RESUMO
An IgG2a monoclonal antibody anti-glucosylceramide was established and termed MEST-2. High performance thin layer chromatography immunostaining, and solid-phase radioimmunoassay showed that MEST-2 reacts with glucosylceramide from yeast and mycelium forms of Paracoccidioides brasiliensis, Histoplasma capsulatum, and Sporothrix schenckii; from hyphae of Aspergillus fumigatus; and from yeast forms of Candida albicans, Cryptococcus neoformans, Cryptococcus laurentii, and Cryptococcus albidus. Studies on the fine specificity of MEST-2 showed that it recognizes the beta-D-glucose residue, and that the 2-hydroxy group present in the fatty acid is an important auxiliary feature for the antibody binding. It was also demonstrated that phosphatidylcholine and ergosterol modulate MEST-2 reactivity to glucosylceramide, by solid-phase radioimmunoassay. Indirect immunofluorescence showed that MEST-2 reacts with the surface of yeast forms of P. brasiliensis, H. capsulatum and S. schenckii. Weak staining of mycelial forms of P. brasiliensis and hyphae of A. fumigatus was also observed. The availability of a monoclonal antibody specific to fungal glucosylceramide, and its potential use in analyzing biological roles attributed to glucosylceramide in fungi are discussed.
Assuntos
Anticorpos Monoclonais/imunologia , Fungos/metabolismo , Glucosilceramidas/imunologia , Plantas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Glucosilceramidas/metabolismo , Metabolismo dos Lipídeos , TemperaturaRESUMO
Cerebroside (monohexosylceramide) components were identified in neutral lipids extracted from both the yeast and mycelial forms of the thermally dimorphic mycopathogen Histoplasma capsulatum. The components were purified from both forms and their structures elucidated by 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and low energy tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Both components were characterized as beta-glucopyranosylceramides (GlcCers) containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base, attached to 18-carbon 2-hydroxy fatty N-acyl components. However, while the fatty acid of the yeast form GlcCer was virtually all N-2'-hydroxyoctadecanoate, the mycelium form GlcCer was characterized by almost exclusive expression of N-2'-hydroxy-(E)-delta(3)-octadecenoate. These results suggest that the yeast-mycelium transition is accompanied by up-regulation of an as yet uncharacterized ceramide or cerebroside 2-hydroxy fatty N-acyl (E)-delta(3)-desaturase activity. They also constitute further evidence for the existence of two distinct pathways for ceramide biosynthesis in fungi, since glycosylinositol phosphorylceramides (GIPCs), the other major class of fungal glycosphingolipids, are found with ceramides consisting of 4-hydroxysphinganine (phytosphingosine) and longer chain 2-hydroxy fatty acids. In addition to identification of the major glucocerebroside components, minor components (< 5%) detectable by molecular weight differences in the ESI-MS profiles were also characterized by tandem ESI-MS/CID-MS analysis. These minor components were identified as variants differing in fatty acyl chain length, or the absence of the sphingoid 9-methyl group or (E)-delta(8)-unsaturation, and are hypothesized to be either biosynthetic intermediates or the result of imperfect chemical transformation by the enzymes responsible for these features. Possible implications of these findings with respect to chemotaxonomy, compartmentalization of fungal glycosphingolipid biosynthetic pathways, and regulation of morphological transitions in H.capsulatum and other dimorphic fungi are discussed.
Assuntos
Cerebrosídeos/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Histoplasma/metabolismo , Configuração de Carboidratos , Cerebrosídeos/química , Cromatografia em Camada Fina , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
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.
Assuntos
Glicoesfingolipídeos/química , Esfingolipídeos/química , Sporothrix/química , Motivos de Aminoácidos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Modelos Químicos , Dados de Sequência Molecular , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Fatores de TempoRESUMO
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.
Assuntos
Ceramidas/química , Esfingolipídeos/química , Sporothrix/química , Agaricus/química , Isótopos de Carbono , Ceramidas/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Molecular , Espectrometria de Massas por Ionização por Electrospray , Esfingolipídeos/isolamento & purificaçãoRESUMO
Major neutral glycosphingolipid components were extracted from Sporothrix schenckii, a dimorphic fungus exhibiting a hyphal saprophytic phase and a yeast parasitic phase responsible for chronic mycotic infections in mammalian hosts. These components, one from the mycelial form and two from the yeast form, were purified and their structures were elucidated by (1)H nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and tandem ESI-MS/MS. All three were characterized as cerebrosides (monohexosylceramides) containing (4E, 8E)-9-methyl-4,8-sphingadienine as the long-chain base attached to N-2'-hydroxyoctadecanoate and N-2'-hydroxy-(E)-Delta(3)-octadecenoate as the fatty acyl components. However, while the mycelial form expressed only beta-glucopyranosylceramide, the yeast form expressed both beta-gluco- and beta-galactopyranosylceramides in approximately equal amounts. In addition, while the glucosylceramides of both mycelial and yeast forms had similar proportions of saturated and (E)-Delta(3) unsaturated 2-hydroxy fatty acid, the galactocerebroside of the yeast form had significantly higher levels of (E)-Delta(3) unsaturation. The differences in cerebroside hexose structure represent a novel type of glycosphingolipid dimorphism not previously reported in fungi. Possible implications of these findings with respect to regulation of morphological transitions in S. schenckii and other dimorphic fungi are discussed.
Assuntos
Cerebrosídeos/metabolismo , Sporothrix/metabolismo , Sporothrix/patogenicidade , Animais , Cerebrosídeos/análise , Cerebrosídeos/química , Glicoesfingolipídeos/análise , Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Sporothrix/crescimento & desenvolvimento , Esporotricose/etiologiaRESUMO
Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.
Assuntos
Aspergillus fumigatus/química , Ácidos Graxos Insaturados/biossíntese , Glicoesfingolipídeos/química , Paracoccidioides/química , Acilação , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Sequência de Carboidratos , Ácidos Graxos/química , Ácidos Graxos Insaturados/química , Galactosilceramidas/metabolismo , Glucosilceramidas/química , Espectrometria de Massas , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidadeRESUMO
Two major acidic glycolipid components (Pb-1 and Pb-2) have been extracted from the mycopathogen Paracoccidioides brasiliensis, a thermally dimorphic fungus endemic to rural areas of South and Central America. Sera of all patients exhibiting paracoccidioidomycosis were found to be reactive with Pb-1, but not with Pb-2; no reactivity was observed with sera of healthy patients or those with histoplasmosis [Toledo, M. S., Suzuki, S., Straus, A. H., and Takahashi, H. K. (1995) J. Med. Vet. Mycol. 33, 247-251]. We report here the complete structure elucidation of both P. brasiliensis glycolipids using monosaccharide, fatty acid, sphingosine, and inositol component analysis by GC-MS; 1H- and 31P NMR spectroscopy; ESI-MS and -MS/CID-MS, linkage analysis, and exoglycosidase digestion. The compounds were found to be glycosylinositol phosphorylceramides (GIPCs) with the following structures: Pb-2, Manpalpha1-->3Manpalpha1-->2Ins1-P-1Cer; and Pb-1, Manpalpha1-->3[Galfbeta1-->6]Manpalpha1-->2Ins1- P-1Cer. The serologically nonreactive Pb-2 appears to be a biosynthetic intermediate between mannosylinositol phosphorylceramide (MIPC), which is widely distributed among fungi, and the antigenic Pb-1. Pb-1 is a novel glycosphingolipid, similar to a triglycosyl IPC (Hc-VI) reported from Histoplasma capsulatum [Barr, K., Laine, R.A, and Lester, R. L. (1984) Biochemistry 23, 5589-5596], but differing in the anomeric configuration of the terminal Galf1-->6 residue, which is immunodominant. The significance of these structures as serological and taxonomic markers, as well as their potential utility as targets for immunodiagnostic agents, is discussed.
Assuntos
Ceramidas/química , Glicoesfingolipídeos/química , Paracoccidioides/química , Fosfatos Açúcares/química , Antígenos de Fungos/química , Ceramidas/imunologia , Galactose/química , Galactose/imunologia , Glicoesfingolipídeos/imunologia , Epitopos Imunodominantes/química , Paracoccidioides/imunologia , Fosfatos Açúcares/imunologiaRESUMO
Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues.
Assuntos
Antígenos de Protozoários/imunologia , Glicoesfingolipídeos/imunologia , Leishmania mexicana/imunologia , Animais , Anticorpos Monoclonais/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues.
Assuntos
Camundongos , Animais , Anticorpos Monoclonais/imunologia , Glicoesfingolipídeos/imunologia , Coração , Técnicas In Vitro , Rim/imunologia , Leishmania mexicana/imunologia , Fígado/imunologia , Pulmão/imunologia , Baço/imunologia , Leishmania mexicana/química , Camundongos Endogâmicos BALB CRESUMO
Twelve different species of neutral monohexosyl ceramides (CMHs) and two species of neutral monohexosyl ceramides were isolated from mycelium and yeast forms of Paracoccidioides brasiliensis, respectively, by a combination of ion-exchange chromatography, HPLC, and HPTLC. The glucosylceramides did not react with sera from patients with paracoccidioidomycosis (PCM). Carbohydrate analysis indicated that CMHs contain glucose. Analysis of 1H-NMR and mass spectrometry data suggest that the structure of the CMHs is Glcp beta 1-->Cer (mycelium forms present 12 different ceramides and yeast forms present 2 different ceramides). The composition of the lipid moieties was analyzed by negative fast atom bombardment mass spectrometry. No glycosphingolipid other than glucosylceramide was detected in P. brasiliensis.
Assuntos
Glucosilceramidas/isolamento & purificação , Paracoccidioides/química , AnimaisRESUMO
Twelve different species of neutral monohexosyl ceramides (CMHs) and two species of neutral monohexosyl ceramides were isolated form mycelium and yeast forms of Paracoccidioides brasiliensis, respectively, by a combination of ion-exchange chromatography, HPLC, and HPTLC. The glucosylceramides did not react with sera from patients with paracoccidioidomycosis (PCM). Carbohydrate analysis indicated that CMHs contain glucose. Analysis of (1)H-NMR and mass spectrometry data suggest that the structure of the CMHs is Glcpbeta1(Cer (mycelium forms present 12 different ceramides and yeast forms present 2 different ceramides). The composition of the lipid moieties was analyzed by negative fast atom bombardment mass spectrometry. No glycosphingolipid other than glucosylceramide was detected in P. brasiliensis.
Assuntos
Animais , Glucosilceramidas/isolamento & purificação , Paracoccidioides/química , Cromatografia , Glucosilceramidas/química , Espectroscopia de Ressonância Magnética , Espectrometria de MassasRESUMO
An acidic glycolipid (Band 1), purified from P. brasiliensis by a combination of ion exchange chromatography, HPLC, and HPTLC, was found to be reactive with sera of all patients with paracoccidioidomycosis (PCM). Monosaccharide analysis of Band 1 yielded mannose and galactose in a 2:1 ratio, while mild acid hydrolysis and mild periodate oxidation/NaB3H4 reduction indicated the presence of a terminal galactofuranose. Preliminary analysis of 1H-NMR and MS data suggests that the structure of the glycan is Galf beta 1-->6(Manp alpha 1-->3)Manp beta 1-->2Ins (Ins = myo-inositol). Removal of the galacto-furanose decreased by 60-80% the reactivity of sera from PCM patients with Band 1, suggesting that this residue is immunodominant. With the presumed absence of galactofuranose in mammalian hosts, compounds containing this residue may be useful targets for therapy of several parasitic and fungal diseases.
Assuntos
Antígenos de Fungos/química , Galactose/análise , Glicolipídeos/química , Paracoccidioides/química , Paracoccidioidomicose/sangue , Antígenos de Fungos/sangue , Antígenos de Fungos/isolamento & purificação , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Ensaio de Imunoadsorção Enzimática , Glicolipídeos/sangue , Glicolipídeos/isolamento & purificação , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Paracoccidioidomicose/imunologia , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
Neutral glycosphingolipids (GSLs) from amastigote forms of Leishmania (L.) amazonensis were isolated, and their structures and biological properties were characterized. Based on various immunochemical methods, these GSLs were shown to be expressed at certain stages of amastigote development. GSLs were extracted and purified from amastigotes of hamster foot lesions by established procedures. Three mouse monoclonal antibodies (MoAbs) specific for carbohydrate epitopes of these GSLs were established, and their inhibition of parasite binding and macrophage invasion was analyzed. MoAb ST-3 inhibited 80% of macrophage invasion by amastigotes and 60% of that by promastigotes. Since GSLs reacting with MoAb ST-3 were found in amastigotes but not in promastigotes, ST-3 reactivity with promastigotes presumably depends on an epitope present on an unidentified promastigote glycoconjugate. MoAbs ST-4 and ST-5 inhibited 60-80% of macrophage invasion by amastigotes but were not effective in preventing macrophage invasion by promastigotes. Fab fragments of ST-3 inhibited invasion of cultured mouse macrophages by amastigotes (80%) or promastigotes (60%). The GSL with the simplest structure recognized by these MoAbs was isolated and characterized (by negative ion fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry of the permethylated compound, degradation with exoglycosidases, and 1H NMR) as the novel globoseries structure Gal beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc beta 1-->Cer, which has beta 1-->3Gal in place of the beta 1-->3GalNAc of globoside. The ceramide contains a 16:0 fatty acid and d18:1 sphingosine as the long chain base. The MoAbs also reacted with a series of GSLs from amastigote forms of L. amazonensis, with longer carbohydrate chains, probably containing identical end groups Gal beta 1-->3Gal alpha 1-->R. Expression of surface GSLs may render amastigote forms more effective than promastigotes in binding and invading host macrophages, thus enhancing the infectious process.