Role of membrane gangliosides in the binding and action of bacterial toxins

Summary Gangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside G_M1. Toxin-resistant cells which lack G_M1 can be sensitized to cholera toxin by treating them with G_M1. Cholera toxin specifically protects G_M1 from cell surface labeling procedures and only G_M1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that G_M1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to G_M1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. G_M1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin ofE. coli is very similar to cholera toxin in both form and function and can also use G_M1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides G_D1b and G_T1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established G_M1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.     

Developmental profiles of gangliosides in mouse and rat cerebral cortex

Summary Developmental profiles of 11 gangliosides, concentration of lipid- and glycoprotein-bound sialic acid, and activity of AChE of the rat and mouse cerebral cortex were followed from the 7^th day of gestation to the 21^st postnatal day.There are three main changes in ganglioside concentration, which are similar in both species. The first occurs from gestation day 10 until birth: parallel to decreased proliferation, cell migration, and neuroblast differentiation, G_M3 and G_D3 in mouse cortex and G_D3 in the rat's decreases in favor of G_Q1b, G_T1b, and G_D1a.The second occurs from birth until the first postnatal week: Parallel to increased growth and arborization of dendrites and axons as well as synaptogenesis in rats and mice, there is a two-fold rise of G_D1a, whereas G_Q1b and G_T1b remain on a nearly constant level. Concomitantly, G_M3 and G_D3 decreases. The third period of ganglioside changes starts in the second postnatal week, parallel to onset of myelination, and is characterized by an increase of G_M1 in parallel with a decrease of the polysialogangliosides G_T1b and G_Q1b.     

Preparation of the tritiated molecular forms of gangliosides with homogeneous long chain base composition

Gangliosides G_M2, G_M1 and G_D1b were radiolabelled at C-6 of the terminal galactose orN-acetylgalactosamine by the galactose oxidase/[³H]NaBH₄ method; gangliosides G_M2, G_M1, Fuc-G_M1 and G_D1a were radiolabelled at C-3 of the long chain base by the 2,3-dichloro-5,6-dicyanobenzoquinone/[³H]NaBH₄ method.By application of an original HPLC procedure, eight different molecular species were prepared from each labelled ganglioside. Each of these species was characterized by the presence of one of the following long chain bases:erythro C18 sphingosine,threo C18 sphingosine,erythro C18 sphinganine,threo C18 sphinganine,erythro C20 sphingosine,threo C20 sphingosine,erythro C20 sphinganine andthreo C20 sphinganine.From G_D1b only the species containing theerythro forms of long chain bases were obtained.The individual molecular species were more than 99% homogeneous and had a radiopurity better than 99%. The molecular species of the same ganglioside, radiolabelled at C-3 of the long chain base, had identical specific radioactivity, namely 1.17, 1.25, 0.85 and 1.28 Ci/mmol for G_M2, G_M1, Fuc-G_M1 and G_D1a respectively. The molecular species of the same ganglioside, radiolabelled at C-6 of terminal galactose orN-acetylgalactosamine, had similar specific radioactivity, namely 1.34–1.40, 1.44–1.51, 1.37–1.44 Ci/mmol for G_M2, G_M1 and G_D1b respectively.     

Electron paramagnetic resonance studies on the fluidity and surface dynamics of egg phosphatidylcholine vesicles containing gangliosides

The influence of different gangliosides (G_M1, G_D1a, G_T1b) on the fluidity and surface dynamics of phosphatidylcholine small unilamellar vesicles was studied by electron paramagnetic resonance. 5-and 16-nitroxystearic acid, sounding respectively the region close to the surface and that close to the hydrophobic core of the vesicle, were employed as spin-label probes. The signals released by 5-nitroxystearic acid showed that the presence of gangliosides reduced the mobility of the hydrocarbon chains around the probe. The effect increased by increasing ganglioside concentration, and diminished from G_M1 to G_D1a and G_T1b. The decrease of membrane fluidity was also monitored by the 16-nitroxystearic acid probe. On addition of Ca²⁺ the fluidity of ganglioside-containing vesicles (as signalled by the 5-nitroxystearic acid probe) promptly decreased, thereafter returning slowly to the original value. It is suggested that gangliosides cause strong side-side head group interactions on the bilayer surface -between ganglioside oligosaccharide chains and between ganglioside and phosphatidylcholine polar portions - which lead the lipid chains to assembly in a more rigid fashion. The influence of Ca²⁺ is interpreted as due to lateral phase separation in the vesicle membrane. This phenomenon can be related to the formation or stabilization of ganglioside clusters on the vesicle surface.     

Specific binding of peanut agglutinin to GM1-doped solid supported lipid bilayers investigated by shear wave resonator measurements

This study deals with the specific interaction between the lectin peanut agglutinin (PNA) from Arachis hypogaea and the ganglioside G_M1 which was incorporated in a solid supported lipid bilayer immobilized on a gold electrode placed on top of an AT-cut quartz crystal. Bilayer formation was reached by self-assembly processes. The first monolayer consists of octanethiol attached to the gold surface via chemisorption and the second monolayer was immobilized by vesicle fusion on the preformed hydrophobic surface. We managed to keep unspecific binding to a minimum by using a phospholipid matrix consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Lectin binding to ganglioside G_M1 containing membranes was determined by a decrease of the resonant frequency of the quartz crystal. The minimum amount of receptor within the membrane which is necessary to obtain a complete protein monolayer was found to be less than 2 mol%. The adsorption isotherm of PNA to G_M1 was recorded and analyzed to be of Langmuir type, exhibiting a binding constant of PNA to the ganglioside of 8.3 ⋅ 10⁵ M^–1. The good agreement of the calculated Langmuir adsorption isotherm with the obtained experimental data implies that protein multilayers are not formed and that interactions between the adsorbents can be neglected. Furthermore, the association constants of two different saccharides, β-Galp-(1 → 3)-GalNAc exhibiting a strong binding to PNA in solution, and β-D-galactose with a much lower affinity were estimated by determining the equilibrium concentration of PNA attached to the surface. Moreover we were able to remove the attached lectin monolayer by digestion of the protein with pronase causing an increase in the resonant frequency which almost reversed the frequency shift to lower frequencies during adsorption. An even more complex system was built up by the use of digoxigenin-labeled PNA which also binds to the solid supported membrane containing the receptor G_M1. The immobilized lectin was recognized by anti-digoxigenin-F_ab-fragments, which is measurable by a further decrease of the resonant frequency. For all binding processes we found larger frequency shifts for a complete protein monolayer than predicted by Sauerbrey's equation, clearly showing that in addition to mass loading viscoelastic changes occur at the lipid-protein interface. Received: 22 July 1996 / Accepted: 12 September 1996     

Activation of human T lymphocytes by ganglioside-containing liposomes

We investigated the in vitro stimulatory effect of ganglioside (G_M3, G_D1a, G_D1b, G_T1b, or G_Q1b)-containing liposomes on human immune cells. The effect of ganglioside-containing liposomes on the concentration of cytoplasmic free calcium ions ([Ca²⁺]₁) in human immunocytes was examined using the confocal laser fluorescence microscopic method. The G_D1a- and G_T1b-containing liposomes significantly increased [Ca²⁺]₁ of human T lymphocytes compared with the G_M3-, G_D1b- and G_Q1b-containing ones. The response of CD8⁺ and CD4⁺ cells was significantly higher than that of CD20⁺ cells. Our results show that the increase in [Ca²⁺]_i may be caused by not the number of sialic acids contained in the gangliosides but the conformation of the sialic acid moiety to protrude exteriorly from the liposomal membrane surface, and that a sort of receptor recognizing the sialic acid moiety exists on human T lymphocytes (both CD8⁺ and CD4⁺ cells), which may be involved in the activation of the cells. The present results are almost the same as those obtained for the rat T lymphocyte system previously reported. This clearly confirms that a sort of ganglioside surely stimulates T lymphocytes directly, which is not species-specific but conserved in humans and rats among animal species.     

STALOSYLGALACTOSYL CERAMTDE AS A SPECIFIC MARKER FOR HUMAN MYELIN AND OLIGODENDROGLIAL PERIKARYA: GANGLIOSIDES OF HUMAN MYELIN, OLIGODENDROGLIA AND NEURONS

Gangliosides were isolated from human brain myelin, oligodendroglia, and neurons. Quantitative analysis revealed the following ganglioside contents: myelin, 2.0; neurons, 1.3; and oligodendroglia, 0.35 μg ganglioside sialic acid per mg protein. Myclin had a relatively simple ganglioside pattern with G_M4 and G_M1 as the predominant ganglioside species. The ganglioside pattern of oligodendroglia was quite complex and it resembled that of whole white matter rather than that of myelin. A high concentration of G_M4 was found in oligodendroglial fractions in addition to G_M1, G_D1a, G_D1b, and G_T1b. The usually- minor brain gangliosides G_M3, G_M2, and G_M3 were also enriched in oligodendroglia. The neuronal ganglioside pattern was generally similar to the pattern of whole gray matter. Both neurons and whole gray matter contained very low amounts of G_M4. These results indicate that G_M4 is specifically localized in myelin and oligodendroglia of the CNS. Evidence is also presented that myelin, but not oligodendroglia, is the major reservoir of human white matter G_M1 and G_M4.     

The detection of picomolar quantities of GD1b,GT1b and GQ1b on thin-layer chromatograms by the direct binding of125I-labeled tetanus toxin,fragment C

The¹²⁵I-labeled fragment C of tetanus toxin was found to bind specifically to the gangliosides G_D1b, G_T1b, and G_Q1b when applied to thin-layer chromatograms on which a mixture of gangliosides had been resolved. As little as 2.5 pmoles of these gangliosides could be detected by this method. In addition to factors determined by the sample, namely the amount and species of gangliosides present, optimal binding of the¹²⁵I-labeled fragment C also depended upon the iodination procedure used to generate the probe, the toxin concentration, and the concentration, buffer type, pH, and ionic strength of the binding solution. This new technique was shown to be a sensitive method for the detection and identification of specific gangliosides originating from extraneural or neural cells.Nomenclature: The gangliosides follow the nomenclature system of Svennerholm [Eur J Biochem (1977) 79:11–21] G_M3 II³NeuAc-LacCer - G_D3 II³(NeuAc)₂-LacCer - G_M1 II³NeuAc-GgOse₄Cer - G_D1a IV³NeuAc, II³NeuAc-GgOse₄Cer - G_D1b II³(NeuAc)₂-GgOse₄Cer - G_T1b IV³NeuAc, II³(NeuAc)²-GgOse₄Cer - G_Q1b IV³(Neu-Ac)₂, II³(NeuAc)₂-GgOse₄Cer - G_P1b IV³(NeuAc)₃, II³(NeuAc)₂-GgOse₄Cer     

High-performance thin-layer chromatography and densitometric determination of brain ganglioside compositions of several species.

Improved resolution of complex brain ganglioside mixtures was achieved by high-performance thin-layer chromatography. The percentage distribution of individual gangliosides was then determined by direct densitometric seanning, employing a transmittance mode, of the resorcinol-positive spots on the plate. As little as 90 pmol (29 ng) of lipid-bound sialic acid could be detected with a good signal-to-noise ratio. A linear detector response was observed up to 3.0 μg of lipid-bound sialic acid. The brain white matter ganglioside patterns of eight animal species, including human, chimpanzee, monkey, chicken, bovine, sheep, and pig, were examined in detail. In addition, human brain gray matter, rat cerebral, rat brain gray matter, and rat cerebellar ganglioside patterns were also studied. Ganglioside G_M4 (G₇) was found to be one of the major components in primate and chicken brain white matter, but it represented only a minor ganglioside in other species. Other major gangliosides in all brain samples studied were G_M1, G_D1a, G_D1b, and G_T1b. G_M1 was more abundant in white matter than in gray matter. G_T1a, a recently discovered ganglioside species, was found in all species examined, but was most abundant in the rat cerebellum. The latter source also contained high proportions of G_T1b and G_Q1b.     

Stimulation of platelet adhesion and activation by ganglioside GD3 adsorbed to plastic

Platelet interaction with gangliosides G_D3, G_M3, G_M1, G_D1a and G_T1b has been investigated. These gangliosides were previously identified in the vessel wall and ganglioside G_D3 was found to accumulate selectively in the intima of atherosclerotic vessels. Gangliosides were adsorbed to plastic and incubated with ⁵¹Cr-labeled platelets. The adhesion of gel-filtered platelets to ganglioside G_D3 was 3–4-times higher than to other immobilized gangliosides and to albumin-treated plastic. As was shown by scanning electron microscopy, G_D3 stimulated intensive spreading of adherent platelets and formation of surface-bound aggregates, while only single unspread platelets were present on the surfaces coated with other gangliosides. G_D3 isolated from milk and from human aorta possess the same stimulating activity. Platelet adhesion to G_D3 decreased significantly in the presence of the stable prostacyclin analogue, carbacyclin.     

Formation and turnover of myelin ganglioside

—In young adult rats, the formation and turnover of G_M1-ganglioside in myelin were compared with the formation and turnover of G_M1-ganglioside in whole brain and of total lipids in whole brain and myelin, after injection of d-[1-¹⁴C]glucosamine. During the first 24 hr after injection, the specific activity of G_M1-ganglioside in myelin was less than 25 per cent of that of G_M1-ganglioside in whole brain. The specific activity of ganglioside in whole brain was maximal at 24 hr and then declined steadily during the next 3 months, whereas the specific activity of G_M1-ganglioside in myelin continued to increase and did not reach a peak until about one month after injection, by which time its specific activity had increased five-fold. Consequently, the specific activity of G_M1-ganglioside in myelin was 50 per cent higher than ganglioside in whole brain after one month. These differences in the formation and turnover of G_M1-ganglioside in myelin and of whole brain are similar to those of other lipids of myelin and of whole brain, indicating that the metabolic activity of myelin ganglioside is similar to myelin lipids, but differs from whole brain lipids or whole brain gangliosides. These data provide additional evidence that ganglioside in myelin is an intrinsic constituent of the myelin sheath. G_T1 (G₁), G_D1b, (G₂), G_D1a (G₃), G_M1 (G₄), G_M2 (G₅), G_M3 (G₆).     

Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid.

Radioactive gangliosides, N-[¹⁴C]-acetylneuraminylgalactosylglucosylceramide ([¹⁴C]G_M3) and N- [¹⁴C]-acetylneuraminylgalactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl]-galactosylglucosylceramide ([¹⁴C]G_D1a), were synthesized from CMP-[¹⁴C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G_M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [¹⁴C]G_M3 and [¹⁴C]G_D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K_m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [¹⁴C]G_M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.     

Topographic studies of gangliosides of intact synaptosomes from rat brain cortex

Gangliosides in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups with galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components fractionated on diatrizoate to give four synaptosomal membrane fractions (A-D) and a mitochondrial pellet (E). Fractions A and B represent synaptosomal plasma membranes. When intact synaptosomes were labeled, the major portion of the total radioactivity incorporated into ganglioside fraction was found to be in G _M1 ³ species. With isolated membrane fractions little selectivity was seen: (1) more label was present compared to intact synaptosomes, and (2) zones corresponding to G_M2, G_M1, G_D1a, G_D1b were the major gangliosides labeled. The results confirm the conclusion that membrane fractions A and B are derived from the exposed synaptosome surface and also show that G_M1 is the major ganglioside species available for enzyme oxidation at the surface.     

Ethidium bromide binding to native and denatured poly(dA)poly(dT)

Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·10⁶ M⁻¹≤K≤1.4·10⁶ M⁻¹, free energy ΔG ^o=−8.7±0.3 kcal/mol, enthalpy ΔH ^o≅0, and entropy ΔS ^o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T _m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10⁻⁵ M EtBr) increased by 17°C as compared with the ΔT _m of free homopolymer, whereas the half-width of the transition (T _m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K ₁=1.7·10⁵ M⁻¹; ΔG ^o=−8.10±0.03 kcal/mol) and weak (K ₂=2.9·10³ M⁻¹; ΔG ^o=−6.0±0.3 kcal/mol).The ΔG ^o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.     

Shedding of Gangliosides by Human Medulloblastoma Cells

Shedding of immunosuppressive gangliosides is an important characteristic of both experimental and human tumors. Using a medulloblastoma cell line, Daoy, with a very high ganglioside expression (141 ± 13 nmol/10⁸cells) and a well-characterized ganglioside complement, we have now studied ganglioside shedding by human brain tumor cells. Shedding of gangliosides, quantified by metabolic radiolabeling, was significant (169 pmol/10⁸cells/h) and was generalized with respect to the major ganglioside carbohydrate structures (G_M2, G_M3, and G_D1a). For each ganglioside, however, shedding was selective for ceramide structures containing shorter fatty acyl chains. Rapid and ceramide-selective shedding was confirmed in two additional human medulloblastoma cell lines, D341 Med and D283 Med (112 and 59 pmol/10⁸cells/h). Significant ganglioside shedding is therefore a common characteristic of human medulloblastoma cells and may influence the biological behavior of this tumor, in view of immunosuppressive and other biological properties of shed gangliosides.     

EFFECT OF LIGHT ON GANGLIOSIDES FROM CALF RETINA AND PHOTORECEPTORS

—The gangliosides of the whole calf retina and the rod outer segments have been analysed. This has been done in two functional states: before and after stimulation by light. After exposure to light no statistically significant change in the gangliosides of the whole retina was observed, but a 40 per cent increase in concentration was found in the rod outer segments. This difference was apparent only when using the same batch of rod outer segments. The major ganglioside in the whole calf retina is G_D3 which accounts for 46 per cent of the total. Three other gangliosides G_D1a, G_D1b and G_T1 are quantitatively important, each being between 12 and 16 per cent. G_Q1, G_M1, and G_M3 are minor constituents. In contrast to the chicken retina, G_M2 was not detected. The ganglioside N-acetylneuraminic acid of the rod outer segments accounts for only 1 per cent of the gangliosides of the whole retina. The composition of the gangliosides in the rod outer segments is essentially the same as that of the whole retina. No difference in the relative proportion of the gangliosides of either the rod outer segments or the whole retina was observed after exposure to light.     

Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium

Gangliosides of the G_M1b-pathway (G_M1b and GalNAc-G_M1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas G_M2 and G_M1 (G_M1a-pathway) occurred only in low amounts [Müthing, J., Peter-Katalini, J., Hanisch, F.-G., Neumann, U. (1991)Glycoconjugate J 8:414–23]. Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed. After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), G_M1b and GalNAc-G_M1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells. The expression of G_M1a was also diminished but not as strongly as that of G_M1b and GalNAc-G_M1b. These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium. On the other hand, a new ganglioside, supposed to represent GalNAc-G_D1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation. These observations reveal that the ganglioside composition ofin vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium. Abbreviations: BSA, bovine serum albumin GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; LDL, low density lipoprotein; NeuAc,N-acetylneuraminic acid; NeuGc,N-glycoloylneuraminic acid. The designation of the following glycosphingolipids follows IUPAC-IUB recommendations. GgOse₃Cer or gangliotriaosylceramide, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide, Gal1-3GalNAc1-4Gla1-4GlcCer; GgOse₅Cer or gangliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₆Cer or gangliohexaosylceramide, Gal1-3GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer or GgOse₆Cer; II³NeuAc-GgOse₃Cer or G_M2; II³NeuAc-GgOse₄Cer or G_M1 or G_M1a; IV³NeuAc-GgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc-GgOse₆Cer or Gal-GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³NeuAc, III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc, II³NeuAc-GgOse₅Cer or GalNAc-G_D1a. Enzymes: Vibrio cholerae andArthrobacter ureafaciens neuraminidase (EC 3.2.1.18).     

Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry

YAC-1 cells were propagated in bioreactors in 11 and 7.51 volumes. The cells were metabolically labelled withd-[1-¹⁴C]galactose andd-[1-¹⁴C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between G_M1 and G_D1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of G_M1b ganglioside. As well as small amounts of G_M2 and G_M1, the major gangliosides found in the complex mixture were G_M1b and GalNAc-G_M1b. The structural heterogeneity of these gangliosides was cased by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C_16:0, C_24:0, C_24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse₅Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse₅Cer backbone. No cross-reaction with G_M1b or GgOse₄Cer was observed. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography, HPTLC, high performance thin layer chromatography; NK, natural killer; SIM, selective ion monitoring; TIC, total ion current. NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUB-IUPAC recommendations. GgOse₃Cer or gangliotriaosylceramide or asialo G_M2, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide or asialo G_M1, Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₅Cer organgliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; II³NeuAc-GgOse₄Cer or G_M1; IV³NeuAcGgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³(NeuAc)₂-GgOse₄Cer or G_D1c; IV³NeuAc,III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc,II³(NeuAc)₂-GgOse₄Cer or G_T1b;Vibrio cholerae and Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18).     

Genetic control of ganglioside biosynthesis in mice

The enzymatic basis for the differences in hepatic ganglioside patterns in the mouse strains C57Bl/6 and Swiss White (SW) was investigated. SW has a “Swiss-type” ganglioside profile, expressing G_M1 ^? and G_D1a ^? in addition to G_M2 ^? as major hepatic gangliosides, whereas C57Bl/6 shows a “G_M2-type” profile, expressing only G_M2 ^? as the major hepatic ganglioside. The enzyme UDP-galactose:G_M2 ganglioside galactosyltransferase (G_M2-GalT), which catalyzes the synthesis of G_M1 ganglioside, showed a four- to fivefold elevation in intact and solubilized liver Golgi membrane fractions of the SW strain compared to C57Bl/6. Crosses between C57Bl/6 and SW produced an F₁ generation with a hepatic ganglioside and enzymatic phenotype intermediate between those of the two parental strains. All three genotypic groups show two forms of the Golgi apparatus enzyme with isoelectric points of 6.5–6.8 and 8.3–9.0. The simplest mode of action of genes which control the enzymatic phenotype that would be consistent with these findings are one or two structural genes or one or two cis-regulatory genes affecting the rate of enzyme synthesis.