Glycosphingolipids of human myometrium and endometrium and their changes during the menstrual cycle, pregnancy and ageing

The glycolipid composition of human myometrium and endometrium was examined at various stages of maturation and reproduction. The major neutral glycolipids of both myometrium and endometrium were identified by high-performance thin-layer chromatography as globo-series glycolipids, Gb3 and Gb4. The major acidic glycolipids (gangliosides) were identified similarly as GM3 and GD3, with lesser amounts of GM1, GD1a, and GT1b. During pregnancy, GD3 expression declined in both myometrium and endometrium, whereas GM3 expression increased. Reciprocal changes in GM3/GD3 expression were mirrored by appropriate changes in the glycosyltransferases required for their synthesis; alpha 2----3sialyltransferase activity increased approximately 3-fold during pregnancy, while alpha 2----8sialyltransferase activity declined to about 20%. The results focus attention on the glycolipids of uterine tissues, their regulation, and their possible role in reproduction and fertility.     

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.     

Adult Rat Retina Interneurons Synthesize GD3: GD3 Expression by These Cells Is Regulated by Cell-Cell Interactions

GD3, a ganglioside of the lactosyl series, is prevalent in rat retina neuronal cells. We studied here whether rat retina neurons synthesize their own surface GD3 or if they acquire it from Müller glia cells. We analyzed the activity of GD3 synthase and the in vivo labeling of gangliosides from N-[3H]acetylmannosamine in adult rat retinas after selective destruction of Müller glia cells with the gliotoxic alpha-D,L-aminoadipate (AAA). Immunostaining of rat retina sections and western blot analysis with an antivimentin antibody confirmed the gliotoxic effect of AAA. Neither GD3 synthase activity nor the in vivo labeling of GD3 and other gangliosides was significantly affected by AAA, indicating that neuronal cells synthesize their own GD3. We next analyzed the regulation of the expression of GD3 by these neurons in culture. About 80% of freshly dissociated cells from retina of 4-day-old rats (R4) immunoexpress surface GD3. After 3 days in dispersed cell culture conditions, GD3 expression was under the limit of detection in 80% of neuronal cells, indicating a failure of these cells to maintain the expression of surface GD3 in these experimental conditions. Most flat Müller glia-derived cells present in these cultures were GD3 positive. Surface GD3 was detected in approximately 60% of neuronal cells dissociated from R4 tissue that was developed in vitro as an organ culture for 3 days. Likewise, approximately 50% of neurites that had grown out from R4 retinal explants within 3 days in culture and whose neuronal character was indicated by immunoexpression of growth-associated protein GAP-43 were GD3 positive. These findings suggest that the tissue organization and/or specific interactions modulate GD3 expression in neuronal cells. Under dispersed-cell culture conditions, c-pathway gangliosides (GQ1c and GT1c), which are built up from the sialylation of GD3 and later completion of the oligosaccharide backbone, were detected in approximately 60% of neuronal cells, suggesting a maintenance of production of GD3 as an intermediate for gangliotetraosyl gangliosides.     

Apoptosis of human breast carcinoma cells in the presence of disialosyl gangliosides: II. Treatment of SKBR3 cells with GD3 and GD1b gangliosides

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells has been monitored during the cells' progression to apoptosis by anti-cancer drugs and inhibitors of the cell surface glycolipids, gangliosides and SA-Le(x) biosyntheses [Basu, S (1991) Glycobiology, 1, 469-475; and ibid, 427-435] in animal tissues and human carcinoma cells, respectively. Induction of apoptosis in cancer cells by cell surface glycolipids in the human breast cancer (SKBR3) cells is the aim in this study. We have employed the disialosyl gangliosides (GD3 and GD1b) to initiate apoptosis in SKBR3 cells grown in culture in the presence of (14)C-L-Serine. At lower concentrations (0-20 microM) of exogenously added non-radioactive GD3, GD1b, or bovine ganglioside mixture (GM1:GD1a:GD1b:GT1a 2:4:4:2), the incorporation of radioactivity in both (14)C-sphingolipid and (14)C-ceramide was higher. However, at higher concentrations (20-100 microM), wherein apoptosis occurred in high frequency, the (14)C-incorporation decreased in both GSLs and ceramide. Apoptosis induction was monitored by the concomitant appearance of caspase-3 activation and the binding of a fluorescent dye PSS-380 to the outer leaflet of phosphatidyl-serine. These results indicated that, in addition to many unknown cell surface glycoconjugates GD3 or GD1b (disialosyl ganglioside) could play an important role in the regulation of breast carcinoma cell death.     

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 and neutral glycosphingolipids of normal tissue and oat cell carcinoma of human lung

Concentration and composition of gangliosides and neutral glycosphingolipids of adult human lung, and lung small cell carcinoma were studied. The structures of the glycolipids were determined by quantitative component determination, enzymic degradation, permethylation and fast atom bombardment mass spectrometry. Adult human lung contained mainly gangliosides with lactosylceramide as the basic core, GM3, GD3 and GT3, and approx. equal proportions (10%) of gangliosides of the gangliotetraosyl- and lactotetraosylceramide series. 18 gangliosides with different carbohydrate moieties were identified: four of them were only found in the tumor tissue. The adult human lung contained 85 nmol (77-120) gangliosides and 140 nmol neutral glycosphingolipids per g wet weight. Globoside was the major neutral glycolipid and there were only minor amounts of glycolipids of the lactotetraose series. In small cell carcinoma tissue the concentration of neutral glycosphingolipids was approximately twice as high than in normal lung tissue, and there was a markedly larger concentration of both lactosylceramide and glycolipids of the lactotetraose series and fucose derivatives of these. The concentration of gangliosides varied between 202 and 415 nmol per g wet weight. Compared to normal lung tissue, the tumor tissue had a lower proportion of GD3, and a higher proportion of complex gangliosides, and they contained five tumor-associated gangliosides: Fuc-GM1, Fuc-GD1b, 3'-LM1, Fuc-3'-LM1 and 6'-nLM1.     

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.     

The glycosphingolipids of human prostate tissue

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

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.     

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.     

Growth- and development-dependent expression of gangliosides in rat hepatocytes and liver tissues.

Expression of gangliosides in the liver was examined in primary cultures of hepatocytes from adult rats and liver tissues from rats of different ages. Hepatocytes were isolated from 7-week-old rat liver and cultured in L-15 medium containing insulin, dexamethasone and 10% fetal bovine serum. Hepatocytes proliferated only on the first day, and then ceased proliferation. The content of GD3 and GD1a increased during the period of active proliferation and reached a nearly constant level, whereas GM1, GD1b, GT1b, and GQ1b gradually increased throughout culture. Addition of EGF to the culture medium caused significant increases in the content of GD3, and to a lesser degree of GM3, but exhibited little effect on the expression of other ganglioside species. The specific induction of GD3 and GM3 expression by EGF was reproduced under serum-free conditions, despite the lack of hepatocyte proliferation. Expression of gangliosides in cultured hepatocytes was also modulated by cell density; higher cell density brought about increased content of GM1, GD1a, GD1b, GT1b, and GQ1b with concomitant reduction of GM3 in cells. The composition of gangliosides in liver tissues demonstrated a unique developmental pattern. GD3 and GD1a were strongly expressed in E-16 embryonic tissue and rapidly decreased with increasing age. GD1b, GT1b, and GQ1b were found only in postnatal liver tissues. These findings suggest that the expression of gangliosides in rat hepatocytes and liver tissues are regulated by growth- and development-dependent factors.     

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

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

GD3 Prevalence in Adult Rat Retina Correlates with the Maintenance of a High GD3-/GM2-Synthase Activity Ratio Throughout Development

Unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosyl ceramide series but also maintain a prevalence of GD3, a ganglioside of the lactosylceramide series characteristic of proliferative neural cells, when they are fully differentiated. We show here that GD3 is prevalent at all developmental periods of the rat retina from birth [50% of total gangliosidic N-acetylneuraminic acid (NeuNAc)] to adult (30% of total gangliosidic NeuNAc). GD3-synthase specific activity increased about 1.5-fold from birth to day 7 and essentially plateaued thereafter. The GD3-/GM2-synthase specific activity ratio was compared in rat and chicken retina at early and late developmental stages. In chicken retina the ratio was about 0.7 at early (when GD3 is prevalent) and decreased to 0.07 at late (when GD1a is prevalent) developmental stages. In rat retina the ratio was about 13 and 6 at, respectively, early and late developmental stages. These findings suggest that the prevalence of GD3 and of other "b" pathway gangliosides in adult rat retina neurons could be due in part to the maintenance of a high GD3-/GM2-synthase activity ratio throughout development of the tissue.     

Gangliosides administration causes sugar moiety-specific enzymatic changes in brain

Exogenous gangliosides are known to affect the metabolism when administered to the body. To study the mechanism of this effect three types of gangliosides were administered intraperitoneally to mice and the changes in the enzyme activity of the cerebral tissues studied. The effect of GM2 from bovine brain was characterized by a decrease in the activity of various aminopeptidases, while GD3 from cow's milk caused an increase in the activity of sugar-related enzymes such as sialidase, glucosidase, and fucosidase. GM3 from horse erythrocytes showed intermediate effects between GM2 and GD3. Multivariate analysis showed that the effects of the three gangliosides are clearly separable statistically. These results which demonstrate the sugar moiety-specificity of gangliosides are discussed in relation to the A and B pathways of ganglioside synthesis.     

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.     

Comparative study of lymph node gangliosides and blood serum of normal and T-lymphotrophic baboons

The gangliosides from the lymph nodes and blood sera of normal and T-lymphomic baboons were studied. In lymph nodes the major gangliosides were identified as GM3 and GD3, those in blood sera--as GM3, GM1 and GD3. Gangliosides GM3 and GD3 contained N-acetyl as well as N-glycoloyl neuraminic acids. In gangliosides isolated from lymph nodes and blood sera of T-lymphomic baboons the levels of N-glycoloyl neuraminic acid markedly exceeded that in normal tissues. In tumour lymph nodes the GM3/GD3 ratio was shifted towards GD3.     

Apoptosis of human breast carcinoma cells in the presence of disialosyl gangliosides: II. Treatment of SKBR3 cells with GD3 and GD1b gangliosides

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells has been monitored during the cells' progression to apoptosis by anti-cancer drugs and inhibitors of the cell surface glycolipids, gangliosides and SA-Le^x biosyntheses [Basu, S (1991) Glycobiology, 1, 469–475; and ibid, 427–435] in animal tissues and human carcinoma cells, respectively. Induction of apoptosis in cancer cells by cell surface glycolipids in the human breast cancer (SKBR3) cells is the aim in this study. We have employed the disialosyl gangliosides (GD3 and GD1b) to initiate apoptosis in SKBR3 cells grown in culture in the presence of ¹⁴C-L-Serine. At lower concentrations (0–20 μM) of exogenously added non-radioactive GD3, GD1b, or bovine ganglioside mixture (GM1:GD1a:GD1b:GT1a 2:4:4:2), the incorporation of radioactivity in both ¹⁴C-sphingolipid and ¹⁴C-ceramide was higher. However, at higher concentrations (20–100 μM), wherein apoptosis occurred in high frequency, the ¹⁴C-incorporation decreased in both GSLs and ceramide. Apoptosis induction was monitored by the concomitant appearance of caspase-3 activation and the binding of a fluorescent dye PSS-380 to the outer leaflet of phosphatidyl-serine. These results indicated that, in addition to many unknown cell surface glycoconjugates GD3 or GD1b (disialosyl ganglioside) could play an important role in the regulation of breast carcinoma cell death. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Different fine binding specificities of monoclonal antibodies to disialosylganglioside GD2

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

Glycolipids of human large intestine: difference in glycolipid expression related to anatomical localization, epithelial/non-epithelial tissue and the ABO, Le and Se phenotypes of the donors

Human large intestine specimens were obtained during elective surgery from donors of known blood group ABO, Lewis and secretor phenotypes. The intestinal epithelial cells were isolated from the non-epithelial tissue in one case and in another case mucosa tissue was obtained by scraping. Total non-acid glycolipid and ganglioside fractions were isolated from the tissue specimens, analyzed by thin-layer chromatography and detected by chemical reagents and autoradiography after staining the plate with various blood group monoclonal antibodies and bacterial toxins. The amount of non-acid glycolipids present in the large intestine epithelial cells was 3.9 micrograms/mg of cell protein and in the non-epithelial tissue 0.39 mg/g dry tissue weight. The epithelial cells contained monoglycosylceramides and blood group Lea pentaglycosylceramides as major compounds together with small amounts of diglycosylceramides. In addition, trace amounts of tri- and tetra-glycosylceramides together with more complex glycolipids were present. The non-epithelial tissue contained mono-, di-, tri- and tetra-glycosylceramides as major non-acid components. Blood group ABH glycolipids were present in trace amounts in the non-epithelial part of the large intestine. Lea pentaglycosylceramide was the major blood group glycolipid present in all Le-positive individuals independent of the secretor status. Leb glycolipids were present in trace amounts in secretor individuals but completely lacking in non-secretors. Trace amounts of X antigens were found in all individuals, while Y antigens were only present in secretor individuals. The Lea, Leb, X and Y glycolipids were located in the epithelial cells. The gangliosides were present mainly in the non-epithelial tissue (65-350 nmol of sialic acid/g dry weight) and only trace amounts (less than 0.014 nmol/mg of cell protein) were found in the epithelial cells. The major gangliosides of the non-epithelial tissue were identified as GM3, GM1, GD3, GD1b, GT1b and GQ1b. In addition, several minor gangliosides were also present. Binding of cholera toxin to the thin-layer plate revealed trace amounts of the GM1 ganglioside in the epithelial cell ganglioside fraction.     

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.