Glycobiology Advance Access originally published online on October 30, 2007
Glycobiology 2008 18(1):84-96; doi:10.1093/glycob/cwm122
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Glycosylinositolphosphoceramides in Aspergillus Fumigatus
2 Unité de Résonance Magnétique Nucléaire des Biomolécules, CNRS URA 2185 Institut Pasteur, 25 rue du Docteur Roux 75724 Paris cedex 15
3 Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq cedex
4 Unité des Aspergillus, Institut Pasteur, 25 rue du Docteur Roux 75724 Paris cedex 15, France
1 To whom correspondence should be addressed: e-mail: tfontain{at}pasteur.fr
Received on August 1, 2007; revised on October 23, 2007; accepted on October 23, 2007
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Abstract |
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Fungal glycosylinositolphosphoceramides (GIPCs) are involved in cell growth and fungal–host interactions. In this study, six GIPCs from the mycelium of the human pathogen Aspergillus fumigatus were purified and characterized using Q-TOF mass spectrometry and 1H, 13C, and 31P NMR. All structures have the same inositolphosphoceramide moiety with the presence of a C18:0-phytosphingosine conjugated to a 2-hydroxylated saturated fatty acid (2-hydroxy-lignoceric acid). The carbohydrate moiety defines two types of GIPC. The first, a mannosylated zwitterionic glycosphingolipid contains a glucosamine residue linked in
1-2 to an inositol ring that has been described in only two other fungal pathogens. The second type of GIPC presents an -Manp-(1
3)--Manp-(12)-IPC common core. A galactofuranose residue is found in four GIPC structures, mainly at the terminal position via a β1-2 linkage. Interestingly, this galactofuranose residue could be substituted by a choline–phosphate group, as observed only in the GIPC of Acremonium sp., a plant pathogen. Key words: Aspergillus fumigatus / galactofuranose / glycosphingolipid / glycosylinositolphosphoceramide / NMR
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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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 β-glucosyl-ceramide and β-galactosyl-ceramide. This type of GSL plays a crucial role in spore germination, hyphal growth, and in the cell cycle (Levery et al. 2002
; Barreto-Bergter et al. 2004; da Silva et al. 2004; Rittershaus et al. 2006). The second class of fungal GSLs is composed of a complex neutral moiety and a saturated ceramide, suggesting a different biosynthetic pathway (Maciel et al. 2002; Aoki, Uchiyama, Yamauchi et al. 2004; Barreto-Bergter et al. 2004). 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 catalyzes the transfer of inositol and phosphate to ceramide is lethal in Saccharomyces and Aspergillus (Nagiec et al. 1997; Cheng et al. 2001; 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 (Barr et al. 1984; Levery et al. 1998, 2001; Heise et al. 2002), (ii) -Man-(1-6)-IPC found in Sporothrix schenckii (Loureiro y Penha et al. 2001; Toledo, Levery, Glushka et al. 2001), and (iii) -GlcN-(1-2)-IPC described in S. schenckii, Acremonium sp., and Aspergillus fumigatus (Toledo, Levery, Straus et al. 2001; Toledo et al. 2007; Aoki, Uchiyama, Itonori et al. 2004). To these core structures, other monosaccharides such as fucose, xylose, galactose, or choline–phosphate could be associated (Jennemann et al. 1999; Heise et al. 2002; Arigi et al. 2007; Gutierrez et al. 2007). The presence of an -Man-(1-4)-IPC sequence has only been reported in Basidiomycetes, outside the three families described suggesting a high level of complexity in the structure of the fungal sphingolipids (Jennemann et al. 2001).
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 (Latgé 1999). Man2-IPC and five other GIPC structures containing additional mannose, galactofuranose, glucosamine, or N-acetylglucosamine residues have been previously identified in A. fumigatus (Levery et al. 2001; Toledo et al. 2007). This fungus also produces a lipogalactomannan linked to the cellular membrane through a GlcN-IPC (Costachel et al. 2005). 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.
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Results |
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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 five glycosphingolipid fractions (Figure 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 the Sabouraud medium of A. fumigatus. Silica gel column chromatography was used to purify the unbound GSL-U and four fractions of negatively charged GSLs (GSL-A, GSL-B, GSL-C, and GSL-D) (Figure 1). The GSL-A and GSL-C fractions contain two molecules. Only one spot was detected in fractions B and D.
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Composition analysis obtained by GLC and GLC-MS revealed the presence of C18:0-phytosphingosine and a 2-monohydroxylated C24:0 fatty acid in all GSL fractions. Minor fatty acids such as 2OH-C25:0 and 2OH-C26:0 were observed in low amounts (data not shown). 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).
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GSL fractions have been analyzed by Q-TOF mass spectrometry to identify the molecular weight of isolated glycosphingolipids; pseudomolecular negative ions m/z are presented in Table I and Figure 2. The pseudomolecular ion at m/z = 1410 obtained with GSL-U fraction corresponded to a glycosphingolipid containing two hexose residues and one hexosamine associated with an inositolphosphoceramide (IPC) whereas the ceramide was composed of a C18:0-phytosphingosine associated with a 2-hydroxylated C24:0 fatty acid (Costachel et al. 2005
). The presence of minor ions that differ by a mass of 14 or 28 reflected the variability in the 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 two, three, or four hexose residues. A fragmentation pseudomolecular ion at m/z = 1411 produced one main ion at m/z = 745 [Hex3-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 (Figure 2; Costachel et al. 2005). 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 two, three, and four 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 (Figure 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 Hex3-IPC. Daughter fragments at m/z 1517 [CH2-CH2-P-Hex3-IPC]–, 910 [(CH3)3N-CH2-CH2-P-Hex3-inositol-P]–, and 851 [CH2-CH2-P-Hex3-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 C6 of a galactose residue in the GSL-D fraction.
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Linkages of monosaccharides were analyzed after methylation (Table II). Methylation of the GSL-U fraction revealed the presence of three 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, Levery, Straus et al. 2001
; Toledo et al. 2007; Aoki, Uchiyama, Itonori et al. 2004), these data suggest the following sequence: Man1–3-Man1–6GlcN. In the GSL-A fraction, the presence of two terminal monosaccharides, a mannose and a galactofuranose, without a disubstituted monosaccharide confirmed the presence of two GSL structures. GSL-B fraction contained three 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 positions 3 and 6 indicated a branched structure with a terminal galactofuranose and/or mannose. In the GSL-D fraction, two 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 methanolysis and trimethysilylation. In agreement with the Q-TOF mass spectra (Figure 2), these data suggest a choline–phosphate–6-Galf–Man–Man sequence.
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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 C18:0 phytosphingosine linked to an unbranched 2-hydroxylated C24:0 fatty acid identified by GLC-MS. The carbon linked to the nitrogen atom in the sphingosine base shifted at 53.36 ppm was located in the 1H 13C HSQC experiment as well as three 1H/13C shifts corresponding to CHOH groups at 3.451/76.01, 3.348/73.65, and 3.835/74.3 (Figure 4). A phosphorus atom was detected in the ceramide moiety attached to the glycosidic one. Indeed, three correlations were observed in the 1H, 31P 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 (Figures 3–5) indicating the sequence inositol–P–ceramide.
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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 1H and 2D 1H, 13C 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 (Figure 3). The 1H and 13C chemical shift analysis and the examination of 3JH,H and 1JC1,H1 values indicated the presence of two
-mannopyranose and one -glucosamine residues (Bock and Pedersen 1974, 1983). The myo-inositol residue was identified from its H2 equatorial proton in the 1,2,3,4,5,6-cyclohexanehexol ring as assessed by the small 3JH1,H2 and 3JH2,H3 values (2.7 Hz) (Table III).
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A NOESY experiment demonstrated a strong correlation between the H1 proton of a mannose residue and the H3 proton of the second mannose residue (Figure 3). Moreover, C1/H3 and H1/C3 correlations between these two mannose residues were observed in the gHMBC experiment (Figure 3), indicating a branched sequence of
-Manp-(13)--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-(16)--GlcNH2 sequence. This linkage was confirmed by a 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 68.29 ppm 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 of a correlation between the C1 carbon of the glucosamine residue and the H2 proton of the inositol residue in the gHMBC experiment, indicating the sequence motif: 6)--GlcNH2-(12)-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-(13)--Manp-(16)--GlcNH2-(12)-Ins-(1O)-P-Cer. This structure has previously been characterized by NMR (Toledo et al. 2007). Differences in 1H and 13C chemical shifts are observed with our results that are associated primarily with the following: (i) the 13C referencing method inducing a shift of 3.11 ppm, (ii) the protonation state of the amine group of the glucosamine residue (Bunel et al. 1993).
GSL-B Fraction.
Among the acidic GIPCs, fraction B was analyzed first because it was the most abundant; it contained only one GSL spot on HPTLC (Figure 1). The chemical shifts and coupling constants of the glycosidic moiety obtained for the GSL-B fraction are shown in Table IV. The 1D 1H and 2D 1H, 13C gHSQC spectra exhibited three signals of equivalent areas in the anomeric region (5.029/103.85 ppm, 5.020/101.71 ppm, and 4.881/108.70 ppm) indicating the presence of three monosaccharide residues (Figure 4, Table IV). For the first two glycosidic residues, protons were assigned from the 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 1H, 13C edited HSQC and H2BC experiments recently described as a useful method for tracing the proton-bearing carbon skeleton of a molecule (Petersen et al. 2006). Sugar rings stereochemistry deduced from 3JH,H coupling constants were consistent with Manp for these two glycosidic residues. Their -configuration was evident from 1JC1,H1 coupling constant values (169.1 and 173.1 Hz). According to the GLC composition and methylation analysis (Tables I and II), the galactose residue was identified in a β-furanosic configuration by its characteristically very low field anomeric 13C resonance at 108.70 ppm (Ritchie et al. 1975). 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 β-galactofuranose, and one inositol residue (Table IV).
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In the NOESY spectrum, the two inter-residue dipolar interactions observed between the H1 proton of the β-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-residue correlations between the β-galactofuranose residue and this mannose residue were observed indicating the β-Galf-(1
2)--Manp motif (Figure 4). This linkage accounts for the 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-(13)--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 81.70 ppm (Table 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-(12)-Ins (Figure 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 (Figure 2, Table I), these NMR data established the structure of GSL-B as β-Galf-(12)--Manp-(13)--Manp-(12)-Ins-(1O)-P-Cer.
GSL-A Fraction.
In agreement with the TLC data, two distinct molecules were observed by NMR in the GSL-A fraction. The chemical shifts and coupling constants of the corresponding glycosidic sequences are shown in Table V. The one-dimensional 1H 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 β-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 (73.47 ppm) upon branching of galactofuranose as observed for the GSL-A2 molecule (77.09 ppm). Moreover, only three correlations were observed in the 1H, 31P HSQC (not shown) between a phosphorus atom (3.099 ppm) and the two methylene protons of the ceramide on one side (4.039 ppm and 3.658 ppm) and the H1 proton of inositol on the other side (3.738 ppm). These data are in agreement with the fact that the two molecules differ only at the nonreducing end of the glycosidic moiety. These NMR experiments indicated the presence of the following two structures: GSL A1, -Manp-(13)--Manp-(12)-Ins-(1O)-P-Cer and GSL A2, β-Galf-(12)--Manp-(13)--Manp-(12)-Ins-(1O)-P-Cer.
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GSL-C Fraction.
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 the 1H, 31P HSQC spectrum (data not shown) corresponding to two distinct phosphorus atoms resonating at 2.478 ppm and 2.370 ppm 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 3.720 ppm and 3.713 ppm, respectively. The one-dimensional 1H-NMR spectrum (not shown) exhibited eight H1 resonances of about equal intensity. In the anomeric region of the 2D 1H, 13C 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 β-Galf-(12)--Manp-(13)--Manp-(12)-Ins. One of the other anomeric signals at 4.799/111.10 ppm corresponded to a second β-galactofuranose residue identified by its characteristic downfield shifted anomeric carbon. The last anomeric signal at 4.640/102.57 ppm was assigned to an -mannopyranose residue. The 1H, 13C edited gHSQC methylene region of the GSL-C fraction compared to that of the GSL-B fraction showed two extra downfield shifted C6 at 68.27 ppm and 69.79 ppm indicating that the two corresponding mannose residues were 6-O-substituted.
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Thus, NMR data allowed the identification of the two following molecules:
GSL-C1, β-Galf(12)--Manp(13)-[-Manp(16)]--Manp-(12)-Ins(1O)-P-Cer and GSL-C2, β-Galf(12)--Manp(13)-[β-Galf(16)]--Manp-(12)-Ins(1O)-P-Cer.
GSL-D Fraction.
The chemical shifts and coupling constants obtained for this fraction are summarized in Table VII. The 1D 1H and 2D 1H, 13C gHSQC spectra exhibited three signals of equivalent areas in the anomeric region (5.108/103.47 ppm, 5.069/102.24 ppm, and 4.939/109.11 ppm), indicating the presence of three monosaccharides (Figure 5). The glycosidic ring spin systems assignment and coupling constants examination permitted the identification of two -mannose residues and one β-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 (Figures 4 and 5), emphasizing the monosaccharide sequence identity with GSL-B. However, differences were observed when comparing 1H, 13C HSQC spectra of the two fractions (Figures 4 and 5). Thus, a large downfield shift at 68.92 ppm and a smaller upfield shift at 72.11 ppm were observed for the C6 and C5 carbons respectively of the β-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 4.127/61.62 ppm, 3.571/68.79 ppm, and 3.169/56.48 ppm respectively. The COSY experiment showed that these methylene carbons were linked together. The characteristic methyl 13C shift is consistent with the MS data (Figure 2), indicating the presence of an N-trimethyl group (Figure 5). In the HMBC spectrum, two 1H, 13C long-range interactions were observed between this N-trimethyl group and the second methylene group indicating the presence of the (CH3)3–N–CH2–CH2 motif. Two sets of correlations were observed in the 1H, 31P HSQC (Figure 5). A first set of three correlations was detected between a phosphorus atom (2.974 ppm) and the two methylene protons of the ceramide on one side (4.094 ppm and 3.660 ppm) and the H1 proton of inositol on the other side (3.756 ppm), corresponding to the IPC. Another set of two correlations was observed between a second phosphorus atom (0.763 ppm) and the two extra methylene protons on the one hand (4.127 ppm) and the H6 protons of β-Galf on the other hand (3.764 ppm). This was in agreement with the chemical shifts observed for C6 and C5 carbons resonances of the β-Galf residue, indicating its substitution by a phosphocholine residue. In addition, the comparison with the 1H 1D and 1H, 13C HSQC spectra of a phosphatidylcholine reference compound (Sigma, St. Louis, MO) 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 fraction corresponded to Cho-P-(O6)-β-Galf-(12)--Manp-(13)--Manp-(12)-Ins(1O)-P-Cer. The substitution of galactofuranose residue by a phosphate group explains the low amount of galactose detected by GLC (Table I).
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Discussion |
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The results presented here and in previous studies show that A. fumigatus produced at least nine GIPCs of different structures (Table VIII). All GSLs have the same ceramide moiety composed of a 2-hydroxylated lignoceric acid (2-OH C24:0) associated with a C18:0-phytosphingosine base. This lipid moiety is common to most fungi species. The glycan part has more variability and two types of GIPCs 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 et al. (2007) and has been described in only two other fungal pathogens, S. schenckii and Acremonium sp. (Toledo, Levery, Glushka et al. 2001; Aoki, Uchiyama, Itonori et al. 2004). Secondly, the five 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 (Levery et al. 2001; Toledo et al. 2007) and other fungal species (Levery et al. 1998, 2001; Bennion et al. 2003; Barr and Lester 1984). No -Man-(1-6)-Inositol as found in S. schenckii (Toledo, Levery, Glushka et al. 2001; Loureiro y Penha et al. 2001) and no -Man-(1-4)-inositol as found in mushrooms (Jennemann et al. 1999) have been observed in A. fumigatus GSLs. Four of the five acidic GSLs analyzed 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 (Barr and Lester 1984; Levery et al. 1998; Toledo et al. 2007). In these later structures, the galactofuranose residue is linked to the first mannose as for the GSL-C2 of A. fumigatus. However, the galactofuranose residue is mainly linked to the terminal nonreduced mannose residue through a β1-2 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, Uchiyama, Itonori et al. 2004). Some discrepancies are seen between our data and the study of Toledo et al. (2007). These authors did not observe the β-galactofuranose linked in β1-2 to the terminal mannose and the choline–phosphate linked to this β-galactofuranose. In contrast, they described the presence of a mannose residue linked in 1-2 to Man2-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.
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GIPCs have been mainly analyzed in human fungal pathogens (A. niger, H. capsulatum, P. brasiliensis, S. schenckii, and C. neoformans), and it has been suggested that GIPCs have an immunological function during fungal infection. The presence of galactofuranose in these GIPCs that is absent in mammals seems to play an important role in fungal–host interactions. Earlier studies have shown that galactofuranose containing molecules of A. fumigatus are extremely antigenic (Latgé et al. 1994
; Costachel et al. 2005; Morelle et al. 2005; Leitao et al. 2003). Galactofuranose residues are also an immunodominant in GSLs of P. brasiliensis and H. capsulatum (Barr and Lester 1984; Levery et al. 1998). In Leishmania major, a monoclonal antibody against a glycolipid containing terminal galactofuranose residue can reduce the macrophage infectivity of this parasite (Suzuki et al. 2002). The expression in infected human tissues of intelectin that recognizes a single terminal galactofuranose residue (Tsuji et al. 2001) is in agreement with the involvement of galactofuranosylated GSLs during infection. Antibodies from patients with aspergillosis recognized the A. fumigatus GIPCs isolated by Toledo et al. (2007); however, the role of A. fumigatus GIPCs in host cellular immunity has not been defined as yet.
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 (Funato et al. 2002; Dickson et al. 2006). 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 (Dickson et al. 2006). Similarly, in Aspergillus, the gene encoding the IPC synthase is essential for fungal growth as well as basA gene encoding a phytosphingosine (Li et al. 2007). Sphingolipids are involved in polarized growth via the control of actin cytoskeleton (Cheng et al. 2001; Hu et al. 2007). This result is in agreement with the susceptibility of Aspergillus species to various inhibitors of a sphingolipid synthesis pathway (Zhong et al. 2000; Li et al. 2007). In A. fumigatus, three types of membrane-anchored molecules present the same IPC lipid moiety: GPI-anchored protein (Fontaine et al. 2003), the lipogalactomannan, a GPI-anchored polysaccharide (Costachel et al. 2005), and GIPCs. In this study, the inositol ring of an 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, two 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 Ca2+ (Beeler et al. 1997), 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 (β1-2mannan linked to MIPC) and decreased virulence of the mutant (Trinel et al. 2002; Mille et al. 2004). 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.
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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 (Hartland et al. 1996
). The mycelium was collected by filtration under vacuum, washed with water, and then disrupted in 200 mM Tris–HCl, 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 x g 10 min at 4°C. Total membranes were then collected by ultracentrifugation at 125,000 x g 60 min at 4°C. Membrane pellet was homogenized in the disruption buffer with a Dounce homogenizer and then centrifuged once more at 125,000 x g for 60 min at 4°C. Pellet was resuspended again in 20 mM Tris–HCl, 2 mM EDTA, pH 8.0 and store at –80°C.
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, and then centrifuged at 10,000 x 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 dialyzed 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 (GE Healthcare Bio-Sciences, Uppsala, Sweden, 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, and then retained products were eluted by a chloroform/methanol/NH4Ac 1 M (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 dialyzed against water and freeze-dried. GIPCs were then purified on a silica 60 column (Merck, Darmstadt, Germany 1.8 x 30 cm), equilibrated in propanol-1/water/NH4OH 30% (85/15/5, v/v/v) and eluted at 25 mL/h. Samples were deposited onto the silica 60 column and eluted by 3 column volumes of propanol-1/water/NH4OH 30% (85/15/5, v/v/v), then 3 volumes of propanol-1/water/NH4OH 30% (80/20/5, v/v/v), and then 3 volumes of propanol-1/water/NH4OH 30% (70/30/5, v/v/v). The presence of carbohydrate in the fraction was detected as described above. Fractions were dialyzed against water and freeze-dried.
Analytical methods
Neutral hexoses were identified by GLC as alditol acetates obtained after hydrolysis (4 N trifluoroacetic acid, 100°C, 4 h) (Sawardeker et al. 1967). Glucosamine and myo-inositol were quantified by GLC-MS after hydrolysis (6 N HCL, 110°C 20 h), N-acetylation and trimethysilyation, using the scyllo-inositol as a standard (Ferguson 1993). 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, 20 h). Fatty acids were extracted with heptane and analyzed after trimethylsilylation. The methanol phase containing the sphingosine base was N-acetylated, trimethylsilylated, and analyzed by GLC-MS. Phosphorylated carbohydrates were identified by GLC-MS after acid methanolysis (1 N HCL in methanol, 80°C, 20 h) and trimethylsilylation (Ferguson 1993). The lipophosphoglycan from L. donovani, a kind gift from Pascale Pescher (Unité de Virulence Parasitaire, Institut Pasteur), was used as a positive control. Methylation of GSL fractions was performed using the sodium hydroxide procedure (Ciucanu and Kerek 1984). 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 analyzed by GLC-MS as polyolacetates (Björndal et al. 1970).
HPTLC.
GIPC fractions were applied to a 10-cm aluminum-backed silica gel 60 (Merck) and developed with chloroform/ methanol/1 M ammonium acetate/NH4OH 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 ionization detector using a capillary column (30 m x 0.25 mm id) filled with a ECTM-1 (Alltech, Templemars, France) under the following conditions: gas vector and pressure, helium 0.7 bar; temperature program 120 to 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, Thermo Electron Corporation, Runcorn, UK) coupled to a CarloErba gas chromatograph (model 8000 top), using a capillary column (30 m x 0.25 mm id) filled with a ECTM-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.
Nanoelectrospray 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 nanoelectrospray ion source (Protana, Odense, Denmark).
The samples in propanol-1/H2O (25/75) dissolved in chloroform/methanol (50/50) were sprayed from gold-coated "medium length" borosilicate capillaries (Protana). A potential of –800 V was applied to the capillary tip. The declustering potential was set at –120 V and the focusing potential was set at –200 V. The molecular ions were then selected in the quadrupole analyzer and partially fragmented in the hexapole collision cell, with the pressure of collision gas (N2) 5.3 x 10–5 Torr (1 Torr = 133.3 Pa). The collision energy 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 multiple channel acquisition (MCA) scans over mass ranges of m/z 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 full width at half maximum (FWHM) was 7000 in the measured mass ranges. External calibration was performed prior to each measure using a 4 pmol/µL solution of taurocholic acid in acetonitrile/water (50/50, v/v) containing 2 mM of ammonium acetate.
NMR Spectroscopy.
NMR spectra were acquired at 50°C on a Varian, Les Ulis, France Inova 500 spectrometer equipped with a triple 1H{13C/15N} resonance 1H PFG probe or an indirect PFG probe for 1H, 31P experiments. For the low-concentration samples, complementary experiments were performed at 35°C on a Varian Inova 600 spectrometer equipped with a cryogenically cooled triple resonance 1H{13C/15N} PFG probe. Samples were dissolved in DMSO-d6 for NMR (99.96% 2H atoms, Euriso-top, CEA, Saclay, France) and transferred in 5 mm Shigemi tubes (Shigemi Inc., Allison Park, PA). D2O (99.97% 2H atoms, Euriso-top) 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 1H 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 0 ppm. 13C chemical shifts were then calculated from the 1H chemical shift and gamma ratio relative to DSS. The 13C/1H gamma ratio of 0.251449530 was used (Wishart et al. 1995). 31P chemical shifts were determined with neat phosphoric acid (Wilmad-Labglas, NJ) by the 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 the glycosidic residues (Rance et al. 1983; Wagner 1983). The intraglycosidic residue spin systems were often completed by mean of a TOCSY experiment with a long mixing time (120 ms) (Griesinger et al. 1988). A 1H–13C edited gHSQC experiment allowed to achieve 13C chemical shifts assignment from previously identified 1H resonances (Willker et al. 1993). Then, 1H, 1H coupling constants analysis from the 1D and/or COSY spectrum (1H 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 3J1,2 values and confirmed by the measurement of the 1JC1H1 heteronuclear coupling constants in the 1H dimension of the undercoupled gHSQC spectrum (1H resolution of 0.6 Hz) or of the gHMBC spectrum (1H resolution of 1.2 Hz) (Willker et al. 1993). Finally, glycosidic linkages were established via through-space dipolar interactions using a 1H–1H NOESY experiment (mixing time of 200 ms) (Macura et al. 1981) and/or via three-bond interglycosidic 1H–13C correlations using a 1H–13C gHMBC experiment (long-range delay of 60 ms). In addition, the branching point between the phospholipid and the glycosidic moieties was identified using the 1H–31P gHSQC.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We thank Professor R. Calderone (Georgetown University Medical center, Washington) for reading of the manuscript.
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Abbreviations |
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COSY, correlation spectroscopy; DMSO, dimethylsulfoxyde; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt; FWHM, full width at half maximum; gHMBC, gradient selected heteronuclear multiple bond correlation; gHSQC, gradient selected heteronuclear single-quantum correlation; GIPC, glycosylinositolphosphoceramide; GLC, gas–liquid chromatography; GLC-MS: gas–liquid chromatography mass spectrometry; GPI, glycosylphosphatidylinositol; GSL, glycosphingolipid; H2BC, heteronuclear two-bond correlation; HPTLC, high-performance thin layer chromatography; IPC, inositolphosphoceramide; MCA, multiple channel acquisition; MS, mass spectrometry; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; PFG, pulsed field gradient; Q-TOF, pulsar quadrupole-time of flight; RELAY 2D, relayed COSY; TOCSY, total correlation spectroscopy
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References |
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