Human brain gangliosides in development, aging and disease.

In this study, brain gangliosides in prenatal and postnatal human life and Alzheimer's disease were analyzed. Immunohistochemically, the presence of the "c"-series of gangliosides (GQ1c) was only registered in the embryonic brain at 5 weeks of gestation. Biochemical results indicated a two-fold increase in ganglioside concentration in the human cortex between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except in the cerebellar cortex, which was characterized by increasing GT1b. During prenatal human development, regional differences in ganglioside composition could only be detected between the cerebrum ("a"-pathway) and the cerebellum ("b"-pathway). Between birth and 20-30 years of age, a cerebral neocortical difference of ganglioside composition occurred, characterized by the lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In the frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in the occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In the cerebellar cortex, GD1b and GT1b fractions decreased with aging. In Alzheimer's disease, we found all ganglio-series gangliosides (GM1, GD1a, GD1b, GT1b) to be decreased in regions (temporal and frontal cortex and nucleus basalis of Meynert) involved in pathogenesis of disease. In addition, in Alzheimer's disease we found simple gangliosides (GN2, GM3) to be elevated in the frontal and parietal cortex, which might correlate accelerated lysosomal degradation of gangliosides and/or astrogliosis occurring during neuronal death.     

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.     

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.     

Gangliosides stimulate astroglial cell proliferation in the absence of serum

Previous studies with microcultures of astroglial (AG) cells from newborn rat cerebrum had shown an ability of gangliosides to interact with AG cells cultured under defined conditions. We have now investigated the capability of gangliosides to stimulate DNA synthesis and cell number increases in similar secondary microcultures of newborn rat cerebrum AG cells. At a concentration of 6 X 10(-5)M, GM1 ganglioside stimulated DNA synthesis and increased cell numbers, with DNA synthesis leading cell increases by 12-24 hr. The ganglioside-induced AG cell proliferative response occurred with GD1a, GD1b and GT1b, GT1b being the most potent at 10(-5)M--while asialo GM1 and sialic acid were without effect. In the standard test cultures, DNA synthesis declined very steeply after the first day, with cell numbers stabilizing at the level reached after 2 days. Ganglioside was not itself responsible for the restricted proliferative response, as serum produced the same behaviors.     

Gangliosides in the human brain development and aging.

In this study, brain gangliosides in prenatal and postnatal human life were analyzed. Immunohistochemically, the presence of "c"-pathway of gangliosides (GQ1c) in embryonic brain was only recorded at 5 weeks of gestation. Biochemical results indicated a twofold increase in human cortex ganglioside concentration between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except cerebellar cortex, which was characterized by increasing GT1b. In this developmental period, GD3 was found to be localized in the ventricular zone of the cortical wall. After birth, GD1b ganglioside in neuropil of granular cell layer corresponding to growing mossy fibers was expressed in cerebellar cortex. Between birth and 20/30 years of age, a cerebral neocortical difference of ganglioside composition was observed, characterized by lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In cerebellar cortex, GD1b and GT1b fractions decreased with aging.     

Liver gangliosides of various animals ranging from fish to mammalian species

Liver gangliosides of different animal species were analyzed. Bony fish liver contained a major ganglioside that migrated faster than GM3 on thin-layer chromatography (TLC). This ganglioside was identified to be GM4 (NeuAc) by methods including product analysis after sialidase treatment and negative-ion electrospray ionization (ESI)-mass spectrometry (MS). The presence of GM4 (NeuGc) in fish liver was also demonstrated. The main ganglioside band of bovine liver consisted of two different molecular species, i.e. GD1a (NeuAc/NeuAc) and GD1a (NeuAc/NeuGc). Major gangliosides of liver tissue exhibited a distinct phylogenetic profile; GM4 was expressed mainly in lower animals such as bony fish and frog liver, whereas mammalian liver showed ganglioside patterns with smaller proportions of monosialo ganglioside species. While c-series gangliosides were consistently expressed in lower animals, they were found only in mammalian liver of particular species. No apparent trend was observed between the concentration of liver gangliosides and the phylogenetic stage of animals. The present study demonstrates the species-specific expression of liver 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.     

Content, pattern and metabolic processing of rat-liver gangliosides during liver regeneration

During rat liver regeneration, the ganglioside content and distribution undergo significant changes after partial hepatectomy; total liver gangliosides increase remarkably till the 4th day after surgery, thereafter progressively decreasing to reach the values of sham-operated controls at the 12th day. The qualitative pattern is characterized by the 95% relative increase of GD1a at the 4th day and the 40% relative decrease of GD1b. In order to investigate the processes of ganglioside penetration into cells, degradation and biosynthesis, radiolabelled GM1 ([Sph-3H] GM1) was administered. One day after hepatectomy the liver uptake and metabolism of exogenous ganglioside were significantly reduced. Three days post-surgery these parameters were restored to control values; however an increased radioactivity incorporation was found in GD1a, thus suggesting an enhancement of its biosynthesis around the 4th day. The data reported here suggest that in the first two days after partial hepatectomy, the ganglioside degradation is reduced with a consequent increase of ganglioside content; later on the catabolic routes normalize and some biosynthetic processes leading to GD1a are enhanced. GD1a seems to be a marker of a peculiar transition phase of liver regeneration.     

Age-related changes in GM1, GD1a, GT1b components of gangliosides in Wistar albino rats

In this study, age-related changes of GM1, GD1a, GT1b fractions of gangliosides were investigated in whole brain of male Wistar albino rats. Insignificant increases were detected in GM1 values from the third to the 24th month, whereas GD1a and GT1b concentrations of ganglioside in 24-month-old rats decreased significantly as compared to 6-month-old rats. Although there were no significant differences in the GD1a/GT1b ratio of any groups, GM1/GD1a and GM1/GT1b ratios were significantly increased as compared to 6-month-old rats. The increase in the ratios of gangliosides are not due to an increase of GM1 fractions; they result from a decrease of GD1a and GT1b fractions of gangliosides. In conclusion, the concentration of ganglioside decreased with ageing.     

Ganglioside stimulation of nerve growth factor synthesis in cultured rat Schwann cells

To investigate effects of gangliosides on nerve growth factor (NGF) synthesis/secretion by Schwann cells, we obtained Schwann cells from dorsal sensory ganglia of one-day old Wistar rats and cultured them with various concentrations of a mixed ganglioside comprising GM1, GD1a, GD1b, and GT1b. NGF synthesis was evaluated by the measurement of NGF concentration in the conditioned medium using an enzyme immunoassay. In the continuous presence of 10(-3) M gangliosides, the NGF concentration in the medium showed a four fold increase on the 4th day, and it then decreased by the 8th day. The present results indicate that gangliosides promote the production/synthesis of NGF by Schwann cells.     

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.     

Studies on the binding substances on human erythrocytes for the heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli

To study the predominant binding substance for the heat-labile enterotoxin (LTc) isolated from chicken enterotoxigenic Escherichia coli, competitive binding assays were performed with neuraminidase-treated human type B erythrocytes and 125I-labeled B subunit of LTc (LTc-B). Of all inhibitors used, the ganglioside GM1 was the most effective in inhibiting the binding of 125I-labeled LTc-B to the erythrocytes. The other gangliosides used as inhibitors, gangliosides GD1b, GD1a, GM2, GT1b and GM3, were about 24, 166, 250, 440 and at least 440 times less reactive than ganglioside GM1, respectively. With glycoproteins as inhibitors, on the other hand, hog A + H, porcine thyroglobulin and bovine salivary mucin were over 10(4) times less potent. No inhibition was obtained by other mono-, di- and polysaccharides at the highest concentrations used. These findings suggest that the predominant binding substance on neuraminidase-treated human type B erythrocytes for the LTc-B is ganglioside GM1 and that the combining site of LTc-B may be specific for the terminal disaccharide (galactose-N-acetyl-D-galactosamine)-linked portion of ganglioside GM1.     

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.     

Isolation and characterization of gangliosides from Trypanosoma brucei

Gangliosides were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated by TLC. G-1 ganglioside had the same TLC migration rate as GM3. In contrast, G-2, G-3, and G-4 gangliosides migrated a little slower than GM1, GD1a, and GD1b, respectively. To characterize the molecular species of gangliosides from T. brucei, G-1, G-2, G-3, and G-4 gangliosides were purified and analyzed by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside showed the reactivity to the monoclonal antibody against ganglioside GM3. G-2 was recognized by the anti-GM1 monoclonal antibody. G-3 showed reaction with the monoclonal antibody to GD1a. G-4 had the reactivity to anti-GD1b monoclonal antibody. Using 4 kinds of monoclonal antibodies, we also studied the expression of GM3, GM1, GD1a, and GD1b in T. brucei parasites. GM3, GM1, GD1a, and GD1b were detected on the cell surface of T. brucei. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-1Cer), GM1 (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), GD1a (NeuAc alpha2-3Gal beta1-3GalNAc beta1-4[NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), and GD1b (Gal beta1-3GalNAc beta1-4[NeuAc alpha2-8NeuAc alpha2-3]Gal beta1-4Glc beta1-1Cer), respectively, and also that they are expressed on the cell surface of T. brucei.     

Changes in the Ganglioside Long-Chain Base Composition of Rat Cerebellar Granule Cells During Differentiation and Aging in Culture

Abstract: Changes in the ganglioside long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total native ganglioside mixtures, extracted from the cells maintained in culture up to 22 days, were fractionated by reversed-phase HPLC, each ganglioside homogeneous in the oligosaccharide chain as well as in the LCB being quantified. Two main LCBs were components of the ganglioside species of cultured cells, the C18:1 LCB and the C20:1 LCB. The content of C20:1 ganglioside molecular species was low and quite constant during differentiation, comprising ∼8% of the total ganglioside species content, the C20:1 LCB appearing to be represented more in the ganglioside of the "b series" (GD1b, GT1b, and GQ1b) than in the "a series" (GM1 and GD1a). During aging in culture, for 8–22 days, the content of the C20:1 species of all gangliosides increased, being more pronounced for GM1 and GD1a.     

Densitometric quantification of brain gangliosides separated by two-dimensional thin layer chromatography

A procedure for accurate densitometric quantification of gangliosides separated by two-dimensional thin layer chromatography is reported. The procedure was set up employing 9 different pure gangliosides and was applied to the analysis of calf and pig brain gangliosides. Silica gel high performance thin layer plates, 10 × 10 cm. were two-dimensionally developed at 18–20 C with the following solvents: chloroform methanol 0.2% aqueous CaCl₂, 50/40/10 by volume, for the first run; n-propanol 17 M NH₄OH/water, 6/2/1 by volume for the second run. Ganglioside spots were visualized by spraying with an Ehrlich reagent, which is specific for sialic acid, and heating at 120 C for 15 min. The spots were quantified by sequential scanning densitometry, linear responses being obtained for ganglioside amounts on the plate ranging from 0.1 to 6 nmol as bound sialic acid. The reproducibility of densitometric responses resulted to be acceptable since the standard deviation values were lower than ± 15% of the mean values also for those ganglioside species contained in minor proportions. The ganglioside mixtures of calf and pig brain were resolved in about 20 spots. Of these 9 corresponded to gangliosides GM3, GM2, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b, which were identified with certainty and quantified. The identification of GM3 (carrying N-glycolylneuraminic acid), GD3, GD1a (carrying N-acetyl- and N-glycolyl-neuraminic acid) and GT1a was only tentative. All the other spots corresponded to unidentified gangliosides, some of them possibly new species.     

Gangliosides as paramyxovirus receptor. Structural requirement of sialo-oligosaccharides in receptors for hemagglutinating virus of Japan (Sendai virus) and Newcastle disease virus

A sensitive assay system for receptor activity of gangliosides to paramyxovirus was developed. This system involves incorporation of gangliosides into neuraminidase-treated chicken erythrocytes (asialoerythrocytes) followed by estimation of virus-mediated agglutination and hemolysis. The asialoerythrocytes coated with I-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer) were effectively agglutinated by hemagglutinating virus of Japan (HVJ, Sendai virus). The hemolysis of the asialoerythrocytes mediated by HVJ was restored to the highest level by labeling the cells with gangliosides possessing lacto-series oligosaccharide chains, i.e., I-active ganglioside, N-acetylneuraminosylparagloboside (SiaPG(NeuAc)), and i-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer). The specific receptor activity of ganglioside GD1a possessing a gangliotetraose chain was lower than those of the gangliosides described above. Gangliosides GM3, GD3, GM1a, GD1b, SiaPG(NeuGc) showed little effect on the restoration of HVJ-mediated hemolysis. On infection with Newcastle disease virus (NDV), the highest specific restoration of lysis was found in chicken asialoerythrocytes coated with SiaPG(NeuAc or NeuGc) and GM3(NeuAc or NeuGc), whereas those coated with I-active ganglioside, GD3, GM1a, and GD1b showed very low NDV-mediated hemolysis. The above results indicate that the determinants of receptor for HVJ contain sialylated branched and/or linear lacto-series oligosaccharides carried by I,i-active gangliosides and SiaPG(NeuAc) and sialosylgangliotetraose chain carried by GD1a. The determinants for NDV are carried by SiaPG(NeuAc or NeuGc) containing linear lacto-series oligosaccharide and GM3(NeuAc or NeuGc). The absence of detectable binding of free oligosaccharides obtained from I-active ganglioside and sialoglycoprotein GP-2 isolated from bovine erythrocyte membranes as HVJ receptor (Suzuki, Y., et al. J. Biochem. (1983) 93, 1621-1633; (1984) 95, 1193-1200) indicates that HVJ recognizes the sialooligosaccharides oriented out of the lipid bilayer in the cell membranes where the hydrophobic ceramide or peptide backbone of the receptor is integrated.     

Isolation and characterization of extremely minor gangliosides, GM1b and GD1 alpha, in adult bovine brains as developmentally regulated antigens

In addition to ganglioside GM1b, an unusual and extremely minor ganglioside, GD1 alpha, was efficiently isolated from bovine brain by combination of Q-Sepharose and Iatrobeads column chromatographies. In the course of purification steps, the presence of the sialidase-labile ganglioside was proved by a highly sensitive TLC/enzyme-immunostaining method. The structure was characterized by gas-liquid chromatography, permethylation study, sialidase degradation, immunostaining with specific antibodies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry. The content of the ganglioside was very small (0.016%) in the total gangliosides. This finding suggests that a synthetic pathway of asialo GM1----GM1b----GD1 alpha may exist in mammalian brains. A monoclonal antibody NA-6 that was obtained by immunizing mice with purified GM1b reacted specifically with GM1b but showed no cross-reactivity with other structurally related gangliosides such as GM1a, GD1a, and so on. Using the method of TLC/immunostaining with NA-6, GM1b was found to be strongly expressed during embryonic days 14-17 in chick brains. Thus, it is assumed that extremely minor gangliosides like GM1b and GD1 alpha found in adult brains are characterized as embryonic molecules.     

Changes in the Ceramide Composition of Rat Forebrain Gangliosides with Age

Five major gangliosides (GM1, GD1a, GD1b, GT1b, and GQ1b) were extracted and isolated by normal-phase HPLC from the forebrain of Sprague-Dawley rats of ages ranging from 3 days to 24 months. Each ganglioside was fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base moiety. At all ages, the C18:1 and C20:1 long-chain base species predominated, whereas the C18:0 and C20:0 ones represented 1-3% of the total. The C18:1 long-chain base species, predominant at 3 days (91-96%), diminished with age and reached, at 2 years, 73%, 65%, 61%, 59%, and 45% of the total for GD1a, GM1, GT1b, GD1b, and GQ1b, respectively. The content of the C20:1 long-chain base species, low at birth (4-9%), increased with age in all gangliosides and reached, at 2 years, 27-55% of the total. The developmental behavior of the ganglioside species containing the C18:1 long-chain base was characterized by the following: (a) a biphasic profile with a maximum around 15 days for GD1a, the most abundant ganglioside at all ages; (b) an increase until 6 months for GM1; (c) a sharp decrease until 30 days, followed by leveling for GT1b; and (d) a low, constant level for GD1b and GQ1b. All the ganglioside species containing the C20:1 long-chain base showed a constant increase during development, the increase being more marked in the first 30 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ganglioside-specific binding protein on rat brain membranes

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