1H-NMR assignments of GM1-oligosaccharide in deuterated water at 500 MHz by two-dimensional spin-echo J-correlated spectroscopy

The ¹H-NMR spectra of the oligosaccharide derived from monosialoganglioside G_M1 (G_M1 = β-d-galactosyl-(1–3)-β-d-N-acetylgalactosaminyl-(1–4)-[α-N-acetylneuraminyl-(2–3)]-β-d-galactosyl-( 1–4)-β-d-glucosylceramide) (G_M1OS) and its reduced form (G_M1OS-R) have been obtained at 500 MHz in D₂O. Through the combined use of one-dimensional and homonuclear two-dimensional spin-echo J-correlated (2D SECSY) spectra of G_M1OS-R, the assignments for the ring protons of G_M1OS are made. Data on chemical shifts and coupling constants of G_M1OS including the α-linked neuraminic acid protons, in aqueous solution, are tabulated. Due to the very small coupling constants (<2 Hz) and the closeness in chemical shifts (<0.04 ppm) for the pair of correlated peaks in the two-dimensional spectrum, the information on the connectivities of the H5 ring protons of the neutral sugar residues is missing. Second-order coupling also blurs this information. Data are compared with those obtained for ganglioside G_M1 in dimethyl sulfoxide (DMSO;the actual composition therein was 97% DMSO-d₆ and 3% D₂O) by T.A.W. Koerner, J. H. Prestegard, P. C. Demou, and R. K. Yu (1983, Biochemistry22, 2676). While the heterogeneity of chemical shifts for the H5, H6a, and H6b protons diminishes in D₂O, that for A-9a and A-9b remains. The latter suggests an intraneuraminic acid conformation involving the glycerol side chain unaffected by the solvent. Moreover, the chemical shifts of the III-1, III-2, and A-4 protons (and perhaps the II-4, IV-2, and A-8 protons) in D₂O exhibit unusual upfield shifts compared with those in DMSO. This indicates that the intramolecular interactions between GalNAc residue III and neuraminic acid present in DMSO are weakened in D₂O. The effect of temperature on the conformation is also examined and appears to be minimal (<0.02 ppm) in the range 22–50 °C.     

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.     

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.     

1H-NMR study of GM2 ganglioside: evidence that an interresidue amide-carboxyl hydrogen bond contributes to stabilization of a preferred conformation

Several properties of the exchangeable amide protons of the ganglioside G_M2 were studied in detail by¹H-NMR spectroscopy in fully deuterated dimethylsulfoxide [²H₆]DMSO/2% H₂O, and compared with data obtained for the simpler constituent glycosphingolipids G_A2 and G_M3. In addition to chemical shifts,³ J _2,HN coupling constants, and temperature shift coefficients, the kinetics of NH/²H chemical exchange were examined by following the disappearance of the amide resonances in [²H₆]DMSO/2%²H₂O. The results included observation of an increase in half-life of theN-acetylgalactosamine acetamido HN by more than an order of magnitude in G_M2 compared to G_A2, attributable to the presence of the additionalN-acetylneuraminic acid residue. Additional one-dimensional dipolar cross relaxation experiments were also performed on nonexchangeable protons of G_M2. The results of all of these experiments support a three-dimensional model for the terminal trisaccharide in which a hydrogen bond is formed between theN-acetylgalactosamine acetamido NH and theN-acetylneuraminic acid carboxyl group. The interaction is proposed to be of the -acceptor type, a possibility which has not yet been explored in the literature on carbohydrates. The proposed model is discussed in comparison with that of Sabesanet al. (1984,Can J Chem 62: 1034–45), and the models of G_M1 proposed more recently by Acquottiet al. (1990,J Am Chem Soc 112:7772–8) and Scarsdaleet al. (1990,Biochemistry 29:9843–55).     

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.     

Interaction of gangliosides with plasma low density lipoproteins

The role of gangliosides in the reception of low density lipoproteins (LDL) was studied using as targets mouse ascites hepatoma 22a (MAH) cells which bind LDL through a specific high affinity receptor. Low density lipoprotein binding and uptake by MAH cells decreased after brief treatment of the cells with neuraminidase to partially remove surface sialic acid residues. The LDL uptake capability of the neuraminidasetreated MAH cells was fully restored after incorporation of exogeneous G_M1- and G_D1a-gangliosides into the cell surface. In contrast, free (extracellular) gangliosides inhibited LDL uptake by native MAH cells. This inhibitory effect was seen at ganglioside concentrations corresponding to the ganglioside content of serum and was most pronounced with gangliosides whose sialic acids were linked to a terminal galactose residue (G_M3, G_D1a, G_T1b) but was smaller or absent with gangliosides whose sialic acids were attached to an internal galactose (G_M1, G_M2). The binding of gangliosides to LDL was structure and concentration dependent, saturable and trypsin sensitive. The LDL-ganglioside interaction was further investigated by steady state fluorescence spectroscopy. Changes in the LDL fluorescence polarization were observed with as little as 0.01 M concentrations of the gangliosides. The magnitude and nature of the effect depended on the type of ganglioside. We conclude that the LDL surface possesses sites recognizing specific carbohydrate sequences of glycoconjugates and that changes in the cell surface concentrations of sialic acids significantly modulate the LDL uptake. It is postulated that shedding of gangliosides into the blood stream may be a factor involved in regulation of cholesterol homeostasis.Abbreviations MAH mouse ascites hepatoma 22a - LDL low density lipoprotein - ASM anthrylvinyl-labeled sphingomyelin [N-12-(9-anthryl-trans-dodecanoyl-sphingosine-1-phosphocholine] - RITC rhodamine isothiocyanate. The designation of gangliosides follows the IUPAC-IUB recommendation [1]: G_M3, II³NeuAc-LacCer, II³-N-acetylneuraminosyllactosylceramide - G_M2 II³-NeuAc-GgOse₃Cer, II³-N-acetylneuraminosylgangliotriaosylceramide - G_M1 II³-NeuAc-GgOse₄Cer, II³-N-acetylneuraminosylgangliotetraosylceramide - G_D1a, II³ IV³(NeuAc)₂-GgOse₄Cer, II³, IV³-di(N-acetylneuraminosyl)gangliotetraosylceramide - G_T1b II³(NeuAc)₂, IV³ NeuAc-GgOse₄Cer, II³-di-N-acetylneuraminosyl, IV³-N-acetylneuraminosylgangliotetraosylceramide     

Proton NMR study of iron(II)-bleomycin: Assignment of resonances by saturation transfer experiments

The assignment of the paramagnetically shifted resonances of the Fe(II)-bleomycin complex in D₂O has been accomplished using the transfer of saturation method. A number of additional resonances arising from labile N$\text{H}$ protons which are shifted by the metal ion are observed in the ¹H spectrum of the complex in H₂O. The temperature dependence of the chemical shifts is consistent with the formation of an isolated 1:1 complex, but does not obey either the Curie Law or the Curie-Weiss Law. The magnitude of the shifts suggests that the valeric acid hydroxyl (or carbonyl) group, the α-amino group, the imidazole N^π, the carbamoyl oxygen, the pyrimidine N₁ and/or the secondary amino group may be coordinated to the iron(II).     

Thermotropic behavior of binary mixtures of diplamitoylphosphatidylcholine and glycosphingolipids in aqueous dispersions

The thermotropic behavior of mixtures of dipalmitoylphosphatidylcholine (DPPC) with natural glycosphingolipids (galactosylceramide, phrenosine, kerasine, glucosylceramide, lactosylceramide, asialo-G_M1, sulfatide, G_M3, G_M1, G_D1a, G_T1b) in dilute aqueous dispersions were studied by high sensitivity differential scanning calorimetry over the entire composition range. The pretransition of DPPC is abolished and the cooperativity of the main transition decreases sharply at mole fractions of glycosphingolipids below 0.2. All systems exhibit non-ideal temperature-composition phase diagrams. The mono- and di-hexosylceramides are easily miscible with DPPC when the proportion of glycosphingolipids in the system is high. A limited quantity (1–6 molecules of DPPC per molecule of glycosphingolipid (GSL) can be incorporated into a homogeneously mixed lipid phase. Domains of DPPC, immiscible with the rest of a mixed GSL-DPPC phase that shows no cooperative phase transition, are established as DPPC exceeds a certain proportion in the system. One negative charge (sulfatide) or four neutral carbohydrate residues (asialo-G_M1) in the oligosaccharide chain of the glycosphingolipids results in phase diagrams exhibiting coexistence of gel and liquid phases over a broad temperature-composition range. Systems containing gangliosides show complex phase diagrams, with more than one phase transition. However, no evidence for phase-separated domains of pure ganglioside species is found. The thermotropic behavior of systems containing DPPC and glycosphingolipids correlates well with their interactions in mixed monolayers at the air/water interface.     

Galactose oxidase action on GM1 ganglioside in micellar and vesicular dispersions

G_M1 ganglioside was dispersed in different membrane-mimicking systems and the effect of dispersion on G_M1 oxidation by galactose oxidase was studied. The following membrane-mimicking systems were used: homogeneous micelles of G_M1; mixed micelles (at different proportions of constituents) of G_M1 with either G_D1a ganglioside (which is resistant to the enzyme), or the non-ionic detergent Triton X-100, or bovine serum albumin; small unilamellar vesicles of egg phosphatidylcholine (PC), containing various proportions of G_M1. As a reference substrate not involved in membranous systems and freely interacting with the enzyme, the oligosaccharide portion of G_M1 (DesG_M1) was employed.The apparent $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the enzyme was dramatically dependent on the type of G_M1 dispersion. The lowest value was recorded on homogeneous micelles of G_M1 and on mixed G_M1-G_D1a micelles. From this value, the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ increased 2-fold with G_M1-bovine serum albumin lipoprotein micelles, up to 1400-fold with mixed G_M1-Triton X-100 (optimal molar ratio, 1:13.8) micelles, and up to 14 000-fold on PC vesicles containing 8 mol% G_M1 (this proportion was optimal for enzyme activity on vesicles). The activity developed on these latter vesicles turned out to be still greater (2-fold) than that displayed on DesG_M1. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ had very similar values in all different membrane systems; in contrast, it was markedly greater on DesG_M1. Both Triton X-100 micelles and PC vesicles did not appreciably alter the kinetics of galactose oxidase action on pure galactose, indicating that the above effects are dependent on the intrinsic characteristics of the membrane-like systems containing gangliosides.     

Substrate specificities of a bacterial sialidase and rat liver ganglioside GM3 sialyltransferase

Sialidases cleave off sialic acid residues from the oligosaccharide chain of gangliosides in their catabolic pathway while sialyltransferases transfer sialic acid to the growing oligosaccharide moiety in ganglioside biosynthesis. Ganglioside G_M3 is a common substrate for both types of enzymes, for sialidase acting on ganglioside G_M3 as well as for ganglioside G_D3 synthase. Therefore, it is possible that both enzymes recognize similar structural features of the sialic acid moiety of their common substrate, ganglioside G_M3. Based on this idea we used a variety of G_M3 derivatives as glycolipid substrates for a bacterial sialidase (Clostridium perfringens) and for G_D3 synthase (of rat liver Golgi vesicles). This study revealed that those G_M3 derivatives that were poorly degraded by sialidase also were hardly recognized by sialyltransferase (G_D3 synthase). This may indicate similarities in the substrate binding sites of these enzymes.     

Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied ‘food grade’ sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC₅₀ value of 5.521 mg/mL is high compared with 0.2811 μg/mL (182.5 ρM or 1.825 × 10⁻¹⁰ M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E^® is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%, 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease.     

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.     

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).     

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids

Multilamellar liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside G_M1, monosialoganglioside G_M2, monosialoganglioside G_M3, disialoganglioside G_D1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distribution of ¹⁴C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome prepartion. Liver uptake of encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from the liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing G_D1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing G_D1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.     

Glycolipids are not extracted from phospholipid bilayers by binding to ferritin-lectin conjugates

A radioactively-labelled glycosphingolipid, asialo-G_M1, has been incorporated into phosphatidylcholine multilamellar vesicles. After incubation with ferritin-Ricinus communis agglutinin 60 (RCA 60) conjugate at different temperatures, the vesicles were separated from the conjugate by discontinuous density gradient ultracentrifugation. Measurement of the distribution of the radioactively-labelled asialo-G_M1 in the pelleted conjugate fraction and freeze-etch electron microscopy of the vesicle fraction indicate that the decrease in labelling of vesicles by ferritin-RCA 60 conjugate with increasing temperatures (Tillack, T.W., Wong, M., Allietta, M. and Thompson, T.E. (1982) Biochim. Biophys. Acta 691, 261–273) reflects a decrease in apparent binding affinity rather than an ability of the conjugate to extract glycolipid from the phospholipid bilayer after binding.     

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     

Association of gangliosides to fibroblasts in culture: A study performed with GM1 [14C]-labelled at the sialic acid acetyl group

The preparation of a G_M1-ganglioside (GM1) [¹⁴C]-labelled in the sialic acid residue is reported. This can be obtained by re-N-acetylation in the presence of [1-¹⁴C]-acetic anhydride, of a GM1 derivative de-N-acetylated specifically on the sialic acid residue by alkaline hydrolysis of GM1 with tetramethylammonium hydroxide. The radiolabelled GM1 is utilized to investigate the binding properties and the mode of interaction of GM1 with cultured fibroblasts. Three different forms of association (one serum-removable, one trypsin-removable and one trypsin-stable) have been recognized to occur in a way that depended on cell culture conditions (presence or absence of fetal calf serum), ganglioside concentration (from, 5×10^–9 M to 10^–4 M) and incubation time (up to 24 h). Some metabolic modifications of GM1 during the period of high cell viability were also investigated.Abbreviations GM1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer - FCS fetal calf serum - EMEM Eaglés Minimum Essential Medium with Earlés salts - PBS Dulbecco phosphate buffered saline without calcium and magnesium     

A high-resolution c.p.-m.a.s. 13C-n.m.r. study of solid-state cyclomaltohexaose inclusion-complexes: Chemical shifts and structure of the host cyclomaltohexaose

High-resolution, solid-state ¹³C-n.m.r. spectra were obtained for several crystalline cyclomaltohexaose inclusion-complexes. The resonances of C-1, C-4, and C-6 of the host were dispersed. The averaged ¹³C shifts of these resonances were in good agreement with the ¹³C shifts observed in solution, where the dispersion due to conformational diversity is expected to be averaged by rapid interconversion of the conformers. This result indicates that the most plausible source of the solid-state ¹³C-shift dispersions of the resonances of C-1 and C-4 is the diversity of conformations about the glycosidic linkage. The molecular origins of conformation-dependent ¹³C shifts are discussed.     

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides G_M1, G_M3, G_D1a, G_D1b, G_T1b and asialo-G_M1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for G_M1 (K_a = 1.8 ⋅ 10⁸M^–1), a lower one for asialo-G_M1 (K_a = 1.0 ⋅ 10⁷ M^–1) and no affinity for G_M3. The C-fragment of tetanus toxin binds to ganglioside G_D1a, G_D1b and G_T1b containing membranes with similar affinity (K_a∼10⁶ M^–1), while no binding was observed with G_M3. Pertussis toxin binds to membranes containing the ganglioside G_D1a with a binding constant of K_a = 1.6 ⋅ 10⁶ M^–1, but only if large amounts (40 mol%) of G_D1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to G_D1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to G_D1a. Received: 14 January 1997 / Accepted: 15 April 1997     

Structure of the "keratosulfate-like" material in liver from a patient with G M1 -gangliosidosis ( -D-galactosidase deficiency)

Large amounts of a glycopeptide containing galactose, $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and threonine in the ratio 4:3:1:1, together with smaller amounts of mannose, fucose, sialic acid, sulfate, serine, and other amino acids were isolated from the liver of a patient with G_M1-gangliosidosis. Treatment with mild alkali and sodium borohydride indicated an $\text{O}$-glycosidic linkage between $\text{N}$-acetylgalactosamine and threonine. All the hexosamine residues were resistant to sodium metaperiodate whereas 2 out of 4 D-galactose residues were destroyed. Further studies indicated that one of the galactose residues was 1→3 linked to $\text{N}$-acetylgalactosamine (as in G_M1) and the other 1→4 linked to $\text{N}$-acetylglucosamine as found in skeletal keratosulfate.