Monocytic cell activation by Nonendotoxic glycoprotein from Prevotella intermedia ATCC 25611 is mediated by toll-like receptor 2. (1/12)

Lipopolysaccharide (LPS) preparations from gram-negative black-pigmented bacteria such as Porphyromonas gingivalis and Prevotella intermedia activate cells from non-LPS-responsive C3H/HeJ mice, but it is still unclear whether this activity is due to the unique structure of LPS or to a minor component(s) responsible for the activity in the preparation. A nonendotoxic glycoprotein with bioactivity against cells from C3H/HeJ mice was purified from a hot phenol-water extract of P. intermedia ATCC 25611 and designated Prevotella glycoprotein (PGP). Treatment of human monocytic THP-1 cells with 22-oxyacalcitriol (OCT) induced maturation and marked expression of CD14 on the cells, but the cells constitutively expressed Toll-like receptor 2 (TLR2) and TLR4 on the cells irrespective of the treatment. PGP induced a high level of interleukin-8 production at doses of 100 ng/ml and higher in OCT-treated THP-1 cells compared with Salmonella LPS, and the production was significantly inhibited by anti-CD14 and anti-TLR2 but not anti-TLR4 antibodies. Consistent with this, TLR2-deficient murine macrophages did not respond to PGP. It was also shown that PGP activity on the THP-1 cells was LPS-binding protein dependent and was inhibited by a synthetic lipid A precursor IV(A). These results indicate that PGP activates monocytic cells in a CD14- and TLR2-dependent manner.  (+info)

Structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells. (2/12)

Novel ZGLs (zwitterionic glycosphingolipids) have been found in and extracted from the mycelia of filamentous fungi ( Acremonium sp.) isolated from soil. Five ZGLs (ZGL1-ZGL5) were structurally elucidated by sugar compositional analysis, methylation analysis, periodate oxidation, matrix-assisted laser-desorption ionization-time-of-flight MS, (1)H-NMR spectroscopy and fast-atom bombardment MS. Their chemical structures were as follows: GlcN(alpha1-2)Ins1-P-1Cer (ZGL1), Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL2), Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL3), PC-->6Man(alpha1-6)GlcN(alpha1-2)Ins1- P -1Cer (ZGL4), and PC-->6Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL5) (where Cer is ceramide and PC is phosphocholine). In addition, one acidic glycosphingolipid, which was the precursor of ZGLs, was also characterized as inositol-phosphoceramide. The core structure of the ZGLs, GlcN(alpha1-2)Ins1- P, is rather different from those found in other fungi, such as Man(alpha1-2)Ins1- P and Man(alpha1-6)Ins1- P. Interestingly, the terminal mannose residue of ZGL4 and ZGL5 was modified further with a PC group. The presence of PC-containing glycosylinositol-phosphoceramides has not been reported previously in any organism. The ceramide constituents of both ZGLs and acidic glycosphingolipid were essentially the same, and consisted of a 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid base and 2-hydroxytetracosanoic acid (>90%) as the major fatty acid. ZGLs were found to cause cell death in suspensions of cultured rice cells. The cell death-inducing activity of ZGLs is probably due to the characteristic glycan moiety of Man(alpha1-6)GlcN, and PC-containing ZGLs had high activity. This study is the first to demonstrate that fungal glycosylinositol-phosphoceramides induce cell death in cultured rice cells.  (+info)

Role of multiple drug resistance protein 1 in neutral but not acidic glycosphingolipid biosynthesis. (3/12)

Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246-6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis.  (+info)

Characterization of two novel pyruvylated glycosphingolipids containing 2'-aminoethylphosphoryl(-->6)-galactose from the nervous system of Aplysia kurodai. (4/12)

Two novel acidic glycosphingolipids containing pyruvylated galactose were purified from the nervous tissue of Aplysia kurodai by successive Iatrobeads column chromatographies. By component analysis, sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structures of these acidic glycosphingolipids, named F-9 and FGL-I, were determined to be: [3,4-O-(S-1-carboxyethylidene)]Gal beta 1-->3 GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2] (2-aminoethylphosphoryl 1-->6)Gal beta 1-->4Glc beta 1-->1ceramide and [3,4-O-(S-1-carboxyethylidene)] Gal beta 1-->3GalNAc alpha 1-->3(Fuc alpha 1-->2)(2-aminoethylphosphonyl-->6 Gal beta 1-->4Glc beta 1-->1ceramide, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, pyruvylated glycosphingolipids containing phosphoethanolamine in addition to or in place of 2-aminoethylphosphonate are present in the nervous system of Aplysia.  (+info)

Characterization of neutral and acidic glycosphingolipids from the lectin-producing mushroom, Polyporus squamosus. (5/12)

The polypore mushroom Polyporus squamosus is the source of a lectin that exhibits a general affinity for terminal beta-galactosides, but appears to have an extended carbohydrate-binding site with high affinity and strict specificity for the nonreducing terminal trisaccharide sequence NeuAcalpha2 --> 6Galbeta1 --> 4Glc/GlcNAc. In considering the possibility that the lectin's in vivo function could involve interaction with an endogenous glycoconjugate, it would clearly be helpful to identify candidate ligands among various classes of carbohydrate-containing materials expressed by P. squamosus. Since evidence has been accumulating that glycosphingolipids (GSLs) may serve as key ligands for some endogenous lectins in animal species, possible similar roles for fungal GSLs could be considered. For this study, total lipids were extracted from mature fruiting body of P. squamosus. Multistep fractionation yielded a major monohexosylceramide (CMH) component and three major glycosylinositol phosphorylceramides (GIPCs) from the neutral and acidic lipids, respectively. These were characterized by a variety of techniques as required, including one- and two-dimensional (1)H- and (13)C-nuclear magnetic resonance (NMR) spectroscopy; electrospray ionization-mass spectrometry (ESI-MS, tandem-MS/collision-induced decay-MS, and ion trap-MS(n)); and component and methylation linkage analysis by gas chromatography-mass spectrometry. The CMH was determined to be glucosylceramide having a typical ceramide consisting of 2-hydroxy fatty-N-acylated (4E,8E)-9-methyl-sphinga-4,8-dienine. The GIPCs were identified as Manalpha1 --> 2Ins1-P-1Cer (Ps-1), Galbeta1 --> 6Manalpha1 --> 2Ins1-P-1Cer (Ps-2), and Manalpha1 --> 3Fucalpha1 --> 2Galalpha1 --> 6Galbeta1 --> 6Manalpha1 -->2Ins1-P-1Cer (Ps-5), respectively (where Ins = myo-inositol, P = phosphodiester, and Cer = ceramide consisting mainly of long-chain 2-hydroxy and 2,3-dihydroxy fatty-N-acylated 4-hydroxy-sphinganines). Of these GSLs, Ps-2 could potentially interact with P. squamosus lectin, and further investigations will focus on determining the binding affinity, if any, of the lectin for the GIPCs isolated from this fungus.  (+info)

Activation of invariant NKT cells by toll-like receptor 9-stimulated dendritic cells requires type I interferon and charged glycosphingolipids. (6/12)

Invariant natural killer T (iNKT) cells are a subset of innate lymphocytes that recognize lipid antigens in the context of CD1d and mediate potent immune regulatory functions via the rapid production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). We investigated whether diverse Toll-like receptor (TLR) signals in myeloid dendritic cells (DCs) could differentially stimulate iNKT cells. Together with the lipopolysaccharide-detecting receptor TLR4, activation of the nucleic acid sensors TLR7 and TLR9 in DCs were particularly potent in stimulating iNKT cells to produce IFN-gamma, but not IL-4. iNKT cell activation in response to TLR9 stimulation required combined synthesis of type I interferon and de novo production of charged beta-linked glycosphingolipid(s) by DCs. In addition, DCs stimulated via TLR9 activated both iNKT cells and NK cells in vivo and protected mice against B16F10-induced melanoma metastases. These data underline the role of TLR9 in iNKT cell activation and might have relevance to infectious diseases and cancer.  (+info)

Invariant Valpha14 natural killer T cell activation by edible mushroom acidic glycosphingolipids. (7/12)

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.  (+info)

Structure of phosphonoglycosphingolipid containing pyruvylated galactose in nerve fibers of Aplysia kurodai. (8/12)

A phosphonoglycosphingolipid, designated as FGL-IIb, was identified in nerve fibers of Aplysia kurodai by two-dimensional thin layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). FGL-IIb was isolated from the nervous system of A. kurodai by Iatrobeads column chromatography using three solvent systems. Pyruvic acid was identified by thin layer chromatography as its 2,4-dinitrophenylhydrazone and established by permethylation studies to be attached as a ketal to O-3 and O-4 of the terminal galactose of the oligosaccharide chain in FGL-IIb. By sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structure of FGL-IIb was concluded to be [3,4-O-(1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3(Fuc alpha 1----2) (2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1ceramide. Its major aliphatic components were palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. This is the first report of the occurrence of pyruvylated galactose as a constituent of animal sphingolipid.  (+info)

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