Occurrence of gangliosides in the common squid and pacific octopus among protostomia

Acidic lipids from tissues of the common squid Todarodes pacificus and the pacific octopus Octopus vulgaris were characterized. Hepatopancreatic tissues of both animals had complex compositions of resorcinol-positive acidic lipids, many of which became reactive with cholera toxin B subunit and anti-G(M1) antibody after in situ treatment with sialidase on TLC. One of the major acidic lipids in squid tissue was isolated and examined for its structure. This acidic lipid was identified to be the ganglioside G(D1a) based upon the susceptibility to sialidases of different substrate specificity, characterization of reaction products, and electrospray ionization-mass spectrometry of the lipid. Hepatopancreatic tissues of squid and octopus also contained acidic lipids that reacted with A2B5, a monoclonal antibody specific to c-series gangliosides. Cerebral ganglia of both animals expressed resorcinol-positive acidic lipids, though their compositional patterns differed from the hepatopancreatic tissues. N-Acetylneuraminic acid was identified as the main species in lipid-bound sialic acid in both tissues. The contents of lipid-bound sialic acid in cerebral ganglia were significantly lower than those of hepatopancreatic tissues in both animals. The present study presents the first evidence for the occurrence of gangliosides in protostomia.     

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.     

Expression of c-series gangliosides in rat hepatocytes and liver tissues

C-series gangliosides in rat hepatocytes and liver tissues were analyzed by thin-layer chromatographic (TLC) immunostaining with the specific monoclonal antibody A2B5. Primary cultures of hepatocytes isolated from adult rats were immunostained positively by A2B5. TLC immunostaining with A2B5 of gangliosides from the cells suggested that rat hepatocytes express c-series gangliosides including GT3, GT1c, GQ1c, and GP1c. Expression of c-series gangliosides in cultured hepatocytes was modulated by growth conditions of cells. The amount of GT3 was increased significantly by epidermal growth factor, while the contents of polysialo species such as GT1c, GQ1c, and GP1c were enhanced by higher cell density in culture. Examination of c-series gangliosides in rat liver tissues showed a unique developmental profile with a shift from GT3-dominant to polysialo species-dominant composition in late embryonic stages. These results suggest that the expression of c-series gangliosides in rat hepatocytes is regulated in a growth- and development-dependent manner.     

A specific loss of C-series gangliosides in pancreas of streptozotocin-induced diabetic rats.

Gangliosides in pancreas, kidney, and liver tissues from streptozotocin-induced diabetic rats were analyzed by methods including thin-layer chromatographic (TLC) immunostaining with a specific monoclonal antibody to c-series gangliosides. In rats suffering diabetes for one month, the composition of major gangliosides in pancreatic tissue was almost identical to control, except for a slight increase in the content of GM3. Though c-series gangliosides such as GT3, GT2, GQ1c, and CP1c were expressed in normal pancreatic tissue, they were practically lost in pancreas of diabetic animals. A specific loss of c-series gangliosides was also observed in pancreatic tissue from rats suffering diabetes only for three days. While the composition of major gangliosides in the kidney did not change, streptozotocin-induced diabetic conditions brought about significant increases in contents of practically all major ganglioside species in liver tissue. No change was observed in the amount and composition of c-series gangliosides in both tissues. These results strongly suggest that c-series gangliosides are specifically localized in pancreatic B cells.     

The specificity of monoclonal antibody A2B5 to c-series gangliosides

To examine the specificity of monoclonal antibody A2B5, four A2B5-reactive gangliosides (designated as G-1, G-2, G-3 and G-4) were purified from bonito fish brain. Ganglioside-1, -2, and -3 migrated above GD1b, below GQ1b, and far below GQ1b on thin-layer chromatography. Ganglioside-4 had the slowest chromatographic mobility and migrated below G-3. The structures of these gangliosides were characterized by overlay analysis with glycolipid-specific ligands, product analysis after sialidase or mild acid treatment, and electrospray ionization-mass spectrometry (ESI-MS). Accordingly, G-1, G-2 and G-3 were identified to be GT3, GQ1c and GP1c, respectively. The ganglioside G-4 was shown to have the following structure: NeuAc-NeuAc-NeuAc-Galbeta1-3Gal NAcbeta1-4(NeuAc-NeuAc-NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer. The antibody A2B5 reacted with these c-series gangliosides, but not with GD3 and other gangliosides and neutral glycosphingolipids. The antigenic epitope for A2B5 was assumed to include the trisialosyl residue connected to the inner galactose of the hemato- or ganglio-type oligosaccharide structure of gangliosides. Phylogenetic analysis of brain gangliosides using the A2B5 preparation demonstrated that c-series gangliosides are enriched in lower animals, especially bony fish of different species. The monoclonal antibody A2B5 would be a useful tool for examining the distribution and function of c-series gangliosides.     

Major and c-series gangliosides in lenticular tissues: mammals to molluscs

Gangliosides of eye lenses were examined in mammals (rat, rabbits, pig, cow), bird (chicken), reptile (terrapin), amphibian (bullfrog), bony fish (red sea bream, bluefin tuna, bonito, Pacific mackerel) and molluscs (common squid, Pacific octopus). Besides the fact that GM3 was the common ganglioside species, the composition of major gangliosides in mammalian eye lenses significantly differed from each other. While gangliotetraose gangliosides were abundant in rat eye lens, they did not constitute major components in porcine and bovine tissues. The c-series ganglioside GT3 was expressed in rat eye lenses but were practically absent in other mammalian tissues. The composition of major gangliosides in eye lenses of lower animals varied from species to species, whereas c-series gangliosides were consistently expressed, showing similar compositional profiles. Our results demonstrate the species-specific compositions of lenticular gangliosides. Evidence was also provided suggesting that eye lenses of common squid (Todarodes pacificus) and Pacific octopus (Octopus vulgaris) express gangliosides including gangliotetraose species and c-series gangliosides.     

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.     

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.     

Effect of different fixatives on immunocytochemical localization of HNK-1-reactive antigens in cerebellum: a method for differentiating the localization of the same carbohydrate epitope on proteins vs lipids.

Monoclonal antibody (MAb) HNK-1 recognizes a carbohydrate epitope present in certain glycolipids, glycoproteins, and proteoglycans. Five different fixation methods, together with biochemical analyses of the antigens, were evaluated to study immunocytochemical localization of this epitope in layers of adult rat cerebellum; 4% paraformaldehyde/0.5% cetylpyridinium chloride was found to be optimal for overall immunoreactivity, and the antigens were apparent in all cerebellar layers. To differentially localize HNK-1-reactive carbohydrate epitope on proteins vs lipids in cerebellar layers, we tested the effect of 0.2%, 2%, or 4% glutaraldehyde combined with 2% paraformaldehyde (GT/PF) on HNK-1 and other MAb-reactive protein and lipid antigens; 2% or 4% GT/PF significantly reduced or abolished immunoreactivity of MAb HNK-1 and 5F9 (reacting with microtubule-associated protein 2) with cerebellar proteins analyzed on Western blots, but did not decrease HNK-1 reactivity to lipid antigens on HPTLC blots. In cerebellar tissue sections, HNK-1 and 5F9 immunoreactivity was reduced after 2% or 4% GT/PF fixation. However, significant amounts of HNK-1 immunoreactivity remained in molecular layer and deep cerebellar nuclei. GT/PF fixation did not cause significant changes in immunoreactivity patterns of other carbohydrate lipid antigens, such as those that react with MAb A2B5, 7A, and WCC4. Therefore, carbohydrate epitope on lipids, as opposed to that on proteins, may be preferentially detectable by immunocytochemistry after fixation with 2% or 4% GT/PF. The selective localization of HNK-1-reactive carbohydrate in the molecular layer and deep cerebellar nuclei with 2% or 4% GT/PF fixation correlates well with the observed presence of HNK-1-reactive lipids in these areas but not in the granular layer and white matter, as determined by microdissection of the individual layers and biochemical analysis. The application of 2% or 4% GT/PF fixation as a general method for differentiating the same carbohydrate epitope on proteins vs lipids in immunocytochemistry for other tissues and other antibodies remains to be further evaluated.     

Gangliosides in rat kidney: composition, distribution, and developmental changes

Gangliosides in rat kidney were analyzed for their composition, regional distribution, and developmental changes. Renal tissue from 7-week-old rats showed a GM3-dominant pattern with GD3 and several minor ganglioside components including GM4, GM2, GD1a, and an unknown ganglioside (ganglioside X). The tissue also contained c-series gangliosides that included GT3 as the main component with GT2 in a lesser amount. Ganglioside analysis of cortical and medullary regions of renal tissue suggested the restricted localization of some gangliosides. While GM4 and GD3 were enriched in the cortical region, GM2 was distributed mainly in the medullary area. Renal gangliosides showed unique developmental profiles during a period from Embryonic Day 20 (E20) to 7 weeks postnatal. The content of renal gangliosides increased from E20, reached the highest around Postnatal Day 1, and thereafter, decreased rapidly to the adult level. The ratio of N-glycolylneuraminic acid to total sialic acids in gangliosides tended to change in inverse proportion to the amount of total sialic acids. The composition of major gangliosides in renal tissues shifted from GD3-dominant to GM3-dominant patterns with advancing ages. While GM1 was expressed only at early stages of the development, GM4, GM2, and ganglioside X appeared after Postnatal Day 3. The expression of c-series gangliosides was less affected through the period examined. These results suggest that gangliosides may be implicated with development and function of rat kidney.     

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.     

A set of glycoproteins recognized by A2B5 antibody with major bands at 55 and 76 kDa is overexpressed in human head and neck squamous cell carcinomas

A2B5 antibody was found to strongly label frozen sections of human head and neck squamous cell carcinomas. The low amount of glycolipids (c-series gangliosides and sulfatides) purified from the same tumors and reactive with A2B5 by immunostaining on thin-layer plates could not account for the high level of tissue labeling. Proteins were extracted from both normal tissues and squamous cell carcinomas and analyzed by Western blot with A2B5 antibody on PVDF membranes. The antibody was found to stain a set of glycoproteins with two major bands at 55 and 76 kDa present in normal tissues and overexpressed in carcinomas. Staining was abolished by prior treatment of the PVDF membranes either with Arthrobacter ureafaciens neuraminidase or with a solution of 10 mM periodate that is known to destroy carbohydrates. Our results show that the carbohydrate epitope recognized by A2B5 antibody can be displayed by both glycolipids and glycoproteins.     

Sulfatide is expressed in neurons and astrocytes and accumulates at degradation deficiency

Few studies of lipid rafts have investigated gangliosides in brain tissue. This study focus on analyses of lipids and the major brain gangliosides (GM1, GD1a, GD1b, GT1b) in human cortex (frontal, temporal) and corresponding detergent resistant membranes (DRMs), i.e. rafts. A high proportion of the gangliosides (18–26%) as well as of cholesterol (21%) and sphingomyelin (38%) was found in rafts, while lower yields was observed for ganglioside GM2 (9%), phospholipids (8%) and in particular proteins (2%). Significant alterations in lipid composition was noticed in rafts from Alzheimer brain tissue. These results show that sphingolipids and cholesterol are major constituents of rafts also in the human brain and that the main brain gangliosides are distributed in rafts to a similar degree. Moreover, lipid rafts might be considered in the pathology of Alzheimer's disease.     

Activities of Five Different Sialyltransferases in Fish and Rat Brains

Abstract: To investigate the role of Sialyltransferases in the metabolism of brain gangliosides, we examined activities of five different Sialyltransferases (GM3-, GD3-, GT3-, GD1a-, and GT1a-synthase) using total membrane preparations from cichlid fish and Sprague-Dawley rat brains, and analyzed the relationship between the enzyme activities and the ganglloside compositions. The patterns of sialyltransferase activities in fish and rat brains differed from each other. In fish brain, the GM3-synthase activity was lower than GD3-synthase activity, whereas the opposite relationship was observed in rat brain. The GT3-synthase reaction with fish brain membranes produced radiolabeled GM3, GD3, and a ganglioside that was identified as GT3 based on mobility on TLC using two different solvent systems. No GT3-synthase activity was detected in rat brain. The GD1a-and GT1a-synthase activities in fish brain were higher than those in rat brain. Although GT1a was a single radiolabeled ganglioside in fish GT1a-synthase reaction, this ganglioside could not be detected in rat brain. The ratios of GM3-, GD3-, GT3-, GD1a-, and GT1a-synthase activities in fish and rat brain were 23:31:4:28:14 and 61:21:0:18:0, respectively. Ganglioside analysis showed that fish brain was enriched with c-series gangliosides including GT3 and polysialo-species, whereas a-and b-se-ries gangliosides were major components in rat brain. These results suggest that the species-specific expression of gangliosides in brain tissues may be regulated, at least in part, at the level of sialyltransferase activities.     

Lipid analyses of human brain rafts

Few studies of lipid rafts have investigated gangliosides in brain tissue. This study focus on analyses of lipids and the major brain gangliosides (GM1, GD1a, GD1b, GT1b) in human cortex (frontal, temporal) and corresponding detergent resistant membranes (DRMs), i.e. rafts. A high proportion of the gangliosides (18–26%) as well as of cholesterol (21%) and sphingomyelin (38%) was found in rafts, while lower yields was observed for ganglioside GM2 (9%), phospholipids (8%) and in particular proteins (2%). Significant alterations in lipid composition was noticed in rafts from Alzheimer brain tissue. These results show that sphingolipids and cholesterol are major constituents of rafts also in the human brain and that the main brain gangliosides are distributed in rafts to a similar degree. Moreover, lipid rafts might be considered in the pathology of Alzheimer's disease.     

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.     

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.     

Ganglioside-dependent factor, inhibiting lipid peroxidation in rat brain synaptosomes.

Preincubation of rat brain synaptosomes with GM1, GD1a or GT1b (10(-10)-10(-6) M), as well as with phorbol 12-myristate, 13-acetate (10(-10)-10(-6) M) was found to have dose dependent inhibitory effect on Fe(2+)-ascorbate induced lipid peroxidation, while penetrating analogue of c-AMP markedly decreased the inhibitory effect of these compounds. In liposomes made of lipids isolated from synaptosomal membranes the degree of inhibition of induced LPO by gangliosides was practically absent. The inhibitory effect of GM1 on lipid peroxidation could not be revealed after thermal denaturation of synaptosomes or after treatment with polymyxin B (inhibitor of lipid-dependent protein kinases). These results and some other data provide evidence for the existence of ganglioside-dependent factor inhibiting lipid peroxidation in brain tissue. It may be suggested to be a protein kinase modulated by gangliosides.     

Monoclonal antibody 18B8, which detects synapse-associated antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer)

By immunofluorescence, mouse monoclonal antibody 18B8 detects developmentally regulated antigens in chick neural retina. In older embryos and in adults these antigens are localized in discrete laminae within the inner and outer synaptic layers. The antibody binds to several gangliosides that undergo both qualitative and quantitative changes during neuronal development (Grunwald, G.B., Fredman, P., Magnani, J.L., Trisler, D., Ginsburg, V., and Nirenberg, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4008-4012). The simplest of these gangliosides was isolated from lipid extracts of 10-day chick embryonic retinas by DEAE-Sepharose and silicic acid column chromatography. About 300 micrograms was obtained from 9.3 g (wet weight) of retina. The isolated ganglioside was identified as GT3 by enzymatic analysis and by a comparison of its properties with the authentic ganglioside. By immunostaining thin-layer chromatograms with antibody 18B8, GT3 was detected in gangliosides from human neural tissue including cerebellum, optic nerve, and spinal cord, but not in gangliosides from human liver, pancreas, small intestine, adrenals, thyroid, or erythrocytes. GT3 was also found in five of seven human melanoma cell lines.     

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.