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

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.     

Novel phosphonoglycosphingolipids containing pyruvylated galactose from the nervous system of Aplysia kurodai

We have reported the existence of a phosphonoglycosphingolipid containing a pyruvylated galactose, FGL-IIb, in nerve fibers of Aplysia kurodai (Araki, S., Abe, S., Ando, S., Kon, K., Fujiwara, N. & Satake, M. (1989) J. Biol. Chem. 264, 19922-19927). We have now isolated two other pyruvylated galactose-containing phosphonoglycosphingolipids, named FGL-V and FGL-IIa, from the nervous tissue of Aplysia, and characterized them as [3,4-O-(S-1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3[6'-O-(2- aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6) Gal beta 1----4Glc beta 1----1 ceramide and [3,4,O-(S-1-carboxyethylidene)] Gal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 11----ceramide, respectively. Their major aliphatic components are palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, the nervous system of Aplysia contains several pyruvylated phosphonoglycolipids.     

Structure of a neutral glycosphingolipid recognized by human antibodies in polyagglutinable erythrocytes from the rare NOR phenotype

NOR is a rare inheritable polyagglutination phenomenon that has been described in two families. Our recent studies on these erythrocytes showed they contained at least two unique neutral glycosphingolipids, and based on their reactivity with Griffonia simplicifolia IB4 (GSL-IB4) isolectin (Kusnierz-Alejska, G., Duk, M., Storry, J. R., Reid, M. E., Wiecek, B., Seyfried, H., and Lisowska, E. (1999) Transfusion 39, 32-38), both oligosaccharide chains terminated with an alpha-galactose residue. The reactivity with GSL-IB4 suggested that these oligosaccharide chains terminated with a Galalpha1-->3Gal- sequence and that anti-NOR agglutinins were common human anti-Galalpha1-->3Gal xenoantibodies. In this report we describe the structure of one NOR component (NOR1) that migrated on thin-layer chromatographic plates in the region of pentaglycosylceramides. Treatment of this sample with alpha-galactosidase and beta-N-acetylhexosaminidase was followed by high-performance thin-layer chromatography with product detection by lectins and the anti-Gb4 monoclonal antibody. The results suggested that NOR1 was an alpha-galactosylated Gb4Cer with a beta-N-acetylhexosaminidase-resistant GalNAc residue. Gas phase disassembly by ion trap mass spectrometry analysis showed the sequence to be Hex1-->4HexN1-->3Hex1-->4Hex1-->4Hex linked to a ceramide composed of C18 sphingosine and a C24 monounsaturated fatty acid. Together these data indicate NOR1 to be a novel Galalpha1-->4GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4 Glc-Cer structure. Additionally it has been shown that NOR glycolipids are recognized by human antibodies that were distinct from the known anti-Galalpha1-->3Gal xenoantibodies.     

Structure of phosphonoglycosphingolipid containing pyruvylated galactose in nerve fibers of Aplysia kurodai

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.     

Defining the carbohydrate specificities of aplysia gonad lectin exhibiting a peculiar D-galacturonic acid affinity

Aplysia gonad lectin (AGL), which has been shown to stimulate mitogenesis in human peripheral lymphocytes, to suppress tumor cells, and to induce neurite outgrowth and improve cell viability in cultured Aplysia neurons, exhibits a peculiar galacturonic acid/galactose specificity. The carbohydrate binding site of this lectin was characterized by enzyme-linked lectino-sorbent assay and by inhibition of AGL-glycan interactions. Examination of the lectin binding with 34 glycans revealed that it reacted strongly with the following glycoforms: most human blood group precursor (equivalent) glycoproteins (gps), two Galalpha1-->4Gal-containing gps, and two d-galacturonic acid (GalUA)-containing polysaccharides (pectins from apple and citrus fruits), but poorly with most human blood group A and H active and sialylated gps. Among the GalUA and mammalian saccharides tested for inhibition of AGL-glycan binding, GalUA mono- to trisaccharides were the most potent ones. They were 8.5 x 10(4) times more active than Gal and about 1.5 x 10(3) more active than the human blood group P(k) active disaccharide (E, Galalpha1-->4Gal). This disaccharide was 6, 28, and 120 times more efficient than Galbeta1-->3GlcNAc(I), Galbeta1-->3GalNAc(T), and Galbeta1--> 4GlcNAc (II), respectively, and 35 and 80 times more active than melibiose (Galalpha1-->6Glc) and human blood group B active disaccharide (Galalpha1-->3Gal), respectively, showing that the decreasing order of the lectin affinity toward alpha-anomers of Gal is alpha1-->4 > alpha1-->6 > alpha1-->3. From the data provided, the carbohydrate specificity of AGL can be defined as GalUAalpha1-->4 trisaccharides to mono GalUA > branched or cluster forms of E, I, and II monomeric E, I, and II, whereas GalNAc is inactive.     

Structures of ceramide tetrasaccharides from various sources: uniqueness of rat kidney ceramide tetrasaccharide

Methylation analysis of ceramide tetrasaccharide isolated from human erythrocytes gave acetates of 2,3,6-tri-O-methylgalactitol and 2,4,6-tri-O-methylgalactitol in a ratio of 1:1, and about 50% of the galactose was oxidized by periodate. Rat kidney ceramide tetrasaccharide gave, in contrast, a much larger proportion of the acetates of 2,4,6-tri-O-methylgalactitol (ratio 1:0.3), and less than 20% of the galactose was oxidized by periodate. Sequential degradation by beta-N-acetylhexosaminidase, alpha-galactosidase, and beta-galactosidase showed ceramide tetrasaccharides to have identical carbohydrate sequences and anomeric structures. The major part of ceramide trihexoside derived from rat kidney ceramide tetrasaccharide migrated on thin-layer chromatography more slowly than that derived from other ceramide tetrasaccharides. The structure of a major part of rat kidney ceramide tetrasaccharide was thus determined to be GalNAcbeta(1-->3)Galalpha(1-->3)Galbeta(1-->4)Glcbeta(1-->1)Cer, whereas other ceramide tetrasaccharides have Galalpha(1-->4) structure at the penultimate residue.     

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.     

Structure of a novel diphosphonoglycosphingolipid isolated from the skin of Aplysia kurodai

A new phosphonoglycosphingolipid containing two 2-aminoethylphosphonate residues was isolated from the skin of Aplysia kurodai, a marine gastropod, using two systems of silicic acid chromatography. By methanolysis, permethylation, mild acid hydrolysis and hydrogen fluoride treatment combined with thin layer chromatography and gas chromatography-mass spectrometry, the new phosphonoglycosphingolipid was shown to be 3-O-MeGal (1----3) GalNAc (1----3) [6'-O-(2-aminoethylphosphonyl) Gal (1----2)] [2-aminoethylphosphonyl (----6)] Gal (1----4) Glc (1----1) ceramide. Most of the fatty acid (90 per cent) was palmitic acid. Octadeca-4-sphingenine and anteiso-nonadeca-4-sphingenine were the major sphingosine bases of the new glycolipid.     

Characterization of a diphosphonopentaosylceramide containing 3-O-methylgalactose from the skin of Aplysia kurodai (sea hare)

The complete structure is proposed for a ceramide (Cer), bis(2-aminoethylphosphono)-pentaoside, isolated from the skin of Aplysia kurodai. This new phosphonoglycosphingolipid was purified using two systems of column chromatography on silicic acid. The purity of the glycolipid was confirmed by thin-layer chromatography, analysis of its composition, and proton magnetic resonance spectrometry. The component carbohydrates were glucose, galactose, N-acetylgalactosamine, and 3-O-methylgalactose. Most (90%) of the fatty acid was palmitic acid and the major sphingosine bases were octadeca-4-sphingenine (51%) and anteisononadeca-4-sphingenine (38%). 2-Aminoethylphosphonyl-6-galactose was identified after its partial hydrolysis. From studies by methanolysis, permethylation, mild acid hydrolysis, hydrogen fluoride treatment, chromium trioxide oxidation combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry, the structure of the glycolipid was concluded to be 3-OMeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)-Gal alpha 1----2](2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1Cer.     

Characterization of neutral and acidic glycosphingolipids from the lectin-producing mushroom, Polyporus squamosus

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.     

Structural characterization of a novel class of glycophosphosphingolipids from the protozoan Leptomonas samueli.

Aqueous phenol extraction of the lower trypanosomatid Leptomonas samueli released into the aqueous layer a chloroform/methanol/water-soluble glycophosphosphingolipid fraction. Alkaline degradation and purification by gel filtration chromatography resulted in a tetrasaccharide (phosphatidylinositol (PI)-oligosaccharide A), and a pentasaccharide (PI-oligosaccharide B), each containing 2 mol of 2-aminoethylphosphonate and 1 mol of phosphate. Nuclear magnetic resonance spectroscopy and fast atom bombardment-mass spectrometry suggested that the structure of PI-oligosaccharide A is [formula: see text] and that of PI-oligosaccharide B is as shown. [formula: see text] Both compounds contain an inositol unit linked to ceramide via a phosphodiester bridge. The major aliphatic components of the ceramide portion are stearic acid, lignoceric acid, and C20-phytosphingosine. These novel glycolipids fall within the glycosylated phosphatidylinositol (GPI) family, since they contain the core structure Man alpha (1-->4)GlcNH2 alpha (1-->6)myo-inositol-1-PO4, which is also found in the glycoinositolphospholipids and lipophosphoglycan of Leishmania spp., the L. major promastigote surface protease, the glycosylphosphatidylinositol anchor of Trypanosoma brucei variant surface glycoprotein, and the lipopeptidophosphoglycan of Trypanosoma cruzi. The glycophosphosphingolipids of Leptomonas have features in common with the glycolipids of both Leishmania and T. cruzi, resembling the former by the alpha (1-->3) linkage of mannose to the GPI core, while the 2-aminoethylphosphonate substituent on O-6 of glucosamine and the presence of ceramide in place of glycerol lipids is more reminiscent of T. cruzi. Thus these data lend some support to the hypothesis that both T. cruzi and Leishmania evolved from a Leptomonas-like ancestor.     

Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols.

A processive diacylglycerol glucosyltransferase has recently been identified from Bacillus subtilis [Jorasch, P., Wolter, F.P., Z?hringer, U., and Heinz, E. (1998) Mol. Microbiol. 29, 419-430]. Now we report the cloning and characterization of two other genes coding for diacylglycerol glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana; only the S. aureus enzyme shows processivity similar to the B. subtilis enzyme. Both glycosyltransferases characterized in this work show unexpected acceptor specificities. We describe the isolation of the ugt106B1 gene (GenBank accession number Y14370) from the genomic DNA of S. aureus and the ugt81A1 cDNA (GenBank accession number AL031004) from A. thaliana by PCR. After cloning and expression of S. aureus Ugt106B1 in Escherichia coli, SDS/PAGE of total cell extracts showed strong expression of a protein having the predicted size of 44 kDa. Thin-layer chromatographic analysis of the lipids extracted from the transformed E. coli cells revealed several new glycolipids and phosphoglycolipids not present in the controls. These lipids were purified from lipid extracts of E. coli cells expressing the S. aureus gene and identified by NMR and mass spectrometry as 1, 2-diacyl-3-[O-beta-D-glucopyranosyl]-sn-glycerol, 1, 2-diacyl-3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyrano-+ ++syl] -sn-glycerol, 1, 2-diacyl-3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-( 1-->6)-O-beta-D-glucopyranosyl]-sn-glycerol, sn-3'-[O-beta-D-glucopyranosyl]-phosphatidylglycerol and sn-3'-[O-(6"'-O-acyl)-beta-D-glucopyranosyl-(1"'-->6")-O-beta-D-gluco pyranosyl]-sn-2'-acyl-phospha-tidylglycerol. A 1, 2-diacyl-3-[O-beta-D-galactopyranosyl]-sn-glycerol was isolated from extracts of E. coli cells expressing the ugt81A1 cDNA from A. thaliana. The enzymatic activities expected to catalyze the synthesis of these compounds were confirmed by in vitro assays with radioactive substrates. Experiments with several of the above described glycolipids as 14C-labeled sugar acceptors and unlabeled UDP-glucose as glucose donor, suggest that the ugt106B1 gene codes for a processive UDP-glucose:1, 2-diacylglycerol-3-beta-D-glucosyltransferase, whereas ugt81A1 codes for a nonprocessive diacylglycerol galactosyltransferase. As shown in additional assays with different lipophilic acceptors, both enzymes use diacylglycerol and ceramide, but Ugt106B1 also accepts glucosyl ceramide as well as cholesterol and cholesterol glucoside as sugar acceptors.     

Structure of a triphosphonopentaosylceramide containing 4-O-methyl-N-acetylglucosamine from the skin of the sea hare, Aplysia kurodai

A novel phosphonoglycosphingolipid named SGL-I containing 3 mol of 2-aminoethylphosphonate residues was isolated from the skin of a sea gastropod, Aplysia kurodai. The saccharide moiety of the glycolipid was characterized as 4-O-methyl-GlcNAc alpha 1----4GalNAc alpha-1----3 [6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6)Gal beta 1----4(2-aminoethylphosphonyl----6) Glc beta 1----1-ceramide. The major aliphatic components of the ceramide portion were palmitic acid, stearic acid, octadeca-4-sphingenine, and anteisononadeca-4-sphingenine. This glycolipid is unique in containing 4-O-methyl-N-acetylglucosamine and 3 mol of 2-aminoethylphosphonate residues, one of which is attached to C-6 of glucose.     

Characterization of novel structures of mannosylinositolphosphorylceramides from the yeast forms of Sporothrix schenckii.

Novel structures of glycoinositolphosphorylceramide (GIPC) from the infective yeast form of Sporothrix schenckii were determined by methylation analysis, mass spectrometry and NMR spectroscopy. The lipid portion was characterized as a ceramide composed of C-18 phytosphingosine N-acylated by either 2-hydroxylignoceric acid (80%), lignoceric (15%) or 2,3-dihydroxylignoceric acids (5%). The ceramide was linked through a phosphodiester to myo-inositol (Ins) which is substituted on position O-6 by an oligomannose chain. GIPC-derived Ins oligomannosides were liberated by ammonolysis and characterized as: Manpalpha1-->6Ins; Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->2Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins. These structures comprise a novel family of fungal GIPC, as they contain the Manpalpha1-->6Ins substructure, which has not previously been characterized unambigously, and may be acylated with a 2,3 dihydroxylignoceric fatty acid, a feature hitherto undescribed in fungal lipids.     

Characterization of glycoinositolphosphoryl ceramide structure mutant strains of Cryptococcus neoformans

In fungi, glycoinositolphosphoryl ceramide (GIPC) biosynthetic pathway produces essential molecules for growth, viability, and virulence. In previous studies, we demonstrated that the opportunistic fungus Cryptococcus neoformans synthesizes a complex family of xylose-(Xyl) branched GIPCs, all of which have not been previously reported in fungi. As an effort to understand the biosynthesis of these sphingolipids, we have now characterized the structures of GIPCs from C. neoformans wild-type (KN99alpha) and mutant strains that lack UDP-Xyl, by disruption of either UDP-glucose dehydrogenase (NE321) or UDP-glucuronic acid decarboxylase (NE178). The structures of GIPCs were determined by a combination of nuclear magnetic resonance (NMR) spectroscopy, tandem mass spectrometry (MS), and gas chromatography-MS. The main and largest GIPC from wild-type strain was identified as an alpha-Manp(1 --> 6)alpha-Manp(1 --> 3)alpha-Manp[beta-Xylp(1 --> 2)]alpha-Manp(1 --> 4)beta-Galp(1 --> 6)alpha-Manp(1 --> 2) Ins-1-P-Ceramide, whereas the most abundant GIPC from both mutant strains was found to be an alpha-Manp(1 --> 3)alpha-Manp(1 --> 4)beta-Galp(1 --> 6)alpha-Manp(1 --> 2)Ins-1-P-Ceramide. The ceramide moieties of C. neoformans wild-type and mutant strains were composed of a C(18) phytosphingosine, which was N-acylated with 2-hydroxy tetra-, or hexacosanoic acid, and 2,3-dihydroxy-tetracosanoic acid. Our structural analysis results indicate that the C. neoformans mutant strains are unable to complete the assembly of the GIPC-oligosaccharide moiety due the absence of Xyl side chain.     

Structural analysis of two glycosphingolipids from the lipopolysaccharide-lacking bacterium Sphingomonas capsulata.

Two glycosphingolipids, GSL-1 and GSL-3, were isolated from Sphingomonas capsulata and studied by methylation analysis, laser desorption mass spectrometry, and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY and heteronuclear 13C,1H COSY experiments. GSL-1 and GSL-3 differ in their carbohydrate part, their structures being alpha-D-GlcpA-(1-->1)-Cer and alpha-D-Galp-(1-->6)-alpha-D-GlcpN-(1-->4)-alpha-D-GlcpA(1-- >1)Cer, respectively. Variations occur in the ceramide of GSL-1 and GSL-3, both having the same long-chain bases, erythro-2-amino-1, 3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1, 3-diol and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol, in the ratios 2.6 : 1 : 3.5 in GSL-1 and 1 : 1.2 : 1.5 in GSL-3. All bases are quantitatively substituted by amide-linked (S)-2-hydroxymyristic acid.     

Structure of triphosphonoglycosphingolipid containing N-acetylgalactosamine 6-O-2-aminoethylphosphonate in the nervous system of Aplysia kurodai

A phosphonoglycosphingolipid, named F-21, was found in the nervous system of Aplysia kurodai by two-dimensional thin-layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). F-21 was isolated from the nervous tissue of Aplysia in this study, and its chemical structure was characterized as follows, where 2-AEP is 2-aminoethylphosphonate. (Formula; see text) The major aliphatic components of the ceramide portion were palmitic acid (75%), stearic acid (22%), octadeca-4-sphingenine (43%), and anteisononadeca-4-sphingenine (54%). Some information on the steric interactions in the sugar moiety was obtained by NMR spectroscopy. The ring protons of the internal galactose, H1, H3, and H4 and the H3 of the side chain galactose were shifted, as compared to the corresponding protons of dephosphonylated F-21. This may indicate the interactions between the 2-AEP residue of N-acetylgalactosamine and the internal galactose and between the N-acetyl group of N-acetylgalactosamine and the side chain galactose, implying a sterically restricted and unique structure that may relate to some biological functions of F-21.     

Structural analysis of a new glycosphingolipid from the lipopolysaccharide-lacking bacterium Sphingomonas adhaesiva.

A new glycosphingolipid, GSL-4B, was isolated from Sphingomonas adhaesiva and found to share the ceramide moiety with GSL-1 and GSL-3 from Sphingomonas capsulata studied earlier [Kawahara, K.; Moll, H.; Knirel, Y. A.; Seydel, U.; Z?hringer, U. Eur. J. Biochem. 2000, 267, 1837-1846]. It is heterogeneous with respect to the long-chain bases erythro-2-amino-1,3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1,3-diol, and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol which in GSL-4B are present in the ratios of 1.1:1.0:1.1, and all bearing amide-linked (S)-2-hydroxymyristic acid. Methylation analysis and MALDI-TOF-MS along with 1H and 13C NMR spectroscopy showed that the carbohydrate part of GSL-4B has the structure of alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-GlcpA-(1-->1)-Cer     

Formation and biological activity of 12-ketoeicosatetraenoic acid in the nervous system of Aplysia

12-Hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE), a lipoxygenase product, simulates the synaptic responses produced by the modulatory transmitter, histamine, and the neuroactive peptide, Phe-Met-Arg-Phe-amide (FMRFamide), in identified neurons of the marine mollusk, Aplysia californica (Piomelli, D., Shapiro, E., Feinmark, S. J., and Schwartz, J. H. (1987) J. Neurosci. 7, 3675-3886; Shapiro, E., Piomelli, D., Feinmark, S., Vogel, S., Chin, G., and Schwartz, J. H. (1988) Cold Spring Harbor Symp. Quant. Biol. 53, in press). The 12-lipoxygenase pathway has not yet been fully characterized, but 12-HPETE is known to be metabolized further. We therefore began to search for other metabolites in order to investigate whether the actions of 12-HPETE might require its conversion to other active products. Here we report the identification of 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE), a metabolite of 12-HPETE formed by Aplysia nervous tissue. This product was identified in incubations of the tissue with arachidonic acid using high performance liquid chromatography, UV spectrometry, and gas chromatography/mass spectrometry. [3H]12-KETE was formed from endogenous lipid stores in nervous tissue, labeled by incubation with [3H]arachidonic acid, when stimulated by application of histamine. In L14 and L10 cells, identified neurons in the abdominal ganglion, applications of 12-KETE elicit changes in membrane potential similar to those evoked by histamine. 12(S)-Hydroxy-5,8,10,14-eicosatetraenoic acid, another metabolite of 12-HPETE, is inactive. These results support the hypothesis that 12-HPETE and its metabolite, 12-KETE, participate in transduction of histamine responses in Aplysia neurons.     

Rapid and sensitive GC/MS characterization of glycolipid released Galalpha1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Galalpha1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Galalpha1,3Gal-containing and blood group- type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix- assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Galalpha1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Galalpha1,3nLc4and Galalpha1,3Lexstructures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Galalpha1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Galalpha1,3nLc6structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ- specific expression of blood group AH antigens in the pig.