Analysis of pyruvic acid acetal containing polysaccharides by methanolysis and reductive cleavage methods

Fontaine, Thierry; Talmont, Franck; Dutton, Guy G.S.; Fournet, Bernard

doi:10.1016/0003-2697(91)90083-6

Cited by 18 publications

(18 citation statements)

References 27 publications

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“…Prior to the methylation procedure, polysaccharides were peracetylated as previously described [50]. Dried sample (2 mg) was methylated by the DMSO/lithium methyl sulfinyl carbanion/ICH 3 procedure [50].…”

Section: Methodsmentioning

confidence: 99%

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Galactosaminogalactan, a New Immunosuppressive Polysaccharide of Aspergillus fumigatus

et al. 2011

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A new polysaccharide secreted by the human opportunistic fungal pathogen Aspergillus fumigatus has been characterized. Carbohydrate analysis using specific chemical degradations, mass spectrometry, 1H and 13C nuclear magnetic resonance showed that this polysaccharide is a linear heterogeneous galactosaminogalactan composed of α1-4 linked galactose and α1-4 linked N-acetylgalactosamine residues where both monosacharides are randomly distributed and where the percentage of galactose per chain varied from 15 to 60%. This polysaccharide is antigenic and is recognized by a majority of the human population irrespectively of the occurrence of an Aspergillus infection. GalNAc oligosaccharides are an essential epitope of the galactosaminogalactan that explains the universal antibody reaction due to cross reactivity with other antigenic molecules containing GalNAc stretches such as the N-glycans of Campylobacter jejuni. The galactosaminogalactan has no protective effect during Aspergillus infections. Most importantly, the polysaccharide promotes fungal development in immunocompetent mice due to its immunosuppressive activity associated with disminished neutrophil infiltrates.

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Section: Methodsmentioning

confidence: 99%

“…Dried sample (2 mg) was methylated by the DMSO/lithium methyl sulfinyl carbanion/ICH 3 procedure [50]. After hydrolysis of the permethylated sample (4 N TFA 100°C, 4 h), borodeuteride-reduction and peracetylation, methyl ethers were identified by GC-MS. Oligosaccharides were permethylated by the DMSO/NaOH/ICH 3 procedure [51].…”

Section: Methodsmentioning

confidence: 99%

Galactosaminogalactan, a New Immunosuppressive Polysaccharide of Aspergillus fumigatus

et al. 2011

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“…Methylation of galactomannan fraction was performed using the lithium methyl sulfinyl carbanion procedure (12). Oligosaccharides were methylated using the sodium hydroxide procedure (13).…”

Section: Methodsmentioning

confidence: 99%

Glycosylphosphatidylinositol-anchored Fungal Polysaccharide in Aspergillus fumigatus

Costachel

Coddeville

Latgé

et al. 2005

Journal of Biological Chemistry

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100

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Galactomannan is a characteristic polysaccharide of the human filamentous fungal pathogen Aspergillus fumigatus that can be used to diagnose invasive aspergillosis. In this study, we report the isolation of a galactomannan fraction associated to membrane preparations from A. fumigatus mycelium by a lipid anchor. Specific chemical and enzymatic degradations and mass spectrometry analysis showed that the lipid anchor is a glycosylphosphatidylinositol (GPI). The lipid part is an inositol phosphoceramide containing mainly C 18 -phytosphingosine and monohydroxylated lignoceric acid (2OH-C 24:0 fatty acid). GPI glycan is a tetramannose structure linked to a glucosamine residue: Man␣1-2Man␣1-2Man␣1-6Man␣1-4GlcN. The galactomannan polymer is linked to the GPI structure throught the mannan chain. The GPI structure is a type 1, closely related to the one previously described for the GPI-anchored proteins of A. fumigatus. This is the first time that a fungal polysaccharide is shown to be GPI-anchored.Aspergillus fumigatus has become over the past decade the most prevalent airborne fungal pathogen causing fatal invasive infections in immunocompromised patients (1). One of the characteristic components of this fungus is composed of a linear mannan chain with a tetra-␣-mannoside repeating unit and side chains of ␤1-5 galactofuranoside residues (2). The galactomannan from A. fumigatus has been described as a free polysaccharide found in the culture medium, and it is also covalently associated to the cell wall through the ␤1-3 glucan net and plays a role in the structural organization of the cell wall (2, 3). A monoclonal antibody directed against the galactofuranose side chain has been used to detect the presence of this polymer in the sera of patient with invasive aspergillosis (4, 5). Biosynthesis of the galactomannan is totally unknown in this fungus. During the search of lipid molecules that could be an anchor for the galactomannan, we isolated a membrane bound fraction that contained galactomannan. We investigated the nature of the membrane anchor of the galactomannan by GLC-MS 2 (gas liquid chromatography-mass spectrometry) and ES-MS-MS (electrospraytandem mass spectrometry) and found that the lipid anchor was a glycosylphosphatidylinositol. EXPERIMENTAL PROCEDURES Fungal Culture and Membrane PreparationA. fumigatus, strain CBS 144-89 was grown in a 15-L fermenter in 2% glucose and 1% mycopeptone (Biokar Diagnostics, Pantin, France) for 24 h at 25°C as described previously (6). Total membrane preparation from mycelium was done as described previously (7). Extraction and Purification of the LipogalactomannanLipids and polar glycolipids were extracted from membrane preparation by an organic extraction. A chloroform/methanol 1/1 (v/v) solution was added to the membrane suspension to reach a final concentration of chloroform/methanol/water (10:10:3 v/v/v). The mixture was stirred for 2 h at room temperature to extract lipids, then centrifuged at 10,000 ϫ g for 10 min. The pellet was resuspended in the same solvent, and the e...

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“…Gas Chromatography (GC)/Gas-Liquid Chromatography (GLC)-Mass Spectrometry (MS)-Vacuum-dried samples (2 mg) were subjected to methylation using the lithium methyl sulfinyl carbanion procedure (29) modified by Fontaine et al (30). Then alditol acetates were obtained after hydrolysis (4 N trifluoroacetic acid, 100°C, 4 h), reduction, and peracetylation of the methylated samples (31).…”

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Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae

Aimanianda

Clavaud

Simenel

et al. 2009

Journal of Biological Chemistry

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127

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Despite its essential role in the yeast cell wall, the exact composition of the ␤-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant ␤-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of ϳ38 kDa and could be hydrolyzed only by ␤-(1,6)-glucanase. Gas chromatographymass spectrometry and NMR ( 1 H and 13 C) analyses confirmed it to be a ␤-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two ␤-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the ␤-(1,6)-glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[ 14 C]glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched ␤-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for ␤-(1,6)-glucan synthetic activity were defined.

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Analysis of pyruvic acid acetal containing polysaccharides by methanolysis and reductive cleavage methods

Cited by 18 publications

References 27 publications

Galactosaminogalactan, a New Immunosuppressive Polysaccharide of Aspergillus fumigatus

Galactosaminogalactan, a New Immunosuppressive Polysaccharide of Aspergillus fumigatus

Glycosylphosphatidylinositol-anchored Fungal Polysaccharide in Aspergillus fumigatus

Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae

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