A revised structure for the disialosyl globo-series gangliosides of human erythrocytes and chicken skeletal muscle.

Disialosyl globo-series gangliosides have previously been isolated from chicken skeletal muscle (E. L. Hogan, R. D. Happel, and J.-L. Chien (1982) Adv. Exp. Med. Biol. 152, 273-278; S. Dasgupta, J.-L. Chien, E. L. Hogan, and H. van Halbeek (1991) J. Lipid Res. 32, 499-506) and human erythrocytes (S. K. Kundu, B. E. Samuelsson, I. Pascher, and D. Marcus (1983) J. Biol. Chem. 258, 13857-13866). In both cases, the structure of this ganglioside was proposed to be NeuAc alpha 2-->3(NeuAc alpha 2-->6)Gal beta 1-->3GalNAc beta 1-->3Gal alpha 1-->Gal alpha 1-->4Gal beta 1-->1Cer (V3NeuAcV6NeuAcGb5Cer). We have reinvestigated the human erythrocyte antigen and now propose an alternative structure differing in the location of the NeuAc alpha 2-->6 residue: NeuAc alpha 2-->3Gal beta 1-->3 (NeuAc alpha 2-->6)GalNAc beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc beta 1-->1 Cer (V3NeuAcIV6NeuAcGb5Cer). This novel structure is supported by results of 1H-NMR spectroscopy, negative ion fast atom bombardment mass spectrometry, and methylation linkage analysis with capillary gas chromatography--mass spectrometry in both electron impact and chemical ionization modes. Furthermore, based on new results from negative ion fast atom bombardment mass spectrometry and linkage analysis, we propose that the chicken skeletal muscle antigen also has this revised structure, differing only in ceramide composition. The terminal tetrasaccharide of these gangliosides is identical to that of GD1 alpha, NeuAc alpha 2-->3Gal beta 1-->3(NeuAc alpha 2-->6)GalNAc beta 1-->4Gal beta 1-->4Glc beta 1-->1 Cer(IV3NeuAcIII6NeuAcGg4Cer), previously identified in a rat ascites hepatoma cell line (T. Taki, Y. Hirabayashi, H. Ishikawa, S. Ando, K. Kon, Y. Tanaka, and M. Matsumoto (1986) J. Biol. Chem. 261, 3075-3078) and a murine lymphoma cell line with low metastatic potential (K. Murayama, S. B. Levery, V. Schirrmacher, and S. Hakomori (1986) Cancer Res. 46, 1395-1402), although they appear to be immunologically distinct.

A disialoganglioside of the globo-series from chicken skeletal muscle.

We have isolated a disialoganglioside of the globo-series from chicken pectoral muscle. The compound was obtained by extraction followed by ion-exchange and silicic acid column chromatography and judged to be pure by thin-layer chromatography in three solvent systems. The structure of the ganglioside was determined by carbohydrate and ceramide composition analysis, sequential exoglycosidase digestion, methylation analysis, and 500-MHz 1H-NMR spectroscopy to be: (formula; see text) Analysis of the ceramide moiety indicated d18:1 sphingosine as the long-chain base, and C16:0, C18:0, C18:1, and C20:0 as the prevalent fatty acids. This glycolipid is only the second ganglioside of the globo-series, and the first disialo member of the series, found in chicken muscle.

Biosynthesis of GM1b and similar neolactoseries gangliosides by a partially purified chicken skeletal muscle sialyltransferase. Effect of sphingomyelin and acetylcholine.

An alpha 2----3 glycolipid galactosyl sialyltransferase (SAT3/4) has been partially purified from embryonic chicken skeletal muscle. It is preserved in 50 mM Hepes buffer (pH 6.8) containing 1% Triton CF-54 and 20% glycerol at -70 degrees C for a period of 6 months without loss of activity. The SAT3+4 preparation transfers sialic acid to nLcOse4Cer, nLcOse6Cer and GgOse4Cer with respective Km values of 1.4, 0.83 and 0.45 mM. The activity is stimulated 2-3-fold at high substrate concentration and 6-8-fold at low substrate concentration; 0.01 and 0.005 mumol for asialo GM1 and 0.025 and 0.01 mumol for other glycolipids in the presence of phosphatidylcholine (PC) and sphingomyelin (SM) at an optimum concentration 0.75%. A higher concentration is inhibitory. SM from chicken muscle is more effective than that from bovine brain and the stimulation is qualitatively proportional to that of the saturated fatty acyl content of SM. Free fatty acids (palmitic and stearic), their sodium salts, other choline compounds including choline chloride, phosphorylcholine and acetylcholine either do not have any effect or are inhibitory. Acetylcholine, even in the presence of SM and PC, is strongly inhibitory (70%).

Sialylation of lacto-N-neotetraosyl ceramide by a solubilized sialyltransferase(s) from chicken skeletal muscle: effect of phosphatidylcholine and sphingomyelin.

The sialytransferase(s) that transfers sialic acid to lacto-N-neotetraosylceramide and other glycosphingolipids with a galactose nonreducing terminus has been successfully solubilized from embryonic chicken skeletal muscle. The enzyme can be stored in 50 mM HEPES (pH 6.8), 1% Triton CF-54, and 20% glycerol at -70 degrees C for as long as six months. Addition of phosphatidylcholine or sphingomyelin (0.167%) readily reactivates the stored inactive enzymes and such activity persists for about two weeks at 0 degrees-4 degrees C with the peak activity occurring at 1 to 2 days. Sphingomyelin from chicken muscle, which contains mainly C16:0 and C18:0, is 2.1-fold more effective than bovine brain sphingomyelin at the same concentration (0.4%).

Free calcium and calmodulin levels in acinar carcinoma and normal acinar cells of rat pancreas.

Exposure of acinar carcinoma cells and normal acinar cells of rat pancreas to the muscarinic agonist drug carbamylcholine stimulated 45Ca2+ outflux from 45Ca2+-labeled cells. More rapid outflux of 45Ca2+ was detected for carcinoma cells following muscarinic stimulation than for normal cells. Direct fluorometric measurement of cytosolic Ca2+ under basal (unstimulated) conditions in quin 2-loaded cells revealed significantly lower concentration of free Ca2+ in carcinoma cells (approximately 180 nM) than in normal cells (approximately 200 nM). Stimulation with 1 mM carbamylcholine increased the cytosolic Ca2+ concentration in carcinoma and normal cells to approximately 1900 nM, after which carcinoma cells removed cytosolic Ca2+ at a faster rate to a post-stimulation plateau concentration of approximately 140 nM, in comparison to normal cells which obtained a post-stimulation plateau concentration of approximately 300 nM. Essentially identical differences between carcinoma and normal cells were detected upon stimulation with the peptidergic agonist cholecystokinin octapeptide. Finally, carcinoma cells demonstrated approximately 3 times greater calmodulin concentration than normal acinar cells. Also, the calmodulin antagonist drug W7 (N-6-(aminohexyl)-5-chloro-1-naphthalene sulfonamide) inhibited the carbamylcholine-induced release of intracellular Ca2+ in acinar carcinoma cells. These results indicate that neoplastic pancreatic acinar cells have retained mechanisms of muscarinic- and peptidergic-stimulated intracellular Ca2+ release, and implicate calmodulin as a regulatory factor in secretagogue activation of intracellular Ca2+ release. We propose that the more rapid decline of intracellular Ca2+ concentration following muscarinic or peptidergic stimulation and the increased intracellular calmodulin concentration indicate calmodulin-mediated down-regulation of free cytosolic Ca2+ in acinar carcinoma cells to levels lower than those of normal cells.

Muscarinic receptor coupling to intracellular calcium release in rat pancreatic acinar carcinoma.

Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cholinergic receptor protein affinity labeled with the muscarinic antagonist [3H]propylbenzilylcholine mustard revealed a major polypeptide with molecular weight of 80,000-83,000 in both acinar carcinoma and normal acinar cells of rat pancreas. Muscarinic receptor protein is therefore conserved in pancreatic acinar carcinoma. A small but significant difference was detected in the affinity of carcinoma cell receptors (Kd approximately 0.6 nM) and normal cell receptors (Kd approximately 0.3 nM) for reversible binding of the muscarinic antagonist drug, N-methylscopolamine. In addition, carcinoma cell muscarinic receptors displayed homogeneous binding of the agonist drugs carbamylcholine (Kd approximately 31 microM) and oxotremorine (Kd approximately 4 microM), whereas normal cell receptors demonstrated heterogeneous binding, with a minor receptor population showing high affinity binding for carbamylcholine (Kd approximately 3 microM) and oxotremorine (Kd approximately 160 nM), and a major population showing low affinity binding for carbamylcholine (Kd approximately 110 microM) and oxotremorine (Kd approximately 18 microM). Both carcinoma and normal cells exhibited concentration-dependent carbamylcholine-stimulated increases in cytosolic free Ca2+, as measured by 45Ca2+ outflux assay and intracellular quin 2 fluorescence. However, carcinoma cells were observed to be more sensitive to Ca2+ mobilizing actions of submaximal carbamylcholine concentrations, demonstrating 50% maximal stimulation of intracellular Ca2+ release at a carbamylcholine concentration (approximately 0.4 microM) approximately one order of magnitude below that seen for normal cells. These results indicate altered muscarinic receptor coupling to intracellular Ca2+ release in acinar carcinoma cells, which manifests as a single activated receptor state for agonist binding, and increased sensitivity of Ca2+ release in response to muscarinic receptor stimulation.

Lipid content of swine influenza and other vaccines.

An analysis of the lipids in swine influenza vaccines was performed, comparing six different lots of swine influenza, other influenza and noninfluenza vaccines. Cholesterol content and phospholipid content varied greatly, but there were no major differences between the types of vaccines. Appreciable amounts of phosphatidylethanolamine were found in only one swine influenza vaccine. The major phospholipids of influenza vaccines were phosphatidylcholine, sphingomyelin and phosphatidic acid. A detectable amount of phosphatidylserine was not found in any swine influenza vaccine, but was present in two of three nonswine influenza vaccines. Only two of six swine influenza vaccines showed trace amounts (less than 0.5 microgram/ml) of ganglioside (GM3). However, larger quantities of galactocerebroside were found (2.24-6.43 micrograms/ml) in all influenza vaccines examined, including swine influenza vaccines.

Sialylation of lacto-N-neohexaosylceramide by sialyltransferase from embryonic chicken muscle.

A sialyltransferase which catalyzes the in vitro biosynthesis of N-acetylneuraminosyllacto-N-neohexaosylceramide from lacto-N-neohexaosylceramide and CMP-NeuAc has been examined in embryonic chicken breast muscle. The maximum enzyme activity was observed in 11-12-day-old embryos. The enzyme has optimum activity at pH 6.8 in the presence of Triton CF-54 and Mg2+. The apparent Km values for lacto-N-neohexaosylceramide and CMP-NeuAc were 0.9 and 0.67 mM, respectively. The enzymic product was characterized by TLC, neuraminidase hydrolysis and permethylation analysis. The structure was identical to authentic N-acetylneuraminosyllacto-N-neohexaosylceramide from chicken muscle. In addition, a disialo derivative has been detected that constitutes 15% of the total radioactivity incorporated. The two sialic acids connected by sialosyl-sialosyl linkage were attached to the terminal galactose residue. To our knowledge, this is the first report of biosynthesis of this disialo compound.

Differentiation of muscarinic cholinergic receptors in acinar carcinoma of rat pancreas.

Analysis of muscarinic cholinergic receptors in pancreatic acinar carcinoma of rat by measurement of N-[methyl-3H]scopolamine binding has revealed a single homogenous population of muscarinic receptors in the tumor. The plasmalemma density (approximately 25 receptors/micron 2 of cell membrane surface) and dissociation constant (approximately 0.4 nM) of muscarinic receptors in acinar carcinoma cells are identical to the density and affinity of muscarinic receptors in normal acinar cells of rat pancreas. Muscarinic receptors in the carcinoma are functionally linked to cholinergic stimulation of cellular Ca2+ release. An equivalent number of functional muscarinic receptors is present in poorly differentiated carcinoma cells which are deficient in zymogen granules and protein secretion, as compared to well-differentiated carcinoma cells which contain granules and secrete protein in response to cholinergic stimulation. These observations indicate that muscarinic cholinergic receptors are displayed in normal fashion on tumor membranes and are fully expressed in carcinoma cells regardless of their degree of secretory development. Expression of muscarinic cholinergic receptors is thus a stable phenotypic property of pancreatic acinar carcinoma cells. This suggests that muscarinic receptor maturation is an early event in the differentiation of pancreatic exocrine cells, preceding acquisition of secretory responsiveness to cholinergic stimulation.

A simplified procedure for the preparation of tritiated GM1 ganglioside and other glycosphingolipids.

The ganglioside II3NeuAc-GgOse4Cer and other glycosphingolipids can be radiolabeled to high specific activity by the galactose oxidase-NaB3H4 procedure, by purifying the oxidized compounds prior to reductive labeling. The oxidized products are separated from nonoxidized compounds and detergents (Triton X-100 and sodium taurocholate) present during the enzymatic oxidation. Since the oxidized derivatives are separated, the final specific activity depends solely upon the specific activity of the NaB3H4 and the reduction conditions.

Glycosphingolipids of chicken skeletal muscle in early development and genetic dystrophy.

The acidic and neutral GSL of chicken pectoral muscle and the activities of relevant sialyltransferase and glycosidases have been examined during embryonic and early post-hatching development. At this stage of myogenesis, a prominent shift to the neutral GSL of longer oligosaccharide length involving Forssman glycolipid most prominently and also globoside and GbOse3Cer occurred but the distribution of muscle-type gangliosides was not obviously affected. The glycosidase and sialyltransferase activities decreased dramatically just prior to or at hatching. The fusion-linked change in GSL suggests a role for terminal galactosamine and/or galactose residues in myoblast aggregation. A parallel developmental study of genetic muscular dystrophy revealed similar GSL levels and enzyme activities. A larger proportion of lactosylceramide in dystrophic muscle throughout development suggests a developmental lag in the mutant.

Novel pentahexosyl ganglioside of the globo series purified from chicken muscle.

A monosialosyl ganglioside, sialosylpentaglycosyl-ceramide, has been isolated from chicken pectoral muscle. This ganglioside is the first to be found which contains the glycosphingolipid structure of the globo series. It was extracted from tissue with a mixture of tetrahydrofuran and aqueous KCl and purified by anion exchange and silicic acid chromatography. Enzymic hydrolysis utilizing specific glycosidases and methylation technique were employed to establish the structure which is proposed to be: NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 3Gal alpha 1 leads to 4Gal beta 1 leads to 4GlcCer. The chain base was predominantly sphingosine and the fatty acids present were mainly stearate and oleate.

Cell differentiation in pancreatic cancer.

Cell differentiation in cancer of the exocrine pancreas is currently under intensive study. In this report, new developments are critically reviewed on the use of transplantable pancreatic carcinoma and the hybridoma technique to define cell membrane changes during pancreatic cancer growth. It is concluded that two categories of plasmalemma structure hold promise as differentiation markers specific for pancreatic cancer: (1) membrane receptors for cholinergic and peptide secretagogues and (2) membrane glycoproteins as detected by monoclonal antibodies. Assay of secretagogue receptors and membrane glycoprotein antigens will be central to elucidation of mechanisms of pancreatic carcinoma cell differentiation (? stem cell differentiation or retrodifferentiation) and, hopefully, will provide tumor-specific or tissue-specific markers for the laboratory diagnosis and monitoring of pancreatic cancer.

Characterization of two gangliosides of the paragloboside series from chicken skeletal muscle.

Two glucosamine-containing gangliosides, a ceramide pentasaccharide and ceramide heptasaccharide, have been purified from the gangliosides of chicken skeletal muscle. The lipids were extracted with a mixture of tetrahydrofuran and aqueous KCl and purified by anion-exchange and silicic acid column chromatography. Their structures were determined by specific enzymatic hydrolysis and methylation analysis. The proposed structures are sialosyl derivates of: (a) paragloboside, and (b) paragloboside with an additional lactosamine disaccharide: (a) NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcCer, (b) NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcCer. The long base was predominantly sphingosine and the acyl groups were mainly palmitate, stearate and oleate.

Isolation and characterization of an endo-beta-galactosidase from a new strain of Escherichia freundii.

A new strain of Escherichia freundii was isolated from human feces. Compared with the previous strain (Kitamikado, M., and Ueno, R. (1970) Bull. Jpn. Soc. Sci. Fish. 36, 1175-1180), this strain releases comparable levels of endo-beta-galactosidase with lower levels of exoglycosidases in the culture medium containing 0.3% of keratan sulfate. Endo-beta-galactosidase was purified by an improved purification procedure involving Amicon H1P10 hollow fiber filtration, QAE-Sephadex A-50, and CM-Sephadex C-50 chromatographies. The purified enzyme is completely free from proteases and exoglycosidases. The general properties of this enzyme are: molecular weight, 28,000; optimal pH, 5.5 TO 5.8; PI, pH 8.0. This enzyme hydrolyzed keratan sulfates isolated from different sources to produce 6-O-sulfo-GlcNAc beta1 leads to 3Gal as the major product. In addition, the specificity of this enzyme toward various glycoconjugates was also studied.

Isolation and characterization of a heptaglycosylceramide from bovine erythrocyte membranes.

A heptaglycosylceramide was isolated from bovine erythrocyte membranes. The structure was characterized to be Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta1-3)Gal(beta 1-4)Glc-NAc(beta 1-4)al(beta 1-4)GlcCer. A hexaglycosylceramide that has the same sequence except for the terminal alpha-galactosyl unit has also been isolated. We have previously found that gangliosides isolated from bovine erythrocyte membranes contain a keratan sulfate type repeating unit --[3Gal(beta 1-4)-GlcNAc beta]--n. This study shows that the keratan sulfate type repeating unit is also present in the neutral glycosphingolipids of bovine erythrocyte membranes.

Occurrence of glycosphingolipids in chicken egg yolk.

Chicken egg yolk was found to contain a unique glycosphingolipid pattern not seen in other types of tissue or cell. These glycosphingolipids were isolated in pure form and their structures established by sequential enzymic hydrolysis and permethylation analysis. The major gangliosides in chicken egg yolk are N-acetylneuraminosylgalactosylceramide, N-acetylneuraminosyl-lactosylceramide and di-N-acetylneuraminosyl-lactosylceramide. The only neutral glycosphingolipid found in chicken egg yolk is galactosylceramide.