2 Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Bloco G, Universidade Federal do Rio de Janeiro, 21944-970, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, Brasil; 3 Laboratory for Molecular Structure, NIBSC, Herts EN6 3QG, UK; and 4 Kennedy Institute for Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, 1 Aspenlea Rd, London W6 8LH, UK
Received on December 21, 2001; revised on February 18, 2002; accepted on February 18, 2002
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Abstract |
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Key words: C. neoformans/glycophosphosphingolipids/mass spectrometry/NMR spectroscopy
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Introduction |
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Some fungal glycans are linked to sphingolipid or sphingophospholipid anchors. Glycoinositolphosphoryl ceramides (GIPCs) are membrane glycolipids containing a phosphodiester linkage between inositol and ceramide, inositol-(1-O)-phosphoryl-(O-1) ceramide constituting a common structural element (Laine and Hsieh, 1987). Phosphorylinositol-containing sphingolipids are not present in animals but have been detected in protozoa (including the soil amoeba Acanthamoeba castellanii), plants, fungi (Lester and Dickson, 1993
), and in the parasitic nematode Ascaris suum (Sugita et al., 1996
). In yeast, phosphorylinositol-containing sphingolipids are essential for growth and viability, despite constituting a low percentage of total cell membrane phospholipids (Dickson and Lester, 1999
). In addition, pathogenic fungi such as C. neoformans, Candida albicans, Aspergillus fumigatus, and Histoplasma capsulatum are killed by inhibitors of inositolphosphoryl ceramide (IPC) synthase, an essential glycosphingolipid biosynthetic enzyme (Takesako et al., 1993
; Mandala et al., 1997
, 1998). Therefore, the biosynthesis of phosphorylinositol-containing sphingolipids is a promising target for the development of more effective antifungal agents (Dickson and Lester, 1999
; Nagiec et al., 1997
).
Ceramide-(phosphorylinositol)2-mannose, ceramide-phosphorylinositol-mannose and ceramide-phosphorylinositol have been identified in C. neoformans (Vincent and Klig, 1995). Similar compounds have been described in Saccharomyces cerevisae (Smith and Lester, 1974
), Neurospora crassa (Lester et al., 1974
), H. capsulatum (Barr et al., 1984
), Phytophthora capsici (Lhomme et al., 1990
), and C. albicans (Wells et al., 1996
). However, more complex GIPC structures have only been found in the mycopathogens Aspergillus niger, H. capsulatum (Lester and Dickson, 1993
), Paracoccidioides brasiliensis (Levery et al., 1998
), and more recently in Sporothrix schenckii (Penha et al., 2000
; Toledo et al., 2001
). In the present study we have isolated, purified, and characterized members of an unusual series of complex GIPCs from C. neoformans, and structural differences between wild-type (WT) encapsulated and mutant acapsular yeast cells are described.
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Results |
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Carbohydrate and lipid composition of WT- and Cap67-GIPCs from C. neoformans
Compositional analysis showed that the isolated GIPCs contained mannose, galactose, xylose, and inositol. The Cap67-GIPCs contained a higher molar ratio of Man compared to the WT (designated here GIPC A) isolate. Glucuronic acid (GlcA), hexosamine, or sialic acid were not detected in any of the preparations (Table I). Gas chromatography (GC) and GCmass spectrometry (GC-MS) analyses of acid methanolysates of the crude GIPCs show the presence of C18 phytosphingosine N-acylated with 2-hydroxy tetra-, penta-, or hexacosanoic fatty acids, and with 2-hydroxy and 2,3-dihydroxy tetra- or penta-cosanoic acids for WT-GIPC A and Cap67-GIPCs respectively (Table I). Due to the absence of authentic standards, these two unusual 2,3-dihydroxy fatty acids were identified by electron ionization mass spectrometry (EI-MS). The EI-MS of 2,3-dihydroxytetracosanoic acid and 2,3-dihydroxypentacosanoic acid was similar to that of 2,3-dihydroxy-12-methyltridecanoate isolated from Legionella pneumophila (Mayberry, 1981), except that the fragments (M-15), (M-59), and (M-161) were shifted up in mass by 140 and 154 units, respectively.
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Tandem electrospray ionization mass spectrometry (ESIMS/MS) of the GIPCs after permethylation enabled their partial characterization. Because the ceramide groups were lost during the methylation procedure, the size heterogeneity attributable to the N-acyl groups present in the native samples was abolished. This loss of ceramide was also observed when methylation was performed using the procedure of Ciucanu and Kerek (1984). The daughter ion spectrum (Figure 4) of the sodium cationized permethylated oligosaccharide from the WT-GIPC A at m/z 1343.5 contained a complete series of Y-type fragment ions (Domon and Costello, 1988). The signals at m/z 1125.5 (Y4
), 1169.5 (Y4ß), and 951.4 (Y4ß/Y4
) were consistent with a Hex-[Pen]-Hex-Hex-Hex-Ins-PO4 structure where the branching point is located at the third Hex unit distal from the Ins residue. Sodium cationized B-type ions were observed down to B2, B4 at m/z 1013.5 being particularly abundant. In addition a ring cleavage 0,3A4 was detected at m/z 897.5 with secondary fragments at m/z 679.4 (Y4
/0,3A4), 505.2 (Y4ß/Y4
/0,3A4), and 315.2 (Y3/0,3A4). The fragmentation patterns of the sodium cationized permethylated GIPC-derived Ins- oligosaccharides (precursor ions at m/z 1547.6, 1751.7, and 1955.9) from the Cap67 mutant were similar (spectra not shown). The major difference was the appearance of 0,3A5, 0,3A6, and 0,3A7 fragments at m/z 1101.6, 1305.6, and 1509.8, respectively, consistent with more complex structures where additional permethylated Hex units (increments of 204 mass units) were linked to the nonreducing terminal Hex residue found in the WT-GIPC A.
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(Structure I ) [Xylp(1-2,3)]Manp(1-4)Galp(1-6)Manp(1-2)Ins
(Structure II) [Xylp(1-2,3)]Manp(1-6)Manp(1-4)Galp(1-2)Ins
To discriminate between these possibilities, the GIPCs were subjected to Smith degradation and the products fractionated on Bio-Gel P-2. The main carbohydrate fraction, eluting at a volume consistent with a Hex disaccharide, was hydrolyzed, and the derivatized products when analyzed by GC and GC-MS showed the presence of Man and threitol in a molar ratio of 1.0:1.0. The ESI mass spectrum of this carbohydrate fraction contained abundant signals at m/z 285 and 307, which correspond to the molecular masses of protonated and sodium cationized mannosyl-threitol, respectively. Because the threitol derives from Gal C-3, 4, 5, and 6 we conclude that the Ins-oligosaccharide structure I is the common core structure of GIPCs from WT and Cap67, consistent with a glycan sequence [Xylp(1-2,3)]Manp(1-4)Galp(1-6)Manp(1-2)Ins.
Linkage of the phosphate group to Ins. The phosphorylation position was deduced by a combination of periodate oxidation and methylation analysis. Oxidation of WT-GIPC A and Cap67-GIPCs (B and D) with NaIO4 and subsequent reduction and hydrolysis yielded a phospho-alcohol that, after treatment with alkaline phosphatase and acetylation, yielded a product that was identified as erythritol tetra-acetate by GC and GC-MS. Because the erythritol is derived from carbons 1, 2, 3, and 6 of the inositol, and methylation analysis showed that the oligosaccharide chain is linked to the Ins O-2, O-1 must carry the phosphate group.
NMR spectroscopy of the Ins-oligosaccharides
Ins-oligosaccharides released from WT-GIPC A and Cap67-GIPCs B and D by ammonolysis were analyzed by nuclear magnetic resonance (NMR) spectroscopy. Proton NMR spectra were assigned from total correlation spectroscopy (TOCSY) experiments, and the 13C spectra through heteronuclear correlation. Information on the linkage and sequence of the sugar residues was obtained from rotating frame nuclear Overhauser enhancement spetroscopy (ROESY) spectra and long-range 1H-13C correlations. Additional 1H and 13C assignments were derived from a two-dimensional heteronuclear single-quantum correlation (HSQC)-TOCSY experiment. Figure 5 shows the 1D spectra, and assignments are summarized in Table III. The sequence of and linkage between the sugar residues was established from interresidue nuclear Overhause effects (NOEs) and long-range transglycosidic 1H13C correlations; these data are summarized in Figure 6. The ß-anomeric configuration of the Gal and Xyl residues was established from the magnitudes of 3JH1,H2, from the chemical shifts of the H-1 and C-1, and from the chemical shifts of H-3 and H-5 (or H-5a), when identified. The -anomeric configurations of the Man residues were established from the chemical shifts of H-1 and C-1 and of H-2, H-3, and H-5. The Ins residue was identified from its lack of an anomeric resonance and by the presence of a low-field H-2 at 4.13 ppm. In the ROESY spectrum, this correlated with Man(1) H-1 at 5.122 or 5.127 ppm, establishing the
-Manp-(1
2)-Ins linkage.
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Compared to the Ins-pentasaccharide from the WT-GIPC A, the Ins-hexasaccharide from the Cap67-GIPC B contained an additional -Man residue with both H-1 and C-1 resonating at relatively high field (4.930 and 101.52 ppm respectively), consistent with the presence of an additional
-Manp-(1
6) residue, and supported by the presence of a low-field methylene resonance at 67.03 ppm, the downfield shift of the Man(3) C-5 (73.24 ppm), and a small change in the chemical shift of the Man(3) H-1. An interresidue NOE is observed between the Man(4) H-1 and a resonance at 3.68 ppm, which in turn correlates with the C-6 at 67.03 ppm in the HSQC, assigned as the Man(3) H-6. This is consistent with extension of the Man arm with an additional
-Manp(1
6)-linked residue.
The Ins-octasaccharide (Cap67-GIPC D) contained two more -Man residues with H-1s at relatively low field (
H/
C 5.161/100.08 and
H/
C 5.045/104.13 ppm). These chemical shifts are consistent with a
-Manp-(1
2)-
-Manp(1
system (Carreira et al., 1996
), and interresidue NOEs were observed between Man(6) H-1 (5.045) and the Man(5) H-1 and H-2. The Man(5) H-1 showed an interresidue NOE to a resonance at 3.773 ppm, assigned as the Man(4) H-6, and consistent with the Ins-octasaccharide being the same as the Ins-hexasaccharide but with an extension of
-Manp-(1
2)-
-Manp-(1
6) sequence (Figure 6).
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Discussion |
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Another interesting structural feature is the extension of the Ins-pentasaccharide by additional Man units in Cap67-GIPCs, but not in the WT. The reason for this is not clear, because identical Ins-pentasaccharide precursors are found in both strains. One possible explanation is that the putative 1
6 mannosyl transferase, which adds the first Man residue, may be inactive in the WT cells, or possibly insufficient levels of GDP-Man are available for its activity, because of depletion by extensive GXM capsular polysaccharide production (Doering, 2000
). Additional structural diversity is apparent in the ceramide domain. In common with the majority of fungi studied so far, the WT synthesizes GIPC based on C18 phytosphingosine predominantly N-acylated with 2-hydroxylignoceric acid, whereas the Cap67 mutant synthesizes GIPCs, which are N-acylated with 2,3-dihydrolignoceric acid or with 2-hydroxylignoceric acid.
The biological reason for the interstrain differences in C. neoformans ceramide composition is unclear. The fact that the in the Cap67 mutant the amount of GIPC is increased four-fold compared with WT-cells is suggestive of an adaptation to compensate for the absence of the GXM capsule. The presence of a higher percentage of 2,3-dihydroxy acids in the acapsular mutant would confer enhanced hydrogen-bonding capacity promoting formation of a thicker and less permeable bilayer (Levine et al., 2000). Studies with S. cerevisiae have shown that plasma membrane sphingophospholipids are essential for growth (Wells and Lester, 1983
), viability (Pinto et al., 1992
), and resistance to environmental stress (Patton et al., 1992
); this is likely to be the case in other fungal species, including C. neoformans. Thus, Luberto et al. (2001)
recently showed that the down-regulation of the IPC synthase gene (IPC1) results in defective growth of Cryptococcus at acidic pH and that the biosynthesis of complex sphingolipids is essential for intracellular growth, suggesting that GIPC could function as virulence factors in C. neoformans.
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Materials and methods |
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Extraction and purification of GIPCs from C. neoformans
GIPCs were extracted with aqueous phenol as previously described (Previato et al., 1990). Briefly, after removal of capsular GXM and GalXM from C. neoformans (250 g wet weight) with 450 ml of 20 mM citrate buffer, pH 7.0 (90 min at 121°C) (Lloyd and Bitoon, 1971
) the residual cells were recovered by centrifugation, (6,000 x g, 15 min) and extracted with 45% (v/v) aqueous phenol at 75°C for 20 min. After cooling and centrifugation (3,000 x g, 30 min) the aqueous layer was dialyzed, freeze-dried, dissolved in water, and applied to a column (2 x 80 cm) of Bio-Gel P-6DG. The excluded material was lyophilized, and the GIPCs recovered by three extractions with 200 ml chloroform/methanol/water (10:10:3, v/v/v). After compositional and structural analyses this was further purified on a Florisil column (1.2 x 40 cm) eluted with chloroform/methanol/water (10:10:3, v/v/v). Fractions of 1 ml were collected and screened for homogeneity by negative-ion MALDI-TOF MS (see following procedures) and HPTLC on Silica gel 60 plates (Merck) using chloroform/methanol/1 M NH4OH (10:10:3, v/v/v) as mobile phase. GIPCs were detected by spraying with orcinol-sulfuric acid (Humbel and Collaert, 1975
).
Carbohydrate and inositol analyses
The monosaccharide composition of GIPCs was determined according to Sweeley et al. (1963)). After methanolysis with 0.5 M HCl in methanol (18 h at 80°C), the mixture was extracted three times with hexane and the methanolic phase neutralized with Ag2CO3. The products were N-acetylated with acetic anhydride (overnight at room temperature in dark), dried under a stream of nitrogen, and treated with bis-(trimethylsilyl) trifluoroacetamide (BSTFA)/pyridine (1:1, v/v, 1 h at room temperature). Trimethylsilyl derivatives were analyzed by GC using a DB-5 fused silica capillary column (25 m x 0.25 mm ID) with hydrogen (10 psi) as the carrier gas. The column temperature was programmed from 120°C to 240°C at 2°C/min. For the analysis of Ins, GIPCs were hydrolyzed with 6 M HCl (18 h at 105°C), dried under reduced pressure, treated with BSTFA/pyridine (1:1, v/v, 1 h at room temperature), and analyzed by GC as described. Peaks were identified by comparison of their retention times to authentic standards and by GC-MS.
Lipid analysis
After methanolysis of the GIPCs with methanolic-HCl (18 h at 80 °C), fatty acid methyl esters were extracted with hexane. The extracts were combined, concentrated under a stream of nitrogen, and analyzed by GC after derivatization with BSTFA/pyridine as described. The column temperature was programmed from 180°C to 280°C at 3°C/min. Peaks were identified by their retention times compared to authentic standards and by GC-MS. For the analysis of long-chain bases, GIPCs were methanolyzed (1 M methanol-HCl made 10 M with respect to water) (Carter and Gaver, 1967) for 18 h at 80 °C. After adjusting the pH to about 11 with aqueous NaOH, long-chain bases were extracted with diethyl ether. The combined extracts were washed with water, dried with anhydrous sodium sulfate, evaporated to dryness, dissolved in methanol, and N-acetylated with acetic anhydride (18 h at room temperature in the dark). The product was treated with BSTFA/pyridine and analyzed by GC and GC-MS as described for the fatty acid methyl esters.
Isolation of Ins-oligosaccharides from GIPCs by ammonolysis
GIPCs were hydrolyzed with 10 M ammonium hydroxide (18 h at 150°C) (Barr and Lester, 1984). After cooling, the hydrolysate was evaporated, dissolved in water, and ultracentrifuged (40,000 x g, 6 h). The supernatant was passed through a reversed-phase LC-18 SPE column (Supelco); the Ins-oligosaccharides was eluted with water and further fractionated on a Bio-Gel P-4 (extra-fine) column (1 x 120 cm). Carbohydrate-containing fractions were pooled, lyophilized, and characterized by methylation analysis and NMR spectroscopy.
Methylation analysis
GIPCs and Ins-oligosaccharides were permethylated by the procedure of Paz Parente et al. (1985) methanolyzed (0.5 M HCl in methanol, 18 h at 80°C), acetylated (acetic anhydride/pyridine 9:1, v/v, 18 h at room temperature) and analyzed by GC and GC-MS on DB-1 fused silica column using a temperature programme from 110°C to 200°C at 2°C/min. The acetylated partially methylated methyl glycosides were initially identified by comparison of their retention times to those of authentic standards and confirmed by GC-MS as described by Fournet et al. (1981)
. The methylated GIPC was also analyzed by positive-ion ESI-MS/MS.
Determination of phosphorylation position of inositol
A modification of the procedure of Sugita et al. (1996) was used to determine the substitution pattern of Ins. GIPCs were oxidized (20 mM NaIO4 in 0.2 M sodium acetate buffer, pH 5, 72 h at 4°C in the dark), reduced with NaBH4, and hydrolyzed (2 M HCl, 2 h at 100°C). The products were chromatographed on Dowex 1X8 column (100200 mesh, Cl form). The phospho-alcohol was eluted with 0.1 M HCl and freeze-dried. After dephosphorylation by incubation with 1 U of alkaline phosphatase from Escherichia coli (type III, Sigma), the product was peracetylated and identified by GC-MS as described. A peracetylated threitol (prepared by periodate oxidation of a purified (1
4)-linked ß-D-galactopyranan kindly provided by Prof. José P. Parente of Núcleo de Pesquisas de Produtos Naturais, UFRJ, Brazil) and erythritol (prepared by acetylation of erythritol from Sigma) were used as standards.
Smith degradation of GIPCs
GIPCs were oxidized with NaIO4 and reduced with NaBH4 as described. The products were hydrolyzed (0.02 M trifluoroacetic acid, 40 min at 100°C), fractionated on a Bio Gel P-2 (extra-fine) column (1.0 x 100 cm) eluted with water. Fractions of 1.0 ml were collected and assayed for carbohydrate (Dubois et al., 1956). A fraction eluting at a volume consistent with that of a Hex disaccharide was isolated, hydrolyzed (2 M trifluoracetic acid, 1 h at 100°C), reduced with NaBH4, and analyzed by GC after peracetylation (as described for methylation analysis). Derivatives were identified by comparison of their retention times with those of mannitol, threitol, erythritol, and glycerol and by GC-MS.
Other analytical methods
Total phosphorus was determined by the method of Ames (1966). The procedure of Lauter and Trams (1962)
was used for the quantitative analysis of the long-chain bases in the methanolysates of GIPCs, using C18 phytosphingosine as standard.
MALDI-TOF-MS and ESI-QTOF-MS
MALDI mass spectra were recorded with a Micromass TofSpec 2E spectrometer, equipped with a 337-nm nitrogen laser. The instrument was operated in the positive ion reflectron mode at 20 kV accelerating voltage with time-lag focusing enabled. Samples were dissolved in 5% formic acid, and 1 µl was mixed with an equal volume of norharmane matrix solution (10 mg/ml in 50% acetonitrile) (Nonami et al., 1998) and air-dried on the stainless steel target. Spectra were externally calibrated using deprotonated molecules of angiotensin I (m/z 1294.670), and ACTH clip 1834 (m/z 2463.183) as references.
For nano-ESI-MS samples were dissolved in 50% (v/v) methanol/0.1% (v/v) aqueous formic acid, loaded into palladium coated borosilicate nanoelectrospray needles (Protana, Odense, Denmark), and mounted in the source of a Micromass Q-Tof hybrid quadrupole/orthogonal acceleration time of flight spectrometer. Stable electrospray was obtained at capillary voltage between 1200 and 1800 V. The collision gas was argon, and collision energies and argon pressure were tuned to optimise the fragmentation pattern of individual precursor ions.
NMR spectroscopy
NMR spectra were obtained on a Varian Unity 500 spectrometer equipped with pulsed field gradients and a 5-mm pulsed field gradient triple resonance probe, at a probe temperature of 30°C, as previously described (Todeschini et al., 2001). Standard pulse sequences were used for 1D proton, TOCSY, ROESY spectra, except for the inclusion of spin-echo sequences into the TOCSY and ROESY pulse programs. Mixing times were 80 ms in TOCSY and 150 ms in ROESY spectra. HSQC spectra were obtained using the sequence of Wider and Wuthrich (1993)
, employing pulsed field gradients to suppress unwanted signals. These were optimized for 1JC,H of 150 Hz (delay = 3.3 ms) or for nJC,H of 20 Hz (delay = 25 ms). A 2D HSQC-TOCSY spectrum, related to the pulse sequence described by Crouch et al. (1990)
but using the Wider and Wuthrich sequence for heteronuclear correlation (optimized for 1JC,H = 150 Hz and with a 20 msec mixing time), was also performed. Proton and 13C chemical shifts were referenced to internal acetone at 2.225 ppm and 31.50 pm respectively.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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