Yoshitaka Nagai

Glycolipids at the cell surface and their biological functions

A large variety of sphingoglycolipids occur at the surface of animal cells. The carbohydrate moieties vary, and each glycolipids may exhibit a special function, as an annular lipid, receptor or surface marker and matrix lipid.

Interaction between Clostridium botulinum neurotoxin and gangliosides

The effect of gangliosides on Clostridium botulinum type A neurotoxin was examined in terms of detoxification. The molar concentrations of gangliosides necessary to detoxify 50% of 1 M Cl. botulinum neurotoxin were as follows: GM1, 2073; GM2, 2439; GM3, 6098; GD1a, 610; GD1b, 488; GT1a, 829; GT1b, 6 and GQ1b, 27. Inhibition by gangliosides of the neurotoxin binding to synaptosomes showed that GT1b was highly effective, but the others were not. Low-temperature treatment inhibited the detoxification of neurotoxin by GT1b and the binding of 125I-labelled neurotoxin to the synaptosome fraction. 125I-labelled neurotoxin was mixed with GM1 or GT1b and their molecular size was estimated by sucrose-density-gradient centrifugation. When 125I-labelled neurotoxin was incubated with GM1, a single radioactive peak having a sedimentation coefficient of 7.3 S appeared. When incubated with GT1b, however, 125I-labelled neurotoxin gave three peaks having sedimentation coefficients 14, 10 and 7.3 S, respectively. The present results indicated that the location and the number of sialic acids in ganglioside molecules are of significance in the detoxification and the binding of Cl. botulinum neurotoxin with ganglioside molecules.

Synthetic Sialyl Compounds as Well as Natural Gangliosides Induce Neuritogenesis in a Mouse Neuroblastoma Cell Line (Neuro2a)

Amphipathic compounds containing N‐acetylneuraminic acid (sialic acid) [for example, D‐N‐acetylneuraminyl‐(α2‐1)‐2S, 3R,4E‐2‐N‐tetracosanoyl sphingenine, sialyl alkyl glycerol ethers, and sialyl cholesterols] induced neuritogenesis in a neuroblastoma cell line (Neuro2a). The sialic acid in the hydrophilic moiety of the compounds is specifically required for neuritogenesis. The requirement for molecular specificity of the hydrophobic moiety, however, is rather low. Regarding the hydrophobic moiety, no preference for cholesterol, alkyl glycerol ether, or ceramide residues was observed as to their neuritogenic activity. Sialyl compounds with α‐ketosidic sialyl linkages were more active than the corresponding β‐anomers. These sialyl compounds induced the growth of only one neurite, but a long one, from the cell body. This type of neuritogenesis is completely different from that induced by compounds capable of elevating the concentration of intracellular cyclic AMP, which induced the appearance of many neurites from a single cell body. Besides this morphological change, the active sialyl compounds also caused a change in the carbohydrate composition of the cell surface. Sialyl compound treatment drastically increased the concentration of peanut agglutinin binding sites.

Identification of glycolipid receptors for Helicobacter pylori by TLC-immunostaining

Helicobacter pylori has been identified as a causative agent in active chronic gastritis. The receptor for this bacteria, however, is not known. It is likely that the receptor molecules may be glycosphingolipids* as shown in the cases of other bacteria. We explored this possibility by a thin‐layer chromatography (TLC)‐immunostaining method. Among glycosphingolipids extracted from human gastric mucosa, intact Helicobacter pylori specifically bound to PSO3‐GalCer and II3NeuAc‐LacCer, whereas no specific binding to neutral glycosphingolipids, which share the same ceramide moiety with PSO3‐GalCer or II3NeuAc‐LacCer, was demonstrated. Sonicated bacteria could still bind to II3NeuAc‐LacCer with comparable affinity. In contrast, the binding of bacteria of PSO3‐GalCer was greatly diminished upon sonication. These results suggest that each of the oligosaccharide moieties of II3NeuAc‐LarCer and PSO3‐GalCer may be specifically recognized by different ligand molecules of Helicobacter pylori.

Ganglioside syndrome, a new autoimmune neurologic disorder, experimentally induced with brain gangliosides.

Rabbits intensively immunized with total bovine brain gangliosides or a certain molecular species of gangliosides, such as GD(1a) and GM(1), respectively, in complete Freuds adjuvant developed a variety of neurologic symptoms and signs which closely resembled experimental allergic encephalomyelitis, i.e., a sluggish righting reflex, muscular weakness, hindleg paralysis (flaccid in the case of GD(1a) and rigid in GM(1)) and body weight loss, frequently followed by death. Histological examinations of GD(1a)-immunized rabbits showed extensive degeneration of the peripheral nerves, including breakdown of myelin and fragmentation of axis cylinders. In central nervous tissues, however, only mild damage was observed in some cases. Similar neurologic symptoms and signs were also induced in guinea pigs. The results suggest that this syndrome might be induced by a cell-mediated autoimmune response to gangliosides.

Structural elucidation of underivatized gangliosides by electrospray-ionization tandem mass spectrometry (ESIMS/MS)

The structural characterization of intact polysialogangliosides by electrospray-ionization mass spectrometry (ESIMS) and tandem mass spectrometry (MS/MS) is described. The negative-ion ESI mass spectra of gangliosides, such as a tetrasialoganglioside GQ1b, showed molecularly related ions with high sensitivity and accuracy. Collision-induced dissociation (CID) MS/MS provided adequate information to characterize the number and positions of sialic acids. ESIMS and CIDMS/MS techniques were applied successfully also in the positive-ion mode for characterization of intact gangliosides. Two isomeric disialogangliosides, GD1a and GD1b, are distinguishable from one another by CIDMS/MS in both negative- and positive-ion modes. The technique is readily applicable to structural analyses such as determining the positional isomers of glycoconjugates.

Solubilization of the enzyme catalyzing CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol and its comparison to CDP-diglyceride:Inositol transferase

Abstract A solubilized preparation with activity for catalyzing the incorporation of free myo-inositol into phosphatidyl inositol was obtained from a rat liver microsomal fraction. The incorporation took place both in the presence and in the absence of cytidine diphosphodiglyceride (CDP-DG). The pH optimum of the incorporation in the absence of CDP-DG was 7.4–7.5, while that of the incorporation in its presence was 8.5–8.6. The incorporation in the absence of CDP-DG was activated by Mn2+ but not by Mg2+, while that in the presence of CDP-DG was activated by either Mn2+ or Mg2+. These results indicated that the incorporation in the absence of CDP-DG and the incorporation in its presence were catalyzed by different enzymes. Before Solubilization, the CDP-DG-independent enzyme was bound to endoplasmic reticulum. The CDP-DG-dependent enzyme also was bound mainly to endoplasmic reticulum and, to a minor extent, to plasma membrane. The CDP-DG-independent enzyme was more easily solubilized by sodium cholate than the CDP-DG-dependent enzyme. There were also differences between these two enzyme activities of the solubilized preparation with respect to their sensitivity to various detergents and their dependence on exogenous lipids. The CDP-DG-independent incorporation was inhibited by CDP-DG, by some nucleotides, and by phosphatidyl serine, while the CDP-DG-dependent incorporation was not inhibited by these substances. Both activities were both inhibited by thiol-reactive compounds.

TLC immunostaining characterization of Clostridium botulinum type A neurotoxin binding to gangliosides and free fatty acids.

TLC GQ1b ganglioside Anti‐neurotoxin antibody Salt effect

Systematic analysis of glycosphingolipids in the human gastrointestinal tract: Enrichment of sulfatides with hydroxylated longer-chain fatty acids in the gastric and duodenal mucosa

The composition of the glycosphingolipids of the human gastrointestinal tract was studied. The major neutral glycosphingolipids were ceramide monohexosides (e.g., GalCer, GlcCer), LacCer, Gb3Cer, Gb4Cer and more polar ones with more than four sugars, whereas neither Gg3Cer nor Gg4Cer were present. The acidic glycosphingolipids consisted of sulfatides and gangliosides such as GM3, GM1, GD3 and GD1a. Also a large amount of sulfatides was found in the gastric mucosa and duodenum. The concentrations of sulfatides in the fundic mucosa, antral mucosa and duodenum amounted to 416.0, 933.8 and 682.9 nmol/g of dry weight, respectively, exceeding those in the gastric mucosa and kidney of other mammals. The major molecular species of the sulfatides were identified as I3SO3-GalCer with hydroxylated longer-chain fatty acids based on the analyses by gas-liquid chromatography and negative ion fast-atom bombardment mass spectrometry. In contrast, gangliosides in these regions showed a tendency to be lower than sulfatides, and the molar ratios of sulfatides to gangliosides were about 2.0, whereas those in other parts were less than 0.5. A high content of sulfatides in the gastric and duodenal mucosa, where mucosa is easily insulted by acid, pepsin and bile salts, may be closely related to their roles in mucosal protection.

Ototoxicity of neomycin and its penetration through the round window membrane into the perilymph.

The ototoxicity of neomycin and its concentration in the perilymph after direct application on the round window membrane were studied. After placing 5 mg of neomycin on the round window membrane of guinea pigs for various time intervals, concentration of the drug in the perilymph was determined by high performance liquid chromatography, and the cochlea was examined by light microscopy. Neomycin penetrated the round window membrane quite easily, and its concentration in the perilymph became extremely high in a short time and then decreased gradually. This indicates that high concentration of neomycin in the perilymph can be attained by application of a small amount of the drug on the round window membrane. Ototoxicity of neomycin was observed after application for 4 hours. Cochlear damage increased as neomycin application time became longer, but no consistent relationship was noted between the concentration of neomycin and the amount of damage. This result is discussed from the point of concentration and persistence of the drug in the inner ear fluids.