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Originally published In Press as doi:10.1074/jbc.M510163200 on October 4, 2005

J. Biol. Chem., Vol. 280, Issue 48, 39835-39842, December 2, 2005
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Glycosylphosphatidylinositol-anchored Fungal Polysaccharide in Aspergillus fumigatus*

Corina Costachel{ddagger}, Bernadette Coddeville§, Jean-Paul Latgé{ddagger}, and Thierry Fontaine{ddagger}1

From the {ddagger}Unité des Aspergillus, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France and the §Laboratoire de Chimie Biologique, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq cedex, France

Received for publication, September 15, 2005 , and in revised form, October 4, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 C18-phytosphingosine and monohydroxylated lignoceric acid (2OH-C24:0 fatty acid). GPI glycan is a tetramannose structure linked to a glucosamine residue: Man{alpha}1–2Man{alpha}1–2Man{alpha}1–6Man{alpha}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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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-{alpha}-mannoside repeating unit and side chains of {beta}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 {beta}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-MS2 (gas liquid chromatography-mass spectrometry) and ES-MS-MS (electrospraytandem mass spectrometry) and found that the lipid anchor was a glycosylphosphatidylinositol.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Fungal Culture and Membrane Preparation
A. 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 Lipogalactomannan
Lipids 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 x g for 10 min. The pellet was resuspended in the same solvent, and the extraction was repeated once. The final pellet was dried, ground, and suspended in 50 ml of 50 mM Tris-HCl, pH 7.5, buffer containing 5 mM sodium azide and incubated with 50 mg of protease from Streptomyces griseus (4.9 units/mg, Sigma, previously incubated for 30 min) at 37 °C for 48 h. The reaction was stopped by heating (5 min at 100 °C). Propan-1-ol was added to the final concentration of 5% and insoluble material was removed by centrifugation (5,000 x g for 10 min). The soluble material was fractionated by hydrophobic interaction chromatography on an octyl-Sepharose column (25 x 1.6 cm, Sigma). The supernatant was deposited onto the column equilibrated with 5% propan-1-ol in 100 mM ammonium acetate at the flow rate of 10 ml/h. After the elution of the unbound fraction, bound products were eluted with a linear gradient of 5–60% propan-1-ol in 100 mM ammonium acetate (300 ml). Carbohydrates were detected by the phenol sulfuric acid procedure (8). Galactofuran was detected by enzyme-linked immunosorbent assay (2). Bound positive fractions were pooled, dialyzed against water, and freeze-dried. Dried residue was solubilized by 50 ml of butanol-saturated water, then five partitions with 20 ml of water-saturated butanol were performed to remove contaminating glycolipids. The absence of protein and glycolipid was observed by SDS-PAGE and TLC, respectively. The aqueous-phase containing the lipogalactomannan was then dialyzed against water and freeze-dried.

Gel Filtration Chromatography
The molecular weight of lipogalactomannan was estimated by gel filtration chromatography on a Superose 12 column (29 x 1.2 cm, Amersham Biosciences) equilibrated with 100 mM ammonium acetate containing 30% (v/v) propan-1-ol at 0.2 ml/min. The products were detected using a refractometer (Gilson, model 132). Dextran T10, T40, and T70 (Amersham Biosciences) were used as molecular weight standards. Products obtained after acetolysis of the lipogalactomannan were separated by gel filtration chromatography on a HW40S column (80 x 1.4 cm, TosoHaas) equilibrated with 0.25% acetic acid at a flow rate of 0.5 ml/min. The products were detected using a refractometer. Maltooligosaccharides (Glucidex 19 from Roquette Frères, Lestrem, France) were used as molecular weight standards.



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  		FIGURE 1.
Purification of a protease digest of a chloroform/methnol extract of a membrane preparation of A. fumigatus mycelium. The extract was added to an octyl-Sepharose column equilibrated with 5% propan-1-ol in 100 mM ammonium acetate. Bound products were eluted with a linear gradient of propan-1-ol (5–60%). Neutral sugars were detected with the phenol/sulfuric colorimetric assay (closed circles) (8), and galactofuranose epitope was detected by enzyme-linked immunosorbent assay (open circles) (2).

 
Analytical Methods
Phosphate was quantified according to Ames (9). Total hexoses were quantified by the phenol-sulfuric acid procedure using mannose and galactose as standards (8). Neutral hexoses were identified by GLC as alditol acetates obtained after hydrolysis (4 N trifluoroacetic acid, 100 °C, for 4 h) (10). Glucosamine and myo-inositol were quantified by GLC-MS after hydrolysis (6 N HCl, 110 °C, 20 h), N-acetylation, and trimethylsilylation, using scyllo-inositol as standard (11). Lipid analysis was performed by GLC-MS on the HF-treated lipogalactomannan (see below). Fatty acids and the sphingosine base were released by methanolysis (1 N HCl in MeOH, 80 °C, 20 h). Fatty acids were extracted with heptane and analyzed by GLC-MS after trimethylsilylation. The sphingosine base containing methanol phase was N-acetylated, trimethylsilylated, and analyzed by GLC-MS (11).

Methylation of galactomannan fraction was performed using the lithium methyl sulfinyl carbanion procedure (12). Oligosaccharides were methylated using the sodium hydroxide procedure (13). Glycolipids, containing a glucosamine residue, were peracetylated with a pyridine/acetic anhydride solution (50/200 µl) overnight at room temperature prior to the methylation procedure. Methyl ethers were analyzed by GLC-MS as polyol acetates (14) and/or as methyl glycosides (15).

Chemical and Enzymatic Treatments
Phosphatidylinositol-specific phospholipase C (PI-PLC) Digestions—1.5 mg of purified lipogalactomannan was dissolved in 100 µlofimidazole/acetate buffer, pH 7.5, containing 5 mM sodium azide and 0.1% Triton X-100 and incubated with 5 µl of PI-PLC (Glyko, 200–300 units/ml) at 37 °C for 24 h. Digested products were deposited onto an octyl-Sepharose column (6 x 1.4 cm) equilibrated with 5% propan-1-ol in 100 mM ammonium acetate. Bound products were then eluted with 50% propan-1-ol in 100 mM ammonium acetate. For the chemical analysis of PI-PLC released lipid moiety, a similar digestion was done on 100 µgof sample without Triton X-100. Released lipid moiety was extracted with water-saturated butanol and analyzed by GLC-MS.

Aqueous HF Treatment—To cleave the phosphoester bond, 100 µgof dried purified lipogalactomannan was treated by 100 µl of 50% aqueous HF for 48 h on an ice bath (11). Released lipids were extracted with water-saturated butanol.

Nitrous Acid Deamination (11)—0.5–1.5 mg of sample was dissolved in 200 µl of 0.1 M sodium acetate, pH 4. Aliquots of 100 µl of freshly prepared 0.5 M sodium nitrite were added at hourly intervals and incubated at 50 °C for 3 h. The reaction mixture was concentrated to dryness. Released PI was extracted with water-saturated butanol and purified on a small silica column (200 µl of Kieselgel 60, 0.063–0.2 mm, Merck (Darmstadt, Germany), in a glass Pasteur pipette) as described previously (7). Water-soluble products were deposited onto an octyl-Sepharose column and eluted as described for extracts submited to PI-PLC digestion.

Partial Acid Hydrolysis—10 mg of lipogalactomannan was treated with 50 mM HCl at 100 °C for 15 h. After neutralization with aqueous ammonia, released glycolipids were isolated by HPLC on a C-4 reverse phase column (10 x 4.6 mm, 120 Å, 5 µm, Uptisphere Interchim) using the following conditions: flow 0.25 ml/min; buffer A, 1% propan-1-ol in 20 mM ammonium acetate; buffer B, 80% propan-1-ol in 20 mM ammonium acetate; gradient 0–10 min, isocratic (100% buffer A); 10–40 min, linear gradient (100% buffer A, 0% buffer B to 0% buffer A, 100% buffer B), 40–50 min isocratic (100% buffer B). Sugars were detected by orcinol sulfuric acid on spotting of fraction (1 µl) on silica sheet. Then fractions containing sugar were analyzed by ES-MS and the methylation procedure.

Jack Bean {alpha}-Mannosidase (JBAM) Degradation—500 µg of HCl-treated lipogalactomannan were dissolved in 200 µl of 50 mM NaOAc, pH 4.0, buffer containing 5 mM sodium azide and incubated with 20 µl of JBAM (95 units/ml, Sigma) at 37 °C for 24 h. Released lipid moiety was extracted with water-saturated butanol and then purified on a small silica column.

Acetolysis—500 µg of purified lipogalactomannan were peracetylated by treatment with formamide/acetic anhydride/pyridine (2/2/1, v/v/v) overnight at room temperature, then dialyzed against water and freezedried. The peracetylated products were treated with 200 µl of an acetic acid/acetic anhydride/sulfuric acid solution (10:10:1 v/v/v) at 37 °C for 7 h (11). The reaction was stopped by addition of 800 µl of an ice-cold pyridine/water mixture (1/3 v/v). Acetolysed products were extracted with chloroform and the organic phase was washed with water and then concentrated to dryness. De-O-acylation was performed in 300 mM NaOH containing NaBH4 (10 mg/ml) overnight at room temperature. The excess of reagent was destroyed by addition of 10% acetic acid. Released lipid moiety was extracted by the butanol-water partitioning.

TLC Analysis—TLC was performed on precoated aluminum-backed silica 60 HPTLC plates (Merck). Plates were developed at room temperature using chloroform/methanol/30% aqueous NH3/1 M NH4Ac/water (180/140/9/9/23 by volume) as the solvent (16). After drying, sugars were revealed by spraying with orcinol/H2SO4 reagent.



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  		FIGURE 2.
Gel filtration analysis on TSK HW40S column of acetolysed products from galactomannan extracts produced by A. fumigatus. A, galactomannan from the culture medium; B, lipogalactomannan from membrane preparation.

 
GLC and Mass Spectrometry—GLC was performed on a Delsi 200 instrument with a flame ionization detector using a capillary column (30 m x 0.25 mm inner diameter) filled with a ECTM-1 (Alltech) under the following conditions: gas vector and pressure, helium 0.7 bar; temperature program 120–180 °C at 2 °C/min, 180–240 °C at 4 °C/min. GLC-MS was performed on an Automass II apparatus (Finigan) coupled to a CarloErba gas chromatograph (model 8000top), using a capillary column (30 m x 0.25 mm inner diameter) filled with a ECTM-1 (Alltech) under the following conditions: gas vector and flow rate, helium 2 ml/min; temperature program for inositol and monosaccharide analysis: 100–200 °C at 5 °C/min, 200–240 °C at 15 °C/min, and 240 °C for 5 min; temperature program for sphingosine base and fatty acid analysis: 100–200 °C at 10 °C/min, 200–260 °C at 15 °C/min, and 260 °C for 13 min.

Electrospray-Mass Spectrometry—All electrospray-mass measurements were carried out in negative-ion mode on a triple quadrupole instrument (Micromass Ltd., Altrincham, UK) fitted with an atmospheric pressure ionization electrospray source. A mixture of polypropylene glycol (range 30–2,000 daltons) was used to calibrate the quadrupole mass spectrometer. The samples were dissolved in chloroform/methanol (1/2) at a concentration of 1–10 pmol/µl. Solutions were infused using a Harvad syringe pump at a flow rate of 3 µl·min–1.Quadrupole was scanned from 400 to 2000 Da with a scan duration of 3–5 s and a scan delay of 0.1 s. The samples were sprayed using 3.25 kV needle voltage, and the declustering (cone) was set at 70 V. For collision-induced dissociation experiments, the pressure of argon in the cell was set at 2.7.10–3 mbar, and the collision energy was set to values ranging from 40 to 70 V depending of the studied daughter ions.

Matrix-assisted Desorption Ionization Time-of-Flight Mass Spectrometry—Mass spectra were measured on a reflectron-type Vision 2000 time of flight mass spectrometer (Finnigan MAT, Bremen, Germany). Samples were mounted on an x,y movable stage allowing irradiation of selected areas. A nitrogen laser with an emission wavelength of 337 nm and 3-ns pulse duration was used. The spectrum was recorded in the positive ion mode and accelerated to an energy of 10 keV before entering the flight tube. Ions were prepared by mixing directly on the target 1 µl of oligosaccharide solution (about 25 pmol) and 1 µl of 2,5-dihydroxybenzoic acid matrix solution (12 mg/ml) dissolved in CH3OH/10 mM NaCl (80:20 v/v).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purification and Composition of the Lipogalactomannan—The crude membrane fraction of A. fumigatus mycelium was treated with a chloroform/methanol/water mixture, and the pellet was digested with a protease mixture. Soluble material was fractionated by a hydrophobic interaction. The major phenol-sulfuric acid-positive peak, bound to the octyl-Sepharose column, was also positive with the anti-galactofuranose antibody detection (Fig. 1). The purified fraction had an apparent molecular mass of 30 kDa on a Superose 12 column (data not shown) and represented 0.04 ± 0.01% of total mycelium dry weight.

Colorimetric assays and GLC analysis showed that the octyl-bound fraction was mainly composed of mannose and galactose residues in a ratio 3:2 and for this reason was called lipogalactomannan (LGM). The LGM fraction contains a small amount of glucosamine, phosphate, myo-inositol, 2-monohydroxy-C24:0 fatty acid, and C18-phytosphingosine (data not shown). The presence of these compounds suggested that the polysaccharide was anchored to the membrane by a GPI anchor (7). A PI-PLC- or HNO2-treated lipogalactomannan did not bind to the octyl-Sepharose column (data not shown). Taken all together, these results showed that the galactomannan moiety was bound to a GPI anchor.

Analysis of the Carbohydrate Structure of the Lipogalactomannan—To determine the monosaccharide linkages, the membrane-bound polysaccharide fraction was permethylated. Methyl ethers analysis revealed the presence of mannose residues monosubstituted in position 2 or 6 and disubstituted in position 2,3 or 2,6 as described for the secreted galactomannan isolated from A. fumigatus culture filtrate (TABLE ONE) (2). Terminal galactofuranose has also been identified. 2,3,6-Tri-O-methyl-1,4,5-tri-O-acetyl-galactitol indicated the presence of galactofuranose residues substituted in position 5 or galactopyranose in position 4. The specific detection with the anti-galactofuran monoclonal antibody and the sensitivity of galactose residue to a mild acid hydrolysis and/or aqueous HF treatment (TABLE ONE) indicated that all galactose residues were in furanic configuration similar to those described in the secreted galactomannan (2).


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  		TABLE ONE
Molar ratio of methyl ethers obtained after permethylation of the native, HF-treated and HCl-treated lipogalactomannan isolated from A. fumigatus membrane preparations

Molar ratio was calculated by GLC-flame ionization detection.

 



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  		FIGURE 3.
ES-MS analysis of PI released by nitrous deamination from the purified lipogalactomannan of A. fumigatus. A, negative ion ES-MS spectrum of PI; B, daughter ion ES-MS-MS spectrum of the ion at 924. P, phosphate group; hexagon represents the inositol ring.

 
To confirm the similarity of structures, acetolysis was performed on the lipogalactomannan and secreted galactomannan. Analysis of released products by gel filtration (Fig. 2), matrix-assisted desorption ionization time-of-flight mass spectrometry, and GLC analysis revealed the same pattern of degraded products such as mainly galactose residue and a tetra-{alpha}1–2-mannoside from both polysaccharide fractions. All these data indicated that the membrane-bound galactomannan from A. fumigatus has the same structure as the soluble polymer.

Analysis of the Lipid Anchor of the Lipogalactomannan—The lipid moiety, released by nitrous acid deamination from the lipogalactomannan, purified by butanol/water partition and by chromatography on a small silica column, was analyzed by ES-MS-MS as described previously (7). The ES-MS spectrum of the PI fraction (Fig. 3A) revealed one principal [M–H]– pseudomolecular ion at m/z = 924 and four minor ions at m/z = 908, 910, 938, and 952. The fragmentation of each PI compounds produced only two daughter ions at m/z = 241 [inositol 1,2-cyclic phosphate]– and at m/z = 259 [inositol monophosphate]– (Fig. 3B). The presence of these daughter ions and the absence of the fatty acid carboxylate ion and/or 1-O-alkylglycerol-2,3-cyclic phosphate ion is characteristic of inositol phosphoceramides (IPCs) (17). From the measured mass and taking into account the identified lipid compounds, we concluded that the main PI structure is made of a C18-phytosphingosine and a 2-monohydroxylated C24:0 fatty acid. The minor components that differ by a mass of 14, 16, or 28 reflected the variability of size of fatty acid or the absence of hydroxyl group that was observed by the fatty acid analysis (data not shown).

To confirm the position of glucosamine residue, the lipogalactomannan fraction was submitted to a mild HCl acid hydrolysis followed by a JBAM digestion. The ES-MS spectrum of the released lipid moiety revealed one principal [M–H]– pseudomolecular ion at m/z = 1086 (Fig. 4A). Except for minor ions due to the variability of the aliphatic chain (m/z = 1072, 1100, and 1114), two minor [M–H]– pseudomolecular ions at m/z = 1248 and 1410 were observed and corresponded to an increase of 162 and 324 suggesting the presence of 1 and 2 hexose residues, respectively. The fragmentation of the ion at m/z = 1086 produced two daughter ions at m/z = 402 [hexosamine-inositol-1,2cyclic phosphate]– and at m/z = 420 [hexosamine-inositol monophosphate]– (Fig. 4B). Since glucosamine has been identified in the LGM fraction, and since the ion mass at m/z = 1086 corresponds to the presence of an hexosamine residue linked to the identified PI, the presence of the two daughter ions is characteristic of the glucosamine linked to the inositol phosphoceramide.



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  		FIGURE 4.
ES-MS analysis of the butanol-soluble products released by mild HCl hydrolysis and jack bean {alpha}-mannosidase digestion from the purified lipogalactomannan of A. fumigatus. A, negative ion ES-MS spectrum; B, daughter ion ES-MS-MS spectrum of the ion at 1086; C, daughter ion ES-MS-MS spectrum of the ion at 1248. P, phosphate group; hexagon represents the inositol ring.

 
The fragmentation of the ion at m/z = 1248 produced also two daughter ions at m/z = 564 [hexose-hexosamine-inositol-1,2cyclic phosphate]– and at m/z = 582 [hexose-hexosamine-inositol-monophosphate]– (Fig. 4C). Since only mannose has been identified as hexose in the butanol phase after the HCl-JBAM treatment, these daughter ions are characteristic of the Man-GlcN-linked to the inositol phosphoceramide.

The LGM fraction was submitted to acetolysis, which cleaves preferentially the 1–6 linkage. The ES-MS of the butanol-soluble-resistant products revealed two main [M–H]–, pseudomolecular ions at m/z = 965 and at m/z = 1290 (Fig. 5A). Minor ions at m/z = 803 and at m/z = 1128 correspond to the loss of 162 from the two main ions, respectively, suggesting the loss of an hexose residue. The fragmentation of the ion at m/z = 1290 produced two main daughter ions at m/z = 606 [hexose-N-acetylhexosamine-inositol-1,2cyclic phosphate]– and at m/z = 624 [hexose-N-acetyl-hexosamine-inositol monophosphate]– (Fig. 5B). Since only mannose was identified as hexose in this fraction, and since acetolysis resulted in the N-acetylation of the glucosamine residue, the daughter ions are characteristic of the Man-GlcNAc linked to the inositolphosphoceramide. The fragmentation of the ion at m/z = 965 produced three daughter ions at m/z 606, at m/z = 624, and at m/z = 420 (Fig. 5C). The ions at m/z 606 and at m/z = 624 are similar to those obtained by the fragmentation of the parental ion at m/z = 1290, indicating a same structure of the glycan part in both parental ions. The mass difference between this both parental ions corresponds to the substitution of the 2-monohydroxyl C24:0 fatty acid by an acetyl group, indicating that the acetolysis cleaved the acetamide linkage. The presence of one mannose residue linked to the N-acetylglucosamine after acetolysis indicated that this first mannose residue is substituted in position 6.

After mild acid hydrolysis (50 mM HCl for 15 h at 100 °C) without JBAM digestion, the ES-MS spectrum of HPLC-purified glycolipids revealed 10 main pseudomolecular ions at m/z = 1086, 1248, 1410, 1572, 1734, 1896, 2058, 2220, 2382, and 2544 (Fig. 6). The ion at m/z = 1086 corresponds to the GlcN-IPC described in the legend to Fig. 4. The increase of ion mass of 162, 324, 486, 648, 810, 972, 1134, 1296, and 1458 indicated the presence of a mixture of oligosaccharides containing 0–9 hexoses linked to the GlcN-IPC. Monosaccharide linkage analysis, done after peracetylation, permethylation, and methanolysis, revealed the presence of terminal glucosamine, 4-substituted glucosamine, terminal mannose, 2-subsituted mannose, 6-substituted mannose residues (TABLE ONE). According to these ES-MS data, the terminal glucosamine residue came from the GlcN-IPC. The presence of the 4-substituted glucosamine residue indicates that the first mannose residue is linked in position 4 to the glucosamine. Taking into account the results from acetolysis experiment, the 6-substituted mannose corresponds to the first mannose residue in the Man2–9GlcN-IPC structures. The quantity of 2-substituted mannose, higher than the one of 6-substituted mannose, indicated that at least the two following mannose residues (third and fourth) are substituted in position 2. All of these mannose residues, removed by a JBAM digestion, are linked in {alpha}-anomeric configuration (data not shown). After mild acid hydrolysis, the presence of structures containing more than 5 mannose residues linked to GlcN-IPC and the absence of phosphoethanolamine group excluded the putative contamination from GPI-anchored proteins (7). The sensitivity of A. fumigatus LGM to the PI-PLC indicated that the linkage between GlcN and inositol is not {alpha}1–2 (18). Taken all together, these results were in agreement with the following GPI structure: galactomannan-Man{alpha}1–2Man{alpha}1–2Man{alpha}1–6Man{alpha}1–4GlcN-inositol-P-ceramide.

The GPI anchor was linked to the {alpha}-mannan chain of the galactomannan, which contains the same repeat unit. The small amount of 2,6-disubstituted mannose (TABLE ONE) suggests the putative presence of a mannose as a side chain in this structure


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
GPI structures have been observed in all eukaryotic cells. In mammalian and plant cells, GPI are involved in the anchoring of proteins in the lipid bilayer by a common core Man3-GlcN-PI. In parasite cells, in addition to GPI-anchored proteins, oligosaccharides, and polysaccharides have also been described to be GPI-anchored. To date, in fungi only proteins had been found to be GPI-anchored (7, 19). Herein, we described the first fungal polysaccharide to be linked to a GPI. Since many other ascomycetes have a galactomannan with similar structures (20–23), it can be expected now that other fungi also possess GPI-anchored polysaccharide. The role of these polysaccharides in fungal life remains to be understood. The lipid moiety of the LGM and proteins GPIs of A. fumigatus is the same ceramide (7). However, anchoring of the GPI to the protein and polysaccharide is different; no phosphoethanolamine group has been identified on the LGM anchor, and the fifth {alpha}1–3linked mannose residue of the GPI structure of anchored proteins is replaced by the galactomannan in the LGM structure. Nevertheless, in contrast to the Leishmania lipophosphoglycan and to the Crithidia fasciculata lipoarabinogalactan, where the second mannose residue is {alpha}1–3-linked (24, 25), it is the first time that a polysaccharide is linked to a GPI type 1 structure, as described for GPI-anchored proteins.



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  		FIGURE 5.
ES-MS analysis of the butanol-soluble products released by acetolysis of the purified lipogalactomannan of A. fumigatus. A, negative ion ES-MS spectrum; B, daughter ion ES-MS-MS spectrum of the ion at 1290; C, daughter ion ES-MS-MS spectrum of the ion at 965. P, phosphate group; hexagon represents the inositol ring.

 



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  		FIGURE 6.
ES-MS analysis of the glycolipides released by mild acid hydrolysis from the purified lipogalactomannan of A. fumigatus (negative ion ES-MS spectrum).

 
IPC structures from the LGM are similar to the glycosylinositol phosphoceramide (GIPC) described in A. fumigatus (26). Numerous GIPCs have been described at the cell membrane of other filamentous fungi and yeast cells but are absent from mammalian cells. The ceramide structure of these glycosphingolipids seems conserved between species with the presence of a phytosphingosine, associated to a saturated fatty acid, mainly the 2-hydroxylignoceric acid (2OH-C24:0) (27–35). In contrast to the lipid moiety, the glycan moiety is heterogenous between fungal species with the presence of mannose, galactose in a pyranic or furanic configuration, and even fucose residue in higher mushrooms (36). Usually, glycans of GIPC contain 2–6 hexose residues. Up to 18 mannose residues have been exceptionnally found in the phospholipomannan of C. albicans (33). Two types of GIPC have been described in fungal cells: (i) MIPC, where the glycan is linked to the IPC through an {alpha}-mannose residue, and (ii) ZGL (zwitterionic glycosphingolipid), where the glycan moiety is linked to the IPC through a glucosamine residue (35, 37). In contrast to LGM structure, in this latter ZGL structure only described in three fungal species, the glucosamine residue is {alpha}1–2-linked to the inositol ring.

The galactomannan from A. fumigatus is mainly constituted by the polymerization of a tetramannoside unit containing two side chains of {beta}-D-galactofuranose residues (2). The presence of a repeat unit suggests a biosynthesis of the tetramannoside on a acceptor prior to the polymerization of the mannan chain. Our data suggest that the acceptor could be a GPI moiety. Enzymes involved in this biochemical pathway are totally unknown. A glycosyltransferase activity able to mannosylate the GlcN-PI, recently observed in an A. fumigatus cellular lysate, could play a role in this biochemical event. This activity, independent of the dolichol phosphomannose synthesis and of the acylation of the inositol ring, is not involved in the biosynthesis of GPI intermediates of GPI-anchored proteins (38). These data suggest that GPI anchor of LGM could be synthesized in an independent biosynthetic pathway using GDP-Man as substrate donor, as it was speculated for ZGL in Acremonium and Trichoderma sp. (35).

The A. fumigatus galactomannan has now been described in three different forms: (i) soluble and recovered in the culture medium (2), which does not contain a lipid anchor (data not shown), (ii) covalently linked to the cell wall through the {beta}1–3 glucan network (3), and (iii) GPI-anchored to the membrane. These forms present the same carbohydrate chemical structure, suggesting a common biosynthetic pathway. The galactomannan seems secreted to the plasma membrane with a GPI anchor. At this stage, the galactomannan could be transfered to {beta}1–3 glucan chain as it has been proposed for some yeast GPI-anchored protein (39), or the galactomannan could be released in the culture medium by a specific glycosidase. Enzymes putatively involved in such in block transfer are actively searched.


   FOOTNOTES
 
* This work was supported in part by a research and development grant awarded by Aventis-Pharma (to J.-P. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 33-1-45-68-82-25; E-mail: tfontain{at}pasteur.fr.

2 The abbreviations used are: GLC-MS, gas liquid chromatography-mass spectrometry; ES-MS-MS, electrospray-tandem mass spectrometry; PI-PLC, phosphatidylinositol-specific phospholipase C; HPLC, high performance liquid chromatography; JBAM, jack bean {alpha}-mannosidase; LGM, lipogalactomannan; IPC, inositol phosphoceramide; GIPC, glycosylinositol phosphoceramide; ZGL, zwitterionic glycosphingolipid. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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