Characterization of a novel triphosphonooctaosylceramide from the eggs of the sea hare, Aplysia kurodai.

We have reported the existence of a triphosphonoglycosphingolipid, EGL-I, in the eggs of a sea gastropod, Aplysia kurodai [Yamada, S., Araki, S., Abe, S., Kon, K., Ando, S., and Satake, M. (1995) J. Biochem. 117, 794-799]. We have now isolated a novel glycosphingolipid, named EGL-II, from the eggs of Aplysia. By component analysis, sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, secondary ion mass spectrometry, and proton magnetic resonance spectrometry, its structure was revealed to be as follows: Galalpha1-->3(GlcNAcalpha1-->2)Galalpha1-->3(3-O-MeGalalpha1-->2)Galalpha1-->3[6'-O-(2-aminoethylphosphonyl)Galalpha1-->2](2-aminoethylphosphonyl-->6)Galbeta1-->4(2-aminoethylphosphonyl-->6)Glcbeta1-->1ceramide. The major aliphatic components of the ceramide are palmitic acid, stearic acid, and anteisononadeca-4-sphingenine.

High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization.

Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.

Conversion of cellular sialic acid expression from N-acetyl- to N-glycolylneuraminic acid using a synthetic precursor, N-glycolylmannosamine pentaacetate: inhibition of myelin-associated glycoprotein binding to neural cells.

Sialic acids are prominent termini of mammalian glycoconjugates and are key binding determinants for cell-cell recog-nition lectins. Binding of the sialic acid-dependent lectin, myelin-associated glycoprotein (MAG), to nerve cells is implicated in the inhibition of nerve regeneration after injury. Therefore, blocking MAG binding to nerve cell sialoglycoconjugates might enhance nerve regeneration. Previously, we reported that certain sialoglycoconjugates bearing N-acetylneuraminic acid (NeuAc) but not N-glycolylneuraminic acid (NeuGc) support MAG binding (Collins et al., 1997a). We now report highly efficient conversion of sialic acids on living neural cells from exclusively NeuAc to predominantly NeuGc using a novel synthetic metabolic precursor, N-glycolylmannosamine pentaacetate (Man-NGc-PA). When NG108-15 neuroblastoma-glioma hybrid cells, which normally express only NeuAc (and bind to MAG), were cultured in the presence of 1 mM ManNGcPA, they expressed 80-90% of their sialic acid precursor pool as NeuGc within 24 h. Within 5 days, 80% of their ganglioside-associated sialic acids and 70% of their glycoprotein-associated sialic acids were converted to NeuGc. Consistent with this result, treatment of NG108-15 cells with ManNGcPA resulted in nearly complete abrogation of MAG binding. These results demonstrate that ManNGcPA treatment efficiently alters the sialic acid structures on living cells, with a commensurate change in recognition by a physiologically important lectin.

Ganglioside composition of GH3 cells: enhancement of fucoganglioside expression by estradiol, epidermal growth factor and insulin.

The GH3 cell line, a bipotential cell line secreting both prolactin (PRL) and growth hormone (GH), is a useful model for investigating GH/PRL cell lineage differentiation and anterior pituitary adenoma formation. In this study, we investigated the ganglioside composition of GH3 cells and identified two fucogangliosides as the major gangliosides expressed by these cells. Analyses by DEAE-Sephadex A-25 and thin-layer chromatography (TLC) revealed that the GH3 cells contained two major gangliosides, designated FG1 and FG2, respectively. Their structures were identified by fast atom bombardment mass spectrometry and proton nuclear magnetic resonance spectrometry: FG1 is IV2FUc alpha,II3NeuAc-GgOse4Cer and FG2 is IV2FUc alpha,IV3Gal alpha,II3NeuAc-GgOse4Cer. Expression of these fucogangliosides was enhanced by chronic treatment with 17 beta-estradiol (1 nM), epidermal growth factor (10 nM) and insulin (300 nM), which induced differentiation of GH3 cells to normal PRL-secreting cells. Interestingly, immunocytochemistry and flow cytometry revealed that the increased expression of these gangliosides reflected a quantitative change inside the cells but not on the cell surface. These results suggest that the intracellular distribution of fucogangliosides is closely related to the differentiation of GH3 cells.

Ganglioside composition of the rat choriocarcinoma cell line, Rcho-1.

The Rcho-1 cell line, originally established from a rat choriocarcinoma, shows differentiation into placental trophoblastic giant cell-like cells and has been used to study the mechanism of placental function control. In the present study, we analysed the ganglioside composition of Rcho-1 cells by HPTLC orcinol/H2So4, TLC/ immunostaining and immunohistochemistry. Rcho-1 cells expressed GM3 and GD3 as the major gangliosides and CTH as major neutral glycolipid when they were cultured in growth medium (20% FCS) or transplanted beneath the kidney capsule. The expression of these gangliosides was strong in the undifferentiated small cells, whereas the completely differentiated giant cells showed poor staining with antibodies against the gangliosides. Under culture conditions to induce cell differentiation using horse serum (1-20% HS), the expression of GD3 was suppressed and re-expressed when the medium was changed to growth medium, suggesting that a change of ganglioside components may trigger and define the direction of terminal differentiation. Thus the composition of glycolipids is conserved in Rcho-1 cells and is similar to that of the rat placenta, where GM3 is dominant in mid-pregnancy and decreased in late pregnancy, whereas GD3 is low in mid-pregnancy and increased in late pregnancy.

Glycosphingolipid composition of rat placenta: changes associated with stage of pregnancy.

The composition of glycolipids and their changes in the placenta were investigated in the normal pregnant rat. Total lipid fractions extracted from the placenta between days 12 and 20 of pregnancy (day 0 = oestrus) were subjected to glycolipid analysis using DEAE-Sephadex chromatography, silica-gel HPLC, silica-gel TLC, TLC/immunostaining, matrix-assisted secondary-ion mass spectrometry in the negative-ion mode and 1H NMR. Glycolipids identified in the rat placenta were: gangliosides GM3 (NeuAcLacCer and NeuGcLacCer) and GD3 (NeuAcNeuAcLacCer, NeuAcNeuGcLacCer and NeuGcNeuAcLacCer), and neutral glycolipids ceramide monosaccharide (CMH) (GlcCer), ceramide disaccharide (CDH) (LacCer), ceramide trisaccharide (CTH) (Gb3Cer) and ceramide tetrasaccharide (CQH) (Gb4Cer). The content of neutral glycolipids was higher than that of gangliosides throughout pregnancy. Of the neutral glycolipids, CMH and CTH predominated and the level of CDH was low at mid-pregnancy. During late pregnancy, CMH and CTH decreased and CDH increased markedly. CQH remained at a low level throughout pregnancy. Of the gangliosides, GM3 was predominant on days 12-16 and then decreased, whereas GD3, which was low on day 12, increased slightly on day 16 and maintained the same level thereafter. Immunohistochemical studies indicated that these changes in the expression of major gangliosides from GM3 to GD3 occurred in labyrinthine trophoblasts. Thus expression of these glycolipids appears to change markedly during pregnancy.

Immunogenic properties of mannose-containing ceramide disaccharide and immunochemical detection of its hapten in the two kinds of crustacean, Euphausia superba and Macrobrachium nipponense.

Antiserum against Man beta 1-4Glc beta 1-1Ceramide (MIOse2Cer), a mannolipid isolated from spermatozoa of the fresh-water bivalve, Hyriopsis schlegellii, has been elicited in rabbits by repeated injection of a mixture of hapten-bovine serum albumin (1:1, mg/ml) with Freund's adjuvant. The specificity of the affinity-purified antibody (immunoglobulin G type) obtained from the serum was examined, using other glycosphingolipids and glyco-proteins structurally related to MIOse2Cer, by means of ELISA and TLC-immunostaining. The purified antibody was highly specific to MIOse2Cer and lacked reactivity with other glycolipids and glycoproteins including glucosylceramide, lactosylceramide, dimannosylglucosylceramide (MIOse3Cer), glucosaminylmannosylglucosylceramide (ArOse3Cer), thyroglobulin and alpha 1-acid glycoprotein. The antibody was found to bind, although less efficiently, to certain other compounds containing the group Man beta 1-4Glc and/or Man beta 1-4GlcNAc at their termini, such as MIOse2-sphingosine and Man beta 1-4GlcNAc beta 1-p-aminobenzoic acid ethylester derivatives. The present antibody was applied to the detection of the natural hapten in crustacean glycolipids. The purified antibody reacted with a neutral glycosphingolipid present in the two kinds of crustacean, Euphausia superba (antarctic krill) and Macrobrachium nipponense (fresh-water shrimp) as shown by TLC-immunostaining. The crustacean glycolipid antigen was isolated and characterized to be the Man beta 1-4Glc-Cer. This is the first report on the presence of a mannose-containing glycosphingolipid in the crustacean.

Polar glycosphingolipids in insect: chemical structures of glycosphingolipid series containing 2'-aminoethylphosphoryl-(----6)-N-acetylglucosamine as a polar group from larvae of the green-bottle fly, Lucilia caesar.

A series of glycosphingolipids containing 2'-aminoethylphosphoryl(----6)-N-acetylglucosamine as a polar group has been demonstrated in larvae of the green-bottle fly, Lucilia caesar. The thin-layer chromatographic pattern of the total polar glycolipid revealed the presence of more than eight components, of which five major components were purified by the use of successive column chromatography on QAE- and DEAE-Sephadex and silicic acid (Iatrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, methylation analysis, and fast atom bombardment mass spectrometry, their structures were deduced to be as follows: 2'-aminoethylphosphoryl----6GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc alpha 1-4GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, Gal beta 1-3GalNAc alpha 1-4GalNAc-beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, and GlcNAc beta 1-3Gal-beta 1-3GalNAc alpha 1-4GalNAc beta 1-4 (2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc-beta 1-Cer. The main molecular species of the ceramide moiety was arachidinyltetradecasphingenine in all of the major glycolipids.

Isolation and characterization of a monosialosylgangliopentaosyl ceramide from Xenopus laevis oocyte.

A monosialosylgangliopentaosyl ceramide was isolated from Xenopus laevis oocytes. It represented 5.8% of the total acidic glycosphingolipids. From the results of sugar-composition analysis, enzymatic hydrolysis, permethylation analysis, and negative ion fast atom bombardment mass spectrometry, the structure of the ganglioside was determined to be as follows: [sequence: see text] The predominant species of fatty acids were alpha-hydroxy fatty acids, h22:0, h24:0, and h24:1. The long chain bases of this ganglioside consisted mainly of d18:1 sphingosine and phytosphingosine. Other acidic glycolipids were also characterized. The most abundant component of acidic glycolipids was sulfatide, which represented 85.7% of the total acidic glycolipid mixture. GM3, GM2, GM1a, and GD1a were also detected.

Characterization of a new phosphonocerebroside, N-methyl-2-aminoethylphosphonylglucosylceramide, from the antarctic krill, Euphausia superba.

A novel phosphonglycosphingolipid was purified from the whole tissue of the antarctic krill, Euphausia superba by successive column chromatography on DEAE- and QAE-Sephadex and silicic acid (Iatrobeads). The structure was elucidated by means of IR, FAB-MS, 1H-NMR, GC and GC-MS analyses of the water-soluble products after complete and partial acid hydrolysis, and methylation analysis of a product of hydrogen fluoride degradation; it was identified to be a phosphonocerebroside, 6'-O-(N-methyl-2-aminoethylphosphonyl)Glcp beta 1----1ceramide. The ceramide moiety was composed of tetradecasphingenine and octadecasphingatriene as the main sphingoids, and monounsaturated C22- and C24-acids and their 2-hydroxy homologues as the major fatty acids.

Characterization of two glucuronic acid-containing glycosphingolipids in larvae of the green-bottle fly, Lucilia caesar.

Two glucuronic acid-containing glycosphingolipids were purified from larvae of the green-bottle fly, Lucilia caesar by DEAE-Sephadex and Iatrobeads column chromatography. Structures of these acidic glycolipids, glycolipids X and Y, were elucidated by means of sugar analysis, permethylation, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and NMR studies. Glycolipid X was determined to have the following structure: GlcA beta 1-3Gal beta 1-3GalNAc alpha 1-4 GalNAc beta 1-4 GlcNAc beta 1-3Man beta 1-4Glc beta 1-1 ceramide. The other acidic glycolipid, glycolipid Y contains a phosphoethanolamine residue linked through the 6-hydroxy group of the N-acetyl-glucosamine unit of glycolipid X. The ceramide moieties were composed of saturated fatty acids (16:0-22:0) and tetradeca- and hexadeca-4-sphingenines. Based on the structural similarity of the ceramide moieties it appears likely that glycolipid X is an intermediate from which glycolipid Y is synthesized by addition of a phosphoethanolamine residue.

Involvement of the acyl chain of ceramide in carbohydrate recognition by an anti-glycolipid monoclonal antibody: the case of an anti-melanoma antibody, M2590, to GM3-ganglioside.

The effect of the chain length of the fatty acid residue of the ceramide moiety of ganglioside GM3 on the binding ability of monoclonal antibody M2590, which is specific for the carbohydrate structure of GM3-ganglioside, was examined by means of a direct binding assay on thin layer chromatography plates (TLC immunostaining) and a quantitative enzyme-linked immunosorbent assay (ELISA). Derivatives of GM3 with a long fatty acid chain reacted with the M2590 antibody, but those with a short fatty acid chain showed no reaction in either assay system. These results suggested that the acyl fatty acid moiety of the ganglioside played an important role in the formation or maintenance of the antigenic structure of the carbohydrate moiety of the ganglioside.

Immunogenic properties of mannose-containing ceramide tetrasaccharide from fresh-water bivalve, Hyriopsis schlegelii.

Antiserum against GlcNAc beta 1----2Man alpha 1----3Man beta 1----4Glc beta 1----1Cer (MlOse4Cer), a mannolipid isolated from spermatozoa of the fresh-water bivalve Hyriopsis schlegelii, was elicited in rabbits by repeated injection of a mixture of hapten-bovine serum albumin with Freund's adjuvant. The specificity of the affinity-purified antibody obtained from the serum was based on two forms of enzyme-immunodetection of its binding to structurally related glycolipids, either adsorbed to microtiter plates or chromatographed on thin-layer plates. The purified antibody exhibited a significant cross-reactivity with GlcNAc beta 1----2Man alpha 1----3(Xyl beta 1----2)Man beta 1----4Glc beta 1----1Cer, (MIXOse5Cer) containing a core structure closely related to MlOse4Cer, but almost unrelated to other glycolipids. Distribution of MlOse4Cer and MlXOse5Cer in various bivalve and snail glycolipid extracts were screened in thin-layer immunobinding assays by using this purified specific antibody. The presence of the glycolipid antigens was limited to certain taxonomic orders of shellfish species.