Isolation and characterization of a tetrasialoganglioside from mouse brain, containing 9-O-acetyl,N-acetylneuraminic acid

A new ganglioside, containing an alkali-labile linkage, was extracted from mouse brain and purified. It represents 3.6% of total lipid-bound sialic acid in the tissue and was obtained in pure form with a yield of about 35%. It contains sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1:1:2:1:4 and, upon exhaustive sialidase treatment gives the monosialoganglioside GM1. Partial acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry and chromium trioxide oxidation studies showed its basic neutral glycosphingolipid core to be ganglio-N-tetraose-ceramide. Three of the four sialic acid residues are N-acetylneuraminic acid and one, as shown by gas-liquid chromatography-mass spectrometry, is 9-O-acetyl,N-acetylneuraminic acid, which contains the alkali labile linkage. 9-O-acetyl,N-acetylneuraminic acid is -ketosidically linked to position 8 of the N-acetylneuraminic acid residue bound to position 3 of the internal galactose. The other two N-acetylneuraminic acid residues form a disialosyl residue linked to position 3 of external galactose. The complete structure of the studied ganglioside is as follows: NeuAc2–8NeuAc2–3Galβ1–3GalNAcβ1–4(9-O-Ac-NeuAca2–8NeuAc2-1′-N-acylsphingosine, and it can be considered as a derivative of the tetrasialoganglioside GQ1b.     

Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs

Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a > GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.     

Rabbit antibodies against the human milk sialyloligosaccharide alditol of LS-tetrasaccharide a (NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH)

The sialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LS-tetrasaccharide a), a minor component of human milk, is obtained in relatively large quantities from autohydrolysates of the major milk disialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc (disialyllacto-N-tetraose). Rabbits immunized with an oligosaccharide-protein conjugate prepared from keyhole limpet hemocyanin and LS-tetrasaccharide a produce antibodies directed against the corresponding oligosaccharide alditol. The anti-LS-tetrasaccharide a sera bind 3H-labeled LS-tetrasaccharide a in a direct-binding radioimmunoassay on nitrocellulose filters. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides. Strong hapten-antibody binding (Ka greater than 10(6) M-1) requires sialic acid linked alpha 2-3 to the nonreducing terminal galactose residue of reduced lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH). Specificities of antibodies prepared against keyhole limpet hemocyanin conjugates of LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) differ only slightly from rabbit antibodies prepared against the corresponding bovine serum albumin conjugates described previously [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59].     

Isolation of three novel cholinergic neuron-specific gangliosides from bovine brain and theirin vitro syntheses

In the present study, three extremely minor but novel Chol-1 antigens, termed X1, X2, and X3 have been isolated from bovine brain gangliosides. Based on the results of sialidase degradation, TLC-immunostaining with anti-Chol-1 antibody and fast atom bombardment mass spectrometry, their chemical structures were identified as: $$\begin{gathered} III^6 NeuAc--GgOse4Cer (X1:GM1\alpha ) \hfill \\ III^6 NeuAc,II^3 NeuAc--GgOse4Cer (X2:GT1a\alpha ) \hfill \\ III^6 NeuAc,II^3 NeuAc--NeuGc--GgOse4Cer (X3:GT1b\alpha ) \hfill \\ \end{gathered} $$ The yields of GM1α, GD1aα, and GT1bα, were approximately 150, 20, and 10 µg, respectively, from 10 g of the bovine brain ganglioside mixture. In conjunction with our previous observations, all gangliosides with anti-Chol-1 reactivity were found to contain a common sialyl α2–6N-acetylgalactosamine residue, indicating that this unique sialyl linkage is the specific antigenic determinant. We subsequently examined the biosyntheses of the three novel Chol-1 gangliosides using rat liver Golgi fraction as an enzyme source. The results showed that GM1α, GD1aα, and GT1bα were synthesized from asialo-GM1, GM1a, and GD1b, respectively, by the action of a GalNAc α2-6sialyltransferase.     

N-Acetylneuraminic acid and N-glycolylneuraminic acid in the O-linked oligosaccharides of a tumor cell glycoprotein. Incorporation and distribution

The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma, which differ in several cell surface properties, contain a major mucin-type glycoprotein, termed ASGP-1. The sialic acid content of MAT-C1 ASGP-1 is 2-3-fold greater than MAT-B1 ASGP-1 (Sherblom, A. P., Buck, R. L., and Carraway, K. L. (1980) J. Biol. Chem. 255, 783-790). Sialic acid analysis demonstrated that, whereas MAT-C1 ASGP-1 contained approximately equal amounts of N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGl), MAT-B1 ASGP-1 was devoid of NeuGl. MAT-B1 microsomes also did not contain NeuGl. MAT-B1 cells incubated with [3H]N-acetylmannosamine did not synthesize either labeled CMP-NeuGl or free NeuGl, even though the CMP-sialic acid synthetase was active with the substrate NeuGl. Thus, MAT-B1 cells may be deficient in the enzyme N-acetylneuraminate monooxygenase. The O-linked oligosaccharides from both MAT-B1 and MAT-C1 ASGP-1 have been shown to contain a core tetrasaccharide Gal(beta 1-4)GlcNAc(beta 1-6)(Gal(beta 1-3]GalNAc in which both galactose residues may be linked to additional sugars (Hull, S. R., Laine, R. A., Kaizu, T., Rodriquez, I., and Carraway, K. L. (1984) J. Biol. Chem. 259, 4866-4877). The distribution of NeuAc and NeuGl between the two galactose termini of the core tetrasaccharide was examined for MAT-C1 ASGP-1. Oligosaccharides were released by alkaline-borohydride treatment of MAT-C1 ASGP-1 which had been labeled with [14C]glucosamine and galactose oxidase/B3H4. Following fractionation by Bio-Gel P-4, DEAE-Sephadex, and high-performance liquid chromatography, oligosaccharides were analyzed for NeuAc and NeuGl and for susceptibility to digestion with beta-galactosidase. Three disialylated oligosaccharides were identified containing 2 mol of NeuAc (5.5% recovery), 2 mol of NeuGl (4.5%), or 1 mol each of NeuAc and NeuGl (11.1%). For monosialylated oligosaccharides, NeuGl appeared preferentially associated with the Gal(beta 1-4)GlcNAc terminus (9.0%), whereas significant amounts of oligosaccharide containing NeuAc at both the Gal(beta 1-3)GalNAc (2.6%) and Gal(beta 1-4)GlcNAc (4.5%) termini were detected. Each of the major qualitative differences between MAT-B1 and MAT-C1 oligosaccharides, including the presence of NeuGl (MAT-C1), sulfate (MAT-B1), and alpha-linked galactose (MAT-B1), occurs at the Gal(beta 1-4)GlcNAc terminus.     

Occurrence of tetra- and pentasaccharides with the sialyl-Le(a) structure in human milk

The occurrence of two novel oligosaccharides in human milk was investigated. These oligosaccharides were purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 500-MHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry, indicated the structures of these compounds to be NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNAc and NeuAc alpha 2----3Gal beta 1----3(Fuc alpha 1----4) GlcNac beta 1----3Gal. This constitutes the first evidence for the occurrence of N-acetylglucosamine or galactose as the reducing-end residue of human milk oligosaccharides. These two oligosaccharides bound MSW 113 to nearly the same extent as sialyl-Lea hexasaccharide but to another sialyl-Le(a) structure-directed monoclonal antibody, NS-19-9, only weakly.     

Biosynthesis of the O-glycosidically linked oligosaccharide chains of fetuin. Indications for an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase with a narrow acceptor specificity in fetal calf liver

Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.     

Occurrence of glycosylation and deglycosylation of exogenously administered ganglioside GM1 in mouse liver.

Ganglioside GM1, 3H-labelled at the level of terminal galactose or of sphingosine, was intravenously injected into Swiss albino mice and some steps in its metabolic fate in the liver were investigated. After administration of [3H]sphingosine-labelled GM1 all major liver gangliosides [GM3, GM2, GM1, GD1a-(NeuAc,NeuGl)] became radioactive, the radioactivity residing in all cases on the sphingosine moiety. The specific radioactivity was highest in GM1, which carried about 53% of the radioactivity incorporated into gangliosides, followed by GM2, with 34.5% of incorporated radioactivity, GM3 and GD1a-(NeuAc,NeuGl), both with about 5% of incorporated radioactivity. After administration of [3H]galactose-labelled GM1 the only radioactive gangliosides present in the liver were GM1 and GD1a-(NeuAc,NeuGl), the former carrying about 95% of the total ganglioside-incorporated radioactivity, the latter about 3%. Both gangliosides were radioactive exclusively in the terminal galactose residue. According to these results exogenously administered GM1, after being taken up by the liver, is mainly degraded to GM2 and GM3, a part being, however, sialylated to GD1a-(NeuAc,NeuGl). All this suggests that exogenous GM1 may be involved in the metabolic routes of endogenous liver gangliosides.     

Newcastle disease virus contains a linkage-specific glycoprotein sialidase. Application to the localization of sialic acid residues in N-linked oligosaccharides of alpha 1-acid glycoprotein

Newcastle disease virus sialidase was found to exhibit strict specificity for hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage contained in glycoprotein oligosaccharides both N-linked to asparagine and O-linked to threonine or serine under conditions that left oligosaccharides containing the NeuAc alpha 2 leads to 2 leads to 6Gal and NeuAc alpha 2 leads to 6GallNAc linkages intact. This was determined, in part, by examining the viral sialidase for its ability to hydrolyze glycoprotein oligosaccharides derivatized with purified sialyltransferases to contain the [14C]NeuAc alpha 2 leads to 3Gal, [14C]NeuAc alpha 2 leads to 6GalNAc, and [14C]NeuAc alpha 2 leads to 6Gal linkages. The viral sialidase was also tested for hydrolysis of the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages on the N-linked oligosaccharides of alpha 1-acid glycoprotein. Selective hydrolysis of the NeuAc alpha 2 leads to 3Gal linkage was shown by periodate oxidation and by 500-MHz 1H-NMR spectroscopy of native and sialidase-treated glycopeptides. The NMR spectra, together with composition data, further indicated that the NeuAc alpha 2 leads to 3Gal and NeuAc alpha 2 leads to 6Gal linkages were localized to specific branches of the major tri- and tetraantennary oligosaccharides of alpha 1-acid glycoprotein. The results indicate that the Newcastle disease virus sialidase can initiate the selective degradation of N-linked oligosaccharide branches containing the NeuAc alpha 2 leads to 3Gal linkage.     

Kinetic parameters of a beta-D-galactoside alpha 2 leads to 6 sialyltransferase from embryonic chicken liver

A beta-D-galactoside alpha 2 leads to 6 sialyltransferase was purified 500-fold in 14% yield from 14-day embryonic chicken liver. Characterization of the product of the sialyltransferase catalysis was accomplished by separation and permethylation of double-labelled ([14C]NeuAc, [3H]Gal) oligosaccharides following their release from the glycoprotein fetuin by hydrazinolysis. The enzyme transfers NeuAc to Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to)R-terminated oligosaccharides; no activity was found towards Gal(beta 1 leads to 3)GalNAc(alpha 1 leads to)R structures. The trisaccharide. NeuAc(alpha 2 leads to 6)Gal(beta 1 leads to 4)Glc, was shown to be a good inhibitor of the sialyltransferase. Kinetic investigations of the enzyme indicate it to have a sequential, random bi-bi mechanism.     

A new monoclonal antibody directed to sialyl alpha 2-3lactoneotetraosylceramide and its application for detection of human gastrointestinal neoplasms

A new monoclonal antibody (NS24) directed to the N-acetylneuraminyl alpha 2-3Gal beta 1-4GlcNAc residue in type II sugar chain of N-acetylneuraminyllactoneotetraosylceramide [sialylparagloboside, IV3(NeuAc)nLc4Cer] was prepared by hybridoma technique. Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, IV3(NeuAc)nLc4Cer, and lipopolysaccharides from Salmonella minnesota R595 were used for immunization with IV3(NeuAc)nLc4Cer isolated from human erythrocytes. This method allowed the fusion of spleen cells of immunized mouse with myeloma cells only three days after immunization. NS24 reacted specifically to both naturally occurring and chemically synthesized IV3-(NeuAc)nLc4Cer, whereas it has no reactivity to structurally related gangliosides, such as IV6(NeuAc)nLc4Cer, N-glycolylneuraminyl alpha 2-3lactoneotetraosylceramide [IV3(NeuGc)-nLc4Cer], i-active ganglioside [VI3(NeuAc)nLc6Cer], I-active ganglioside [VIII3(NeuAc)-VI3(NeuAc)IV6kladoLc8Cer], GM4(NeuAc), GM3(NeuAc), GM3(NeuGc), GM1b(NeuAc), GD3-(NeuAc), other ganglio-series gangliosides, sulfatide, and paragloboside (nLc4Cer). Synthetic N-acetylneuraminyl alpha 2-3lactotetraosylceramide [IV3(NeuAc)Lc4Cer] and its asialo-derivative (Lc4Cer) carrying type I sugar chain also showed no reaction with NS24. One to 100 pmol of IV3(NeuAc)nLc4Cer was detected dose-dependently by a thin-layer chromatography/enzyme immunostaining procedure. Human gastric carcinomas showed positive reactions with NS24 immunochemically and histochemically. NS24 reacted preferentially with poorly differentiated adenocarcinomas rather than well differentiated ones.     

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.     

Identification of N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine as a major adduct in rat liver DNA after treatment with the carcinogens, N,N-dimethylnitrosamine or 1,2-dimethylhydrazine

Human Tamm-Horsfall urinary glycoprotein from an individual of the blood group Sd(a+) phenotype was tritium-labelled by treatment with galactose oxidase and sodium boro[3H]hydride and was then digested with endo-beta-galactosidase. A series of dialysable, labelled fragments was released from which a pentasaccharide was isolated that strongly inhibited the agglutination of Sd(a+) red cells by human anti-Sda serum and hence contained the Sda determinant structure. Reduction, methylation analysis and sequential exo-glycosidase digestion established the structure of the pentasaccharide as: GalNAc beta(1 leads to 4)[NeuAc(2 leads to 3)]Gal beta(1 leads to 4)GlcNAc beta(1 leads to 3)Gal     

Biosynthesis of a disialylated sequence in N-linked oligosaccharides: identification of an N-acetylglucosaminide (alpha 2----6)-sialyltransferase in Golgi apparatus from rat liver

Rat liver Golgi apparatus are shown to have a CMP-N-acetylneuraminate: N-acetylglucosaminide (alpha 2----6)-sialyltransferase which catalyzes the conversion of the human milk oligosaccharide LS-tetrasaccharide-a (NeuAc alpha 2----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----4Glc) to disialyllacto -N- tetraose containing the terminal sequence: (formula: see text) found in N-linked oligosaccharides of glycoproteins. The N-acetylglucosaminide (alpha 2----6)-sialyltransferase has a marked preference for the sequence NeuAc alpha 2----3-Gal beta 1---- 3GlcNAc as an acceptor substrate. Thus, the order of addition of the two sialic acids in the disialylated structure shown above is proposed to be first the terminal sialic acid in the NeuAc alpha 2----3Gal linkage followed by the internal sialic acid in the NeuAc alpha 2---- 6GlcNAc linkage. Sialylation in vitro of the type 1 branches (Gal beta 1---- 3GlcNAc -) of the N-linked oligosaccharides of asialo prothrombin to produce the same disialylated sequence is also demonstrated.     

N-Acetylneuraminic acid uptake in Pasteurella (Mannheimia) haemolytica A2 occurs by an inducible and specific transport system.

The N-acetylneuraminic acid (NeuAc) transport system of Pasteurella (Mannheimia) haemolytica A2 was studied when this bacterium was grown in both complex and chemically defined media. Kinetic measurements were carried out at 37 degrees C in 50 mM Tris-HCl buffer, pH 8.0, containing 50 microg/ml bovine serum albumin. Under these conditions, the uptake rate was linear for at least 3 min and the calculated K(m) for NeuAc was 0.1 microM. The transport rate was increased by the addition of several cations and was inhibited by sodium arsenite (95%), N,N'-dicyclohexyl-carbodiimide (50%), and 2,4-dinitrophenol (40%) at final concentration of 1 mM (each). These results support the notion that NeuAc uptake is an active sugar cation symporter. Study of specificities showed that glucosamine, mannose and mannosamine inhibited the transport of NeuAc in this bacterium. Analysis of expression revealed that the NeuAc transport system was induced by NeuAc and by the simultaneous presence of glucose and galactose in the growth medium.     

GM1b and GM1b-GalNAc: major gangliosides of murine-derived macrophage-like WEHI-3 cells.

WEHI-3 cells, derived from a BALB/c mouse, are a myelomonocytic leukemic cell line with macrophage-like properties. We have isolated, purified and characterized the monosialogangliosides from WEHI-3 cells by 1D-HPTLC, 2D-HPTLC, enzymatic degradation, HPTLC-immunostaining, gas-liquid chromatography and fast atom bombardment-mass spectrometry (FAB-MS). Quantitative 2D-HPTLC shows two monosialogangliosides are the major components, constituting 77% of the total, with a third monosialoganglioside being 3%. The two major components were identified as (NeuAc)GM1b and (NeuAc)GM1b-GalNAc and the minor component as (NeuAc)GM1b-GalNAc-Gal. The presence of GM1b in this myelomonocytic cell line is consistent with its presence in other murine immune cells and tissues. GM1b-GalNAc and GM1b-GalNAc-Gal have been reported in T-lineage cells but not in resident or stimulated murine macrophages. Each of these monosialogangliosides belongs to the asialoGM1 synthetic pathway. Preliminary results indicate a disialo member of this pathway, GDlc, may also be present as a minor component. This ganglioside pathway, containing species which are not sialylated on the internal galactose, appears to be dominant in and may be characteristic of murine immune cells.     

Characterization of two molecular species GD3 ganglioside from bovine buttermilk

Two gangliosides, representing 85% of total lipid-bound sialic acid, have been isolated from bovine buttermilk and characterized. Both contained long-chain base, glucose, galactose and sialic acid in the molar ratio 1:1:1:2, and gave, upon sialidase treatment, a neutral glycolipid, characterized as lactosylceramide. Partial acid hydrolysis, permethylation analysis and chromium trioxide oxidation indicated their basic oligosaccharide portion to be NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4Glc. The difference between the two forms was exclusively in the ceramide moiety of the molecule, one containing mainly long-chain (C22-C25) fatty acids and an equimolar proportion of C16 and C18 long-chain bases, and the other mainly palmitic acid and C18 long-chain base.     

Isolation and structural elucidation of a novel type of ganglioside, deaminated neuraminic acid (KDN)-containing glycosphingolipid, from rainbow trout sperm. The first example of the natural occurrence of KDN-ganglioside, (KDN)GM3

Rainbow trout sperm contained almost exclusively monoanionic ganglioside fraction as a major acidic glycosphingolipid. Two monoacidic gangliosides were isolated and purified in this study and designated as sperm ganglioside 1 and 2 (sg-1 and sg-2). The two gangliosides, sg-1 and sg-2, contained the same neutral sugars, galactose and glucose in molar ratio of 1:1 and no GalNAc except for the presence of N-acetyl-neuraminic acid (NeuAc) in sg-1 and deaminated neuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) in sg-2. The complete structures of these gangliosides were determined by a combination of methylation analysis, fast atom bombardment mass spectrometry, 400-MHz one- and two-dimensional 1H nuclear magnetic resonance spectroscopy, fatty acid analysis, and endoglycoceramidase digestion NeuAc alpha 2----3Gal beta 1----4Glc beta 1----Cer sg-1 [(NeuAc)GM3] KDN alpha 2----3Gal beta 1----4Glc beta 1----Cer sg-2 [(KDN)GM3] where, for both sg-1 and sg-2, the ceramide moieties (Cer) were found to be made up of 4-sphingenine and mainly C16:0 fatty acid (palmitate; 95%) with a minor amount of C24:1 fatty acyl chain (nervonate, 5%). The structure of sg-2 is novel and represents the first example of a new class of gangliosides, i.e. KDN-gangliosides.     

Occurrence of a new hematoside in the kidney of guinea pig

We have isolated a new hematoside from guinea pig kidney. Like the usual hematoside (II3NeuAc LacCer), isolated from human erythrocytes, this new hematoside contained glucose, galactose, and N-acetylneuraminic acid in an equimolar proportion. By thin-layer chromatography (TLC), however, it migrated faster than the usual hematoside. After mild alkaline hydrolysis the TLC mobility of this ganglioside became identical to that of the usual hematoside. The sialic acid in this ganglioside was susceptible to Clostridial neuraminidase. Based on TLC mobility and the results of periodate oxidation, the sialic acid of the new hematoside was identified as 9-O-acetyl-N-acetylneuraminic acid. Therefore, the structure of this new hematoside is 9-O-Ac-NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GLc beta 1 leads to 1'Cer.