Monoclonal antibody R24 distinguishes between different N-acetyl- and N-glycolylneuraminic acid derivatives of ganglioside GD3

Monoclonal antibody (MAb) R24 was previously shown to be directed toward ganglioside GD3 [Pukel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155, 1133-1147]. The structural specificity of the MAb has now been further characterized based on binding to structurally related glycolipids, including four GD3 derivatives with different N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) substituents. Three assay systems (enzyme immunostaining on thin-layer chromatography, enzyme-linked immunosorbent assay, and immune adherence inhibition assay) were used. MAb R24 was found to react with (NeuAc-NeuAc-)GD3 and (NeuAc-NeuGc-)GD3 but not with (NeuGc-NeuAc-)GD3 or (NeuGc-NeuGc-)GD3. These results clearly indicate that the outer sialic acid (Sia) moiety of GD3 is crucial and must be a NeuAc residue, while the inner sialic acid is less involved in binding to the MAb and can be either NeuAc or NeuGc. The MAb was also found to cross-react weakly with two gangliosides, GT1a and GQ1b, but none of other gangliosides nor neutral glycolipids tested reacted. These findings suggest that the epitope detected by MAb R24 is the trisaccharide structure NeuAc alpha 2----8Sia alpha 2----3Gal-, which must be in a terminal position.     

Different fine binding specificities of monoclonal antibodies to disialosylganglioside GD2

The fine structural specificities of six monoclonal antibodies (MAbs) to ganglioside GD2, GalNAc beta 1----4(NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4Glc-Cer, were studied. The binding specificities of these MAbs were found to differ from each other by virtue of their binding to structurally related authentic standard glycolipids as revealed by three different assay systems, including enzyme immunostaining on thin-layer chromatography, enzyme-linked immunosorbent assay, and immune adherence inhibition assay. The MAbs examined could be divided into three binding types. MAbs A1-201, A1-410, and A1-425 bound specifically to ganglioside GD2 and none of the other gangliosides tested. Two other MAbs (A1-245 and A1-267) reacted not only with GD2, but also with several other gangliosides having the sequence NeuAc alpha 2----8NeuAc alpha 2----3Gal (GD3, GD1b, GT1a, GT1b, and GQ1b). The reactivities with these gangliosides varied to some degree. In addition, these MAbs were found to react with both GD3(NeuAc-NeuAc) and GD3(NeuGc-NeuAc), but not with GD3(NeuAc-NeuGc) or GD3(NeuGc-NeuGc). The last MAb (A1-287) also reacted with several other gangliosides but with lower avidity than A1-245 and A1-267. These findings suggest that each MAb to ganglioside GD2 may have an individual binding specificity and avidity. These MAbs represent potentially useful reagents for analyzing the function of GD2 on cell surface membranes, and provide a system for precisely studying the interactions between an anti-ganglioside antibody and the binding epitope of the antigenic determinant.     

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.     

Identification of N-glycolylneuraminic acid-containing gangliosides of cat and sheep erythrocytes. 252Cf fission fragment ionization mass spectrometry in the analysis of glycosphingolipids

A number of gangliosides were isolated from cat and sheep erythrocytes for use in analyzing the specificity of a panel of human anti-heterophile monoclonal antibodies. The structures of these compounds were determined by a combination of different procedures, including sugar analysis, glycosidase treatment, periodate oxidation, TLC immunostaining, methylation analysis, and mass spectrometry. These methods identified the cat erythrocytes gangliosides (C1 and C2) as N-glycolylneuraminic acid (NeuGc)-containing hematosides; C1 was shown to be NeuGc alpha 2----8NeuGc alpha 2----3Gal beta I----4Glc-Cer [NeuGc)2GD3) and C2 to be NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)GD3). The two sheep gangliosides (S1 and S2) were found to be novel glycolipids based on the paragloboside sequence; S1 was identified as NeuGc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuGc)2-disialylparagloboside) and S2 as NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)-disialylparagloboside). Structural analysis of these compounds was aided by the use of 252Cf fission fragment ionization time-of-flight mass spectrometry. This method provided easily interpretable spectra on methylated derivatives which were particularly useful in determining the sialic acid composition of the gangliosides and the sequence of their disialosyl side chains.     

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of the trisialogangliosides from bovine adrenal medulla

Trisialogangliosides were isolated from bovine adrenal medulla by DEAE-Sephadex A-25 and Iatrobeads column chromatography. Their structures were elucidated by sugar analysis, neuraminidase digestion, and permethylation studies. The complete structures of trisialogangliosides, A to D, were identified as follows. A: GT1b, IV3NeuAc, II3 (NeuAc)2-GgOse4Cer. B: GT1b(NeuAc/NeuAc-NeuGc-); IV3NeuAc, II3 (NeuAc alpha 2-8 NeuGc-)GgOse4Cer. C: GT1b (NeuGc/NeuAc-NeuAc-); IV3NeuGc, II3 (NeuAc alpha 2-8 NeuAc-)GgOse4Cer. D: GT1b (NeuAc/NeuGc-NeuGc-); IV3NeuAc, II3 (NeuGc alpha 2-8 NeuGc-)GgOse4Cer. Gangliosides B, C, and D, which contain N-glycolylneuraminic acid, have not previously been reported in the literature.     

Generation of one set of monoclonal antibodies specific for b-pathway ganglio-series gangliosides.

We established six murine monoclonal antibodies (MAbs) specific for b-pathway ganglio-series gangliosides by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota mutant R595. The binding specificities of these MAbs were determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatogram. These six MAbs, designated GGB19, GMR2, GMR7, GGR12, GMR5, and GGR13 reacted strongly with the gangliosides GD3, O-Ac-GD3, GD2, GD1b, GT1b, and GQ1b, respectively, that were used as immunogens. All these MAbs except GGB19 showed highly restricted binding specificities, reacting only with the immunizing ganglioside. None of other various authentic gangliosides or neutral glycolipids were recognized. On the other hand, MAb GGB19 exhibited a broader specificity, cross-reacting weakly with O-Ac-GD3, GQ1b, and GT1a, but not with other gangliosides or neutral glycolipids. Using these MAbs, we determined the expression of these gangliosides, especially GD1b, GT1b, and GQ1b on mouse, rat, and human leukemia cells. GD1b was expressed on rat leukemia cells, but not on mouse and human leukemia cells tested. Neither GT1b nor GQ1b was detected in these cell lines.     

Acidic glycosphingolipids of human amnion

Six major acidic glycosphingolipids were isolated from human amnion using DEAE Sephadex A-25 and silica beads column chromatography. The structures of these glycosphingolipids were determined by methylation analysis, TLC immunostaining and/or negative ion FAB-MS, and were concluded to be II3 alpha NeuAcLacCer(GM3), IV3 alpha NeuAcnLc4-Cer (sialyl[alpha 2-3]paragloboside), IV6 alpha NeuAcnLc4Cer (sialyl[alpha 2-6]paragloboside), IV3 alpha NeuAcIII4 alpha FucLc4Cer (sialyl Lea), VI3 alpha NeuAcnLc6Cer (i-ganglioside) and II3 alpha (NeuAc alpha 2----8NeuAc)LacCer (GD3). In addition, several minor glycosphingolipids were detected with specific monoclonal antibodies, including glycolipids with NeuAc alpha 2-3Gal beta 1-4GlcNAc-beta 1- or NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1- determinant. Our results show that the glycosphingolipids of human amnion are characterized by having mainly type II chain analogues and onco-fetal antigens.     

Adhesion of K99 fimbriated Escherichia coli to pig intestinal epithelium: correlation of adhesive and non-adhesive phenotypes with the sialoglycolipid content.

Evidence for the existence of two phenotypes of piglets born to experimental herds was obtained based on the susceptibility of intestinal brush borders to adhesion of K99-positive Escherichia coli. The enterocytes of the K99-receptive piglets displayed a characteristic sialoglycolipid pattern, with a higher content of the monosialoglycolipids II3NeuGc-LacCer (GM3Gc), IV3NeuGc-nLcOse4Cer (SPGGc) and IV3NeuAc-nLcOse4Cer (SPG) and the oligosialogangliosides IV3NeuAc,II3NeuAc-GgOse4Cer (GD1a), II3(NeuAc)2-GgOse3Cer (GD2), II3(NeuAc)2-GgOse4Cer (GD1b) and IV3NeuAc,II3(NeuAc)2-GgOse4Cer (GT1b) when compared to the gangliosides of non-receptive piglets. The gangliosides from enterocytes of the non-receptive piglets were mainly the monosialogangliosides II3NeuAc-GgOse3Cer (GM2) and II3NeuAc-LacCer (GM3), only traces of the other sialoglycolipids being detected. Adhesion of 14C-labelled K99-positive E. coli cells to the piglet small intestinal sialoglycolipids, as tested by the thin-layer chromatogram overlay assay, revealed that the receptive enterocyte membrane was richer in glycolipids containing K99 receptor structures than the non-receptive enterocyte. Adhesion of K99-positive E. coli correlated with the degree of sialylation of the brush border glycolipids.     

Glycosphingolipids of normal bovine and enzootic bovine leukosis lymph node cells

We analyzed glycosphingolipids from normal lymph node cells of seven cattle and lymph node cells of eight cattle with enzootic bovine leukosis. The neutral glycosphingolipids and gangliosides were analyzed by thin-layer chromatography. Both normal and tumorous lymph node cells had GlcCer, LacCer, and GbOse3Cer as major neutral glycosphingolipids. In the ganglioside fraction, GM3 was the predominant component in both normal and tumorous lymph node cells, and another component, ganglioside Gx fraction, was also prominent in tumorous lymph node cells. The structure of this ganglioside Gx fraction was elucidated by thin-layer chromatography, sugar analysis, neuraminidase digestion, and permethylation studies. This ganglioside Gx fraction was found to be a mixture of four ganglioside species. The structures of individual gangliosides Gx (1 to 4) were characterized as follows. 1: GD3, NeuAc alpha 2-8NeuAc alpha 2-3Gal1-4Glc-Cer. 2: GD3, NeuAc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. 3: GD3, NeuGc alpha 2-8NeuAc alpha 2-3Gal1-Glc-Cer. 4: GD3, NeuGc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. These GD3 species may be formed as a result of the induced synthesis inassociation with malignant transformation.     

A sialidase-susceptible ganglioside, IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer, is a major disialoganglioside in WHT/Ht mouse thymoma and thymocytes.

The disialogangliosides of WHT/Ht mouse thymomas, which were obtained by subcutaneous transplantation of a thymoma that developed spontaneously in a WHT/Ht mouse, were purified and characterized. From the results of sugar-composition analysis, a permethylation study, enzymatic hydrolysis followed by TLC-immunostaining, negative-ion fast atom bombardment mass spectrometry (FAB/MS), and 1H-NMR spectroscopy, the structure of one of the five purified disialogangliosides was determined to be IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer. The other 4 disialogangliosides were tentatively characterized on the basis of sialidase treatment followed by TLC-immunostaining with cholera toxin B subunit and anti-Gg4Cer antibody to be IV alpha(NeuAc alpha-NeuGc)-Gg4Cer, IV alpha(NeuGc alpha-NeuAc)-Gg4Cer, IV alpha NeuAc,II3 alpha NeuAc-Gg4Cer, and IV alpha NeuGc,II3 alpha NeuGc-Gg4Cer. In addition, another component exhibiting one spot on TLC was a mixture of IV alpha NeuGc,II3 alpha NeuAc-Gg4Cer and IV alpha NeuAc,II3 alpha NeuGc-Gg4Cer. Then the occurrence of these gangliosides in WHT/Ht mouse thymocytes was examined. As one of two major disialogangliosides, the thymocytes contained IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer, which was characterized with a mass spectrum and mass chromatograms obtained by micro high-performance liquid chromatography-FAB/MS. The other major disialoganglioside was tentatively characterized to be II3 alpha-(NeuGc alpha-NeuGc)-Gg4Cer by sialidase treatment followed by TLC-immunostaining. A sialidase-susceptible monosialoganglioside, IV3 alpha NeuGc-Gg4Cer [GM1b(NeuGc)], had been reported to be characteristic of mouse immune tissues [Nakamura, K. et al. (1988) J. Biochem, 103, 201-208]. Taken together, the results suggest that the pathway from Gg4Cer to IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer through GM1b(NeuGc) is quite active in mouse immune tissues.     

Novel polysialogangliosides of skate brain structural determination of tetra, penta and hexasialogangliosides with a NeuAc-GalNAc linkage.

The gangliosides in the brain of a cartilaginous fish, skate (Bathyraja smirnovi), have been isolated and characterized by means of methylation analysis, antibody binding, enzymatic hydrolysis and MALDI-TOF MS. In addition to gangliosides with known structures (GM2, fucosyl-GM1, GD3, GD2, GT3 and GT2), five polysialogangliosides were isolated and characterized as having the following structures. (1) IV3NeuAc, III6NeuAc, II3NeuAc-Gg4Cer; (2) IV3NeuAc2, III6NeuAc, II3NeuAc-Gg4Cer; (3) IV3NeuAc, III6NeuAc, II3NeuAc2-Gg4Cer; (4) IV3NeuAc, III6NeuAc, II3NeuAc3-Gg4Cer; and (5) IV3NeuAc2, III6NeuAc, II3NeuAc3-Gg4Cer. These structures are 'hybrid-type' which comprise combinations of alpha-series and either a, b or c-series structures. Three gangliosides (2), (4) and (5), were novel. The main features of the ganglioside composition of skate brain were an abundance of gangliotriaosyl species, a lack of gangliotetraosyl species (except fucosyl-GM1), and an abundance of hybrid-types. These characteristics closely resemble those in shark brain which we reported previously [Nakamura, K., Tamai, Y. & Kasama, T. (1997) Neurochem. Int. 30, 593-604]. Two of the hybrid-type gangliosides (1) and (4), were examined for their neuritogenic activity toward cultured neuronal cells (Neuro-2A), and were found to have more potent activity than nonhybrid-type gangliosides such as GM1.     

Monoclonal antibody detects monosialogangliosides having a sialic acid alpha 2----3-galactosyl residue

A murine monoclonal antibody (mAb), designated mAb 202, was generated using a human melanoma cell line, UCLASO-M14 as the immunogen. mAb 202 reacted with two (GM2 and GM3) of the four (GM2, GM3, GD2, and GD3) gangliosides expressed by M14. Several authentic monosialogangliosides, including GM4, GM3, GM2, GM1, GM1b, and sialylparagloboside were then tested for their binding to 202 mAb by the immune adherence inhibition assay, TLC-enzyme immunostaining, and enzyme-linked immunosorbent assay. All showed positive binding but in varying degrees. GM4 showed the strongest affinity. No significant differences of reactivity were observed between the sialic acid derivatives, N-acetyl and N-glycolyl, in these gangliosides. Disialogangliosides such as GD3, GD2, GD1a, and GD1b, trisialoganglioside GT1b, and neutral glycolipids including GlcCer, GalCer, LacCer, GbOs3Cer, GbOs4Cer, GgOs3Cer, GgOs4Cer, and nLcOs4Cer were all negative. These results indicate that the 202 mAb detects sialyl alpha 2----3Gal residue in the monosialoganglioside, irrespective of the internal structure. Since GM4 is not expressed by M14 cells, the terminal disaccharide (sialyl alpha 2----3Gal) in GM3 and/or GM2 must have been the epitope responsible for the generation of the antibody.     

Isolation and characterization of gangliosides from Trypanosoma brucei

Gangliosides were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated by TLC. G-1 ganglioside had the same TLC migration rate as GM3. In contrast, G-2, G-3, and G-4 gangliosides migrated a little slower than GM1, GD1a, and GD1b, respectively. To characterize the molecular species of gangliosides from T. brucei, G-1, G-2, G-3, and G-4 gangliosides were purified and analyzed by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside showed the reactivity to the monoclonal antibody against ganglioside GM3. G-2 was recognized by the anti-GM1 monoclonal antibody. G-3 showed reaction with the monoclonal antibody to GD1a. G-4 had the reactivity to anti-GD1b monoclonal antibody. Using 4 kinds of monoclonal antibodies, we also studied the expression of GM3, GM1, GD1a, and GD1b in T. brucei parasites. GM3, GM1, GD1a, and GD1b were detected on the cell surface of T. brucei. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-1Cer), GM1 (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), GD1a (NeuAc alpha2-3Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), and GD1b (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-8NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), respectively, and also that they are expressed on the cell surface of T. brucei.     

Gangliosides GM2, IV4GalNAcGM1b, and IV4GalNAcGC1a as antigens for monoclonal immunoglobulin M in neuropathy associated with gammopathy

It was previously reported that monoclonal IgM from two patients with gammopathy and neuropathy showed similar specificity by reacting with the same group of unidentified minor components in the ganglioside fractions of human nervous tissues (Ilyas, A. A., Quarles, R. H., Dalakas, M. C., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6697-6700). Enzymatic degradation, ion-exchange chromatography, and immunostaining of purified ganglioside standards on thin-layer chromatograms have now revealed that the antigenic glycolipids recognized by the IgM from these patients are gangliosides GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1Cer(GM2), GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer (IV4GalNAcGM1b), and GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-3GalNAc beta 1-4 beta Gal(3-2 alpha NeuAc)beta 1-4Glc beta 1-1-Cer (IV4GalNAcGD1a). The monoclonal IgM appears to be reacting with the terminal [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-] moiety shared by these three gangliosides and is a useful probe for detecting small amounts of GM2, IV4GalNAcGM1b, IV4GalNAcGD1a, and other gangliosides with the same terminal sugar configuration in tissues. Species distribution studies using the antibody revealed that GM2 is present in the brains and nerves of all species examined, while IV4GalNAcGM1b and IV4GalNAcGD1a exhibit some striking species specificity. GM2, but not IV4GalNAcGD1a, is enriched in purified myelin from human brain.     

Neutral glycosphingolipids and gangliosides from spleen T lymphoblasts of genetically different inbred mouse strains

The gangliosides GM1b, GalNAc-GM1b and GD1α are typical compounds of concanavalin A stimulated splenic T lymphoblasts of CBA/J inbred mice. Their structural characterization has been described in previous studies. The intention of this work was the comparative TLC immunostaining analysis of the glycosphingolipid composition of lectin stimulated splenic T lymphoblasts obtained from six genetically different inbred mouse strains. The strains examined were AKR, BALB/c, C57BL/6, CBA/J, DBA/2 and WHT/Ht, which are commonly used for biochemical and immunological studies. The neutral glycosphingolipid GgOse4Cer, the precursor for GM1b-type gangliosides, was expressed by all six strains investigated. AKR, C57BL/6 and DBA/2 showed high and BALB/c, CBA/J and WHT/Ht diminished expression in T lymphoblasts, based on single cell calculation. The gangliosides GM1b and GalNAc-GM1b, elongation products of GgOse4Cer, displayed strain-specific differences in their intensities, which were found to correlate with the intensities of GgOse4Cer expression of the same strains. Concerning sialic acid substitution of gangliosides, GM1b and GalNAc-GM1b predominantly carry N-acetylneuraminic acid, whereas choleragenoid receptors GM1a and Gal-GalNAc-GM1b, which are also expressed by all six strains, are characterized by dominance of N-glycolylneuraminic acid. Two highly polar gangliosides, designated with X and Y, which have not been previously recognized in murine lymphoid tissue, were detected by positive anti-GalNAc-GM1b antibody and choleragenoid binding, respectively. Both gangliosides were restricted to AKR, DBA/2 and C57BL/6 mice. The other three strains BALB/c, CBA/J and WHT/Ht are lacking these structures. In summary, the GM1b-type pathway is quite active in all six strains analysed in this study. Strain-specific genetic variations in T lymphoblast gangliosides were observed with the occurrence of gangliosides X and Y. This study and data from other groups strongly indicate for GM1b-type gangliosides a functional association with T cell activation and leukocyte mediated reactions. Abbreviations: ConA, concanavalin A; GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) [48] and the ganglioside nomenclature system of Svennerholm [49] for GM1a-type gangliosides. Glucosylceramide or GlcCer, Glcβ1-1Cer; lactosylceramide or LacCer, Galβ1-4Glcβ1-1Cer; gangliotriaosylceramide or GgOse3Cer or Gg3, GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliotetraosylceramide or GgOse4Cer or Gg4, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliopentaosylceramide or GgOse5Cer, GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliohexaosylceramide or GgOse6Cer, Galβ1-3GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer. GM3, II3NeuAc-LacCer; GM1 or GM1a, II3NeuAc-GgOse4Cer; GM1b, IV3NeuAc-GgOse4Cer; GalNAc-GM1b, IV3NeuAc-GgOse5Cer; GD1a, IV3NeuAc, II3NeuAc-GgOse4Cer; GD1b, II3(NeuAc)2-GgOse4Cer; GD1c, IV3(NeuAc)2-GgOse4Cer; GD1α, IV3NeuAc, III6NeuAc-GgOse4Cer. Only NeuAc-substituted gangliosides are presented in this list of abbreviations This revised version was published online in November 2006 with corrections to the Cover Date.     

Escherichia coli K99 binds to N-glycolylsialoparagloboside and N-glycolyl-GM3 found in piglet small intestine

Escherichia coli K12, which possess the K99 plasmid and synthesize K99 fimbriae (E. coli K99), cause severe neonatal diarrhea in piglets, calves, and lambs but not in humans. The organism binds specifically and with high affinity to only two glycolipids in piglet intestinal mucosa as demonstrated by overlaying glycolipid chromatograms with 125I-labeled bacteria. These glycolipids, which are N-glycolyl-GM3 (NeuGc alpha 2-3Gal beta 1-4Glc beta 1-1Cer) and N-glycolylsialoparagloboside (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), occur at about 13 and 0.3 micrograms per gram wet weight of mucosa, respectively. E. coli K99 grown at 18 degrees C, a temperature at which the K99 fimbriae are not expressed, do not bind to these glycolipids. Of the standard glycolipids tested in solid phase binding assays, E. coli K99 binds with highest affinity to N-glycolylsialoparagloboside, with less affinity to N-glycolyl-GM3, and with very low affinity to N-acetylsialoparagloboside. The bacteria do not bind to GM3 (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer), GM2 (GalNAc beta 1-4[Neu-Ac alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), GM1 (Gal beta 1-3GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc beta 1-1Cer), or several other N-acetylsialic acid-containing gangliosides and neutral glycolipids at the levels tested. N-Glycolylsialyl residues are found in the glycoproteins and glycolipids of piglets, calves, and lambs but not in the glycoproteins and glycolipids of humans. Possibly this distribution of sialyl derivatives explains the host range of infection by the organism.     

High-performance liquid chromatography-mass spectrometry of glycosphingolipids: II. Application to neutral glycolipids and monosialogangliosides

We previously reported a method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of molecular species of GlcCer and IV3 beta Gal-Gb4Cer [M. Suzuki et al. (1989) J. Biochem. 105, 829-833]. In this paper, we report a modification of this HPLC/FAB/MS method, which was used for the separation and characterization of neutral glycosphingolipids (GlcCer, LacCer, Gb3Cer, Gb4Cer, and IV3 alpha GalNAc-Gb4Cer) and monosialogangliosides [GM3(NeuAc or NeuGc), GM2 (NeuAc or NeuGc), and GM1 (NeuAc or NeuGc)]. Mixtures of the purified neutral glycolipids and monosialogangliosides were subjected to HPLC on a silica gel column, with programmed elution with isopropanol-n-hexane-water, with or without ammonium hydroxide. In order to obtain mass spectra and mass chromatograms of individual components, effluent from the HPLC column was mixed with a methanol solution of triethanolamine, which was used as the matrix for the FAB ionization, and one-thirtieth of the effluent mixture was introduced into a mass spectrometer through a frit interface. A mixture of the five neutral glycolipids, 5 micrograms of each, gave five peaks on a mass chromatogram obtained by monitoring of the corresponding major pseudo-molecular ions. A mixture of the six monosialogangliosides, 5 micrograms of each, gave six peaks on a mass chromatogram obtained by monitoring of the major pseudo-molecular ions, indicating that GM3, GM2, and GM1 were clearly separated, and that separation due to differences in sialic acid species was also achieved. In the mass spectra of the neutral glycolipids and monosialogangliosides, pseudo-molecular ions and fragment ions due to the elimination of sugar moieties were clearly detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

The glycosphingolipids of human prostate tissue

Neutral glycolipids and gangliosides from surgical samples of benign human prostate tissue were analyzed by chemical, enzymatic and immunostaining procedures. The neutral glycolipids consisted of ceramide mono-, di-, tri- and tetrahexosides of the globo series plus paragloboside. The monosialoganglioside fraction contained GM3 and GM1 plus multiple species of monosialylated lactosamine-containing structures, including sialyl-alpha-2----3paragloboside plus at least two compounds having a non-reducing terminal sialyl alpha 2----6Gal linkage. The disialoganglioside fraction contained GD3 as the major component plus GD1a, GD2 and GD1b. GT1b was the major trisialoganglioside.     

Immunohistochemical localization of minor gangliosides in the rat central nervous system

We previously described the differential distribution of majorgangliosides (GM1, GD1a, GD1b, GT1b and GQ1b) in adult rat braindetected by specific antibodies (Kotani,M., Kawashima,I., Ozawa,I.,Terashima,T. and Tai,T. Glycobiology, 3, 137–146, 1993).We report here the distribution of minor gangliosides in theadult rat brain by an immunofluorescence technique with mousemonoclonal antibodies (MAbs). Ten MAbs (GMR6, GMB28, GMR11,GMR19, GMR2, GMR7, GGR51, AMR10, NGR54 and NGR53) that specificallyrecognize GM3, GM2, GT1a, GD3, O-Acdisialoganglioside, GD2,GM1b, GM4, IV³NeuAc