Carbohydrate analysis of chicken heart glycolipids

Neutral and acidic glycolipids were extracted from chicken hearts. The neutral and acidic compounds were separated by preparative thin-layer chromatography into eight and two fractions, respectively. Total hydrolysis by mineral acid, permethylation analysis, and sequential cleavage with exoglycosidases showed the presence of glycolipids that belong to the globo- and gala-oligosaccharide series, i.e., the monohexosylceramides Glc-Cer and Gal-Cer, the dihexosylceramides Gal beta 1-4Glc-Cer and Gal alpha 1-4Gal-Cer, the tetrahexosylceramides GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal-Cer (III3GalNAc alpha-Ga3Cer) and four subfractions of the Forssman glycolipid GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. With the notable exception of III3GalNAc alpha 1-Ga3Cer, all glycolipids with terminal GalNAc alpha 1-3GalNAc1 reacted on thin-layer chromatograms with a monoclonal anti-Forssman antibody. The major components of the acidic fraction glycolipids were characterized as the lactose-based gangliosides Glac1 (GM3) and Glac2 (GD3).     

Glycolipid composition of a mutant cell line of mouse FM3A cells, and the effect of exogenous glycolipids on cell growth

Had-1 isolated from mouse mammary tumour FM3A cells as a non-permissive cell line to Newcastle disease virus infection is deficient in NDV receptors, and galactosylation of the complex type sugar chains of the glycoproteins is extensively reduced compared to FM3A cells. It is also deficient in UDP-galactose transport into Golgi vesicles. The major neutral glycolipids in FM3A is Lac-Cer, whereas, in Had-1 cell, Glc-Cer is the major glycolipid and the concentration of neutral glycolipids is one-tenth as low as that in FM3A. GM3, GD3 and sialyl i- and I-type lactosaminylceramide are the gangliosides present in both FM3A and Had-1, although their presence in both cells is only in traces. Had-1 contains relatively high N-glycolyl-neuraminic acid. Among the several glycolipids tested, Lac-Cer, Gg-4-Cer and Glc-Cer showed inhibitory effect on proliferation of Had-1 cells, but did not show any appreciable effect on that of FM3A cells. Lac-Cer had the most potent inhibitory effect and this inhibitory effect was completely reversible. While mice injected with 5 x 10(6) cells of FM3A died in one month, those injected of Had-1 cells at the same dose survived for more than 6 months. Thus glycolipids on the cell surface play an essential role during cell growth both in vivo and in vitro.     

Evidence for sialidase hydrolyzing gangliosides GM2 and GM1 in rat liver plasma membrane

Rat liver plasma membrane removed sialic acid from mixed bovine brain gangliosides more efficiently than from sialyllactose and orosomucoid with an optimal pH of 4.5. When individual gangliosides, each labeled with [14C]sialic acid or [3H]sphingosine, were tested, not only GD1a and GM3 but also GM2 and GM1, both of which had been considered to resist mammalian sialidases, were desialylated. The products of GM2 and GM1 hydrolysis were identified as asialo-GM2 and asialo-GM1, respectively, by thin-layer chromatography.     

The occurrence of glycosphingolipids containing mannose in the sea-water bivalve, Meretrix lusoria (Hamaguri)

Total neutral and acidic glycosphingolipids were prepared from whole tissues of the sea-water bivalve, Meretrix lusoria, and the former preparation was further fractionated into subgroups by silicic acid column chromatography. The fractions obtained as mono-(ceramide monosaccharide, CMS), di-(CDS) and triglycosylceramides (CTS) were characterized by thin-layer chromatography, partial hydrolysis with exoglycosidases, methylation studies, CrO3 oxidation, and GLC analysis of the component sugars, fatty acids and long-chain bases. The following structures are proposed: Gal-Cer and Glc-Cer for CMS, Gal(beta 1----4)Glc-Cer and Man(beta 1----4)Glc-Cer (MlOse2Cer) for CDS, Man(alpha 1----3)Man(beta 1----4)Glc-Cer (MlOse3Cer) and Gal(alpha 1----3)Man(beta 1----4)Glc-Cer (II3 alpha Gal-MlOse2Cer) for CTS. To our knowledge II3 alpha Gal-MlOse2Cer has not previously been reported. The fatty acid composition of CMS, CDS, and CTS consisted almost entirely of saturated C16-C24 acids with large amounts of 2-hydroxypalmitic acid and 2-hydroxystearic acid. The long-chain bases consisted of 4-sphingenine and 4,8-sphingadienine. More complex neutral glycolipids than CTS, as well as an acidic glycolipid, were examined by TLC and GLC of the constituent sugars, and an immunochemical technique.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of N-glycolyneuraminic acid-containing glycosphingolipids from a Marek's disease lymphoma-derived chicken cell line, MSB1, as tumor-associated heterophile Hanganutziu-Deicher antigens

Heterophile, Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid-containing glycosphingolipids (GSLs) were detected as tumor-associated foreign antigens of a Marek's disease lymphoma-derived cell line, MSB1, by enzyme-immunoassay with chicken antibody against N-glycolylneuraminyl-lactosylceramide (anti-NeuGc-LacCer). At least three species of HD antigen-active GSLs were detected by two-dimensional thin-layer chromatography (TLC) combined with enzyme-immunoassay. The reactivities of the GSLs with anti-NeuGc-LacCer, their behaviors on two-dimensional TLC and the results of an endo-beta-galactosidase digestion study indicated that these three GSLs were NeuGc-LacCer (NeuGc alpha 2-2Gal beta 1-4Glc-Cer), NeuGc-nLcOse4Cer (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and NeuGc-nLcOse6Cer (NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer).     

Isolation and characterization of ganglioside GM1b from normal human brain

A sialidase-susceptible monosialoganglioside was isolated from normal human brain by DEAE-Sephadex A-25 and Iatrobeads column chromatography. The yield of this ganglioside was about 6 mg per whole brain. Its structure was elucidated by sugar analysis, sialidase digestion, permethylation, and proton NMR studies. This ganglioside had carbohydrate, fatty acid, and long-chain base compositions identical to those of brain GM1a. However, the sialosyl residue was found to be linked (alpha 2-3) to the terminal galactosyl residue of the asialo-GM1a backbone. The complete structure of this ganglioside was therefore identified as GM1b or IV3NeuAcGgOse4Cer.     

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.     

The Degradative Pathway of Gangliosides GM1 and GM2 in Neuro2a Cells by Sialidase

Abstract: Gangliosides GM1 [³H-labeled at the sphingosine (Sph) moiety] and GM2 [³H-labeled at the Sph or N -acetylgalactosamine (GalNAc) moiety] were administered to cultured Neuro2a cells for varying pulse (1–4 h) and chase (up to 4 h) periods, and their metabolic processing was followed. The main and earliest formed ³H-metabolites of [ Sph -³H]GM1 were GM2, asialo-GM1, asialo-GM2, and lactose-ceramide, and those of [ Sph -³H]GM2 were asialo-GM2 and lactose-ceramide. The asialo-GM1 and asialo-GM2 formed were isolated and chemically characterized. [³H]Asialo-GM2 was produced in identical amounts after treatment with equimolar [ Sph -³H]GM2 and [ GalNAc -³H]GM2. At low temperature or in the presence of chloroquine, the formation of all ³H-metabolites, including asialo-GM2 and asialo-GM1, was undetectable, indicating that ganglioside metabolic processing was an endocytosis- and lysosome-dependent process. These results demonstrate that in Neuro2a cells exogenous GM1 (and GM2) is mainly degraded through the pathway GM1 → GM2 → asialo-GM2 →→ Sph, with a minor fraction of GM1 undergoing degradation with the sequence GM1 → asialo-GM1 → asialo-GM2 →→ Sph. These findings are consistent with the hypothesis that Neuro2a cells contain a sialidase (likely of lysosomal nature) affecting ganglioside GM1 and GM2. The sialidase-mediated degradative pathway of GM1 and GM2 in Neuro2a cells might be related to the tumoral nature of these cells.     

Biosynthesis of Lewis fucolipid antigens in human colorectal carcinoma cells. Partial characterization of LcOse4Cer and H-1 fucolipid fucosyl transferase acceptor activities

Purified glycolipids were tested for their ability to serve as acceptors of [14C]fucose from GDP-[14C]fucose as catalyzed by cell-free extracts and purified membrane fractions of human colorectal carcinoma cells, SW1116, cultured in serum-free medium. Purified lactotetraosyl ceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or LcOse4Cer) and H-1 glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or IV2 Fuc alpha LcOse4Cer) stimulated incorporation of radioactivity into lipid-soluble glycolipid at a rate greater than ten times that of Lea glycolipid [Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4 Fuc alpha LcOse4Cer]. The enzymatic activities in crude and purified membrane fractions were optimized for substrate concentrations (glycolipid and GDP-fucose), detergent requirement (taurocholate), pH, time and protein. The radioactive product of H-1 fucosylation migrated as discrete and distinct bands on high-performance thin-layer chromatograms (HPTLC). Evidence for their identity with Leb fucolipid described previously [Fuc alpha 1----2Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4IV2 (Fuc alpha) LcOse4Cer] is presented. The radioactive product of LcOse4Cer fucosylation was mainly Lea fucolipid as determined by co-migration with authentic Lea fucolipid in three HPTLC systems as native and acetylated derivatives. Our results also indicated a low level of H-1 and Leb glycolipid synthesis from LcOse4Cer. On the basis of the optima, linearity for time, and enzyme-limiting conditions, we obtained a 12-19-fold purification of the LcOse4Cer and H-1 fucosyl transferase acceptor activities in three peaks of a sucrose gradient. The peak with the highest specific activity (peak 3) was highest in density and in Na+, K+, ATPase specific activity, although NADH-cytochrome-c reductase and UDP-GalNac transferase were also present in peak 3. The apparent Km values of LcOse4Cer acceptor activity and H-1 acceptor activity in peak 3 were significantly different (p less than 0.01) by statistical tests, 2.4 microM and 0.5 microM, respectively. These apparent Km values were much lower (10(3) X) and the pH optima were lower (4.8-5.3), than the corresponding properties reported for the alpha 1----3/alpha 1----4 fucosyl transferase purified from human milk. Our results suggest a role for the non-glycosidic moieties of the acceptors and/or the tissue-specific or primitive expression of these fucosyl transferase activities.     

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

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

Evidence for the presence of two separate protein activators for the enzymic hydrolysis of GM1 and GM2 gangliosides.

Two different protein activators were isolated simultaneously from human liver for the enzymic hydrolysis of GM1 (Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-galactosidase and GM2 (GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-hexosaminidase A. The hydrolysis of GM1 is stimulated only by the GM1-specific activator which has very little effect on the hydrolysis of GM2. The same is also true for the hydrolysis of GM2. The antiserum raised against GM1 activator did not cross-react with GM2 activator and vice versa. These results suggest the presence of two different activators for the separate hydrolysis of GM1 and GM2. In connection with the enzymic hydrolysis of GM1 and GM2, we found that the hydrolysis of GM2 by human hepatic beta-N-acetylhexosaminidase A was severely inhibited by a buffer of high ionic strength, whereas no such inhibition was observed in the hydrolysis of GM1 by beta-galactosidase.     

Biosynthesis of glycosphingolipids in cultured mouse neuroblastoma cells. Precursor-product relationships among sialoglycosphingolipids.

The reaction sequence for the biosynthesis of gangliosides by mouse neuroblastoma cells has been investigated by studying the pattern of incorporation of labeled precursors into sialoglycosphingolipids. Cultured NB41A cells incorporated N-[3H]acetylmannosamine into the sialic acid moiety of GM3 in less than 10 min. Labeled GM2 was not detected in cells incubated for less than 30 min, while measurable radioactivity did not appear in GM1 until after 60 to 90 min. Analogous experiments were carried out using [14C]galactose. No significant amount of labeled hexose was incorporated into asialo-GM2 during 60 min of culture. These studies are in accord with results of previous studies on glycosyltransferases of NB41A cells (Kemp, S. F., and Stoolmiller, A. C. (1976), J. Neurochem. 26, 723-732), and further support the concept that the pathway of synthesis of gangliosides proceeds via GM3 leads to GM2 leads to GM1.     

Favorable and unfavorable lateral interactions of ceramide, neutral glycosphingolipids and gangliosides in mixed monolayers

Interactions among four natural neutral sphingolipids (ceramide, glucosyl-ceramide, lactosyl-ceramide and asialo-GM1) and six gangliosides (GM3, GM2, GM1, GD3, GD1a and GT1b) were studied in binary Langmuir monolayers at the air-buffer interface in terms of their molecular packing, compressibility, dipole potential and mixing behavior. The changes of surface organization can be grouped into three sets: (a) binary films of neutral GSLs, and of the latter with ceramide, exhibit thermodynamically unfavorable mixing with mean molecular area expansions and dipole moment hyperpolarization; (b) mixed monolayers of ceramide, or of GlcCer, and gangliosides occur with thermodynamically favorable interactions leading to mean molecular area condensation and depolarisation; (c) binary mixtures of LacCer or Gg4Cer with gangliosides, and all ganglioside species among them, revealed molecular immiscibility characterized by additive mean molecular area and dipole potential, with composition-independent constant collapse pressure. These results disclose basic tendencies of GSLs to molecularly mix or demix, leading to their surface segregation, which may underlay vectorial separation of their specific biosynthetic pathways.     

Pseudomonas aeruginosa and Pseudomonas cepacia isolated from cystic fibrosis patients bind specifically to gangliotetraosylceramide (asialo GM1) and gangliotriaosylceramide (asialo GM2)

Pseudomonas aeruginosa infection in the lungs is a leading cause of death of patients with cystic fibrosis, yet a specific receptor that mediates adhesion of the bacteria to host tissue has not been identified. To examine the possible role of carbohydrates for bacterial adhesion, two species of Pseudomonas isolated from patients with cystic fibrosis were studied for binding to glycolipids. P. aeruginosa and P. cepacia labeled with 125I were layered on thin-layer chromatograms of separated glycolipids and bound bacteria were detected by autoradiography. Both isolates bound specifically to asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) and asialo GM2 (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer) but not to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), globoside (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer), paragloboside (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), or several other glycolipids that were tested. Asialo GM1 and asialo GM2 bound the bacteria equally well, exhibiting similar binding curves in solid-phase binding assays with a detection limit of 200 ng of either glycolipid. Both isolates also did not bind to GM1, GM2, or GDla suggesting that substitution of the glycolipids with sialosyl residues prevents binding. As the Pseudomonas do not bind to lactosylceramide, the beta-N-acetylgalactosamine residue, positioned internally in asialo GM1 and terminally in asialo GM2, is probably required for binding. beta-N-Acetylgalactosamine itself, however, is not sufficient as the bacteria do not bind to globoside or to the Forssman glycolipid. These data suggest that P. aeruginosa and P. cepacia recognize at least terminal or internal GalNAc beta 1-4Gal sequences in glycolipids which may be receptors for these pathogenic bacteria.     

Purification, characterization, and cDNA cloning of alpha-N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera

We report here the purification, characterization, and cDNA cloning of a novel N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera. The purified lectin showed 19-kDa, 41-kDa, and 60-kDa protein bands on SDS-PAGE, possibly corresponding to a monomer, homodimer, and homotrimer. Interestingly, on 4-20% native PAGE the lectin showed at least nine protein bands, among which oligomers containing six to nine subunits had potent hemagglutination activity for sheep erythrocytes. The hemagglutination activity of the lectin was specifically inhibited by N-acetylgalactosamine, Tn antigen, and blood group A trisaccharide, but not by N-acetylglucosamine, galactose, galactosamine, or blood group B trisaccharide. The specificity of the lectin was further examined using various glycosphingolipids and biotin-labeled lectin. The lectin was found to bind to Gb5Cer, but not Gb4Cer, Gb3Cer, GM1a, GM2, or asialo-GM2, indicating that the lectin specifically binds to the terminal alpha-GalNAc at the nonreducing end. The hemagglutination activity of the lectin was completely abolished by chelation with EDTA or EGTA and completely restored by the addition of CaCl(2). cDNA cloning of the lectin showed that the protein is composed of 168 amino acids, including a signal sequence of 18 residues, and possesses the typical C-type lectin motif. These findings indicate that the protein is a C-type lectin. The recombinant lectin, produced in a soluble form by Escherichia coli, showed binding activity for asialomucin in the presence of Ca(2+) but no hemagglutination.     

Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice

The apolipoprotein E gene knockout (apoE-/-) mouse develops atherosclerosis that shares many features of human atherosclerosis. Increased levels of glycosphingolipid (GSL) have been reported in human atherosclerotic lesions; however, GSL levels have not been studied in the apoE-/- mouse. Here we used HPLC methods to analyze serum and aortic GSL levels in apoE-/- and C57BL/6J control mice. The concentrations of glucosyl ceramide (GlcCer), lactosyl ceramide (LacCer), GalNAcbeta1-4Galbeta1-4Glc-Cer (GA2), and ceramide trihexoside (CTH) were increased by approximately 7-fold in the apoE-/- mouse serum compared with controls. The major serum ganglioside, N-glycolyl GalNAcbeta1-4[NeuNAcalpha2-3]Galbeta1-4Glc-Cer (N-glycolyl GM2), was increased in concentration by approximately 3-fold. A redistribution of GSLs from HDL to VLDL populations was also observed in the apoE-/- mice. These changes were accompanied by an increase in the levels of GSLs in the aortic sinus and arch of the apoE-/- mice. The spectrum of gangliosides present in the aortic tissues was more complex than that found in the lipoproteins, with the latter represented almost entirely by N-glycolyl GM2 and the former comprised of NeuNAcalpha2-3Galbeta1-4Glc-Cer (GM3), GM2, N-glycolyl GM2, GM1, GD3, and GD1a. In conclusion, neutral GSL and ganglioside levels were increased in the serum and aortae of apoE-/- mice compared with controls, and this was associated with a preferential redistribution of GSL to the proatherogenic lipoprotein populations. The apoE-/- mouse therefore represents a useful model to study the potential role of GSL metabolism in atherogenesis.     

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.     

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.     

Occurrence of ceramide-glycanase in the earthworm, Lumbricus terrestris

We have detected the presence of ceramide-glycanase in the earthworm, Lumbricus terrestris. We have also devised a simple method for the preparation of this enzyme from the earthworm. This enzyme cleaved the linkage between the ceramide and the glycan chain in LacCer, GbOse3Cer, GbOse4Cer, GbOse5Cer, GM3, GM2, GM1 and GD1a. By using tritium-labeled GM2 as substrate, the optimum pH of this enzyme was found to be between pH 4 and 4.5. In the earthworm, the ceramide-glycanase was mainly found in the muscle. The intestine was found to contain a very low level of this enzyme. Because of their easy availability, earthworms should become a convenient source for the preparation of ceramide-glycanase.     

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.