Effects of pH on the binding of Clostridium botulinum type E derivative toxin to gangliosides and phospholipids

Abstract Clostridium botulinum type E derivative toxin directly bound to gangliosides G_T1b, G_D1a, and G_Q1b but not to G_M1 or G_D1b at pH 5.0 or above, At the same pH values, it bound to negatively charged phospholipids but not to noncharged ones. At pH 4.0, it bound to any of gangliosides and phospholipids including G_M1, G_D1a, and non-charged phospholipids. It bound to ceramide, a hydrophobic component of ganglioside and also to sphingomyelin, a phospholipid containing a ceramide moiety, only at pH 4.0. It bound to ceramide and sphingomyelin less firmly than to other phospholipids at pH 4.0. We assume that botulinum toxin adheres to the neural cell surface mainly by sialic acid-specific and charge-dependent binding possibly aided by nonspecific hydrophobic(toxin)-hydrophobic(lipids, mainly phospholipids) interaction.     

Cellular localization of gangliosides in the developing mouse cerebellum: analysis using the weaver mutant

Abstract: The distribution of gangliosides was studied in the weaver ( wv/wv ) mutant mouse, where the vast majority of postmitotic granule cell neurons die prior to their differentiation. The wv mutation also shows a dosage effect, as granule cell migration is slowed or retarded in the + /wv heterozygotes. By correlating changes in ganglioside composition with the well-documented histological events that occur during cerebellar development in the normal (+/+), heterozygous ( +/wv ), and weaver ( wv/ wv ) mutant mice, information was obtained on the cellular localization and function of gangliosides. Ganglioside G_M1 may be enriched in granule cell growth cones and play an important role in neurite outgrowth. A striking accumulation of G_M1, which may result from altered metabolism, occurred in the adult wvlwv mice. G_D3 was heavily concentrated in undifferentiated granule cells, but was rapidly displaced by the more complex gangliosides during differentiation. G_D1a became enriched in granule cells during formation of synaptic and dendritic membranes, whereas G_T1a appeared enriched in Purkinje cell synaptic spines. A possible fucose-containing ganglioside was quantitated only in the wvlwv mice. Ganglioside G_T1b became enriched in granule cells during synaptogenesis, whereas G_Q1b became enriched in these cells after synaptogenesis. The concentrations of G_T1b and especially G_Q1b increased continuously with age. Our results provide further evidence for a differential cellular enrichment of gangliosides in the mouse cerebellum and also suggest that certain gangliosides may be differentially distributed within the membranes of these cells at various stages of development.     

GANGLIOSIDE COMPOSITION AND BIOSYNTHESIS IN CULTURED CELLS DERIVED FROM CNS

Abstract— Cultured mouse neuroblastoma cells (clone N18) contained a homologous series of gangliosides, G_M3, G_M2, G_M1 and G_D1a; the total lipid bound sialic acid (LBSA) was 3.3 nmol per mg of protein, of which G_D1a comprised two-thirds. In contrast, neonatal hamster astrocytes (clone NN) and human glioblastoma cells (Cox clone) contained mainly G_M3, which represented 95% of the 2 nmol of LBSA per mg protein in these cells. When the cells were grown in the presence of [¹⁴C]galactose, label was incorporated into all of the gangliosides isolated from the cells. The labeling pattern corresponded to the ganglioside composition of the cell lines; G_D1a was more extensively labeled in N18 cells and G_M3 was the major labeled ganglioside extracted from glial cells. In addition to in rivo biosynthesis, in vitro synthesis of gangliosides was also determined. The activities of five glycosyltransferases of the ganglioside biosynthetic pathway were measured in homogenates of the three cell lines. The neuroblastoma cells contained all five enzyme activities whereas the two glial cell lines were deficient in UDP- N -acetylgalactosamine: G_M3 N -acetylgalactosaminyltransferase activity, which catalyzes the synthesis of G_M2 from G_M3. The results indicated that cells of neuronal origin contain the more complex gangliosides associated with CNS and the requisite biosynthetic enzymes and that cells of glial origin are missing these complex gangliosides and the key glycosyltransferase required for their synthesis.     

Myelin Gangliosides in Vertebrates

Abstract: A phylogenetic survey of brain myelin ganglioside patterns and concentrations has been carried out on 16 vertebrate species. Gangliosides were isolated from purified myelin and found to vary in concentration from 25 μg of sialic acid per 100 mg of myeh for goldfish to a value of 395 for turkey. The latter species had approximately equivalent amounts of G_M1 and G_M4 as the two major gangliosides. The 11 mammals studied all had G_M1 as the major ganglioside, with variable amounts of G_M4; rhesus monkey and human had 20-25% G_M4, whereas the others had less than 10%. Amphibia and fish myelin contained the least total ganglioside, with patterns that showed relatively little G_M1 and no detectable G_M4. Alligator myelin was unique in having a total concentration as high as the avian species, but a pattern with predominantly diand trisialo gangliosides.     

Enhancement by Gangliosides of the Binding of Serotonin to Serotonin Binding Protein

Abstract: Several gangliosides, especially G_D3 (disialosyllactosyl ceramide) in the presence of another lipid (lecithin) were found to enhance the binding of serotonin to serotonin binding protein (SBP) severalfold. In our conditions, this enhancement was linear to a concentration of 2.7 × 10⁻⁶I G_D3 and a three- to fivefold increase in binding capacity of SBP was obtained with 8.8 × 10⁻⁶ M. The addition of this ganglioside led to an increase of serotonin binding sites, but not to an increase in the affinity of SBP to serotonin. Optimal binding capacity was found with a ratio of lecithin to ganglioside of 6: 1 (w/w). No binding was found in the absence of either SBP or Fe²⁺ (binding of serotonin to SBP is dependent on Fe²⁺). Other glycosphingolipids (sulfatide, G_D1a, G_D1b, G_M1) showed lesser effects at low concentration, whereas asialo-G_M1, cytolipin H, galactocerebroside and G_M3 had insignificant effects. Since earlier studies suggested a storage role for serotonin binding protein, the interaction of gangliosides with this protein may regulate the concentration of the biogenic amine in the synapse.     

Preparation and Properties of Antibodies to GD3 and GM1 Gangliosides

Abstract: Gangliosides G_D3 and G_M1 were coupled to proteins by their car-boxyl groups and antisera were raised against the complexes. Anti-ganglioside antibodies were isolated by affinity chromatography on ganglioside-amino-propyl silica gel columns and the specificity of the antibodies was determined by a quantitative microcomplement fixation assay. Antibodies to G_D3 were highly specific and did not crossreact with G_M3, lactosyl ceramide, or other glycolipids. Purified antibodies to G_M1, in contrast, crossreacted with asialo-G_M1, G_D1b and to a lesser extent, G_M2 and asialo-G_M2. A derivative of G_M1, containing a C-7 sialic acid residue produced by periodate oxidation, reacted with the anti-G_M1 antibodies almost as readily as with G_M1. The specificities of anti-G_M1 antibodies elicited by the covalent ganglioside-protein complexes were similar to those produced by immunization with noncovalent complexes of G_M1 and methylated bovine serum albumin. The ganglioside-protein complexes described here should be useful for preparing antibodies to polysialo-gangliosides that contain neuraminidase-sensitive linkages.     

Neurites and Somata of Sensory and Sympathetic Neurons in Culture Contain Multiple Species of Ganglio sides

Abstract: We have used tissue culture methods to study the capacity of sensory and sympathetic neurons to synthesize gangliosides. Under appropriate culture conditions, explants of dorsal root or superior cervical ganglia generate an extensive halo of ncurites, which is substantially free of contaminating cells. The cultures incorporate enough [³H]glucosamine into glycolipids to allow biochemical characterization. Gangliosides synthesized by the cells are extracted and freed of radioactive precursors and other lipids by column chromatography. Synthesized material comigrates on (thin-layer chromatograms with the gangliosides, G_Q, G_T, G_D1a, G^D1b, and G_M1, In addition, a substantial amount of unidentified labeled material migrates in a region between the mono- and disialo bands. The ganglioside profiles show that a similar spectrum of gangliosides is found on the neurites and somata of a particular class of neuron. Furthermore, the ganglioside compositions of the two types of neuron studied appear to be similar. We conclude that both sensory and adrenergic autonomic neurons synthesize gangliosides of each of the major classes and that representatives of each class are found in both somata and neurites. Dispersed cell cultures of the superior cervical ganglion synthesize a repertoire of gangliosides similar or identical to that of explant cultures.     

Myelin Gangliosides: An Unusual Pattern in the Avian Central Nervous System

Abstract: Gangliosides were isolated from purified myelin obtained from brain and spinal cord of mature chickens and pigeons. Total concentrations were approximately two- to fivefold greater than for previously reported mammalian species, and their patterns also differed in containing significantly more sialosylgalactosylceramide (G_M4). The latter comprised one-third to one-fourth of total myelin ganglioside, approximately equivalent to G_M1 (II³NeuNAc-GgOse₄Cer). As in mammals, G_M4 of avian CNS appeared to be localized in myelin. Fatty acids of this ganglioside included both the hydroxy- and unsubstituted types. and, long-chain bases were almost entirely C₁₈. Ganglioside G_M1 split into two closely migrating bands on TLC, the slower of which resembled mammalian G_M1 in having stearate as the main fatty acid with a measurable amount (10%) of C₂₀-sphingosine; the faster band had predominantly longer-chain fatty acids and very little C₂₀-sphingosine.     

THE EFFECT OF DEVELOPMENT ON THE GANGLIOSIDES OF RAT AND PIG BRAIN

Abstract— The ganglioside content of the forebrain, brain stem and cerebellum have been studied, in the rat at various ages from 1 day to 27 months, and in the pig at various ages from 93 days gestation to 30 months. Each part of the brain was analysed for total ganglioside NANA and for four major gangliosides (G_Ml, G_D1a, G_Dlb and G_T1 in the nomenclature of S vennerholm , 1963). In the rat forebrain, the concentration of ganglioside NANA rose rapidly between 1 and 21 days after birth, fell to 3 months and subsequently rose to a mature value at 6 months. In the rat cerebellum, the peak concentration was reached at 2 months and the lower adult value at 9 months, whilst in the brain stern, the concentration rose more slowly and had a broad peak from 15 days to 2 months. Values are also given for the changes in the total amounts in each brain part. The changes in the concentrations and total amounts of ganglioside NANA, in the three parts of the pig brain were, on the whole, similar to those in rat brain except that the percentage distribution of the major gangliosides had almost attained the mature pattern at birth. In the forebrain of both species, the disialoganglioside, G_D1a, accounted for the highest percentage of the total gangliosides. The results are discussed with respect to their possible structural significance.     

GANGLIOSIDES IN DEVELOPING MOUSE BRAIN MYELIN

Abstract— Gangliosides were isolated from myelin prepared from mouse brains of different ages (23 to 490 days). Quantitative estimation of lipid-bound sialic acid levels indicated a gradual increase from 560 μg/g of myelin at 23 days to about 1200 μg/g of myelin at older ages. The major ganglioside in all myelin preparations was the monosialoganglioside G₄ (G_M1). However, considerable amounts of di- and trisialo species also were found in myelin from young animals. In contrast to human myelin in which the monosialoganglioside, sialosylgalactosylceramide (G₇) was highly enriched (L edeen et al. , 1973), a much smaller enrichment of this ganglioside was noticed in mouse brain myelin. Ganglioside G₇ was not detectable in myelin until the animals were 35 days old, and showed a slight increase with increasing age after that. The results strongly indicated that the concentration of G₇ in myelin is species specific and age dependent. The study also demonstrated that the ganglioside accretion in developing mouse brain myelin was attributable to the enrichment of monosialogangliosides G₄ (G_M1), G₅ (G_M2) and G₇ at the expense of polysialogangliosides.     

Monoclonal antibodies against pertussis toxin subunits

Abstract Twenty monoclonal antibodies (mAbs) reacting with cholera toxin (CT) of Vibrio cholerae strain 569B were characterized in cross-section and G_M1 ganglioside inhibition assays. MAbs were characterized by reaction with CT and Escherichia coli heat-labile porcine strain (LT_p) and human strain (LTh) enterotoxins, and by G_M1 ganglioside inhibition of mAb binding. Eight of 10 CT-A specific and 3 of 10 CT-B-specific mAbs cross-reacted with LTh and LTp. G_M1 ganglioside inhibited reactions of the CT-B cross-reacting antibodies. Results showed that these epitodes common to the B subunit of CT and LT are located in or near the G_M1 ganglioside binding region, and that the G_M1 ganglioside-binding region of LT differs from that of CT.     

Neurological Mutation Characterized by Dysmyelination in NCTR-Balb/C Mouse with Lysosomal Lipid Storage Disease

Abstract: Morphological and biochemical studies were performed on the CNS of neurologically affected NCTR Balb/C mouse. Histological and electron microscopic techniques demonstrated severe myelin deficiency in the affected brains. Neither the presence of lipid-containing macrophages nor reactive gliosis was apparent. Analysis of myelin-associated lipids and proteins revealed prominent depletion of galactocerebroside, sulfatide, and proteolipid proteins. In contrast to the scarcity of myelin specific constituents a marked accumulation of G_M2 and G_M3 gangliosides and several neutral glycolipids, i.e., glucocerebroside, lactosylceramide, gangliotriaosylceramide, and gangliotetraosylceramide were found in affected CNS. These abnormalities were already apparent in 12-day-old pups as well as in 65-day-old mice. A significant deficit in the proportion of long-chain fatty acids (C₂₄), notable in both normal and α-hydroxy acids of cerebrosides from affected white matter, was measured. The lack of reactive gliosis, the observed depletion of galactocerebroside and sulfatide at the early age of 12 days, and the relative decrease in long-chain fatty acids in affected CNS strongly suggest a defect in myelinogenesis in this mutant rather than a secondary process of myelin breakdown.     

LOCALIZATION, SOLUBILIZATION AND PROPERTIES GANGLIOSIDE TRANSFERASES IN RAT BRAIN OF N-ACETYLGALACTOSAMINYL AND GALACTOSYL GANGLIOSIDE TRANSFERASES IN RAT BRAIN

Abstract— The subcellular distributions of UDP-N-acetylgalactosamine: G_M3 N-acetyl-galactosaminyl transferase and UDP-galactose: G_M2 galactosyl transferase, two enzymes involved in the biosynthesis of gangliosides, were determined in the 7-day-old rat brain by means of synaptosomal fractionation techniques. The enzymes were located on the synaptic membranes and appeared to be closely associated with gangliosides and acetylcholinesterase. Solubilization of the transferase enzymes from the microsomal particles was achieved and differed from the solubilization of acetylcholinesterase and of the total membrane protein. Competition studies suggest that the ^N-acetylgalactosaminyl transferase involved in the formation of G_M2 from G_M3 is different from the N-acetylgalactosaminyl transferase involved in the formation of GalNAoGal-Glc-ceramide from Gal-Glc-ceramide, whereas in contrast, both the formation of G_M1 from G_M2 and of Gal-GalNAc-Gal-Glcceramide from GalNAc-Gal-Glc-ceramide appear to be catalysed by the same galactosyl transferase.     

Sandhoff disease: ganglioside GM and asialo-GM2 accumulation in the cerebrospinal fluid

In an earlier investigation it was found that ganglioside G_M2 the typical neuronal storage product in GMz-gangliosidosis, was accumulated also in the cerebrospinal fluid (CSF) of a Tay-Sachs patient. The asialo derivative of ganglioside G_M2, G_M2, which is a concomitant storage product in the brain of these patients, was not detected in the CSF of the patient investigated (BERNHEIMER, 1968). In the present account, a report is made on the accumulation of ganglioside G_M2 and of G_M2 in the CSF of a patient affected by Sandhoff disease.     

EFFECT OF DEVELOPMENT ON THE GANGLIOSIDES OF HUMAN BRAIN

Abstract— The ganglioside content and composition of brains from twenty-five human fetuses, three new-born babies and ten children, were studied. The ages ranged from 13 weeks gestation to 26 months postpartum. Each brain was divided into forebrain. cerebellum and brain stem. The concentration of total gangliosides rose to a plateau at different stages of development in the different parts, whereas the total amount reached a constant value at 9 months of age in each part. The developmental profile of individual gangliosides differed in the different parts of the brain. Thus, in the forebrain G_D1a. and in the cerebellum G_D1a rose to become the major gangliosides. The brain stem showed little change in its ganglioside pattern during the developmental period studied. The possible significance of these charges in the gangliosides during development is discussed.     

STUDIES ON THE BIOSYNTHESIS OF GLYCO-SPHINGOLIPIDS IN CULTURED MOUSE NEUROBLASTOMA CELLS: CHARACTERIZATION AND ACCEPTOR SPECIFICITIES OF N-ACETYLNEURAMINYL- AND N-ACETYLGALACTOSAMINYLTRANSFERASES

Abstract— The pathway of biosynthesis of N -acetylgalactosamine-containing gangliosides in mouse neuroblastoma has been studied using NB41A cells grown in monolayer tissue culture. Cell-free enzyme preparations catalyzed the transfer of NeuNAc from CMP-NeuNAc to lactosylceramide (GL-2a), to form G_M3. Asialo-G_M2 was neither an acceptor nor a competitive inhibitor of the sialyltransferase (CMP-NeuNAc: GL-2a N-acetylneuraminyltransferase, EC 2.4.99.-) under a variety of experimental conditions. Enzyme preparations also contained an N -acetylgalactosaminyltransferase (UDP-GalNAc. G_M3 N -acetylgalactosaminyltransferase, EC 2.4.1.-) which catalyzed the conversion of G_M3 to G_M2. No significant transfer of N -acetylgalactosamine to GL-2a could be demonstrated. The results of the glycosyltransferase assays support the concept that the first NeuNAc of brain gangliosides is introduced into GL-2a. The present data suggests that the occurrence of asialo-G_M2 in NB41A cells under some culture conditions is a consequence of the catabolism of higher gangliosides.     

Modifications of Ganglioside Patterns in Human Meningiomas

Abstract: Ganglioside content and distribution were determined in control meninges and in 30 human meningiomas belonging to four different histological types. Irrespective of the histological classification all meningiomas showed a ganglioside content significantly higher than that of control meninges. The analysis of ganglioside distribution in each meningioma showed that in the majority of the cases the increase of ganglioside content was primarily the result of selective accumulation of ganglioside G_M3; in the remaining cases ganglioside G_M1 was present in a significantly higher amount than in the control dura mater and leptomeninges. A common feature of both types of meningiomas is a simplification of ganglioside pattern, with a shift from the polysialylated to the monosialylated forms. A tentative classification of meningiomas into "G_M3-rich" and "G_M1-rich" types, together with an explanation for the selective accumulation of these two types of ganglioside, is proposed.     

Retinal Gangliosides in RCS Mutant Rats

Abstract: The distribution of retinal gangliosides was studied in normal and mutant rats with retinal dystrophy at 30 and 180 days of age. The loss of photoreceptor cells in the retinal dystrophic RCS rats was not associated with a significant reduction in the relative distribution of any of the major retinal gangliosides. The loss of photoreceptors, however, caused a marked increase in total retinal ganglioside concentration. These findings suggest that photoreceptor cells contain a low concentration of gangliosides and that no major retinal ganglioside is localized or concentrated in these cells. The cellular localization and function of the most abundant retinal ganglioside, G_D3, is discussed.     

DEVELOPMENTAL PATTERNS OF GANGLIOSIDES AND OF PHOSPHOLIPIDS IN CHICK RETINA AND BRAIN

Abstract— The changes in phospholipids and gangliosides during ontogenesis of chick retina have been compared with those in brain. Three phases of accumulation of ganglioside NeuNAc in the retina were detected. In contrast, brain NeuNAc rapidly increased during embryonic life until hatching, followed by a slower increase up to the adult stage. The phospholipid changes in retina and in brain occur in a-similar manner to the variations observed for gangliosides, however in retina the changes of phospholipid content are less marked than in brain, during embryonic life. There were marked changes in the retina and brain ganglioside patterns with age. G _{d 3} and G _{d 1b} decreased rapidly in per cent; correspondingly, G _{d 1a} increased during embryonic life and became the major ganglioside in place of G _{d 3}. There was a similarity between ganglioside patterns of chick retina and brain. Except for some slight variations during embryonic life, the retinal phospholipid pattern did not change noticeably.     

Effect of Malnutrition and Subsequent Rehabilitation on the Development of Mouse Brain Myelin

Abstract: Malnutrition in mice from birth resulted in myelin of brain having higher than normal molar proportions of cholesterol and phospholipids relative to a molar unit of cerebroside + sulphatide. This was found at all ages between 20 and 60 days, and the molar ratio of these lipids in older animals was comparable to that in the younger controls. The phospholipid and the ganglioside patterns were also immature for age. The phospholipid composition was characterized by lower molar proportions of ethanolamine phosphoglyceride (EPG) and sphingomyelin (SPh) and higher proportion of choline phosphoglyceride (CPG), and the ganglioside pattern was characterized by higher molar proportions of the disialogangliosides G_Dla and G_Dlb and markedly lower proportion of the monosialoganglioside G_M1. Malnutrition imposed from 30 days of age did not affect the contents of the major lipids (and so their molar ratio), but within the phospholipids there was a small but significant deficit of SPh, which was compensated by a higher content of CPG. The ganglioside pattern was as if the animals were malnourished from birth. Nutritional rehabilitation up to 60 days of age subsequent to malnutrition for the first 30 days fully corrected the ganglioside pattern, but not the molar ratio, of the major lipids (because of persistent deficit in cerebroside + sulphatide) and the composition of the phospholipids (because of small but significant deficit of SPh). The results indicate that malnutrition instituted at any time during the entire programme of myelination can affect one or other aspect of myelin development, and nutritional rehabilitation of animals malnourished in early life cannot fully correct this developmental gap.