Catherine Simenel

Bernadette Coddeville

Muriel Delepierre

Jean-Paul Latgé

Thierry Fontaine
email: tfontain@pasteur.fr

HSQC C1H1 Ins C2H2 Ins C3H3 -3)--Man P-CH2 Cer C6H6'H6 GlcNH2 1 Glycosylinositolphosphoceramides in Aspergillus fumigatus

Keywords: aspergillus fumigatus, glycosylinositolphosphoceramide, NMR, galactofuranose, glycosphingolipid Glycobiology

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Glycosylinositolphosphoceramides in Aspergillus

fumigatus.

Introduction

Glycosphingolipids (GSLs) are membrane glycolipids found in all eukaryotic cells that are composed of a carbohydrate and a ceramide hydrophobic moiety. In yeast and fungi, three types of GSLs have been identified. The first class is represented by neutral -glucosylceramide and -galactosyl-ceramide. This type of GSL plays a crucial role in spore germination, hyphal growth and in the cell cycle [START_REF] Levery | Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth[END_REF][START_REF] Da Silva | Glucosylceramides in Colletotrichum gloeosporioides are involved in the differentiation of conidia into mycelial cells[END_REF][START_REF] Da Silva | Glucosylceramides in Colletotrichum gloeosporioides are involved in the differentiation of conidia into mycelial cells[END_REF][START_REF] Rittershaus | Glucosylceramide synthase in an essential regulator of pathogenicity of Cryptococcus neoformans[END_REF]. The second class of fungal GSLs is composed of a complex neutral moiety and a saturated ceramide, suggesting a different biosynthetic pathway [START_REF] Maciel | Glycosphingolipids from Magnaporthe grisea cells: expression of a ceramide dihexoside presenting phytosphingosine as the long-chain base[END_REF]Aoki et al., 2004b;[START_REF] Da Silva | Glucosylceramides in Colletotrichum gloeosporioides are involved in the differentiation of conidia into mycelial cells[END_REF].

The third class is composed of glycosylinositolphosphoceramides (GIPCs) which are acidic GSLs containing a phosphodiester linkage between inositol and ceramide. In contrast to cerebrosides, these phosphorylinositol-containing sphingolipids are not present in mammals but have been detected in protozoa, plants, fungi and nematodes. The sphingolipids are essential for fungal growth since the deletion of inositolphosphorylceramide (IPC) synthase that catalyses the transfer of inositol and phosphate to ceramide is lethal in Saccharomyces and Aspergillus [START_REF] Nagiec | Sphingolipid synthesis as a target for antifungal drugs. Complementation of the inositol phosphorylceramide synthase defect in a mutant strain of Saccharomyces cerevisiae by the AUR1 gene[END_REF][START_REF] Cheng | Cell cycle progression and cell polarity require sphingolipid biosynthesis in Aspergillus nidulans[END_REF]Hu et al, 2007). In filamentous fungi, the ceramide moiety is composed of a phytosphingosine associated with a saturated long chain fatty acid containing 18 to 26 carbon atoms with or without a hydroxyl group in position 2. The carbohydrate moiety is more variable. Three types of GIPCs have been mainly identified based on the monosaccharide and its linkage to the inositol ring: i) -Man-(1-2)-IPC found in numerous species [START_REF] Barr | Occurrence of novel antigenic phosphoinositol-containing sphingolipids in the pathogenic yeast Histoplasma capsulatum[END_REF][START_REF] Levery | Structure elucidation of sphingolipids from the mycopathogen Paracoccidioides brasiliensis: an immunodominant beta-galactofuranose residue is carried by a novel glycosylinositol phosphorylceramide antigen[END_REF]2001;[START_REF] Heise | Molecular analysis of a novel family of complex glycoinositolphosphoryl ceramides from Cryptococcus neoformans: structural differences between encapsulated and acapsular yeast forms[END_REF], ii) -Man-(1-6)-IPC, found in Sporothrix schenckii, [START_REF] Loureiro Y Penha | Characterization of novel structures of mannosylinositolphosphorylceramides from the yeast forms of Sporothrix schenckii[END_REF]Toledo et al., 2001a) iii) -GlcN-(1-2)-IPC described in S. schenckii, Acremonium sp. and Aspergillus fumigatus (Toledo et al., 2001b;2007;Aoki et al., 2004a). To these core structures, other monosaccharides such as fucose, xylose, galactose or choline-phosphate 4 could be associated [START_REF] Jennemann | Novel glycoinositolphosphosphingolipids, basidiolipids, from Agaricus[END_REF][START_REF] Heise | Molecular analysis of a novel family of complex glycoinositolphosphoryl ceramides from Cryptococcus neoformans: structural differences between encapsulated and acapsular yeast forms[END_REF][START_REF] Arigi | Characterization of neutral and acidic glycosphingolipids from the lectin-producing mushroom, Polyporus squamosus[END_REF][START_REF] Gutierrez | Characterization of glycoinositolphosphoryl ceramide structure mutant strains of Cryptococcus neoformans[END_REF]. The presence of an -Man-(1-4)-IPC sequence has been only reported in Basidiomycetes, outside the 3 families described suggesting a high level of complexity in the structure of the fungal sphingolipids [START_REF] Jennemann | Glycoinositolphosphosphingolipids (basidiolipids) of higher mushrooms[END_REF].

A. fumigatus is a saprophytic, filamentous fungus found in most environments where it plays an important role in the recycling of organic materials. A. fumigatus is also an opportunistic pathogen responsible for severe pulmonary diseases, particularly in immunocompromised patients [START_REF] Latgé | Aspergillus fumigatus and aspergillosis[END_REF]. Man 2 -IPC and five other GIPC structures containing additional mannose, galactofuranose, glucosamine or N-acetylglucosamine residues have been previously identified in A. fumigatus [START_REF] Levery | Comparative analysis of glycosylinositol phosphorylceramides from fungi by electrospray tandem mass spectrometry with low-energy collision-induced dissociation of Li(+) adduct ions[END_REF][START_REF] Toledo | Sphingolipids of the mycopathogen Aspergillus fumigatus: characterization of glycosylinositol phosphorylceramide antigens with Manp(alpha 1-2)Ins and GlcpN(alpha 1-2)Ins core motifs[END_REF]. This fungus also produces a lipogalactomannan linked to the cellular membrane through a GlcN-IPC [START_REF] Costachel | Glycosylphosphatidylinositolanchored fungal polysaccharide in Aspergillus fumigatus[END_REF]. As an effort to identify cell surface glycans and antigens, four new structures of GIPC were isolated from membrane preparations of A. fumigatus mycelium and chemically characterized using mass spectrometry and NMR analysis.

Results

The crude membrane preparation of A. fumigatus mycelium was treated with chloroform/methanol/water, then with a butanol/water partition to recover a glycolipid preparation. Two liquid chromatographic steps on a DEAE-Sephadex column and Silica 60 column were used to separate 5 glycosphingolipid fractions (Fig. 1). The total amount of GSLs represented around 0.02 % +/-0.005 of the total mycelium dry weight. DEAE anion exchange chromatography yielded an unbound glycosphingolipid (GSL-U) and negatively charged GSLs. Similar amounts of unbound and bound fractions were purified from a 15-L fermentor in Sabouraud medium of A. fumigatus. Silica gel column chromatography was used to purify the unbound GSL-U and 4 fractions of negatively charged GSLs (GSL-A, GSL-B, Mannose and myo-inositol were identified in all GSL fractions, whereas glucosamine was detected only in the GSL-U fraction and galactose only in negatively-charged GSL fractions (GSL-A to D) (Table I).

GSL fractions have been analysed by Q-TOF mass spectrometry to identify the molecular weight of isolated glycosphingolipids; pseudomolecular negative ions m/z are presented in Table 1 and Fig. 2. The pseudomolecular ion at m/z = 1410 obtained with GSL-U fraction corresponded to a glycosphingolipid containing 2 hexose residues and 1 hexosamine associated with an inositolphosphoceramide (IPC) whereas the ceramide was composed of a C 18:0 -phytosphingosine associated with a 2-hydroxylated C 24:0 fatty acid [START_REF] Costachel | Glycosylphosphatidylinositolanchored fungal polysaccharide in Aspergillus fumigatus[END_REF]. The presence of minor ions that differ by a mass of 14 or 28 reflected the variability in size of fatty acids. Pseudomolecular ions at m/z = 1249, 1411, 1573, obtained with GSL-A, B, C fractions were compatible with a similar IPC with the presence of 2, 3 or 4 hexose residues.

A fragmentation pseudomolecular ion at m/z = 1411 produced one main ion at m/z = 745 [Hex 3 -inositol-phosphate] -, ions at m/z = 241 and 259 corresponding to inositol-phosphate and ions at m/z = 79 and 97 corresponding to free phosphate (Fig. 2) [START_REF] Costachel | Glycosylphosphatidylinositolanchored fungal polysaccharide in Aspergillus fumigatus[END_REF].

Fragmentation of pseudomolecular ions at m/z = 1249, 1411, 1573 gave similar patterns of fragmentation with the loss of 666 corresponding to the ceramide moiety (data not shown), characterizing a GIPC and indicating the presence of 2, 3 and 4 hexose residues linked to IPC.

In contrast, the fragmentation of a pseudomolecular ion at m/z = 1576 in the GSL-D fraction did not produce similar patterns of daughter ions, indicating structural modifications (Fig. 2). The composition of this GSL-D fraction indicated the presence of the same IPC structure with mannose residues. Indeed, a pseudomolecular mass at m/z = 1576 did not correspond to a classical mannosylated IPC, but instead, the presence of a choline-phosphate linked to a Hex 3 -IPC. Daughter fragments at m/z 1517 [CH 2 -CH 2 -P-Hex 3 -IPC] -, 910 [(CH 3 ) 3 N-CH 2 -CH 2 -P-Hex 3 -inositol-P] -, 851 [CH 2 -CH 2 -P-Hex 3 -inositol-P] -were in agreement with the presence of a choline-phosphate substituent. Moreover, GLC-MS analysis of the sugar-phosphate following methanolysis and trimethylsilylation of the GSL-D fraction permitted the identification of a monosaccharide-6-phosphate with the same retention time as the galactose-6-phosphate obtained from the lipophosphoglycan of Leishmania donovani (data not shown).

These data suggest the presence of phosphocholine linked to the C-6 of a galactose residue in the GSL-D fraction.

Linkages of monosaccharides were analysed after methylation (Table II). Methylation of the GSL-U fraction revealed the presence of 3 methyl ethers corresponding to a terminal mannose, a mannose substituted in position 3, and a glucosamine substituted in position 6. In agreement with Q-TOF mass spectra and to GSL described in fungal species (Toledo et al., 2001b;2007;Aoki et al., 2004a), these data suggest the following sequence: Man1-3-Man1-6GlcN. In the GSL-A fraction, the presence of 2 terminal monosaccharides, a mannose and a galactofuranose, without a disubstituted monosaccharide confirmed the presence of two GSL structures. GSL-B fraction contained 3 major methyl ethers with a terminal galactofuranose, indicating, in agreement with the Q-TOF mass spectra, a Gal-Man-Man sequence. In the GSL-C fraction, a disubstituted mannose in position 3 and 6 indicated a branched structure with a terminal galactofuranose and/or mannose. In the GSL-D fraction, 2 main methyl ethers corresponding to monosubstituted mannoses have been identified, but no terminal monosaccharide. A stronger acid hydrolysis yielded a 2,3,5-tri-O-methyl hexitol that should correspond to a galactofuranose substituted in position 6 by a phosphate identified after In agreement with the Q-TOF mass spectra (Fig. 2), these data suggest a choline-phosphate-6-Galf-Man-Man sequence.

NMR analysis of the five glycosphingolipid fractions

The GSL structures of A. fumigatus mycelium were elucidated by analysis of homonuclear and heteronuclear two-dimensional NMR experiments.

In agreement with GLC and MS data (Table I), NMR analysis confirmed the presence of an identical ceramide structure in all GSL fractions. From signal integration in the 1D spectrum of the highest concentrated GSL-B fraction, the total number of carbons of the two aliphatic chains was estimated to be 42. A methylene/methyl ratio of 17.5 was in agreement with a C 18:0 phytosphingosine linked to an unbranched 2-hydroxylated C 24:0 fatty acid identified by GLC-MS. The carbon linked to the nitrogen atom in the sphingosine base shifted at 53.36ppm was located in the 1 H 13 C HSQC experiment as well as three 1 H/ 13 C shifts corresponding to CHOH groups at 3.451/76.01, 3.348/73.65 and 3.835/74.3 (Fig. 4). A phosphorus atom was detected in the ceramide moiety attached to the glycosidic one. Indeed, three correlations were observed in the 1 H, 31 P HSQC spectrum between the phosphorus atom and two methylene protons of the ceramide on one side and the H1 proton of inositol on the other side (Fig. 3,4, 5) indicating the sequence inositol-P-ceramide.

GSL-U fraction

The chemical shifts and coupling constants of the glycosidic moiety obtained for the GSL-U fraction are shown in Table III. The 1D 1 H and 2D 1 H, 13 C gHSQC spectra showed three signals in the anomeric region, in equal proportions as deduced from integration in the 1D spectrum, indicating the presence of three monosaccharide residues (Fig. 3). The 1 H and 13 C chemical shift analysis and the examination of 3 J H,H and 1 J C1,H1 values indicated the presence of 2 -mannopyranose and one -glucosamine residues [START_REF] Bock | A study of 13 CH coupling constants in hexopyranoses[END_REF][START_REF] Wagner | Two-dimensional relayed coherence transfer spectroscopy of a protein[END_REF].

The myo-inositol residue was identified from its H2 equatorial proton in the 1,2,3,4,5,6cyclohexanehexol ring as assessed by the small 3 J H1,H2 and 3 J H2,H3 values (2,7Hz)(Table III).

A NOESY experiment demonstrated a strong correlation between the H1 proton of a mannose residue with the H3 proton of the second mannose residue (Fig. 3). Moreover, C1/H3 and H1/C3 correlations between these two mannose residues were observed in the gHMBC experiment (Fig. 3), indicating a branched sequence of -Manp-(1 3)--Manp. This sequence accounts for the downfield chemical shift of the C3 at 82.24 ppm of the second mannose residue (Table III). Similarly, dipolar interactions between the H1 proton of the second mannose residue and H6/H6' protons of the glucosamine residue were observed in the NOESY experiment suggesting a 3)--Manp-(1 6)--GlcNH 2 sequence. This linkage was confirmed by an gHMBC experiment with the observation of the correlations H1/C6 and C1/H6H6' between the second mannose residue and the glucosamine residue. This is in agreement with a C6 downfield chemical shift at 68.29ppm observed for the glucosamine residue. A strong interaction was also observed in the NOESY experiment between the anomeric proton of the glucosamine residue and the H2 proton of the inositol residue, corroborated by the presence in the gHMBC experiment of a correlation between the C1 carbon of the glucosamine residue and the H2 proton of the inositol residue, indicating the sequence motif: 6)--GlcNH 2 -(1 2)-Ins, in agreement with the downfield shift at 85.22 ppm of the inositol C2 carbon. Along with the MS data, the NMR experiments established the following structure:

-

Manp-(1 3)--Manp-(1 6)--GlcNH 2 -(1 2)-Ins-(1 O)-P-Cer.
This structure has previously been characterized by NMR [START_REF] Toledo | Sphingolipids of the mycopathogen Aspergillus fumigatus: characterization of glycosylinositol phosphorylceramide antigens with Manp(alpha 1-2)Ins and GlcpN(alpha 1-2)Ins core motifs[END_REF]. Differences in 1 H and 13 C chemical shifts are observed with our results that are associated primarily with the following: i) the 13 C referencing method inducing a shift of 3.11ppm, ii) the protonation state of the amine group of the glucosamine residue [START_REF] Bunel | Structures of complexes of cobalt(III) and glucosamine. Applications of molecular mechanics and NMR spectroscopy[END_REF]. Among the acidic GIPCs, fractions B was analysed first because it was the most abundant; it contained only one GSL spot on HPTLC (Fig. 1). The chemical shifts and coupling constants of the glycosidic moiety obtained for the GSL-B fraction are shown in Table IV. The 1D 1 H and2D 1 IV). For the two first glycosidic residues, protons were assigned from anomeric proton only up to H3 and H4 respectively using relayed COSY experiments. The missing ring protons partly located in the TOCSY experiment were fully identified using the combination of 1 H, 13 C edited HSQC and H2BC experiments recently described as a useful method for tracing the proton-bearing carbon skeleton of a molecule [START_REF] Petersen | H2BC: a new technique for NMR analysis of complex carbohydrates[END_REF]. Sugar rings stereochemistry deduced from 3 J H,H coupling constants were consistent with Manp for these two glycosidic residues. Their -configuration was evident from 1 J C1,H1 coupling constant values (169.1 and 173.1Hz). According to the GLC composition and methylation analysis (Tables I andII), the galactose residue was identified in a -furanosic configuration by its characteristic very low field anomeric 13 C resonance at 108.70ppm [START_REF] Ritchie | Carbon-13 chemical shifts of furanosides and cyclopentanols. Configurational and conformational influences[END_REF]. The myo-inositol residue was identified as previously for the GSL-U fraction. The NMR structural analysis of this fraction explicitly indicated the presence of two -mannose, one -galactofuranose and one inositol residues (Table IV).

In the NOESY spectrum, the two inter-residue dipolar interactions observed between the H1 proton of the -galactofuranose residue and the H1 and H2 protons of a mannose residue did not allow the characterization of a branching point. In the gHMBC spectrum, only H1/C2 and C1/H2 inter-residues correlations between the -galactofuranose residue and this mannose residue were observed indicating the -Galf-(1 2)--Manp motif (Fig. 4). This linkage IV). The strong H1/H2 interaction between the second mannose residue and the inositol residue observed in the NOESY spectrum and the double correlation H1/C2 and C1/H2 observed in the gHMBC experiment between these two residues indicate the following sequence motif: 3)--Manp-(1 2)-Ins (Fig. 4). This is in agreement with the downfield shift of the inositol C2 carbon resonating at 80.91 ppm (Table IV). Together with methylation and MS data (Fig. 2, Table I), these NMR data established the

structure of GSL-B as -Galf-(1 2)--Manp-(1 3)--Manp-(1 2)-Ins-(1 O)-P-Cer.

GSL-A fraction.

In agreement with the TLC data, two distinct molecules were observed by NMR in the GSL- In this fraction, two different molecules were also observed which differ in their glycosidic sequences (Table VI). The presence of two different molecules was also confirmed by the observation of two close sets of three correlations in the1 H, 31 P HSQC spectrum (data not shown) corresponding to two distinct phosphorus atoms resonating at 2.478ppm and 2.370ppm and interacting with the two methylene protons of the ceramide ( =4.002 -3.667 ppm and =3.997 -3.670 ppm respectively) and the H1 proton of myo-inositol at 3.720ppm Thus, NMR data allowed the identification of the two following molecules :

GSL-C1, -Galf(1 2)--Manp(1 3)-[ -Manp(1 6)]--Manp-(1 2)-Ins(1 O)-P-Cer and GSL-C2, -Galf(1 2)--Manp(1 3)-[ -Galf(1 6)]--Manp-(1 2)-Ins(1 O)-P-Cer.
GSL-D fraction.

The chemical shifts and coupling constants obtained for this fraction are summarized in the presence of three monosaccharides (Fig. 5). The glycosidic ring spin systems assignment and coupling constants examination permitted the identification of two -mannose residues and one -galactofuranose residue as in the GSL-B fraction. Moreover, the NOESY and gHMBC spectra showed the same inter-residue connectivities as those observed for the GSL-B fraction (Fig. 4 and5), emphasizing the monosaccharide sequence identity with GSL-B. However, differences were observed when comparing 1 H, 13 C HSQC spectra of the two fractions (Fig. 4 and5). Thus, a large downfield shift at 68.92ppm and a smaller upfield shift at 72.11ppm were observed for the C6 and C5 carbons respectively of the -Galf residue for the GSL-D fraction, indicating a 6-O substitution for this residue. Furthermore, in a gHSQC experiment, two new methylene carbons and one trimethyl group have been identified at 4.127/61.62ppm, 3.571/68.79ppm and 3.169/56.48ppm respectively. The COSY experiment showed that these methylene carbons were linked together. The characteristic methyl 13 C shift is consistent with the MS data (Fig. 2), indicating the presence of an N-trimethyl group (Fig. 5). In the HMBC spectrum, two 1 H, 13 C long range interactions were observed between this Ntrimethyl group and the second methylene group indicating the presence of the (CH 3 ) 3 -N- of galactose detected by GLC (Table 1).

Discussion

The results presented here and in previous studies show that A. fumigatus produced at least 9

GIPCs of different structure (Table VIII). All GSLs have the same ceramide moiety composed of a 2-hydroxylated lignoceric acid (2-OH C 24:0 ) associated with a C 18:0phytosphingosine base. This lipid moiety is common to most fungi species. The glycan part has more variability and 2 types of GIPC have been isolated from A. fumigatus mycelium.

First, a zwitterionic GSL that is the major GIPC from A. fumigatus mycelium contains a glucosamine residue linked in 1-2 to the inositol ring. This unusual carbohydrate sequence has been recently described in A. fumigatus by Toledo and his colleagues [START_REF] Toledo | Sphingolipids of the mycopathogen Aspergillus fumigatus: characterization of glycosylinositol phosphorylceramide antigens with Manp(alpha 1-2)Ins and GlcpN(alpha 1-2)Ins core motifs[END_REF] F o r P e e r R e v i e w 14 and has been described in only two other fungal pathogens, S. schenckii and Acremonium sp. (Toledo et al., 2001a;Aoki et al., 2004a). Secondly, the 5 other acidic GSL structures contain a common sequence -Man-(1-3)--Man-(1-2)-Inositol. This sequence has been previously described in A. fumigatus GSL [START_REF] Levery | Comparative analysis of glycosylinositol phosphorylceramides from fungi by electrospray tandem mass spectrometry with low-energy collision-induced dissociation of Li(+) adduct ions[END_REF][START_REF] Toledo | Sphingolipids of the mycopathogen Aspergillus fumigatus: characterization of glycosylinositol phosphorylceramide antigens with Manp(alpha 1-2)Ins and GlcpN(alpha 1-2)Ins core motifs[END_REF] and other fungal species [START_REF] Levery | Structure elucidation of sphingolipids from the mycopathogen Paracoccidioides brasiliensis: an immunodominant beta-galactofuranose residue is carried by a novel glycosylinositol phosphorylceramide antigen[END_REF]2001;[START_REF] Bennion | Glycosphingolipids of the model fungus Aspergillus nidulans: characterization of GIPCs with oligo-alpha-mannose-type glycans[END_REF][START_REF] Barr | Occurrence of novel antigenic phosphoinositol-containing sphingolipids in the pathogenic yeast Histoplasma capsulatum[END_REF]. No -Man-(1-6)-Inositol as found in S. schenckii (Toledo et al., 2001a;[START_REF] Loureiro Y Penha | Characterization of novel structures of mannosylinositolphosphorylceramides from the yeast forms of Sporothrix schenckii[END_REF] and no -Man-(1-4)-Inositol as found in mushrooms [START_REF] Jennemann | Novel glycoinositolphosphosphingolipids, basidiolipids, from Agaricus[END_REF] have been observed in A. fumigatus GSLs. Four of the five acidic GSLs analysed contained galactofuranose in A.

fumigatus. The presence of galactofuranose in GSLs has been reported in human pathogens such as Histoplasma capsulatum, Paracoccidioïdes brasiliensis, A. fumigatus [START_REF] Barr | Occurrence of novel antigenic phosphoinositol-containing sphingolipids in the pathogenic yeast Histoplasma capsulatum[END_REF][START_REF] Levery | Structure elucidation of sphingolipids from the mycopathogen Paracoccidioides brasiliensis: an immunodominant beta-galactofuranose residue is carried by a novel glycosylinositol phosphorylceramide antigen[END_REF][START_REF] Toledo | Sphingolipids of the mycopathogen Aspergillus fumigatus: characterization of glycosylinositol phosphorylceramide antigens with Manp(alpha 1-2)Ins and GlcpN(alpha 1-2)Ins core motifs[END_REF]. In these later structures, the galactofuranose residue is linked to the first mannose like for the GSL-C2 of A. fumigatus. However, the galactofuranose residue is mainly linked to the terminal non-reduced mannose residue through a 1-2 linkage (GSL-B). Surprisingly, a choline-phosphate group has been localized

to the terminal galactofuranose residue (GSL-D). A choline phosphate in a GSL structure has also been found in Acremonium sp., however it is linked to a mannose residue instead of a galactofuranose residue (Aoki et al., 2004a). Some discrepancies are seen between our data and the study of Toledo [START_REF] Toledo | Sphingolipids of the mycopathogen Aspergillus fumigatus: characterization of glycosylinositol phosphorylceramide antigens with Manp(alpha 1-2)Ins and GlcpN(alpha 1-2)Ins core motifs[END_REF]. These authors did not observe the galactofuranose linked in 1-2 to the terminal mannose and the choline-phosphate linked to this galactofuranose. In contrast, they described the presence of a mannose residue linked in 1-2 to Man 2 -IPC that we did not observe. These differences are not explained and could be due to the use of different growth conditions or to different strains. [START_REF] Latgé | Chemical and immunological characterization of the extracellular galactomannan of Aspergillus fumigatus[END_REF][START_REF] Costachel | Glycosylphosphatidylinositolanchored fungal polysaccharide in Aspergillus fumigatus[END_REF][START_REF] Mille | Inactivation of CaMIT1 inhibits Candida albicans[END_REF][START_REF] Leitao | Beta-galactofuranose-containing O-linked oligosaccharides present in the cell wall peptidogalactomannan of Aspergillus fumigatus contain immunodominant epitopes[END_REF]. Galactofuranose residues are also an immunodominant in GSLs of P. brasiliensis and H. capsulatum [START_REF] Barr | Occurrence of novel antigenic phosphoinositol-containing sphingolipids in the pathogenic yeast Histoplasma capsulatum[END_REF][START_REF] Levery | Structure elucidation of sphingolipids from the mycopathogen Paracoccidioides brasiliensis: an immunodominant beta-galactofuranose residue is carried by a novel glycosylinositol phosphorylceramide antigen[END_REF]. In Leishmania major, a monoclonal antibody against a glycolipid containing terminal galactofuranose residue can reduce the macrophage infectivity of this parasite [START_REF] Suzuki | Role of beta-Dgalactofuranose in Leishmania major macrophage invasion[END_REF]. The expression in infected human tissues of intelectin that recognizes a single terminal galactofuranose residue [START_REF] Tsuji | Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall[END_REF] is in agreement with the involvement of galactofuranosylated GSLs during infection. Antibodies from patients with aspergillosis recognized the A. fumigatus GIPCs isolated by [START_REF] Toledo | Sphingolipids of the mycopathogen Aspergillus fumigatus: characterization of glycosylinositol phosphorylceramide antigens with Manp(alpha 1-2)Ins and GlcpN(alpha 1-2)Ins core motifs[END_REF], however, the role of A. fumigatus GIPCs in host cellular immunity has not been defined as yet.

GIPCs have been mainly analysed in human fungal

The biosynthetic pathway of sphingolipids begins in the endoplasmic reticulum where it proceeds to the formation of the ceramide moiety. A linkage between the carbohydrate and phytosphingosine occurs in the Golgi apparatus [START_REF] Funato | Biosynthesis and trafficking of sphingolipids in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Dickson | Functions and metabolism of sphingolipids in Saccharomyces cerevisiae[END_REF]. In yeast, ceramide biosynthesis and inositol addition are essential for growth. Indeed, GIPCs are involved in cell regulation, cell polarity, stress response, trafficking and cell wall integrity [START_REF] Dickson | Functions and metabolism of sphingolipids in Saccharomyces cerevisiae[END_REF]. Similarly, in Aspergillus, the gene encoding the IPC synthase is essential for fungal growth as well as basA gene-encoding a phytosphingosine [START_REF] Li | basA regulates cell wall organization and asexual/sexual sporulation ratio in Aspergillus nidulans[END_REF]. Sphingolipids are involved in polarised growth via the control of actin cytoskeleton [START_REF] Cheng | Cell cycle progression and cell polarity require sphingolipid biosynthesis in Aspergillus nidulans[END_REF]Hu et al., 2007). This result is in agreement with the susceptibility of Aspergillus species to various inhibitors of sphingolipid synthesis pathway [START_REF] Zhong | Inhibition of inositol phosphorylceramide synthase by aureobasidin A in Candida and Aspergillus species[END_REF][START_REF] Li | basA regulates cell wall organization and asexual/sexual sporulation ratio in Aspergillus nidulans[END_REF]. In A. fumigatus, 3 types of membrane anchored molecules present the same inositol-phosphoceramide lipid moiety: GPI-anchored protein [START_REF] Fontaine | Structures of the glycosylphosphatidylinositol membrane anchors from Aspergillus fumigatus membrane proteins[END_REF]; 16 the lipogalactomannan, a GPI-anchored polysaccharide [START_REF] Costachel | Glycosylphosphatidylinositolanchored fungal polysaccharide in Aspergillus fumigatus[END_REF] and GIPCs. In this study, the inositol ring of IPC structure could be substituted in position 2 by a mannose or a glucosamine residue. In contrast, previous studies suggested that the glucosamine residue is linked in 1-6 to the inositol ring in the GPI-anchor structures from GPI-protein or from the lipogalactomannan In S. cerevisiae, 2 homologous genes of IPC mannosyltransferases are involved in the addition of the first mannose residue to the inositol ring. The deletion of both genes is not lethal, but mutants are sensitive to the external Ca 2+ [START_REF] Beeler | SUR1 (CSG1/BCL21), a gene necessary for growth of Saccharomyces cerevisiae in the presence of high Ca2+ concentrations at 37 degrees C, is required for mannosylation of inositolphosphorylceramide[END_REF], suggesting a role for the GSLs in cellular stress response. In C. albicans, the deletion of the MIT1, an IPC mannosyltransferase homologue induced the absence of MIPC, M(IP)2C and the phospholipomannan ( 1-2mannan linked to MIPC) and decreased virulence of the mutant [START_REF] Trinel | Candida albicans phospholipomannan, a new member of the fungal mannose inositol phosphoceramide family[END_REF][START_REF] Mille | Inactivation of CaMIT1 inhibits Candida albicans[END_REF]. In A. fumigatus, no such mutant has been described, so the relevance of these GIPCs during A. fumigatus growth and host-pathogen interactions is still unknown.

Materials and methods

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 [START_REF] Hartland | A novel beta-(1-3)-glucanosyltransferase from the cell wall of Aspergillus fumigatus[END_REF]. The mycelium was collected by filtration under vacuum, washed with water, and then disrupted in 200 mM TrisHCl, 20 mM EDTA, pH 8.0, 1 mM PMSF buffer at 4°C with glass beads (1 mm, diameter) in a Dyno mill apparatus (W. A. Bachofen AG, Basel, Switzerland). The cell wall was removed by centrifugation at 10,000 g 10 min at 4°C. Total membranes were then collected by ultracentrifugation at 125,000 g 60 min at 4°C. Membrane pellet was homogenized in the disruption buffer with a Dounce homogenizer and then

Extraction and purification of glycosylinositolphosphatidylceramides

A chloroform/methanol mixture was added to the membrane suspension (20 mg of protein/ml) to obtain a chloroform/methanol/membrane ratio of 10/10/3 respectively. The mixture was stirred for 2 h at room temperature, then centrifuged at 10,000 g for 10 min. The pellet was resuspended in a chloroform/methanol/water (10/10/3, v/v/v) mixture, and the extraction was repeated once. Pooled supernatants were concentrated under vacuum with a rotavapor, and the residue was submitted to a butanol/water partition. The water phase containing GIPCs was dialysed against water and freeze-dried. The residue was dissolved in chloroform/methanol/water (10/15/4, v/v/v) and applied onto a DEAE-Sephadex A-25 column (Pharmacia, 2 x 15 cm) equilibrated in the same solvent at the flow rate of 30 ml/h.

Unbound products were eluted with 2 column volumes of solvent, then retained products were eluted by a chloroform/methanol/NH 4 Ac 1M (10/15/4) solvent. Carbohydrates were detected by spraying with orcinol sulfuric acid on spot of 2 µl of different fractions on silica sheets.

Fractions containing sugars were concentrated and then dialysed against water and freezedried. GIPCs were then purified on a silica 60 column (Merck, 1.8 x 30 cm) equilibrated in propanol-1/water/NH 4 OH 30% (85/15/5, v/v/v) and eluted at 25ml/h. Samples were deposited onto the silica 60 column and eluted by 3 column volumes of propanol-1/water/NH 4 OH 30%

(85/15/5, v/v/v), then 3 volumes of propanol-1/water/NH 4 OH 30% (80/20/5, v/v/v), then 3 volumes of propanol-1/water/NH 4 OH 30% (70/30/5, v/v/v). The presence of carbohydrate in the fraction was detected as described above. Fractions were dialysed against water and freeze-dried. Neutral hexoses were identified by GLC as alditol acetates obtained after hydrolysis (4 N trifluoroacetic acid, 100°C, 4h.) [START_REF] Sawardeker | Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography[END_REF]. Glucosamine and myo-inositol were quantified by GLC-MS after hydrolysis (6 N HCL, 110°C 20h), N-acetylation and trimethysilyation, using the scyllo-inositol as a standard [START_REF] Ferguson | GPI-membrane anchors: isolation and analysis in Glycobiology, a practical approach[END_REF]. Lipid analysis was performed by GLC-MS on HF-treated GIPC fraction (aqueous 50% HF, 2 days on ice). Fatty acids and sphingosine bases were released by methanolysis (1 N HCl in MeOH, 80°C, 20h).

Fatty acids were extracted with heptane and analysed after trimethylsilylation. The methanol phase containing the sphingosine base was N-acetylated, trimethylsilylated and analysed by GLC-MS. Phosphorylated carbohydrates were identified by GLC-MS after acid methanolysis

(1 N HCL in methanol, 80°C, 20h) and trimethylsilylation [START_REF] Ferguson | GPI-membrane anchors: isolation and analysis in Glycobiology, a practical approach[END_REF]. The lipophosphoglycan from Leishmania donovani, a kind gift from Pascale Pescher (Unité de Virulence Parasitaire, Institut Pasteur) was used as positive control. Methylation of GSL fractions was performed using the sodium hydroxide procedure [START_REF] Ciucanu | A simple and rapid method for the permethylation of carbohydrates[END_REF].

GSL containing a glucosamine residue was peracetylated with a pyridine/acetic anhydride solution (50/200 µl) overnight at room temperature prior to the methylation procedure.

Methyl ethers were analysed by GLC-MS as polyolacetates [START_REF] Björndal | Gas-liquid chromatography and mass spectrometry in methylation analysis of polysaccharides[END_REF].

HPTLC. GIPC fractions were applied to a 10 cm aluminium-backed silica gel 60 (Merck) and developed with chloroform/methanol/1 M ammonium acetate/NH 4 OH 30%/water (180/140/9/9/23). Sugars were detected with orcinol-sulfuric acid.

GLC and GLC-Mass spectrometry. GLC was performed on a Delsi 200 instrument with a flame ionisation detector using a capillary column (30 m x 0.25 mm id) filled with a EC TM -1 (Alltech) under the following conditions: gas vector and pressure, helium 0.7 bar; temperature France) was added in order to exchange sugars hydroxyl protons. Since the proton chemical shift of the residual signal of DMSO-d6 depends on temperature and water content, external referencing was applied for 1 H chemical shifts using a capillary containing a freshly prepared solution of 20 mM DSS in DMSO-d6 containing less than 0,01% of water. The DSS methyl resonance was set to 0ppm. 13 C chemical shifts were then calculated from 1 H chemical shift and gamma ratio relative to DSS. 13 C/ 1 H gamma ratio of 0.251449530 was used [START_REF] Wishart | 1H, 13C and 15N chemical shift referencing in biomolecular NMR[END_REF]. 31 P chemical shifts were determined with neat phosphoric acid (Wilmad-Labglas, New Jersey, USA) by substitution method.

For all GSL fractions, the same general strategy was adopted for assignment of nuclei. First, the proton resonances were assigned using two-dimensional COSY and RELAY experiments with one to three relays to follow connectivities from the anomeric proton up to the H5 proton of most of glycosidic residues [START_REF] Rance | Improved spectral resolution in COSY 1H NMR spectra of proteins via double quantum filtering[END_REF][START_REF] Wagner | Two-dimensional relayed coherence transfer spectroscopy of a protein[END_REF]. The intra glycosidic residue spin systems were often completed by mean of a TOCSY experiment with a long mixing time (120ms) [START_REF] Griesinger | Clean TOCSY for proton spin system identification in macromolecules[END_REF]. A 1 H-13 C edited gHSQC experiment allowed to achieve 13 C chemical shifts assignment from previously identified 1 H resonances [START_REF] Willker | Gradient selection in inverse heteronuclear correlation spectroscopy[END_REF]. Then, 1 H, 1 H coupling constants analysis from 1D and/or COSY spectrum ( 1 H resolution of 0.1 Hz and 1,6Hz respectively) was used to assess the identity of monosaccharide residues.

Moreover, the anomeric configuration of monosaccharide residues was established from knowledge of 3 J 1,2 values and confirmed by the measurement of the 1 J C1H1 heteronuclear coupling constants in the 1 H dimension of the undecoupled gHSQC spectrum ( 1 H resolution of 0.6Hz) or of the gHMBC spectrum ( 1 H resolution of 1.2Hz) [START_REF] Willker | Gradient selection in inverse heteronuclear correlation spectroscopy[END_REF]. Finally, glycosidic linkages were established via through-space dipolar interactions using 1 H-1 H NOESY experiment (mixing time of 200ms) [START_REF] Macura | Two-dimensional chemical exchange and cross-relaxation spectroscopy of coupled nuclear spins[END_REF] and/or via three-bond interglycosidic 1 H-13 C correlations using 1 H-13 C gHMBC experiment (long range delay of 60ms). In addition, the branching point between the phospholipid and the glycosidic moieties was identified using the 1 H-31 P gHSQC. III. 1 H and 13 C NMR chemical shifts (ppm) and coupling constants (J H,H and 1 J C1H1 , Hz)

F

for the glycan sequence of the GSL-U fraction.

- Hz) for the glycan sequence of the GSL-B fraction.

Manp-(1-3)--Manp-(1-6)--GlcNH2-(1-2)-Ins-(1-O)-P-Cer H 1 H 2 H 3 H 4 H 5 H 6 -H 6' 3 J 1,2 3 J 2,3 3 J 3,4 3 J 4,5 3 J 5,6 -3 J 5,6' 2 J 6,6' C 1 C 2 C 3 C 4 C 5 C 6 1 J C1H1
- for the two glycan sequences of the GSL-A fraction.

Galf-(1-2)--Manp-(1-3)--Manp-(1-2)-Ins-(1-O)-P-Cer H 1 H 2 H 3 H 4 H 5 H 6 -H 6' 3 J 1,2 3 J 2,3 3 J 3,4 3 J 4,5 3 J 5,6 -3 J 5,6' 2 J 6,6' C 1 C 2 C 3 C 4 C 5 C 6 1 J C1H1

GSL-A1

: -Manp-(1-3)--Manp-(1-2)-Ins(1-O)-P-Cer GSL-A2: -Galf-(1-2)--Manp-(1-3)--Manp-(1-2)-Ins(1-O)-P-Cer H 1 H 2 H 3 H 4 H 5 H 6 -H 6' 3 J 1,2 3 J 2,3 3 J 3,4 3 J 4,5 3 J 5,6 -3 J 5,6' C 1 C 2 C 3 C 4 C 5 C 6 1 J C1H1

  Figure 1

  , GSL-D) (Fig.1). The GSL-A and GSL-C fractions contain two molecules. Only one spot was detected in fractions B and D.Composition analysis obtained by GLC and GLC-MS revealed the presence of C 18:0phytosphingosine and a 2-monohydroxylated C 24:0 fatty acid in all GSL fractions. Minor fatty acids such as 2OH-C 25:0 and 2OH-C 26:0 were observed in low amounts (data not shown).

  H, 13 C gHSQC spectra exhibited three signals of equivalent areas in the anomeric region (5.029/103.85ppm, 5.020/101.71ppm and 4.881/108.70ppm) indicating the presence of three monosaccharide residues (Fig. 4, Table

  downfield chemical shift of the mannose C2 carbon at 77,08 ppm. In the NOESY spectrum, the H1/H3 interaction between the latter mannose residue and the second mannose residue indicates a 2)--Manp-(1 3)--Manp linkage. This glycosidic sequence was confirmed by H1/C3 and C1/H3 correlations between these two mannose residues observed in the gHMBC experiments but also by the downfield chemical shift of C3 of the second mannose residue at 81.70ppm (Table

  A fraction. The chemical shifts and coupling constants of the corresponding glycosidic sequences are shown in TableV. The one-dimensional 1 H spectrum (not shown) exhibited three signals in the anomeric region with areas in the ratio 2/1.2/2. The sugar spin systems assignment and the sequential glycosidic analysis revealed that these signals correspond to five protons belonging to two different molecules present in about equivalent amounts. The GSL-A2 molecule was identical to the GSL-B one. The GSL-A1 structure displayed the identical dimannoside core as GSL-A2 without the -galactofuranose residue at the nonreducing end. Thus, the C2 carbon resonance of the first mannose residue was not downfield shifted for the GSL-A1 molecule (73.47ppm) upon branching of galactofuranose as observed for the GSL-A2 molecule (77.09ppm). Moreover, only three correlations were observed in the ceramide on one side (4.039ppm and 3.658ppm) and the H1 proton of inositol on the other side (3.738ppm). These data are in agreement with the fact that the two molecules differ only at the non-reducing end of the glycosidic moiety. These NMR experiments indicated the presence of the two following structures: GSL A1, -Manp-(1 3)--Manp-(1 2)-Ins-(1 O)-P-Cer and GSL A2, -Galf-(1 2)--Manp-(1 3)--Manp-(1 2)-Ins-(1 O)-P-Cer GSL-C fraction.

  and 3.713ppm, respectively. The one-dimensional 1 H-NMR spectrum (not shown) exhibited eight H1 resonances of about equal intensity. In the anomeric region of the 2D 1 H, 13 C gHSQC spectrum (not shown), six resonances among the eight were grouped two by two that resembled the anomeric region of the GSL-B fraction. Indeed, the sugar spin systems assignment and the sequential glycosidic analysis corresponded to the same glycosidic sequence which is -Galf-(1 2)--Manp-(1 3)--Manp-(1 2)-Ins. One of the other anomeric signals at 4.799/111.10 ppm corresponded to a second -galactofuranose residue identified by its characteristic downfield shifted anomeric carbon. The last anomeric signal at 4.640/102.57ppm was assigned to a -mannopyranose residue. The 1 H, 13 C edited gHSQC methylene region of the GSL-C fraction compared to that of the GSL-B fraction showed two C6 at 68.27ppm and 69.79ppm indicating that the two corresponding mannose residues were 6-O-substituted.

  CH 2 motif. Two sets of correlations were observed in the 1 H, 31 P HSQC (Fig.5). A first set of three correlations was detected between a phosphorus atom (2.974ppm) and the two methylene protons of the ceramide on one side (4.094ppm and 3.660ppm) and the H1 proton of inositol on the other side (3.756ppm), corresponding to the IPC. Another set of two correlations was observed between a second phosphorus atom (0.763ppm) and the two extra methylene protons on the one hand (4.127ppm) and the H6 protons of -Galf on the other hand (3.764ppm). This was in agreement with the chemical shifts observed for C6 and C5 carbons resonances of the -Galf residue, indicating its substitution by a phosphocholine residue. In addition, the comparison with the 1 H 1D and 1 H, 13 C HSQC spectra of a phosphatidylcholine reference compound (Sigma) confirmed the resonances assignment of the phosphocholine (not shown). Thus, the NMR analysis, in agreement with methanolysis/trimethylsylation, methylation and MS data, showed that the GSL-D fractioncorresponded to Cho-P-(O 6)--Galf-(1 2)--Manp-(1 3)--Manp-(1 2)-Ins(1 O)-P-Cer. The substitution of galactofuranose residue by a phosphate group explanes the low amont

  at 125,000 g for 60 min at 4°C. Pellet was resuspended again in 20 mM TrisHCl, 2 mM EDTA, pH 8.0 and store at -80°C.

  180°C at 2°C/min and 180 to 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 id) filled with a EC TM -1 (Alltech) under the following conditions : gas vector and flow rate, helium 1.2 ml/min; temperature program for inositol and monosaccharide analysis : 100 to 200°C at 5°C/min, 200 to 240°C at 15°C/min and 240°C for 5 min; temperature program for sphingosine base and fatty acid analysis : 100 to 200°C at 10°C/min, 200 to 260°C at 15°C/min and 260°C for 13 min.Nano-electrospray mass spectrometry. Mass spectrometric analyses were performed in the negative mode using a Q-STAR Pulsar quadrupole time-of-flight (Q-TOF) mass spectrometer (AB/MDS Sciex, Toronto, Canada) equipped with a nano-electrospray ion source (Protana, Odense, Denmark).The samples in propanol-1/H 2 O (25/75) dissolved in chloroform/methanol (50/50) were sprayed from gold-coated 'medium length' borosilicate capillaries (Protana, Odense, Denmark). A potential of -800 V was applied to the capillary tip. The declustering potential was set at -120V and the focusing potential was set at -200V. The molecular ions were then selected in the quadrupole analyzer and partially fragmented in the hexapole collision cell, with the pressure of collision gas (N 2 ) 5.3 x 10 -5 Torr (1Torr = 133.3 Pa). The collision energy was varied between -40 and -80 eV depending on the sample. For the recording of conventional mass spectra, TOF data were acquired by accumulation of 10 MCA (multiple channel acquisition) scans over mass ranges of m/z ranges 500-1800 Daltons for MS analyses and over mass ranges of m/z 50-1800 for MS/MS analyses. Data acquisition was optimized to supply the highest possible resolution and the best signal-to-noise ratio, even in the case of low abundance signals. Typically, the FWHM (full width at half maximum) was 7000 in the measured mass ranges. External calibration was performed prior to each measure using a

Figure 2 :

 2 Figure 2: Nano-electrospray mass spectrometry analysis of GSL-B and GSL-D fractions of A.

Figure 3 :

 3 Figure3: NMR spectra of GSL-U isolated from A. fumigatus mycelium. 1 H-13 C HSQC, 1 H-1 H NOESY, 1 H-13 C HMBC and 1 H-31 P HSQC.

Figure 4 :

 4 Figure4: NMR spectra of GSL-B from A. fumigatus mycelium. 1 H-13 C HSQC, 1 H-1 H NOESY, 1 H-13 C HMBC and 1 H-31 P HSQC.

Figure 5 :

 5 Figure5: NMR spectra of GSL-D from A. fumigatus mycelium. 1 H-13 C HSQC, 1 H-1 H NOESY, 1 H-13 C HMBC and 1 H-31 P HSQC.

  TableVI. 1 H and 13 C NMR chemical shifts (ppm) and coupling constants (J H,H and 1 J C1H1 , Hz) for the two glycan sequences of the GSL-C fraction.GSL-C1: -Galf(1-2)--Manp(1-3)-[ -Manp (1-6)]--Manp-(1-2)-Ins(1-O)-P-Cer GSL-C2: -Galf(1-2)--Manp(1-3)-[ -Galf (1-6)]--Manp-(1-2)-Ins(1-O)-P-Cer

Table VII .

 VII The 1D 1 H and 2D 1 H, 13 C gHSQC spectra exhibited three signals of equivalent areas in the anomeric region (5.108/103.47ppm, 5.069/102.24ppm and 4.939/109.11ppm), indicating

  Figure 1: HPTLC of GSL fractions (U, A, B, C, D) isolated from A. fumigatus mycelium.

	TLC was developed on 10-cm aluminium-coated silica gel 60 with chloroform/methanol/1 M
	ammonium acetate/NH 4 OH 30%/water (180/140/9/9/23). Sugars were detected using orcinol
	sulfuric acid.
	o r F o r F o r F o r F o r F o r F o r F o r F o r F o r
	P P P P P P P P P P
	e e e e e e e e e e
	e r e r e r e r e r e r e r e r e r e r
	R e v R e v R e v R e v R e v R e v R e v R e v R e v R e v
	i e i e i e i e i e i e i e i e i e i e
	w w w w w w w w w w

Table IV .

 IV 1 H and 13 C NMR chemical shifts (ppm) and coupling constants (J H,H , 1 J C1H1 and J H,P

		4.925	3.781	3.606	3.440	3.468	3.524-3.695
	1.7 105.26 169.6 4.666 1.8 103.51 167.6 F -3)--Man-(1-6)--Man-(1-3)-o r	3 73.56 3.932 3.1 72.54	10 74.07 3.656 9.1 82.24	10 70.57 3.576 9.7 69.06	4.9 76.66 3.468 nm -5.0 76.80	10.9 64.41 3.518-3.665 12.00 64.41
	-6)--GlcNH2-(1-2)--2)-myo-Ins-(1-O)-P	5.222 3.5 101.32 P 2.923 10.2 57.91 e nm 3.825 4.168 2.7 2.7 e r 3.652 10.0 73.83 3.278 10.0	3.320 10.8 72.97 3.371 8.5	3.994 0 -5.2 74.30 3.048 3 J 5,6 =8.5	3.594-3.799 11.0 68.29 3.776 3 J 6,1 =8.8
	nm : not measured	79.86 J H,P =10.4	85.22	73.38 R e v 75.18	78.03	75.11
					i e
					w
				35

Table V .

 V 1 H and 13 C NMR chemical shifts (ppm) and coupling constants (J H,H and 1 J C1H1 , Hz)

		4.881	3.788	3.808	3.936	3.525	3.377-3.401
	2.1 108.70 173.6 5.020 1.5 101.71 F -2)--Man-(1-3)--Galf-(1-2)-o r	3.2 83.78 3.917 3.8 77.08	5.2 80.10 3.607 8.9 72.86	3.2 87.25 3.332 9.4 71.05	6.7-6.0 73.90 3.584 5.0-5.0 76.30	10.9 65.85 3.508-3.578 11.2 64.00
	-3)--Man-(1-2)-	169.1 5.029 P 3.927 1.5 3.3 103.85 72.60 e 173.1 3.727 3.977 e r 3.659 9.4 81.70 3.216	3.608 10.2 68.81 3.351	3.867 5.8-3.5 76.16 2.948	3.488-3.561 11.5 64.13 3.485
	-2)-myo-Ins-(1-O)-P	2.4 78.99 139.3 J H1,P =9.2	2.6 80.91	11.3 73.63 R e 75.29 8.9 v	3 J 5,6 =8.9 78.56	3 J 1,6 =9.4 75.49
					i e
					w
				36

Table VII

 VII . 1 H,13 C and 31 P NMR chemical shifts (ppm) and coupling constants (J H,H , 1 J C1H1 and J H,P , Hz) for the glycan sequence and for the phosphocholine sequence of the GSL-D

	fraction.
	Cho-P-(O-6)--Galf-(1-2)--Manp-(1-3)--Manp-(1-2)-Ins(1-O)-P-Cer
	Glycan moiety	H 1	H 2	H 3	H 4	H 5	H 6 -H 6'
		3 J 1,2 H 1	3 J 2,3 H 2	3 J 3,4 H 3	3 J 4,5 H 4	3 J 5,6 -3 J 5,6' H 5	H 6 -H 6'
		C 1 1 J C1H1 3 J 1,2	C 2 3 J 2,3	C 3 3 J 3,4	C 4 J 4,5 3	C 5 3 J 5,6 -3 J 5,6'	C 6
	GSL-C1	C 1	C 2	C 3	C 4	C 5	C 6
		4.877 P	3.777	3.800	3.933	3.518	3.360-3.392 P
	1.1 108.47 174.7 4.989 3.2 101.72 169.3 4.939 1,8 o F -Galf-(1-2)--2)--Manp-(1-3)-r 109.11 P-(O-6)--Galf-(1-2)-	2.9 83.60 3.933 3.5 76.81 3.894 83.74	79.93 3.604 72.77 3.898 79.46	87.07 3.325 70.79 3.974 86.66	73.72 3.565 76.29 3.696 72.11	65.72 3.507-3.599 3.451*-3.595* 63.74 64.17* 3.764 0.763 68.92
	-Manp-(1-6)--3,6)--Manp-(1-2)--2)--Manp-(1-3)-	4.640 1.5 2.5 102.57 169.1 4.982 3.2 103.83 174.6 P 5.069 e 3.642 3.1 73.20 3.924 2.5 72.43 3.964 1.6 5.2 102.24 77.25 e r 3.506 73.72 3.621 82.02 3.672 9.6 73.19 5.108 3.959 3.745	3.366 70.07 3.632 68.27 3.413 9.6 71.07 3.647	3.380 76.51 4.021 74.31 3.643 76.70 3.958	3.445-3.602 64.17 3.486-3.702 68.27 J H1,P =7.5 3.651-3.562 64.14 3.566
	3.706 weak 78.92 4.877 1.1 108.47 174.7 5.003 2.5 101.63 169.3 1.9 103.47 3.756 2.4 79.07 2.974 J H1,P =8.6 Choline-phosphate substitution -2)-myo-Ins-(1-O)-P GSL-C2 -Galf-(1-2)--2)--Manp-(1-3)--3)--Man-(1-2)--2)-myo-Ins-(1-O)-P 4.799 1 H 1.9 2.5 111.1 13 C -Galf-(1-6)-31 P	3.949 4.8 80.70 3.777 2.9 83.60 3.916 3.4 76.81 4.7 72.66 R 3.208 8.5 73.43 3.800 79.93 3.594 72.77 82.26 e 68.87 3.337 9.0 75.15 3.933 87.07 3.325 70.79 3.982 3.237 3.433 v 2.5 9.4 9.1 i 80.43 74.07 75.25 e w 2.936 3 J 5,6 = 8.8 78.49 3.518 73.72 3.565 76.29 76.22 2.973 3 J 5,6 =9.1 79.00 3.806 3.779 3.759 3.486 3.7 84.25 79.87 85.71 73.78	3.480 3 J 6,1 = 9.4 75.42 3.360-3.392 65.72 3.507-3.599 3.451*-3.595* 63.74 64.29 3.538 3 J 1,6 =9.2 75.74 64.17* 3.507-3.599 3.451*-3.595* 63.74
	CH 2 OP -3,6)--Manp-(1-2)-	171.3 4.982 4.127 2.6 103.83 61.62 0.763	3.921 2.9 72.43	3.643 10.2 81.58	3.581 10.2 68.87	4.013 6.6 74.97	64.17* 3.429-3.747 69.79
	-2)-myo-Ins-(1-O)-P CH 2 N	174.6 3.699 weak 3.571 68.79	3.967 4.8	3.203 8.5	3.337 9.0	2.936 3 J 5,6 = 8.8	3.468 3 J 6,1 = 9.4
	* possible chemical shift N(CH 3 ) 3	78.93 3.169 56.48	81.13	73.43	75.15	78.49	75.42
			38

H, 31 P HSQC (not shown) between a phosphorus atom (3.099ppm) and the two methylene

phospholipomannan beta-mannosylation, reduces virulence, and alters cell wall protein beta-mannosylation. J Biol Chem. 279:47952-47960.

Acknowledgments

We thank Professor R. Calderone (Georgetown University Medical center, Washington, USA) for reading of the manuscript.

Abbreviations

Methyl ethers a Linkages Glycosphingolipid fractions [START_REF] Toledo | Sphingolipids of the mycopathogen Aspergillus fumigatus: characterization of glycosylinositol phosphorylceramide antigens with Manp(alpha 1-2)Ins and GlcpN(alpha 1-2)Ins core motifs[END_REF] this study -Man-(1-3)--Man-(1-2)-Ins-P-cer [START_REF] Levery | Comparative analysis of glycosylinositol phosphorylceramides from fungi by electrospray tandem mass spectrometry with low-energy collision-induced dissociation of Li(+) adduct ions[END_REF][START_REF] Toledo | Sphingolipids of the mycopathogen Aspergillus fumigatus: characterization of glycosylinositol phosphorylceramide antigens with Manp(alpha 1-2)Ins and GlcpN(alpha 1-2)Ins core motifs[END_REF] this study