Sphingolipids of the mycopathogen Sporothrix schenckii: identification of a glycosylinositol phosphorylceramide with novel core GlcNH(2)alpha1-->2Ins motif

Acidic glycosphingolipid components were extracted from the yeast form of the dimorphic mycopathogen Sporothrix schenckii. Two minor and the major fraction from the yeast form (Ss-Y1, -Y2, and -Y6, respectively) have been isolated. By a combination of 1- and 2-D 1H-nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and gas chromatography/mass spectrometry (GC/MS), Ss-Y6 was determined to be triglycosylinositol phosphorylceramide with a novel glycan structure, Manalpha1-->3Manalpha1-->6GlcNH(2)alpha1-->2Ins1-P-1Cer (where Ins=myo-inositol, P=phosphodiester). While the GlcNH(2)alpha1-->6Ins1-P- motif is found widely distributed in eukaryotic GPI anchors, the linkage GlcNH(2)alpha1-->2Ins1-P- has not been previously observed in any glycolipid. Ss-Y1 and Ss-Y2 were both found to have the known glycan structure Manalpha1-->3Manalpha1-->2Ins1-P-1Cer. Together with the results of a prior study [Toledo et al. (2001) Biochem. Biophys. Res. Commun. 280, 19-24] which showed that the mycelium form expresses GIPCs with the structures Manalpha1-->6Ins1-P-1Cer and Manalpha1-->3Manalpha1-->6Ins1-P-1Cer, these results demonstrate that S. schenckii can synthesize glycosylinositol phosphorylceramides with at least three different core linkages.     

Structure elucidation of sphingolipids from the mycopathogen Sporothrix schenckii: identification of novel glycosylinositol phosphorylceramides with core manalpha1-->6Ins linkage

Acidic glycosphingolipid components were extracted from the mycelium form of the thermally dimorphic mycopathogen Sporothrix schenckii. Two fractions from the mycelium form (Ss-M1 and Ss-M2), having the highest Rf values on HPTLC analysis, were isolated and their structures elucidated by 1- and 2-D 13C- and 1H-nuclear magnetic resonance spectroscopy, and electrospray ionization mass spectrometry with lithium adduction of molecular ions. The structures of Ss-M1 and Ss-M2 were determined to be Manalpha1-->Ins1-P-1Cer and Manalpha1--> 3Manalpha1-->Ins1-P-1Cer, respectively (where Ins = myo-inositol, P = phosphodiester). The Manalpha1-->6Ins motif is found normally in diacylglycerol-based glycophosphatidylinositols of Mycobacteria, but this is the first unambiguous identification of the same linkage making up the core structure of fungal glycosylinositol phosphorylceramides (GIPCs). These results are discussed in relation to the structures of GIPCs of other mycopathogens, including Histoplasma capsulatum and Paracoccidioides brasiliensis.     

Glycosphingolipids of the model fungus Aspergillus nidulans: characterization of GIPCs with oligo-alpha-mannose-type glycans

Aspergillus nidulans is a well-established nonpathogenic laboratory model for the opportunistic mycopathogen, A. fumigatus. Some recent studies have focused on possible functional roles of glycosphingolipids (GSLs) in these fungi. It has been demonstrated that biosynthesis of glycosylinositol phosphorylceramides (GIPCs) is required for normal cell cycle progression and polarized growth in A. nidulans (Cheng, J., T.-S. Park, A. S. Fischl, and X. S. Ye. 2001. Mol. Cell Biol. 21: 6198-6209); however, the structures of A. nidulans GIPCs were not addressed in that study, nor were the functional significance of individual structural variants and the downstream steps in their biosynthesis. To initiate such studies, acidic GSL components (designated An-2, -3, and -5) were isolated from A. nidulans and subjected to structural characterization by a combination of one-dimensional (1-D) and 2-D NMR spectroscopy, electrospray ionization-mass spectrometry (ESI-MS), ESI-MS/collision-induced decomposition-MS (MS/CID-MS), ESI-pseudo-[CID-MS]2, and gas chromatography-MS methods. All three were determined to be GIPCs, with mannose as the only monosaccharide present in the headgroup glycans; An-2 and An-3 were identified as di- and trimannosyl inositol phosphorylceramides (IPCs) with the structures Man alpha 1-->3Man alpha 1-->2Ins1-P-1Cer and Man alpha 1-->3(Man alpha 1-->6)Man alpha 1-->2Ins1-P-1Cer, respectively (where Ins = myo-inositol, P = phosphodiester, and Cer = ceramide). An-5 was partially characterized, and is proposed to be a pentamannosyl IPC, based on the trimannosyl core structure of An-3.     

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 elucidation of neutral, di-, tri-, and tetraglycosylceramides from High Five cells: identification of a novel (non-arthro-series) glycosphingolipid pathway

The major neutral glycosphingolipids (GSLs) of High Five insect cells have been extracted, purified, and characterized. It was anticipated that GSLs from High Five cells would follow the arthro-series pathway, known to be expressed by both insects and nematodes at least through the common tetraglycosylceramide (Glcbeta1Cer --> Manbeta4Glcbeta1Cer [MacCer] --> GlcNAcbeta3Manbeta4Glcbeta1Cer [At(3)Cer] --> GalNAcbeta4- GlcNAcbeta3Manbeta4Glcbeta1Cer [At(4)Cer]). Surprisingly, the structures of the major neutral High Five GSLs already diverge from the arthro-series pathway at the level of the triglycosylceramide. Studies by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI-MS) showed the structure of the predominant High Five triglycosylceramide to be Galbeta3Manbeta4Glcbeta1Cer, whereas the predominant tetraglycosylceramide was characterized as GalNAcalpha4Galbeta3Manbeta4- Glcbeta1Cer. Both of these structures are novel products for any cell or organism so far studied. The GalNAcalpha4 and Galbeta3 units are found in insect GSLs, but always as the fifth and sixth residues linked to GalNAcbeta4 in the arthro-series penta- and hexaglycosylceramide structures (At(5)Cer and At(6)Cer, respectively). The structure of the High Five tetraglycosylceramide thus requires a reversal of the usual order of action of the glycosyltransferases adding the GalNAcalpha4 and Galbeta3 residues in dipteran GSL biosynthesis and implies the existence of an insect Galbeta3-T capable of using Manbeta4Glcbeta1Cer as a substrate with high efficiency. The results demonstrate the potential appearance of unexpected glycoconjugate biosynthetic products even in widely used but unexamined systems, as well as a potential for core switching based on MacCer, as observed in mammalian cells based on the common LacCer intermediate.     

Studies on Galalpha3-binding proteins: comparison of the glycosphingolipid binding specificities of Marasmius oreades lectin and Euonymus europaeus lectin

The carbohydrate binding preferences of the Galalpha3Galbeta4 GlcNAc-binding lectins from Marasmius oreades and Euonymus europaeus were examined by binding to glycosphingolipids on thin-layer chromatograms and in microtiter wells. The M. oreades lectin bound to Galalpha3-terminated glycosphingolipids with a preference for type 2 chains. The B6 type 2 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) was preferred over the B5 glycosphingolipid (Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), suggesting that the alpha2-linked Fuc is accommodated in the carbohydrate binding site, providing additional interactions. The lectin from E. europaeus had broader binding specificity. The B6 type 2 glycosphingolipid was the best ligand also for this lectin, but binding to the B6 type 1 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer) was also obtained. Furthermore, the H5 type 2 glycosphingolipid (Fucalpha2Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), devoid of a terminal alpha3-linked Gal, was preferred over the the B5 glycosphingolipid, demonstrating a significant contribution to the binding affinity by the alpha2-linked Fuc. The more tolerant nature of the lectin from E. europaeus was also demonstrated by the binding of this lectin, but not the M. oreades lectin, to the x2 glycosphingolipid (GalNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. The A6 type 2 glycosphingolipid (GalNAcalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GalNAcalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer were not recognized by the lectins despite the interaction with B6 type 2 glycosphingolipid and the B5 glycosphingolipid. These observations are explained by the absolute requirement of a free hydroxyl in the 2-position of Galalpha3 and that the E. europaea lectin can accommodate a GlcNAc acetamido moiety close to this position by reorienting the terminal sugar, whereas the M. oreades lectin cannot.     

Purification and characterization of a Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc-specific lectin from the fruiting body of the polypore mushroom Polyporus squamosus

A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on beta-D-galactosyl-Synsorb and ion-exchange chromatography on DEAE-Sephacel. Gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing indicated that the native lectin, designated P. squamosus agglutinin, is composed of two identical 28-kDa subunits associated by noncovalent bonds. P. squamosus agglutinin agglutinated human A, B, and O and rabbit red blood cells but precipitated only with human alpha(2)-macroglobulin, of many glycoproteins and polysaccharides tested. The detailed carbohydrate binding properties of the purified lectin were elucidated using three different approaches, i.e. precipitation inhibition assay (in solution binding assay), fluorescence quenching studies, and glycolipid binding by lectin staining on high-performance thin layer chromatography (solid-phase binding assay). Based on the results obtained by these assays, we conclude that although the P. squamosus lectin binds beta-D-galactosides, it has an extended carbohydrate-combining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Acalpha2, 6Galbeta1,4Glc/GlcNAc (6'-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for alpha2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research.     

Preferential binding of the anticancer drug rViscumin (recombinant mistletoe lectin) to terminally alpha2-6-sialylated neolacto-series gangliosides

Production of biochemically defined recombinant mistletoe lectin was achieved by cloning and separate expression of the single catalytically active A-chain and the B-chain with carbohydrate binding properties in Escherichia coli, yielding an active heterodimeric protein named rViscumin (Eck et al. [1999] Eur. J. Biochem., 265, 788-797). Employing solid phase binding assays, rViscumin was shown to preferentially bind to terminally alpha2-6-sialylated neolacto-series gangliosides IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer isolated from human granulocytes. Only marginal binding of rViscumin to galactose-terminated neutral GSLs was determined, whereas reinvestigation of ricin specificity demonstrated this lectin as a galactose-binding protein. Human promyelotic HL-60 cells exhibited an IC(50) value (half maximum cytotoxicity) of 1.16 pM and human bladder carcinoma 5637 cells of 12.1 pM rViscumin; CHO-K1 cells were resistant to rViscumin treatment up to a concentration of 5.26 nM tested. Quantification of the predominant receptor ganglioside IV(6)Neu5Ac-nLc4Cer by means of a specific anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibody revealed 3.68 x 10(6) and 1.54 x 10(6) receptor molecules per HL-60 and 5637 cell, respectively; CHO-K1 cells were negative, lacking alpha2-6-sialylated gangliosides. The data imply a direct correlation of rViscumin cytotoxicity and the expression of receptor ganglioside. Moreover, CHO-K1 cells were rendered susceptible toward rViscumin cytotoxicity after exogenous application of human granulocyte gangliosides. Thus, (1) rViscumin has to be considered as a sialic acid-specific rather than a galactose-specific type II ribosome-inactivating protein, and (2) neolacto-series gangliosides with Neu5Acalpha2-6Galbeta1-4GlcNAc-terminus are true functional and physiologically relevant rViscumin receptors.     

Newly discovered neutral glycosphingolipids in aureobasidin A-resistant zygomycetes: Identification of a novel family of Gala-series glycolipids with core Gal alpha 1-6Gal beta 1-6Gal beta sequences

We found for the first time that Zygomycetes species showed resistance to Aureobasidin A, an antifungal agent. A novel family of neutral glycosphingolipids (GSLs) was found in these fungi and isolated from Mucor hiemalis, which is a typical Zygomycetes species. Their structures were completely determined by compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry, and (1)H NMR spectroscopy. They were as follows: Gal beta 1-6Gal beta 1-1Cer (CDS), Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTS), Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTeS), and Gal alpha 1-6Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CPS). The ceramide moieties of these GSLs consist of 24:0, 25:0, and 26:0 2-hydroxy acids as major fatty acids and 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid. However, the glycosylinositolphosphoceramide families that are the major GSLs components in fungi were not detected in Zygomycetes at all. This seems to be the reason that Aureobasidin A is not effective for Zygomycetes as an antifungal agent. Our results indicate that the biosynthetic pathway for GSLs in Zygomycetes is significantly different from those in other fungi and suggest that any inhibitor of this pathway may be effective for mucormycosis, which is a serious pathogenic disease for humans.     

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.     

A chimeric lectin formed from Bauhinia purpurea lectin and Lens culinaris lectin recognizes a unique carbohydrate structure

Lectins are carbohydrate-binding proteins widely used in biochemical, immunochemical, and histochemical studies. Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose. Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-143 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells. The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns. Glycopeptides carrying three constitutive carbohydrate sequences of Galbeta1-3GalNAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin. In contrast, hybrid-type and high mannose-type glycopeptides with a Manalpha1-6(Manalpha1-3)Manalpha1-6Man sequence were found to have a moderate affinity for the chimeric lectin. This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues. Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies.     

Poly-N-acetyllactosamine synthesis in branched N-glycans is controlled by complemental branch specificity of I-extension enzyme and beta1,4-galactosyltransferase I.

Poly-N-acetyllactosamine is a unique carbohydrate that can carry various functional oligosaccharides, such as sialyl Lewis X. It has been shown that the amount of poly-N-acetyllactosamine is increased in N-glycans, when they contain Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->4GlcNAcbeta1 -->2)Manalpha1-->6 branched structure. To determine how this increased synthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and beta1, 4galactosyltransferase I (beta4Gal-TI). First, N-acetyllactosamine repeats were more readily added to the branched acceptor than the summation of poly-N-acetyllactosamines formed individually on each unbranched acceptor. Surprisingly, poly-N-acetyllactosamine was more efficiently formed on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain than in Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R, due to preferential action of iGnT on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain. On the other hand, galactosylation was much more efficient on beta1,6-linked GlcNAc than beta1,2-linked GlcNAc, preferentially forming Galbeta1-->4GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalph a1-->6Manbeta -->R. Starting with this preformed acceptor, N-acetyllactosamine repeats were added almost equally to Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R and Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and beta4Gal-TI leads to efficient and equal addition of N-acetyllactosamine repeats on both side chains of GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalpha1-->6Manbet a-->R structure, which is consistent with the structures found in nature. The results also suggest that the addition of Galbeta1-->4GlcNAcbeta1-->6 side chain on Galbeta1-->4GlcNAcbeta1-->2Man-->R side chain converts the acceptor to one that is much more favorable for iGnT and beta4Gal-TI.     

Isolectins from Solanum tuberosum with different detailed carbohydrate binding specificities: unexpected recognition of lactosylceramide by N-acetyllactosamine-binding lectins

Glycosphingolipid recognition by two isolectins from Solanum tuberosum was compared by the chromatogram binding assay. One lectin (PL-I) was isolated from potato tubers by affinity chromatography, and identified by MALDI-TOF mass spectrometry as a homodimer with a subunit molecular mass of 63,000. The other (PL-II) was a commercial lectin, characterized as two homodimeric isolectins with subunit molecular masses of 52,000 and 55,000, respectively. Both lectins recognized N-acetyllactosamine-containing glycosphingolipids, but the fine details of their carbohydrate binding specificities differed. PL-II preferentially bound to glycosphingolipids with N-acetyllactosamine branches, as Galbeta4GlcNAcbeta6(Galbeta4GlcNAcbeta3)Galbeta4Glcbeta1C er. PL-I also recognized this glycosphingolipid, but bound equally well to the linear glycosphingolipid Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. Neolactotetraosylceramide and the B5 pentaglycosylceramide were also bound by PL-I, while other glycosphingolipids with only one N-acetyllactosamine unit were non-binding. Surprisingly, both lectins also bound to lactosylceramide, with an absolute requirement for sphingosine and non-hydroxy fatty acids. The inhibition of binding to both lactosylceramide and N-acetyllactosamine-containing glycosphingolipids by N-acetylchitotetraose suggests that lactosylceramide is also accomodated within the N-acetylchitotetraose/N-acetyllactosamine-binding sites of the lectins. Through docking of glycosphingolipids onto a three-dimensional model of the PL-I hevein binding domain, a Galbeta4GlcNAcbeta3Galbeta4 binding epitope was defined. Furthermore, direct involvement of the ceramide in the binding of lactosylceramide was suggested.     

Characterization of neutral glycosphingolipids from porcine erythrocyte membranes

1. Six neutral GSL fractions were purified from porcine erythrocyte membranes. 2. They were identified to be LacCer (14% of total neutral GSLs), 2-hydroxy acid-rich and -poor Gb3Cer (3 and 7%, respectively) and Gb4Cer (71%) by means of NMR spectrometry. 3. Monohexosylceramides (5%) were composed of GlcCer and GalCer with near amount. 4. All these GSL classes contained a high concentration (more than 20% of total acids in each class) of 2-hydroxy fatty acids. 5. GalCer and GlcCer contained considerable amounts of C16- and C18-acids, and of C18-phytosphingosine, whereas C24-acids and C18-sphingosine were predominant in the other GSLs. 6. A minor GSL fraction (less than 1% of total neutral GSLs) which migrated more slowly than Gb5Cer on a thin layer plate and composed of several GSL components contained L-fucose.     

Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry

The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin ( approximately 25 microg) onto porous glass beads in a tubing column (9.8 microl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc containing oligosaccharides with K(d) values near 10 microM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.     

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus

Acidic glycosphingolipid components were extracted from the opportunistic mycopathogen Aspergillus fumigatus and identified as inositol phosphorylceramide and glycosylinositol phosphorylceramides (GIPCs). Using nuclear magnetic resonance sppectroscopy, mass spectrometry, and other techniques, the structures of six major components were elucidated as Ins-P-Cer (Af-0), Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-2), Manp(alpha1-->2)Manp(alpha1-->3)Manp(alpha1-->2)Ins-P-Cer (Af-3a), Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)-Ins-P-Cer (Af-3b), Manp(alpha1-->2)-Manp(alpha1-->3)[Galf(beta1-->6)]Manp(alpha1-->2)Ins-P-Cer (Af-4), and Manp(alpha1-->3)Manp(alpha1-->6)GlcpN(alpha1-->2)Ins-P-Cer (Af-3c) (where Ins = myo-inositol and P = phosphodiester). A minor A. fumigatus GIPC was also identified as the N-acetylated version of Af-3c (Af-3c*), which suggests that formation of the GlcNalpha1-->2Ins linkage may proceed by a two-step process, similar to the GlcNalpha1-->6Ins linkage in glycosylphosphatidylinositol (GPI) anchors (transfer of GlcNAc, followed by enzymatic de-N-acetylation). The glycosylinositol of Af-3b, which bears a distinctive branching Galf(beta1-->6) residue, is identical to that of a GIPC isolated previously from the dimorphic mycopathogen Paracoccidioides brasiliensis (designated Pb-3), but components Af-3a and Af-4 have novel structures. Overlay immunostaining of A. fumigatus GIPCs separated on thin-layer chromatograms was used to assess their reactivity against sera from a patient with aspergillosis and against a murine monoclonal antibody (MEST-1) shown previously to react with the Galf(beta1-->6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.     

Preparation of homogenous oligosaccharide chains from glycosphingolipids

After the discovery of glycosphingolipid (GSL) glycan detaching enzymes, Rhodococcal endoglycoceramidase (EGCase) and leech ceramide glycanase (CGase), the method for enzymatically releasing glycans from GSLs has become the method of choice for preparing intact ceramide-free oligosaccharide chains from GSLs. This paper describes (1) the preparation of the intact oligosaccharides from GM1 (II³NeuAcGgOse₄Cer) and GbOse₄Cer as examples to show the use of CGase to prepare intact glycan chains from GSLs, and (2) the specificity and detergent requirements of Rhodococcal EGCases for the release of glycan chains from different GSLs.     

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.     

Urinary oligosaccharides of fucosidosis. Evidence of the occurrence of X-antigenic determinant in serum-type sugar chains of glycoproteins.

Urine of a fucosidosis patient contained a large amount of fucosyl oligosaccharides and fucose-rich glycopeptides. Six major oligosaccharides were purified by a combination of Bio-Gel P-2 and P-4 column chromatographies and paper chromatography. Structural studies by sequential exoglycosidase digestion and by methylation analysis revealed that their structures were as follows: Fucalpha1 leads to 6GlcNAc, Fucalpha1 leads to 2Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 2Manalpha1 leads to 3Manbeta1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to3)GlcNAcbeta1 leads to 2Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc, and Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 6Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc. In additon, the structure of a minor decasaccharide was found to be Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to [Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to]Manbeta1 leads to 4GlcNAc.     

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.