Galβ 1 → 4GlcNAc) which in contrast is the sole species in human erythrocyte and dog small intestine.     

Surface carbohydrates of hamster fibroblasts. I. Chemical characterization of surface-labeled glycosphingolipids and aspecific ceramide tetrasaccharide for transformants.

1. Neutral glycosphingolipids of hamster fibroblast NIL cells have been characterized as follows: glucosylceramide, lactosylceramide (betaGall yields 4Glc yields Cer), a digalactosylceramide (alphaGall yields 4betaGal yields Cer), a trihexosylceramide (alphaGall yields 4betaGall yields 4Glc yields Cer), two kinds of ceramide tetrasaccharides (A: alphaGa1NAcl yields 3betaGalNAcl yields 3alphaGall yields 4betaGall yields 1Cer, a new type of Forssman active glycolipid; B: globoside, betaGalNAcl yields 3alphaGall yields 4betaGall yields 4betaGlc yields Cer), and a ceramide pentasaccharide having a classical structure for Forssman antigen (alphaGalNAcl yields 3betaGalNAcl yields 3alphaGall yields 4betaGall yields 4Glc yields Cer). 2. Neutral glycosphingolipids of polyoma virus-transformed NIL cells (NILpy) have been characterized as having an additional ceramide tetrasaccharide which was absent in normal NIL cells. The structure of this specific glycolipid was identified as lacto-N-neotetraosylceramide (betaGall yields 4betaGlc-NAcl yields 3betaGall yields 4Glc yields Cer). Chemical quantities of ceramide tetra- and pentasaccharides in NILpy cells were much lower than in NIL cells. 3. All of these glycolipids, except glucosylceramide and lactosylceramide, were labeled externally by galactose oxidase and tritiated borohydride according to the method previously described (GAHMBERG, C. G, and HAKOMORI, S. (1973) J. Biol. Chem. 248, 4311-4317). The specific activities of the label in glycolipid of NIHpy cells were much greater than that in NIL cells, i.e. reactivity of glycolipids with galactose oxidase in NIHpy cells was much higher than for NIL cells. The surface label in glycolipids was cell cycle-dependent in NIL cells, and a remarkable exposure of a galactosyl residue of a ceramide tetrasaccharide was demonstrated only on the surface of NILpy cells, due to the presence of lacto-N-neotetraosylceramide.