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.     

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.     

Generation of murine monoclonal antibodies specific for N-glycolylneuraminic acid-containing gangliosides.

We generated two murine monoclonal antibodies (MAbs) specific for mono- and disialylgangliosides having N-glycolylneuraminic acid (NeuGc) as their sialic acid moiety, respectively, by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. By use of a wide variety of glycolipids, including NeuGc-containing gangliosides, the precise structures recognized by these two antibodies were elucidated through enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. One MAb, GMR8, which was generated by immunizing the mice with purified GM3(NeuGc), reacted specifically with gangliosides having NeuGc alpha 2----3Gal- terminal structures, such as GM3(NeuGc), IV3NeuGc alpha-Gg4Cer, IV3NeuGc alpha-nLc4Cer, V3NeuGc alpha-Gb5Cer, and GD1a(NeuGc, NeuGc). None of the other gangliosides having internal NeuGc alpha2----3Gal- sequences, such as GM2(NeuGc) and GM1(NeuGc), nor corresponding gangliosides having NeuAc alpha 2----3Gal- sequences, nor neutral glycolipids were recognized. Thus, the epitope structures recognized by the MAb were found to be strictly NeuGc alpha 2----3Gal- terminal structures. In contrast, the other MAb, GMR3, which was generated by immunizing the mice with purified GD3(NeuGc-NeuGc-) adsorbed to the bacteria, reacted specifically with gangliosides having NeuGc alpha 2----8NeuGc alpha 2----3Gal- terminal sequences, such as GD3(NeuGc-NeuGc-), IV3NeuGc alpha 2-Gg4Cer, IV3NeuGc alpha 2-nLc4Cer, and V3NeuGc alpha 2-Gb5Cer, but did not react with corresponding gangliosides having NeuAc as their sialic acid moiety or with the neutral glycolipids tested. The epitope structures recognized by the MAb were suggested to be NeuGc alpha 2----8NeuGc alpha 2----3Gal- terminal structures. Using these MAbs, we determined the distribution of such gangliosides in the spleen, kidney, and liver of several mice strains. Novel gangliosides reactive with these MAbs were detected in these tissues.     

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.     

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.     

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.     

Composition of fatty acids and sphingosine bases of placental gangliosides

The fatty acids and sphingosine bases from major placenta gangliosides (NeuAcLacCer, IV3NeuAc-nLc4Cer, VI3NeuAc-nLc6Cer, (NeuAc)2LacCer, II3IV3(NeuAc)2Gg4Cer and VI3NeuAc, IV6(II3NeuAc-nLcNAc)-nLc6Cer) were studied. The C18-sphingenine was shown to be present in all ganglioside fractions; fraction GD1a contained, in addition, C20-sphingenine. Saturated fatty acids were identified as major fatty acid fragments. The content of long-chain acids (22-25 C-atoms) in the monosialogangliosides was much higher than that in disialogangliosides.     

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.     

Glycosphingolipids of porcine blood: human blood group A and H antigens with type 1 chain in erythrocytes and plasma

Glycosphingolipids were purified from porcine erythrocytes and plasma. Two minor glycolipids with human blood group A and H antigenicities were found in both sources as components. The two antigenic glycolipids were identified as a hexaglycosylceramide (IV3 alpha GalNAc,IV2 alpha Fuc-Lc4Cer) for the A antigen and pentaglycosylceramide (IV2 alpha Fuc-Lc4Cer) for the H antigen and belonged to lactoseries (type 1 sugar chain) in contrast to those with neolacto core (type 2 sugar chain) in human erythrocytes, thereby endorsing biochemically the previous serological observations that the A antigen on porcine erythrocytes is uptake from plasma, probably the H antigen being the case. In addition to major glycolipids of globoseries in red cells and plasma, a variety of acidic glycolipids including two classes of sulphatides (sulphated galactosylceramide and sulphated lactosylceramide) and five classes of gangliosides (GM3, GD3, GM1, fucosyl GM1 and GD1a) containing N-acetylneuraminic acid and N-glycolylneuraminic acid were obtained from plasma.     

Structural characterization of the disialogangliosides of murine peritoneal macrophages

Sialoglycosphingolipids (gangliosides) have been increasinglyimplicated as regulators of membrane signaling events. Macrophageganglioside patterns dramatically increase in complexity whenmurine peritoneal macrophages are stimulated in vivo with theappearance of the sialidase-sensitive monosialoganglioside GMlb(cisGMl) as a major component Gangliosides from stimulated murineperitoneal macrophages were separated into monosialo and polysialofractions and the polysialo fraction structurally characterizedby enzymatic, chemical, and mass spectra methods. All detectablecomponents of the polysialo fraction were determined to be disialogangliosides.Treatment of the polysialo fraction with Clostridium perfringenssiali-dase produced mostly the sialidase-resistant monosialoganglioside,GMIa, and a minor amount of asiaJoGMI. Perio-date oxidationand mass spectrometry analyses demonstrated the lack of tandemdisialo moieties which indicated the absence of GD1b or GD1c(GDI) entities. The combined data showed the major disialogangliosidesconsisted of GDla entities comprising IV3-NeuAc,II3NeuAc-GgOse4Cer,IV3-NeuGc,II3NeuAc-GgOse4Cer, IV3NeuAc,II3NeuGc-GgOse4Cer, andIV3-NeuGc,II3NeuGc-GgOse4Cer. Minor components consisted ofGDl entities, IV3NeuAc, III6NeuAcGgOse4Cer, IV3NeuGc, III6NeuGcGgOse4Cer,and also positional iso-mer(s) of GDl(NeuAc, NeuGc). These isomericcomponents were identified by collision analysis and tandemmass spectrometry. Consistent with previous analyses, the cer-amideportion of all polysialo (disialo) gangliosides contained solelyC18 sphingosine with C16 and C24 fatty acid moieties. Theseresults, combined with the previous characterization of macrophagemonosialogangliosides, indicate normal murine macrophage gangliosidebiosynthesis proceeds along the "a" ganglioside pathway, e.g.,GM3GM2GMlaGDl, and the proposed asialogan-glioside or "" pathway,asialoGMlGMlbGDl. The presence of totally sialidase-sensitivegangliosides appears to be characteristic of functional murineperitoneal macrophages while they are reduced in geneticallyimpaired cells. ganglioside GDla ganglioside GDl murine macrophages tandem mass spectrometry collision induced dis-association electrospray ionization     

Alpha-fucosidase-ganglioside interactions. Action of alpha-L-fucosidase from the hepatopancreas of Octopus vulgaris on a fucose-containing ganglioside (Fuc-GM1).

alpha-L-Fucosidase, prepared in highly purified form (Mr 70 000-74 000) from Octopus hepatopancreas, was able to hydrolyse a fucose-containing ganglioside, namely Fuc-GM1 (II3NeuAc,IV2Fuc-GgOse4-Cer). The enzyme showed an irregular kinetic behaviour (v/[S] and v/[E] relationships following sigmoidal curves) when working on micellar Fuc-GM1 (Mr of the micelle 500 000), but obeyed regular hyperbolic kinetics when acting on low-Mr substances. It was observed that, on incubation with micellar Fuc-GM1 under the conditions used for the enzyme assay, Octopus alpha-L-fucosidase produced a ganglioside-enzyme complex that was catalytically inactive. This complex had an Mr exceeding 500 000 and a ganglioside/protein ratio of 4:1 (w/w), which is consistent with a stoichiometric combination of one ganglioside micelle with two enzyme molecules. Inactivation of alpha-L-fucosidase by formation of the corresponding complexes was also obtained with micellar gangliosides GM1 (II3NeuAc-GgOse4-Cer), GD1a (II3NeuAc,IV3NeuAc-GgOse4-Cer) and GT1b [II3(NeuAc)2,IV3-NeuAc-GgOse4-Cer], which are not substrates for the enzyme, indicating that the ganglioside micelles per se act as enzyme inhibitors. However, alpha-L-fucosidase easily forms a Fuc-GM1-alpha-L-fucosidase complex, displaying regular Michaelis-Menten kinetics. Therefore the anomalous behaviour exhibited by alpha-L-fucosidase on micellar Fuc-GM1 is likely due to formation of the complex, which separates the fucosyl linkage from the active site of the complexed enzyme, but makes it available to the enzyme in the free form.     

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.     

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.     

Ganglioside-specific binding protein on rat brain membranes

A derivative of ganglioside GT1b (IV3NeuAc,II3(NeuAc)2-GgOse4) with an active ester in its lipid portion was synthesized and covalently attached to bovine serum albumin (BSA). The conjugate, having four GT1b molecules per albumin molecule [GT1b)4BSA) was radioiodinated and used to probe rat brain membranes for ganglioside binding proteins. A ganglioside-specific, high affinity (KD = 2-4 nM), saturable (Bmax = 13-20 pmol/mg membrane protein) binding site for 125I-(GT1b)4BSA was demonstrated on detergent-solubilized rat brain membranes adsorbed to filters. 125I-(GT1b)4BSA binding was tissue-specific (more than 35-fold greater to brain than to liver membranes) and was nearly eliminated by pretreatment of brain membrane-adsorbed filters with trypsin (1 microgram/ml). Underivatized gangliosides added as mixed detergent-lipid micelles blocked 125I-(GT1b)4BSA binding to brain membranes; structurally related GQ1b, GT1b, and GD1b were the most potent (half-maximal inhibition at 70-110 nM), while half-maximal inhibition by other gangliosides (GD3, GD1a, GM3, GM2, and GM1) required 5-20-fold higher concentrations. Other sphingolipids, neutral glycosphingolipids, and glycoproteins were poor inhibitors, and treatment of (GT1b)4BSA with neuraminidase attenuated its binding. Although most phospholipids were noninhibitory, phosphatidylinositol and phosphatidylglycerol inhibited half-maximally at 400-600 nM. However, inhibition of 125I-(GT1b)4BSA binding by gangliosides was competitive and reversible while that by phosphatidylinositol and phosphatidylglycerol was not. Ganglioside-protein conjugate binding reveals ganglioside-specific brain membrane receptors.     

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.     

Identification of novel gangliosides containing lactosaminyl-GM1 structure from rat spleen

Two novel monosialogangliosides were isolated from rat spleen. The structures of the gangliosides (shown below, where NeuNGc is N-glycolylneuraminic acid) were characterized by compositional analysis, methylation analysis, hydrolyses with exoglycosidases, direct probe fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectrometry. A novel structure common to both gangliosides was N-acetyllactosaminyl-GM1 LacN-GM1; where GM1 is II3NeuAc-GgOse4Cer), and this is the first paper to report the occurrence of a new group of gangliosides. [formula: see text] Furthermore, in a monosialoganglioside fraction of rat spleen, the occurrence of a ganglioside having two lactosamine units (Gal alpha 1-3(Gal beta 1-4GlcNAc beta 1-3)2-GM1(NeuAc) (alpha Gal-(LacN)2-GM1] was suggested. These gangliosides have a unique structure, which includes the ganglio series ganglioside core and the extended modification characteristic of the lacto series.     

Susceptibility of infant mice to F5 (K99)E. coli infection: Differences in glycosyltransferase activities in intestinal mucosa of inbred CBA and DBA/2 strains

EnterotoxigenicEscherichia coli (ETEC) strains expressing F5 (K99) fimbriae cause diarrhoea in the young animal through adhesion to specific sialoglycolipids of the small intestine surface. We studied here an infant mouse diarrhoea model, as CBA infant mice are susceptible to F5-positive ETEC infection, whereas DBA/2 ones are resistant. In an attempt to determine an enzymatic basis for susceptibility and resistance, we investigated the intestine ganglioside pattern in relation to the activity of glycosyltransferases responsible for the globo- and ganglio-series. We observed that the intestine of susceptible CBA infant mice displayed a characteristic sialoglycolipid pattern containing mainly the F5 receptors. The two murine strains differed in the relative activities of galactosyltransferases (GbOse₃Cer and GM1 synthases),N-acetylgalactosylaminyltransferases (GA2 and GM2 synthases) and sialyltransferases (GM3 and GD3 synthases). An elevated GM3-synthase activity was observed in the intestine of susceptible CBA infant mice, at the age of high susceptibility. Hence, we conclude that the marked specificity of mouse type correlated with susceptibility and resistance to F5-positive ETEC infection which could be controlled through the regulation of glycosyltransferase activities.Abbreviations NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - Glc glucose - GalNAc N-acetylgalactosamine - Gal galactose - Car ceramide - LacCer lactosylceramide (Galß-4Glcß1-1Cer) - GA2 asialo-GM2 (GgOse₃Cer) - GA1 asialo-GM1 (GgOse₄Cer) - NeuAc/NeuGc-GMla II³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/NeuGc-GM1a IV³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/NeuGc-GM2 II³ NeuAc/neuGc-GgOse₃Cer - NeuAc/NeuGc-GM3, II³ NeuAc/NeuGc-LacCer; NeuAc/NeuGc-GD1a, IV³ NeuAc/NeuGc, II³ NeuAc/NeuGc-GgOse₄Cer; NeuAc/NeuGc-GD1b II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GD1c IV³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GD2, II³ (NeuAc/NeuGc)₂-GgOse₃Cer; NeuAc/NeuGc-GD3, II³ (NeuAc/NeuGc)₂-Lac Cer; NeuAc/NeuGcGT1a IV³ (NeuAc/NeuGc)₂, II³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/neuGc-GT1b IV³ NeuAc/NeuGc, II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GT1c II³ (NeuAc/NeuGc)₃-GgOse₄Cer; NeuAc/NeuGc-GT2, II³ (NeuAc/NeuGc)₃-GgOse₃Cer - NeuAc/NeuGc-GT3 II³ (NeuAc/NeuGc)₃-Lac Cer - NeuAc/NeuGc-GQ1b IV³ (NeuAc/NeuGc)₂, II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GQ1c IV³ NeuAc/NeuGc, II³ (NeuAc/NeuGc)₃-GgOse₄Cer - NeuAc/NeuGc-GP1c IV³ (NeuAc/NeuGc)₂, II³ (NeuAc/NeuGc)₃-GgOse₄Cer - GD, GT and GQ di-, tri- and tetra-sialoglangliosides. NeuGc-SPG, IV³ NeuGc-nLcOse₄Cer. Glycosyltransferases assayed in this work areN-acetylgalactosaminyltransferases - UDP-GalNAc lactosylceramide 1-4N-acetylgalactosaminyltransferase or GA2 synthase (EC 2.4.1-) and UDP-GalNAc:(N-acetylneuraminyl)-lactosylceramide 1-4N-acetylgalactosaminyltransferase or GM2 synthase (EC 2.4.1.92) - sialyltransferases CMP-N-acetylneuraminate: lactosylceramide 2–3 sialyltransferase (sialyltransferases I and IV) or GM3 synthase (EC 2.4.99.-) and CMP-N-acetylneuraminate:(N-acetylneuraminyl) lactosylceramide 2-8 sialyltransferase (sialyltransferase II) or GD3 synthase (EC 24.99.8) - galactosyltransferases UDP-galactose:N-acetylgalactosaminyl-(N-acetylneuraminyl) lactosylceramide 1-3 galactosyltransferase (galactosyltransferase II) or GM1a synthase (EC 2.4.1.62) and UDP-galactose:lactosylceramide 1-4 galactosyltransferase or GbOse₃Cer synthase (EC 2.4.1-)     

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.     

A trisialoganglioside containing a sialyl alpha 2-6 N-acetylgalactosamine residue is a cholinergic-specific antigen, Chol-1 alpha.

Cholinergic-specific antigens termed the Chol-1 family have been suggested to be of a ganglioside nature by Richardson et al. (J. Neurochem. 38, 1605-1614, 1982). Two molecular species of polysialogangliosides among bovine brain gangliosides were found to react with anti-Chol-1 alpha antiserum. One of them, Chol-1 alpha-a, was isolated and characterized as a trisialoganglioside containing the gangliotetraose backbone in which 1 mol of sialic acid was attached to each of the reducing end galactose, N-acetylgalactosamine and internal galactose residues, respectively. The chemical structure of Chol-1 alpha-a was determined for the first time, being as follows: IV3NeuAc III6NeuAc II3NeuAc-GgOse4 Cer.