Enzymatically synthesized exopolysaccharide of a probiotic strain Leuconostoc mesenteroides NTM048 shows adjuvant activity to promote IgA antibody responses.

Leuconostoc mesenteroides strain NTM048 produces an exopolysaccharide (EPS; glucose polymers 94% and fructose polymers 6%) with adjuvanticity for mucosal vaccination. Strain NTM048 includes three putative EPS-synthesizing genes, gtf1 and gtf2 for synthesizing glucose polymers, and lvnS for synthesizing fructose polymer. To elucidate the key polymer structure for adjuvanticity, two genes, gtf1 and gtf2, which were annotated as glycoside hydrolase family 70 enzyme genes, were expressed in Escherichia coli. Glycosyl-linkage composition analysis and NMR analysis showed that the recombinant enzyme Gtf1 produced a soluble form of α-1,6-glucan, whereas the recombinant enzyme Gtf2 produced glucans with approximately equal percentages of α-1,6- and α-1,3-glucose residues both in the supernatant (S-glucan) and as a precipitate (P-glucan). Comparison of polysaccharides synthesized by Gtf1, Gtf2, and LvnS revealed that Gtf2-S-glucan, which was produced in the supernatant by Gtf2 and formed particles of 7.8 µm, possessed 1.8-fold higher ability to stimulate IgA production from murine Peyer's patch cells than native NTM048 EPS. Evaluation of adjuvanticity by intranasal administration of mice with an antigen (ovalbumin) and Gtf2-S-glucan or NTM048 EPS showed that Gtf2-S-glucan induced the production of higher antigen-specific antibodies in the airway mucosa and plasma, suggesting a pivotal role of Gtf2-S-glucan in the adjuvanticity of NTM048 EPS.

Structural analysis of neutral glycosphingolipids from the silkworm Bombyx mori and the difference in ceramide composition between larvae and pupae.

Glycosphingolipids (GSLs) from the silkworm Bombyx mori were identified and GSL expression patterns between larvae and pupae were compared. The structural analysis of neutral GSLs from dried pupae revealed the following predominant species: Glcß1Cer, Manß4Glcß1Cer, GlcNAcß3Manß4Glcß1Cer, Galß3Manß4Glcß1Cer, GalNAcα4Galß3Manß4Glcß1Cer, GlcNAcß3Galß3Manß4Glcß1Cer, Galα4Galß3Manß4Glcß1Cer and (GalNAcα4)1-4 GalNAcα4Galß3Manß4Glcß1Cer. Lin-ear elongation of α4-GalNAc was observed at the non-reducing end of Galß3Manß4Glcß1Cer with up to five GalNAc repeats. The arthro-series GSL GlcNAcß3Manß4Glcß1Cer, a characteristic GSL-glycan sequence of other Arthropoda, was detected in silkworms. The main ceramide species in each purified GSL fraction were h20:0-d14:1 and h22:0-d14:1. GSL expression patterns in larvae and pupae were compared using thin-layer chromatography, which demonstrated differences among acidic, polar and neutral GSL fractions, while the zwitterionic fraction showed no difference. Neutral GSLs such as ceramides di-, tri- and tetrasaccharides in larvae showed less abundant than those in pupae. MALDI-TOF MS analysis revealed that larval GSLs contained four types of ceramide species, whereas pupal GSLs contained only two types. The structural analysis of neutral GSLs from silkworms revealed a novel series of GSLs. The comparison of GSL expression patterns between larvae and pupae demonstrated differences in several fractions. Alterations in GSL ceramide composition between larvae and pupae were observed by MALDI-TOF MS analysis.

Characterization of a Novel Rhamnose-containing Acidic Glycosphingolipid from the Ascidian Halocynthia aurantium.

Halocynthia aurantium, an edible ascidian species belonging to Urochordata, was subjected to structural characterization of acidic glycosphingolipids to investigate these molecules in ascidians: sulfatide from Ciona intestinalis and the glucuronic acid-containing acidic glycosphingolipid from H. roretzi. Acidic glycosphingolipids containing three or five sugars were isolated from soft parts of the ascidian H. aurantium by chloroform-methanol extraction, mild-alkaline hydrolysis, precipitation with cold acetone, and subsequent column chromatography using a DEAE-Sephadex A-25 column, a Florisil column, and an Iatrobead column. The structures of these glycosphingolipids were determined by methylation studies, sugar analysis, fatty acid analysis, sphingoid analysis, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. A novel glucuronic acid-containing glycosphingolipid having a rhamnose residue was identified as Rhaα1-3GlcNAcß1-3Galß1-4(Fucα1-3)GlcAß1-Cer (UGL-2). This novel structure is particularly unusual given that it contains both a rhamnose residue and a reducing terminal glucuronic acid residue within a single molecule. Rhamnose is a characteristic sugar, which is a component of cell wall pectin in plants and exopolysaccharides in bacteria. Ascidians acquired the cellulose synthase gene via lateral gene transfer, and therefore, it can be speculated that they also acquired the rhamnosyltransferase gene in the same manner. We also detected Galß1-4(Fucα1-3)GlcAß1-Cer (UGL-1), which was already identified in another ascidian, H. roretzi.

Structural analysis of cerebrosides from Aspergillus fungi: the existence of galactosylceramide in A. oryzae.

Glucosylceramide and galactosylceramide were detected in three Aspergillus species: Aspergillus oryzae, Aspergillus sojae and Aspergillus. awamori, using borate-coated TLC. The cerebrosides from A. oryzae were further purified by ion exchange and iatrobeads column chromatographies with or without borate, and determined the composition of sugar, fatty acid and sphingoid base by GC/MS, MALDI-TOF/MS and (1)H-NMR. We identified them as ß-glucosylceramide and ß-galactosylceramide. The ceramide moiety of both cerebrosides consisted mainly of 2-hydroxystearic acid and either 9-methyl-octadeca-4, 8-sphingadienine or octadeca-4, 8-sphingadienine. To our knowledge, this is the first study to provide evidence for the presence of ß-galactosylceramide in A. oryzae.

Structural elucidation of the neutral glycosphingolipids, mono-, di-, tri- and tetraglycosylceramides from the marine crab Erimacrus isenbeckii.

The neutral glycosphingolipids, mono-, di-, tri- and tetraglycosylceramides (GL-1, GL-2, GL-3, GL-4a and GL-4b), were identified from whole tissues of the marine crab Erimacrus isenbeckii by successive column chromatography with ion exchange Sephadex (QAE-Sephadex), magnesium silicate (Florisil) and silicic acid (Iatrobeads) resins. Through component analysis, sugar analysis, methylation studies, exoglycosidase cleavage, and various chromatographic and spectrometric techniques, their structures were proposed to be as follows: GL-1, Glcß1-1Cer; GL-2, Manß1-4Glcß1-1Cer; GL-3, Galß1-3Manß1-4Glcß1-1Cer; and GL-4a and GL-4b, Gal3Meα1-4Galß1-3Manß1-4Glcß1-1Cer. The main molecular species of the aliphatic moiety in each purified glycolipid were 18:0, 22:0, 22:1-d14:1 (fatty acid-sphingoid) and 18:0-d16:1 for GL-1; 18:0-d16:1 and 22:1-d14:1, d16:1 for GL-2; 22:1, 24:1-d16:1 for GL-3; 22:1, 24:1-d16:1 for GL-4a; and h22:1, h24:1-d16:1 for GL-4b, respectively. By immunological detection, an arthro-series glycosphingolipid (At3Cer; GlcNAcß1-3Manß1-4Glcß1-1Cer) was also detected as a minor component. The characteristic arthro-series glycosphingolipid has been observed in most animals belonging to the phylum Arthropoda.

Novel neogala-series glycosphingolipids with a terminal glucose residue from the fungus Mariannaea elegans.

Glycosphingolipids (GSLs) are essential membrane components of eukaryotic cells. Recently, a new type of fungal neogala-series GSL was identified in aureobasidin A-resistant fungi. In this study, we analyzed GSLs from four pathogenic fungal strains belonging to the order Hypocreales, and found that Mariannaea elegans contained both acidic GSLs and neutral GSLs with mono- and di-saccharides. The structures of the neutral GSLs of M. elegans were determined by compositional sugar, fatty acid, and sphingoid analyses by GC/MS, MALDI time-of-flight/MS, and 1H NMR. The ceramide moiety of Glcß1-Cer consisted mainly of the 2-hydroxylated C18:0-fatty acid 9-methyl-octadeca-4-sphinganine or 9-methyl-octadeca-4,8-sphingadienine. In contrast, the ceramides of Galß1-6Galß1-Cer and Glc1-6Galß1-Cer consisted mainly of saturated 2-hydroxylated C24:0-fatty acids and C18:0-phytosphingosine. To our knowledge, Glc1-6Galß1-Cer is a novel GSL in fungi, and M. elegans is the first example of an aureobasidin A-sensitive fungus that possesses fungal neogala series GSLs.

Biochemical studies on sphingolipids of Artemia franciscana: complex neutral glycosphingolipids.

Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcß1-3GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer, GalNAcß1-4(Fucα1-3)GlcNAcß1-3GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer, and GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III(3)(GlcNAcα2Fucα)-At4Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important.

Dengue virus type 2 recognizes the carbohydrate moiety of neutral glycosphingolipids in mammalian and mosquito cells.

Dengue viruses infect cells by attaching to a surface receptor which remains unknown. The putative receptor molecules of dengue virus type 2 on the surface of mosquito (AP-61) and mammalian (LLC-MK2) cell lines were investigated. The immunochemical detection and structural analysis of carbohydrates demonstrated that the neutral glycosphingolipids, L-3 (GlcNAcß1-3Manß1-4Glcß1-1'Cer) in AP-61 cells, and nLc(4) Cer (Galß1-4GlcNAcß1-3Galß1-4Glcß1-1'Cer) in LLC-MK2 cells were recognized by the virus. These findings strongly suggest that neutral glycosphingolipids share the key determinant for virus binding and that the ß-GlcNAc residue may play an important role in dengue virus binding to the host cell surface.

Biochemical studies on sphingolipids of Artemia franciscana: novel neutral glycosphingolipids.

Neutral glycosphingolipids containing one to six sugars in their oligosaccharide chains have been isolated from cysts of the brine shrimp Artemia franciscana. The structures of these glycolipids were identified by methylation analysis, partial acid hydrolysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance spectroscopy to be Glcß1-Cer, Manß1-4Glcß1-Cer, Fucα1-3Manß1-4Glcß1-Cer, GlcNAcß1-3Manß1-4Glcß1-Cer, GlcNAcα1-2Fucα1-3Manß1-4Glcß1-Cer, GalNAcß1-4GlcNAcß1-3Manß1-4Glcß1-Cer, GalNAcß1-4(Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer (CPS), and GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer (CHS). Two glycosphingolipids, CPS and CHS, were characterized as novel structures. Because Artemia contains a certain series of glycosphingolipids (-Fucα3Manß4GlcßCer), which differ from the core sugar sequences reported thus far, we tentatively designated the glycosphingolipids characterized as nonarthro-series ones. Furthermore, CHS exhibited a hybrid structure of arthro-series and nonarthro-series sugar chain. Two novel glycosphingolipids were characterized from the brine shrimp Artemia franciscana; one was composed of arthrotetraose and a branching fucose attached to N-acetylglucosamine residue, and the other was composed of CPS with an additional N-acetylglucosamine residue attached to the branching fucose.

Biochemical studies on sphingolipid of Artemia franciscana (I) isolation and characterization of sphingomyelin.

Sphingomyelin was isolated from cysts of the brine shrimp Artemia franciscana using QAE-Sephadex A25, Florisil and Iatrobeads column chromatographies. The chemical structure was identified using thin-layer chromatography, gas-liquid chromatography, infrared spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The ceramide moiety of sphingomyelin consisted of stearic, arachidic, and behenic acids as fatty acids, and hexadeca-4- and heptadeca-4-sphingenines as sphingoids. By comparative analysis, the ceramide component of Artemia sphingomyelin appears unique in invertebrates and vertebrates. Biological functions of sphingomyelin have largely been investigated using mammalian-derived sphingomyelin. In mammals, a wide variety of molecular species of sphingomyelins have been reported, especially derived from nerve tissue, while the lower animal Artemia contains this unusual sphingomyelin perhaps because of having a much simpler nervous system. The purified unusual sphingomyelin derived from Artemia franciscana might be a very useful tool in elucidating the functions and mechanisms of action of this mediator.

Invariant Valpha14 natural killer T cell activation by edible mushroom acidic glycosphingolipids.

Invariant natural killer T (iNKT) cells regulate multi-immune response through Th1/Th2 cytokine release triggered by the recognition of CD1d-restricted glycosphingolipid antigens. Here we report that acidic glycosphingolipids (AGLs) of mushroom (Hypsizigus marmoreus and Pleurotus eryngii) presented by murine CD1d-transfected rat basophilic leukocytes induced interleukin-2 (IL-2) release from iNKT hybridoma cells. AGL-1, one of the AGLs, containing mannose at the non-reducing ends, induced CD1d-dependent IL-2 release. Al-though alpha-galactosylceramide (alpha-GalCer) presented by CD11c-positive cells induced both interferon-gamma (IFN-gamma) and IL-4 release, all of AGLs presented by CD11c-positive cells and AGL-1 presented by B cells induced IL-4 release from iNKT hybridoma cells. A single intravenous injection of AGLs into B6 mice induced only a little elevation of IL-4 in serum but repeated intravenous injection of AGLs induced prolonged retention of IL-4 in serum; therefore, these results suggested that edible mushroom AGLs might contribute to the retention of immunohomeostasis through the minimum induction of iNKT cell activation in vivo.

Novel neogala-series glycosphingolipids with terminal mannose and glucose residues from Hirsutella rhossiliensis, an aureobasidin A-resistant ascomycete fungus.

Hirsutella rhossiliensis, a nematophagous fungus belonging to the Ascomycota, is resistant to aureobasidin A (AbA). In this fungus, the biosynthetic pathway leading to mannosylinositolphosphoceramides, which is inhibited by AbA, was not detected. Instead, this fungus contains neutral complex glycosphingolipids (GSLs) and monoglycosylceramides. Except for monoglycosylceramides, neutral GSLs share a neogala-series core structure, Galbeta1-6Galbeta1-Cer. Among the GSLs of H. rhossiliensis, three novel GSLs with terminal Man and Glc residues on the sugar chain were elucidated. We analyzed GSL structure using compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry (MALDI-TOF MS), and (1)H nuclear magnetic resonance spectroscopy (NMR). The following structures were determined: Manalpha1-3Galbeta1-6Galbeta1-6Galbeta1-Cer; Glcalpha1-2Galbeta1-6Galbeta1-6Galbeta1-Cer; and Manalpha1-3Galbeta1-6(Glcalpha1-4)Galbeta1-6Galbeta1-Cer. In the ceramides, the fatty acids were predominantly saturated h24:0-acids and the sphingoids were predominately t18:0- or t18:1-sphingoids. In contrast, the ceramides of Glcbeta1-Cer contained d18:2- and d19:2-sphingoids. These findings indicate the presence of a novel biosynthetic pathway of neogala-series GSLs in fungi.

Sphingomyelins in four ascidians, Ciona intestinalis, Halocynthia roretzi, Halocynthia aurantium, and Styela clava.

Sphingomyelin is rarely found in lower animals, while sphingophospholipid is a characteristic of higher animals. In this study, sphingomyelin was first isolated and characterized from ascidian Ciona intestinalis. Ascidian sphingomyelin was prepared using ion exchange (QAE-Sphadex-A25) and silicic acid (Florisil and Iatrobeads) column chromatographies. The chemical structure was characterized by fatty acid analysis, sphingoid analysis, hydrogen fluoride degradation, acid hydrolysis, enzymatic hydrolysis, infrared analysis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ceramide moieties of C. intestinalis sphingomyelin consisted primarily of C16:0, C18:0, and C18:1 fatty acids and d18:2 sphingadiene. Furthermore, sphingomyelins were isolated and characterized from 3 other ascidians, Halocynthia roretzi, Halocynthia aurantium, and Styela clava using the same methods. Comparative analysis of the sphingomyelin structures in 4 ascidian species-C. intestinalis (Enterogona) and H. roretzi, H. aurantium, and S. clava (Pleurogona)-revealed that the major fatty acid composition of the ceramides was similar, and that they differed in minor components.

Phosphocholine-containing glycosyl inositol-phosphoceramides from Trichoderma viride induce defense responses in cultured rice cells.

We isolated two major zwitterionic glycosphingolipids (ZGLs) from the phytopathogenic filamentous fungus Trichoderma viride. Structural analyses showed that the ZGLs (designated Tv-ZGL2 and Tv-ZGL3) were the same as the glycosphingolipids ZGL2 and ZGL4 from Acremonium sp., which are described in our previous paper. ZGLs have the following structure: Man(alpha1-6)GlcN(alpha1-2)Ins-P-Cer (Tv-ZGL2) and phosphocholine (PC)-->6Man(alpha1-6)GlcN(alpha1-2)Ins-P-Cer (Tv-ZGL3). To determine whether these ZGLs have functional roles in plant-fungus interaction, we tested to determine whether they would induce defense responses in cultured rice cells. We found that T. viride's ZGLs elicited expression of the PAL and PBZ1 genes, both of which are associated with pathogen resistance. Tv-ZGL2 induced cell death at a moderate rate. Tv-ZGL3, which contains a PC moiety, induced a high level of cell death in rice cells.

Mushroom acidic glycosphingolipid induction of cytokine secretion from murine T cells and proliferation of NK1.1 alpha/beta TCR-double positive cells in vitro.

Interferon (IFN)-gamma and interleukin (IL)-4 regulate many types of immune responses. Here we report that acidic glycosphingolipids (AGLs) of Hypsizigus marmoreus and Pleurotus eryngii induced secretion of IFN- gamma and IL-4 from T cells in a CD11c-positive cell-dependent manner similar to that of alpha-galactosylceramide (alpha-GalCer) and isoglobotriaosylceramide (iGb3), although activated T cells by AGLs showed less secretion of cytokine than those activated by alpha-GalCer. In addition, stimulation of these mushroom AGLs induced proliferation of NK1.1 alpha/beta TCR-double positive cells in splenocytes. Administration of a mixture of alpha-GalCer and AGLs affected the stimulation of alpha-GalCer and generally induced a subtle Th1 bias for splenocytes but induced an extreme Th2 bias for thymocytes. These results suggested that edible mushroom AGLs contribute to immunomodulation.

Structural characterization of glycosylinositolphospholipids with a blood group type B sugar unit from the edible mushroom, Hypsizygus marmoreus.

Edible fungi, mushrooms, are a popular food in Japan and over 15 cultured mushroom species are available at the food markets. Recently, constituents or ingredients of edible mushrooms have drawn attention because possibilities have been seen for their medical usage. Mycoglycolipids (basidiolipids) of higher mushrooms have been characterized as glycosylinositolphosphoceramides, having a common core structure of Manalpha1-2Ins1-[PO(4)]-Cer and extensions of Man, Gal, and/or Fuc sugar moieties. Seven mycoglycolipids were purified from the edible mushroom Hypsizygus marmoreus by successive column chromatography on ion exchange Sephadex (DEAE-Sephadex) and silicic acid (Iatrobeads). Their structures were characterized to be Ins1-[PO(4)]-Cer (AGL0), Manalpha1-2Ins1-[PO(4)]-Cer (AGL1), Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL2), Fucalpha1- 2Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL3), Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL4), Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL5), and Galalpha1-2Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL6) by sugar compositional analysis, methylation analysis, periodate oxidation, partial acid hydrolysis, enzymatic hydrolysis, immunochemical analysis, gas-liquid chromatography (GC), gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and (1)H-nuclear magnetic resonance spectroscopy (NMR). Ceramide constituents of their mycoglycolipids were composed of phytosphingosine as the sole sphingoid, and mainly 2-hydroxy C22:0 and C24:0 acids as the fatty acids. By immunochemical detection, the terminal structure of AGL4, Galalpha1-3(Fucalpha1-2)Galbeta-, was shown to have blood group type B activity. Galalpha1-2 and its repeating sequence in AGL5 and AGL6 are novel structures on the nonreducing sugar end in mycoglycolipids. These two mycoglycolipids in H. marmoreus distinguish it from other basidiomycetes.

A Novel sulfatide, GlcCer-I6 sulfate, from the ascidian Ciona intestinalis.

A novel sulfated glycosphingolipid, SGL-1, was isolated from the ascidian Ciona intestinalis, prepared from chloroform/methanol extracts and fractionated successively on DEAE Sephadex-A25, Florisil and Iatrobeads column chromatographies. Chemical structural analysis was performed using methylation analysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-NMR spectroscopy. This chemical structure is presented as GlcCer I(6)-Sulfate. The ceramide moiety was specified by t16:0, t17:0, br,t17:0, t18:0 and br,t18:0 as sphingoids, and 2-hydroxy, saturated fatty acids as represented by docosanoic and tetracosanoic acids.

Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi.

A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galbeta1-4(Fucalpha1-3)GlcAbeta1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C16-, C17-, and C18-phytosphingosines as major components.

Identification and characterization of carbohydrate molecules in mammalian cells recognized by dengue virus type 2.

The interaction between cell surface receptors and the envelope glycoprotein (EGP) on the viral membrane surface is the initial step of Dengue virus infection. To understand the host range, tissue tropism, and virulence of this pathogen, it is critical to elucidate the molecular mechanisms of the interaction of EGP with receptor molecules. Here, using a TLC/virus-binding assay, we isolated and characterized a carbohydrate molecule on mammalian cell surfaces that is recognized by dengue virus type 2 (DEN2). Structural determination by immunochemical methods showed that the carbohydrate structure of the purified glycosphingolipid was neolactotetraosylceramide (nLc4Cer). This glycosphingolipid was expressed on the cell surface of susceptible cells, such as human erythroleukemia K562 and baby hamster kidney BHK-21. All serotypes of DEN viruses, DEN1 to DEN4, reacted with nLc4Cer, and the non-reducing terminal disaccharide residue Galbeta1-4GlcNAcbeta1- was found to be a critical determinant for the binding of DEN2. Chemically synthesized derivatives carrying multiple carbohydrate residues of nLc4, but not nLc4 oligosaccharide, inhibited DEN2 infection of BHK-21 cells. These findings strongly suggested that multivalent nLc4 oligosaccharide could act as a competitive inhibitor against the binding of DEN2 to the host cells.

Myelin-associated glycoprotein (Siglec-4) expression is progressively and selectively decreased in the brains of mice lacking complex gangliosides.

Myelin-associated glycoprotein (MAG, Siglec-4) is a quantitatively minor membrane component expressed preferentially on the innermost myelin wrap, adjacent to the axon. It stabilizes myelin-axon interactions by binding to complementary ligands on the axolemma. MAG, a member of the Siglec family of sialic acid-binding lectins, binds specifically to gangliosides GD1a and GT1b, which are the major sialoglycoconjugates on mammalian axons. Mice with a disrupted Galgt1 gene lack UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase (GM2/GD2 synthase) and fail to express complex brain gangliosides, including GD1a and GT1b, instead expressing a comparable amount of the simpler gangliosides GM3, GD3, and O-acetyl-GD3. Galgt1-null mice produce similar amounts of total myelin compared to wild-type mice, but as the mice age, they exhibit axon degeneration and dysmyelination with accompanying motor behavioral deficits. Here we report that Galgt1-null mice display progressive and selective loss of MAG from the brain. At 1.5 months of age, MAG expression was similar in Galgt1-null and wild-type mice. However, by 6 months of age MAG was decreased approximately 60% and at 12 months of age approximately 70% in Galgt1-null mice compared to wild-type littermates. Expression of the major myelin proteins (myelin basic protein and proteolipid protein) was not reduced in Galgt1-null mice compared to wild type. MAG mRNA expression was the same in 12-month-old Galgt1-null compared to wild-type mice, an age at which MAG protein expression was markedly reduced. We conclude that the maintenance of MAG protein levels depends on the presence of complex gangliosides, perhaps due to enhanced stability when MAG on myelin binds to its complementary ligands, GD1a and GT1b, on the apposing axon surface.