Monoclonal antibody A2B5, which detects cell surface antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer) and to its 9-O-acetylated derivative

The monoclonal antibody A2B5 recognizes antigens at the surface of neuronal and glial cells but also at the surface of thymus epithelia and pancreatic islet cells. Although these antigens have been characterized as polysialogangliosides, A2B5 also reacts with other unidentified gangliosides. In order to characterize further the epitope of A2B5, two new ganglioside antigens isolated from chicken brain are identified in this study. One is the ganglioside NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide (GT3) and the other is a 9-O-acetylated derivative of GT3). This derivative was purified from 10-day embryonic chicken brain. Acetyl groups substituted on sialic acid were removed either by alkali treatment or by incubation with influenza virus C, which contains receptor-destroying enzyme (a neuraminidate 9-O-acetyl esterase). The product of alkali treatment or viral action was detected by the antibody 18B8 which is specific for GT3. The deacetylated product still reacts with A2B5. These data and the results of mild oxidation of the antigen with sodium periodate suggest that the epitope recognized by antibody A2B5 contains the trisialyl structure found in GT3 but does not include the polyalcohol chain of the terminal sialic acid which can be oxidized by periodate or acetylated without modifying the affinity for the antibody. The epitope recognized by A2B5 is different from the epitope recognized by the antibody 18B8 in that 18B8 requires the three sialic acids with an intact and unsubstituted polyalcohol chain. Antibody 18B8 does not bind to 9-O-acetylated GT3 or GT3 oxidized by sodium periodate.     

Developmental changes of C-series polysialogangliosides in chick brains revealed by mouse monoclonal antibodies M6704 and M7103 with different epitope specificities

We have established many mouse monoclonal antibodies detecting developmentally regulated antigens in chicken embryonic neural tissues of the otic vesicles and neural tube by immunizing mice with the membrane fraction of the neural tube and somite prepared from 3-d chick embryos. Among them, three monoclonal antibodies (MAbs) M6703, M6704, and M7103 were shown to react with the gangliosides isolated from chicken embryonic brains. The precise specificity of the antibodies was determined mainly by enzyme-immunostaining on thin layer plates. MAbs M6703 and M6704 bound to C-series polysialogangliosides including GT3, GT2, GT1c, GQ1c, and GP1c isolated from cod fish brains, but never to A-series, B-series, or X-series gangliosides. On the other hand, MAb M7103 antibody has a rather narrow specificity, reacting with GT1c, GQ1c, and GP1c, but not with ganglioside GT3. This indicates that the epitopes defined by these two MAbs are not with ganglioside GT3. This indicates that the epitopes defined by these two MAbs are different from each other. MAbs M6703 and M6704 recognize a trisialosyl residue, NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3, while M7103 requires both a trisialosyl residue and the gangliotetraosy backbone structure for binding. As compared to similar MAbs which have been reported in the literature. M6703 and M6704 are unique in that they react equally with all of the C-series gangliosides and belong to the IgG3 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibody Leo Mel 3, which inhibits killing of human melanoma cells by anomalous killer cells, binds to a sugar sequence in GD2 (II3(NeuAc)2-GgOse3Cer) and several other gangliosides

Human anomalous killer (AK) cells lyse freshly isolated human melanoma cells which are insensitive to human natural killer cell-mediated lysis. Monoclonal antibody Leo Mel 3, an IgM (k), produced by a hybridoma obtained from a mouse immunized with human melanoma cells, binds to melanoma cells and inhibits their conjugate formation with AK cells as well as their AK cell-mediated lysis. Other IgM antibodies from the same fusion that bind melanoma cells do not inhibit (Werkmeister, J. A., Triglia, T., Andrews, P., and Burns, G. F. (1985) J. Immunol. 135, 689-695). Leo Mel 3 binds several different gangliosides from melanoma cells, as determined by immunostaining thin layer chromatograms. Binding is abolished by treatment of the gangliosides with neuraminidase. In solid-phase radioimmunoassay, Leo Mel 3 binds strongly to ganglioside GD2 and less strongly to gangliosides GT3, GD3, and GQ1b. It does not bind to other gangliosides including GM1, GM2, GM3, GD1a, GD1b, and GT1b. Thus, the epitope recognized by antibody Leo Mel 3 is found in the sugar sequence of ganglioside GD2, GalNAc beta 1-4[NeuAc alpha 2-8NeuAc alpha 2-3]Gal beta 1-4Glc beta 1 .... This sequence may contain a target in melanoma cells recognized by AK cells.     

Gangliosides of rat eye lens: a severe reduction in the content of C-series gangliosides following streptozotocin treatment

Gangliosides of eye lenses from normal and experimentally induced diabetic rats were investigated by methods including glycolipid-overlay techniques. Adult rat eye lens showed a complex ganglioside pattern that consisted of six major ganglioside components. These gangliosides were identified as GM3, GD3, GD1a, GD1b, GT1b, and GQ1b based upon their reactivity to anti-GM1 antibody after in situ sialidase treatment and mobility on thin-layer chromatography (TLC). Gangliosides in eye lens were further characterized by TLC-immunostaining with A2B5, a specific monoclonal antibody directed toward c-series gangliosides. Eye lens contained GT3 as the main c-series ganglioside component. Unexpectedly, the relative concentration of GT3 in total gangliosides of eye lens was highest among neural and extra-neural tissues examined. Administration of streptozotocin to rats caused a severe reduction in the GT3 content in eye lenses as early as day 3 without apparent changes in the composition of major gangliosides. Alloxan failed to produce such an effect despite producing similar hyperglycemic conditions. These results suggest that rat eye lens probably contains a streptozotocin-susceptible cell type(s), which is highly enriched with c-series gangliosides.     

Isolated Bovine Spinal Motoneurons Have Specific Ganglioside Antigens Recognized by Sera from Patients with Motor Neuron Disease and Motor Neuropathy

The gangliosides GM1 and GD1b have recently been reported to be potential target antigens in human motor neuron disease (MND) or motor neuropathy. The mechanism for selective motoneuron and motor nerve impairment by the antibodies directed against these gangliosides, however, is not fully understood. We recently investigated the ganglioside composition of isolated bovine spinal motoneurons and found that the ganglioside pattern of the isolated motoneurons was extremely complex. GM1, GD1a, GD1b, and GT1b, which are major ganglioside components of CNS tissues, were only minor species in motoneurons. Among the various ganglioside species in motoneurons, several were immunoreactive to sera from patients with MND and motor neuropathy. One of these gangliosides was purified from bovine spinal cord and characterized as N-glycolylneuraminic acid-containing GM1 [GM1(NeuGc)] by compositional analysis, fast atom bombardment mass spectra, and the use of specific antibodies. Among seven sera with anti-GM1 antibody activities, five sera reacted with GM1(NeuGc) and two did not. Two other gangliosides, which were recognized by another patient's serum, appeared to be specific for motoneurons. We conclude that motoneurons contained, in addition to the known ganglioside antigens GM1 and GD1b, other specific ganglioside antigens that could be recognized by sera from patients with MND and motor neuropathy.     

Antibodies against ganglioside GT3 in the sera of patients with type I diabetes mellitus

Clinical and experimental data support the concept that type I diabetes mellitus results from autoimmune destruction of pancreatic beta cells. Although both proteins and glycolipids are targets of anti-islet cell antibodies, the Ag have not been purified or characterized. Previously, we observed that rat insulinoma (RIN) cell lines varied in their reactivity with both human antibodies and murine mAb A2B5, which binds to polysialo gangliosides. To determine the chemical basis of the varied immunoreactivity, we analyzed the glycosphingolipids of 5 RIN lines. Glycolipids bound by two mAb and by antibodies in the sera of type I diabetics were identified. The more immunoreactive RIN lines contained a much higher content of gangliosides and a higher proportion of complex gangliosides. The major gangliosides were GM3, GD3, and GT3. By high performance TLC immunostaining, we demonstrated that A2B5 and R2D6, an anti-beta cell murine mAb, bound most strongly to ganglioside GT3. The binding of human sera to gangliosides was analyzed by an ELISA assay. Although both normal and diabetic sera contained antibodies to various glycolipids, binding to GT3 was significantly elevated in 31 new-onset type I diabetics (p less than 0.001). The presence of the GT3 trisialosyl epitope on human islet cells was shown by immunofluorescent staining by both R2D6 and A2B5. These findings support previous suggestions that gangliosides play an important role in the immunopathology of type I diabetes, and identify for the first time a specific ganglioside Ag that is the target for autoantibodies in a subset of diabetic patients.     

Proton NMR and fast-atom bombardment mass spectrometry analysis of the melanoma-associated ganglioside 9-O-acetyl-GD3

A glycolipid antigen, detected by a monoclonal antibody (ME 311) obtained by immunizing mice with a human metastatic melanoma cell line (WM 46), was isolated and structurally characterized. Using immunostaining on thin-layer chromatograms for monitoring, 1.0 mg of a pure alkali-labile disialoganglioside was obtained from 23 g of packed melanoma cells (WM 164). Fractionation of the lipid extract was done on DEAE-Sepharose columns into total disialogangliosides which were repeatedly separated by high-pressure liquid chromatography. On mild alkaline treatment, the ganglioside was converted to a slower migrating species identical with a ganglioside GD3 isolated from the same source (Neu5Ac alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-cer-amide) and specifically detected by monoclonal antibody R24. Comparison of the two gangliosides by fast-atom bombardment mass spectrometry (revealing an acetyl group on the terminal sialic acid on the alkali-labile species) and by 1H NMR (indicating the position of the acetyl group) suggested the following structure: Neu5,9Ac2 alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-ceramide. This is identical with a ganglioside proposed earlier to exist in melanoma cells (Cheresh, D. A., Varki, A. P., Varki, N. M., Stallcup, W. B., Levine, J., and Reisfeld, R. A. (1984) J. Biol. Chem. 259, 7453-7459). Immunostaining with ME 311 antibody of cell extracts on thin-layer chromatography chromatograms revealed only this ganglioside in the melanoma cells, while normal human brain was negative. However, in one of the total ganglioside extracts tested for presence of binding with antibody ME 311, three gangliosides were found to bind. No evidence was obtained for the presence of the antigenic epitope in mucins or glycoproteins of the melanoma cells.     

Characterization of nuclear gangliosides in rat brain: concentration, composition, and developmental changes

Nuclear gangliosides were characterized using two distinct fractions of large (N1) and small (N2) nuclear populations from rat brain. The ganglioside concentration of N1 nuclei from adult rat brain was 0.92 microg sialic acid/mg protein, which was about 3.8 times higher than that of N2 nuclei. N1 and N2 nuclear gangliosides showed similar compositional profiles; they contained major gangliosides of GM1, GD1a, GD1b, and GT1b, with GM3 in lesser amounts. c-Series gangliosides such as GT3, GQ1c, and GP1c were also detected in both nuclear preparations. Nuclear localization of gangliosides was confirmed by immunofluorescence with anti-GM1 antibody, cholera toxin B subunit, and c-series ganglioside-specific monoclonal antibody A2B5. Developmental changes of nuclear gangliosides were examined using rats of different ages ranging from embryonic day 14 (E14) to postnatal 7 weeks. The concentration of N1 nuclear gangliosides changed only slightly during development and did not correlate with that of whole-brain gangliosides. The developmental pattern of ganglioside composition of N1 nuclei was also distinguished from that of microsomal membranes; the ganglioside changes in N1 nuclei included reduced expression of di- and polysialogangliosides at E16 and higher proportions of GM3 at early and late stages of the period. These findings suggest that gangliosides in nuclear membranes are developmentally regulated in a distinct manner in brain cells.     

Inhibitory effects of gangliosides on immune reactions of antibodies to neutral glycolipids in liposomes

Specific immune damage to liposomes containing Forssman or globoside glycolipid was inhibited when the liposomes also contained ganglioside. The activity of a human monoclonal Waldenstr?m macroglobulin antibody to Forssman glycolipid was inhibited by each of three gangliosides tested, GM3, GD1a and GD1b. Inhibition of the monoclonal antibody was dependent on the amount of ganglioside in the liposomes, and was diminished by reducing the relative amount of ganglioside. Inhibition also correlated positively with the number of ganglioside sialic acid groups, with inhibition by GT1b greater than GD1a greater than GM3. Naturally occurring human antibodies to globoside glycolipid were detected in 18% (9 out of 50) of normal human sera tested. Immune damage to liposomes induced by each of the three highest-reacting human anti-globoside sera was blocked by liposomal GM3. We conclude that gangliosides can strongly influence immune damage to membranes induced by antibody interactions with adjacent neutral glycolipids.     

Tissue-specific expression of c-series gangliosides in the extraneural system

c-Series gangliosides in extraneural tissues from young and adult rats were examined using thin-layer chromatographic (TLC) immunostaining with a specific monoclonal antibody A2B5. The composition of c-series gangliosides significantly differed among tissues. In adult rats, while liver tissue contained GT1c, GQ1c, and GP1c, renal tissue had GT3 as the major c-series ganglioside with GT2 in a lesser amount. Pancreatic tissue expressed c-series gangliosides that consisted of GT3, GT2, GQ1c, and GP1c. In other tissues including adrenal, thyroid, and eye lens, GT3 constituted the main c-series ganglioside species. While total ganglioside contents of extraneural tissues were much lower than that of brain tissue, the proportions of c-series gangliosides to total gangliosides were higher in many extraneural tissues. Interestingly, eye lens had the highest GT3 content among rat tissues examined. The compositions and concentrations of c-series gangliosides in liver and kidney significantly differed between 5-day-old and 7-week-old rats, suggesting the development-dependent expression of c-series gangliosides in these tissues. These results suggest that the expression of c-series gangliosides in extraneural tissues is regulated in a tissue-specific manner.     

Binding specificities of heat-labile enterotoxins isolated from porcine and human enterotoxigenic Escherichia coli for different gangliosides

The binding specificities of heat-labile enterotoxins (LTp and LTh) isolated from porcine and human enterotoxigenic Escherichia coli on human erythrocytes were studied by competitive binding assays using different gangliosides as inhibitors. The binding of 125I-labeled LTp to neuraminidase-treated human type A erythrocytes was most effectively inhibited by ganglioside GM1. Ganglioside GM1 was 11 and 105 times more potent than gangliosides GD1b and GM2, respectively. Gangliosides GD1a, GT1b, and GM3 were much less potent. Similar results were also obtained in competitive binding assays with the 125I-labeled B subunit of LTh and neuraminidase-treated human type B erythrocytes, and in those with 3H-labeled ganglioside GM1 and LTp-coupled Sepharose 4B. The binding of 3H-labeled ganglioside GM1 to LTp was not effectively inhibited by galactose-beta(1----3)N-acetyl-D-galactosamine at the highest concentration used. These findings suggest that the combining sites of LTp and LTh may be specific for at least the galactose-N-acetyl-D-galactosamine-galactose (N-acetyl-neuraminic acid) portion of ganglioside GM1.     

A new ganglioside of the lactotetraose series, GalNAc-3'-isoLM1, detected in human meconium

A monoclonal antibody produced by immunization with cells of the human glioma cell line D-54 MG reacted with ganglioside GM2. The binding epitope of the antibody was found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal. Immunological detection of glycolipid antigens on thin-layer plates with this monoclonal antibody, DMAb-1, revealed the presence of a new ganglioside. This ganglioside, co-migrating with NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer(6'-LM1) and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GalNAc beta 1-4Gla beta 1-4Glc beta 1-1Cer (GalNAc-isoGM1) at chromatographic separation was isolated from human meconium. Its structure was determined by permethylation and fast atom bombardment-mass spectometry analyses. The new ganglioside was found to be a combination of the lacto and ganglio series gangliosides, and the structure found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GlcNAc alpha 1-3Gal beta 1-4Glc beta 1-1Cer(GalNAc-3'-isoLM1).     

Specific ganglioside changes in extraneural tissues of adult rats with hypothyroidism

Adults rats with hypothyroidism were prepared by administration of 6-propyl-2-thiouracil (PTU) or methimazole, and the tissues were examined for their gangliosides through methods including glycolipid-overlay techniques. Normal thyroid tissue contained GM3, GD3, and GD1a as the major gangliosides, with GM1, GD1b, GT1b, and GQ1b in lesser amounts. The goitrous tissue of PTU-induced hypothyroid rats had higher concentrations of GM1 and GD1a with a concomitant decrease of GM3. The amount of GT3 in thyroid tissue was increased in hypothyroid animals. While normal liver tissue had a complex ganglioside pattern with a- and b-series gangliosides, the PTU-induced hypothyroid tissue showed a simpler ganglioside profile that consisted mainly of a-series gangliosides with almost undetectable amounts of b-series gangliosides. The expression of c-series gangliosides was suppressed in the hypothyroid liver tissue. Heart tissue had higher contents of GM3 and GT3 than control. No apparent change was observed in the compositions of major and c-series gangliosides in other extraneural tissues (i.e., kidney, lung, spleen, thymus, pancreas, testis, skeletal muscle, and eye lenses), and neural tissues (i.e., cerebrum and cerebellum) from PTU-induced hypothyroid rats. The ganglioside changes of thyroid, liver, and heart tissues were reproduced in corresponding tissues of methimazole-induced hypothyroid rats. These results suggest that hypothyroid conditions affect the biosynthesis and expression of gangliosides in specific tissue and cell types.     

Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM1, GM3, GD1A and GT1B

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.     

Regulation of Ganglioside Composition and Synthesis Is Different in Developing Chick Retinal Pigment Epithelium and Neural Retina

Abstract: We examined the immunocytochemical expression of GM3 and QD3 in 3-day-old chick embryo retinal pigment epithelium (RPE) and neural retina (NR). We also compared the composition of gangliosides and the activities of key ganglioside glycosyltransferases of the RPE and NR of 8-, 12-, and 15-day old embryos. The immunocytochemical studies in 3-day-old embryos showed heavy expression of GM3 and GD3 at the inner and outer layers of the optic vesicle that are the precursors of the RPE and NR, respectively. The compositional and enzymatic studies showed pronounced differences between RPE and NR of 8-day and older embryos. HPTLC showed that at 8 days the major species were GM3 and GD3 in RPE and GD3 and GT3 in NR. As development proceeded, GD3 decreased in both tissues, GM3 became the major ganglioside in RPE, and ganglio-series gangliosides (mainly GD1a) became the major species in NR. At 15 days the major species were GD1 a in NR and GM3 in RPE. Enzyme determinations showed that whereas in RPE from 12-day-old embryos GM2 synthase was under the limit of detection and GD3 synthase activity was about sixfold lower than GM3 synthase, in NR the activities of GM3 and GD3 synthases were similar and both six-to ninefold lower than GM2 synthase. These results evidence a markedly different modulation of the ganglioside glycosylating system in cells of a common origin that through distinct differentiation pathways originate two closely related tissues of the optic system. In addition, they reinforce the relevance of the relative activities of key transferases in determining the pattern of gangliosides in different cell types.     

Gangliosides in normal human serum. Concentration, pattern and transport by lipoproteins

The total content and pattern of gangliosides were determined in the unfractionated sera of 11 healthy human adults and in isolated lipoproteins. The total content of lipid-bound sialic acid was 10.5 +/- 3.2 nmol/ml serum. The ganglioside profile consisted of more than ten different components. The major ganglioside was GM3, followed by GD3, GD1a, GM2, GT1b, MG-3 (sialosyllactoneotetraosylceramide), GD1b and GQ1b. Traces of four additional gangliosides could not be quantified reliably. Ganglioside patterns did not vary in sera taken from healthy adults of different age and sex. Approximately 98% of human serum gangliosides were transported by serum lipoproteins, predominantly by LDL (66%), followed by HDL (25%) and VLDL (7%). The quantitative distribution of individual gangliosides in VLDL and LDL was almost the same as that in the unfractionated serum; some differences existed with the ganglioside profile in HDL.     

Effects of Brefeldin A on Synthesis and Intracellular Transport of Ganglioside GT3 by Chick Embryo Retina Cells

Abstract: Ganglioside GT3 is the precursor of c-series gangliosides. It is synthesized by sialylation of GD3 and is expressed in nervous tissue of birds and mammals at early stages of development. In this study we examined the sub-Golgi location of GT3 synthesis and the mechanism of its transport from the site of synthesis to the plasma membrane in chicken embryo retina cells in culture. Neural retina cells from 10-day-old chick embryo were cultured with [³H]galactose in the absence (control cells) or in the presence of 1 µg/ml brefeldin A (BFA). At the end of the labeling period, the fraction of labeled gangliosides transported to the plasma membrane was determined. For this, cells were treated with C . perfringens neuraminidase in conditions to desialylate only those gangliosides that were transported to the plasma membrane and consequently accessible to the enzyme. After neuraminidase treatment of cells, gangliosides were isolated, purified, and the pattern of radioactivity analyzed by HPTLC-fluorography. It was found that BFA blocked the synthesis of complex gangliosides without affecting the synthesis of GM3, GD3, and GT3. Furthermore, in BFA-treated cells, GM3, GD3, and GT3 were protected from the action of added neuraminidase, indicating an intracellular localization and, hence, an inhibition of their transport to the plasma membrane. The results indicate that synthesis of the first intermediates of a-, b-, and c- series gangliosides occurs in a proximal Golgi compartment and that the proximal Golgi-synthesized gangliosides (GM3, GD3, and GT3) use a transport mechanism that is dependent on ADP ribosylation factor and coatomer proteins.     

Human cancer-associated gangliosides defined by a monoclonal antibody (IB9) directed to sialosyl alpha 2 leads to 6 galactosyl residue: a preliminary note

A new monoclonal antibody (IB9) was prepared by hybridoma technique directed specifically to sialosyl alpha 2 leads to 6 galactosyl residue. With this reagent, accumulation of two major gangliosides in human colonic and liver adenocarcinoma has been detected, and these gangliosides were isolated and characterized as structures A and B (below). Another ganglioside with a ceramide nonasaccharide structure, which reacted to anti-X-hapten antibody after desialylation, was also isolated and partially characterized. (formula; see text) These gangliosides were absent or present in very small quantity in normal tissue and may represent human cancer-associated markers.     

Biosynthesis and Expression of Gangliosides During Differentiation of Chick Embryo Retina Cells In Vitro

Cells from neural retina from 7-day chick embryos were cultured on polylysine-coated dishes up to 7 days. The small, round-shaped cells at seeding differentiated progressively, and after 4 days in vitro the majority had enlarged bodies and abundant processes. The content of protein and DNA was essentially unchanged during the entire period of culture. The incorporation of radioactivity from [3H]glucosamine into gangliosides declined slightly, reaching about 65% of the initial values at the end of the culture period. The proliferating activity measured by the incorporation of [3H]thymidine into DNA decreased to 10% or less of the initial value after 3 days in vitro. Almost at the same chronological times as in ovo, the synthesis of GD3 and of a ganglioside partially identified as GT3 decreased from 70 and 19% of the total incorporation into gangliosides in the first 20 h of culture to about 7 and 5%, respectively, after 3 days in vitro. Conversely, the synthesis of GD1a increased from about 6% at the beginning to about 70% at the end of the culture times. Immunocytochemical analyses of the expression of gangliotetraosyl gangliosides in cultured cells showed that these gangliosides appeared in the bodies and processes of cells having neuronal morphology; very little immunostaining of the scarce flattened cells, probably Müller cells, was found. The results indicate that the changes in ganglioside metabolism, which lead to decreased synthesis of gangliosides lacking the galactosyl-N-acetyl-galactosaminyl disaccharide end and to increased synthesis of gangliotetraosyl gangliosides, occur in cells that in culture differentiate into neurons.     

Trisialosyllactosylceramide (GT3) is a ganglioside of human lung

A trisialoganglioside not found before in human tissue was isolated from showed this to be II3(NeuAc)3-LacCer or GT3. The ganglioside constituted 40% of the trisialogangliosides of human lung or 0.2 nmol ganglioside per g tissue.