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Originally published In Press as doi:10.1194/jlr.M700149-JLR200 on May 8, 2007

Papers In Press, published online ahead of print August 1, 2007
J. Lipid Res., doi:10.1194/jlr.M700149-JLR200
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Journal of Lipid Research, Vol. 48, 1801-1824, August 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatusboxs

Marcos S. Toledo*, Steven B. Levery1,{dagger}, Beau Bennion{dagger}, Luciana L. Guimaraes*, Sherry A. Castle{dagger}, Rebecca Lindsey§, Michelle Momany§, Chaeho Park**, Anita H. Straus* and Helio K. Takahashi1,*

* Department of Biochemistry, Universidade Federal de São Paulo/Escola Paulista de Medicina, 04023-900 São Paulo, Brazil
{dagger} Department of Chemistry, University of New Hampshire, Durham, NH 03824-3598
§ Department of Plant Biology, University of Georgia, Athens, GA 30602-7271
** Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229

boxs The online version of this article (available at http://www.jlr.org) contains additional two figures and one scheme. Back

Published, JLR Papers in Press, May 8, 2007.

2 An abbreviated structural notation is used in the Results and Discussion sections of this paper, wherein the pyranose ring form is assumed unless otherwise designated (e.g., Galf = galactofuranosyl); in many cases, the anomeric carbon number, arrow, and parenthesis denoting linkage are assumed and omitted [e.g., Manp({alpha}1->3)Manp({alpha}1->2)Ins = Man{alpha}3Man{alpha}2Ins]. In some cases, Man may be further abbreviated as M, Ins as I, and Cer as C. Back

3 We previously designated the P. brasiliensis GIPCs Man{alpha}3Man{alpha}2IPC and Man{alpha}3(Galfß6)Man{alpha}2IPC as Pb-2 and Pb-1, respectively (30), but here we refer to them as Pb-2 and Pb-3, respectively, correlating systematically with the number of monosaccharide residues in each and their retention factor (Rf) values in HPTLC. Back

4 Although Barr, Laine, and Lester (21) did not specify precisely the linkage between Man and Ins in their compounds, but characterized it as Man{alpha}1->2/6Ins, NMR spectroscopy of H. capsulatum GIPCs (S. B. Levery, M. S. Toledo, A. H. Straus, and H. K. Takahashi, unpublished data) shows that the linkage is Man{alpha}1->2Ins1-P-1Cer, as reported previously for GIPCs of P. brasiliensis (30) and many other fungi (40, 52, 56). Back

1 To whom correspondence should be addressed. e-mail: slevery{at}cisunix.unh.edu (S.B.L.); takahashi.bioq{at}epm.br (H.K.T.)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Acidic glycosphingolipid components were extracted from the opportunistic mycopathogen Aspergillus fumigatus and identified as inositol phosphorylceramide and glycosylinositol phosphorylceramides (GIPCs). Using nuclear magnetic resonance sppectroscopy, mass spectrometry, and other techniques, the structures of six major components were elucidated as Ins-P-Cer (Af-0), Manp({alpha}1->3)Manp({alpha}1->2)Ins-P-Cer (Af-2), Manp({alpha}1->2)Manp({alpha}1->3)Manp({alpha}1->2)Ins-P-Cer (Af-3a), Manp({alpha}1->3)[Galf(ß1->6)]Manp({alpha}1->2)-Ins-P-Cer (Af-3b), Manp({alpha}1->2)-Manp({alpha}1->3)[Galf(ß1->6)]Manp({alpha}1->2)Ins-P-Cer (Af-4), and Manp({alpha}1->3)Manp({alpha}1->6)GlcpN({alpha}1->2)Ins-P-Cer (Af-3c) (where Ins = myo-inositol and P = phosphodiester). A minor A. fumigatus GIPC was also identified as the N-acetylated version of Af-3c (Af-3c*), which suggests that formation of the GlcN{alpha}1->2Ins linkage may proceed by a two-step process, similar to the GlcN{alpha}1->6Ins linkage in glycosylphosphatidylinositol (GPI) anchors (transfer of GlcNAc, followed by enzymatic de-N-acetylation). The glycosylinositol of Af-3b, which bears a distinctive branching Galf(ß1->6) residue, is identical to that of a GIPC isolated previously from the dimorphic mycopathogen Paracoccidioides brasiliensis (designated Pb-3), but components Af-3a and Af-4 have novel structures. Overlay immunostaining of A. fumigatus GIPCs separated on thin-layer chromatograms was used to assess their reactivity against sera from a patient with aspergillosis and against a murine monoclonal antibody (MEST-1) shown previously to react with the Galf(ß1->6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.

Supplementary key words fungus • sphingolipid • glycolipid • galactofuranose • monoclonal antibody • electrospray ionization • ion trap • mass spectrometry • NMR spectroscopy

Abbreviations: 1-D and 2-D, one-dimensional and two-dimensional; CID, collision-induced decomposition; COSY, correlation spectroscopy; D2O, deuterium oxide; ESI, electrospray ionization; GIPC, glycosylinositol phosphorylceramide; GPI, glycosylphosphatidylinositol; HPTLC, high-performance thin-layer chromatography; HSQC, heteronuclear single-quantum correlation; Ins, myo-inositol; IPC, inositol phosphorylceramide; MALDI-TOF, matrix-assisted laser desorption time-of-flight; Rf, retention factor; TOCSY, total correlation spectroscopy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Of >185 fungal species to which humans are exposed, aspergilli are perhaps the most common; conidia (spores) from a variety of Aspergillus species, such as A. versicolor, A. niger, A. terreus, A. flavus, and A. fumigatus, can be found at high concentrations in both rural and urban environments, outdoors as well as within human habitations (1). Of these species, A. fumigatus is considered the most pathogenic and is now recognized as the most prevalent airborne fungal pathogen in developed countries (2, 3), even in hospital environments, where it may contribute only a small fraction of the aerial spore count (4). Along with the general increase in the number of immunocompromised patients as a result of the use of corticoids (5), organ transplants, human immunodeficiency virus infection, and cancer (6), the high thermotolerance of A. fumigatus, which is able to grow in temperatures varying from <20°C to at least 50°C, as well as its high sporulating capacity and the small size of its spores (2–3 µm) (1), have contributed to the prevalence of A. fumigatus in invasive and noninvasive human aspergillosis worldwide.

Currently, the most commonly used drugs for the treatment of aspergillosis are amphotericin B, which binds membrane sterol and creates transmembrane channels leading to an increase in membrane permeability to monovalent cations (7), and itraconazole, which competes for oxygen at the catalytic heme site of cytochrome P450/lanosterol 14{alpha}-demethylase, leading to an inhibition of ergosterol biosynthesis and the accumulation of 14{alpha}-methylated sterols in the fungal plasma membrane (8). However, these two drugs have major disadvantages, including the nephrotoxicity of amphotericin B and the lack of itraconazole preparations for intravenous application, limiting its use in patients with impaired bowel absorption. In addition, there are increasing reports documenting the development of resistance to these antifungal drugs among a variety of species, including Aspergillus (8). Thus, there exists a compelling interest in the discovery of new targets for the development of antifungal therapeutics: novel functional molecules and the pathways by which they are generated and interact with other components in processes essential for fungal reproduction and growth or host colonization.

Studies carried out during the 1990s demonstrated that many species of fungi are vulnerable to inhibitors of sphingolipid biosynthesis (reviewed in Ref. 9). This has stimulated increased interest in the structure, biosynthesis, and functional roles of fungal sphingolipids as potential targets for antifungal agents. A particularly interesting target is the fungal inositol phosphorylceramide (IPC) synthase (10), inhibitors of which are highly toxic to many mycopathogens but exhibit low toxicity in mammals (11–13). IPC synthase, generally believed to be encoded by orthologous AUR1/IPC1 genes discovered in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida species, Aspergillus species, and Cryptococcus neoformans (10, 14–18), catalyzes the transfer of myo-inositol-1-phosphate from phosphatidylinositol to ceramide. Neither IPC, IPC synthase, nor any orthologous AUR1/IPC1 genes have been found in mammals, consistent with the lack of susceptibility of mammalian species to inhibitors of this enzyme.

Further processing of IPC by glycosyltransferases yields glycosylinositol phosphorylceramides (GIPCs), a class of complex anionic glycosphingolipids widely distributed among fungal species (19). Isolation and detailed characterization of GIPCs from a variety of fungi has begun to reveal an extensive structural diversity (see Discussion), the expression of which is considerably dependent on species and, apparently, at least in some mycopathogens, strongly regulated during morphogenesis (20–24). A total of ~30 distinct GIPC structures have been identified to date, with the discovery rate increasing during the last 5–6 years, based on further elaboration of three well-confirmed core structures distinguishable at the monoglycosyl level (Scheme 1 ).


Figure 15
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  		Scheme 1. Biosynthesis of fungal GIPCs, starting with transfer of myo-inositol-l-O-phosphate (IP) from diacylglycerol (DAG) moiety of phosphatidylinositol to ceramide, catalyzed by AUR1 gene encoded IPC synthase. The observation of three intermediate core structures diverging at the monosaccharide level implies the existence of at least three distict glycosyltransferases capable of using IPC as the acceptor: (i) Mana{alpha}2-T (function requires two genes in Saccharomyces cerevisiae, SUR1/CSG1 and CSG2), making Man{alpha}1->2InsPCer (M{alpha}2IPC); (ii) Man{alpha}6-T, making Man{alpha}->6InsPCer (M{alpha}6IPC); (iii) GlcNAc{alpha}2-T, followed by action of de-N-acetylase, making GlcN{alpha}1->2InsPCer (GlcN{alpha}2IPC).

 
Antigenic glycoside determinants are expressed on some fungal GIPC components, suggesting their potential as targets for immunodiagnostic reagents and the possibility of therapy based on stimulation of the mammalian humoral response (20, 21, 25–29). Elucidation of such immunological interactions calls for further detailed knowledge of the structures of these compounds, which is still limited compared with what is known about glycosphingolipids of animal species.

Here, we report on the structure elucidation of GIPCs from A. fumigatus. Among other observations, these studies revealed the presence of GIPCs containing an antigenic branched ±R->Manp({alpha}1->3)[Galf(ß1->6)]Manp({alpha}1->2)Ins structural motif. Although one GIPC antigen isolated from A. fumigatus, Manp({alpha}1->3)[Galf(ß1->6)]Manp({alpha}1->2)InsPCer (Af-3b), was characterized previously as a major GIPC component of the dimorphic mycopathogen Paracoccidioides brasiliensis (30), other GIPCs characterized here, including structural isomers and derivatives of Af-3b, have not been identified previously. A third major triglycosyl-IPC component of A. fumigatus was characterized as Manp({alpha}1->3)Manp({alpha}1->6)GlcpN({alpha}1->2)Ins1-P-1Cer (Af-3c); in addition, a minor component with an N-acetylated GlcN residue was identified (Af-3c*). Although the de-N-acetylated GlcpN({alpha}1->2)Ins1-P-1-Cer core has been reported in GIPCs of several fungi (23, 31), this is the first time a GIPC containing a GlcpNAc({alpha}1->2)Ins1-P-1Cer core, a presumptive intermediate in this biosynthetic pathway, has been identified.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Fungal isolates and growth conditions
A. fumigatus American Type Culture Collection strain 9197 was used for most preparations. Also used was A. fumigatus strain 237, originally cultured from open lung biopsy from a patient with invasive aspergillosis at Hope Hospital in Manchester, UK (a gift of Dr. David Holden, Hammersmith Hospital, London, UK). Approximately 5 x 109 spores were inoculated to 200 ml or 1 liter of complete medium (1% glucose, 0.2% peptone, 0.1% yeast extract, 0.1% casamino acids, nitrate salts, trace elements, and 0.01% vitamins, pH 6.5; trace elements, vitamins, nitrate salts, and amino acid supplements are described in the appendix to Ref. 32), incubated at 30°C or 37°C with shaking (200 rpm) for 24 h, filtered, and processed as described. Yields were 6–8 g wet weight per 200 ml of medium or 25–35 g wet weight per 1 liter of medium.

Solvents for extraction and anion-exchange chromatography
Solvent A consisted of isopropanol-hexane-water (55:20:25, v/v/v; upper phase discarded). Solvent B consisted of chloroform-methanol (2:1, v/v). Solvent C consisted of chloroform-methanol-water (30:60:8, v/v/v).

High-performance thin-layer chromatography
Analytical high-performance thin-layer chromatography (HPTLC) was performed on silica gel 60 plates (E. Merck, Darmstadt, Germany) using chloroform-methanol-water [50:47:14 (v/v/v), containing 0.038% (w/v) CaCl2; solvent D] as the mobile phase. Lipid samples were dissolved in solvent B and applied by streaking from 5 µl Micro-caps (Drummond, Broomall, PA). Detection was with Bial's orcinol reagent [0.55% (w/v) orcinol and 5.5% (v/v) H2SO4 in ethanol-water (9:1, v/v); the plate is sprayed and heated briefly to ~200–250°C].

HPTLC immunostaining
Immunostaining of purified fungal GIPCs separated by HPTLC was performed by the procedure of Magnani, Smith, and Ginsburg (33), as modified by Kannagi et al. (34), with some additional changes to the HPTLC protocol. Briefly, separation by HPTLC was carried out in three steps. After application of GIPCs, the HPTLC plates were developed twice with chloroform-methanol (4:1, v/v; solvent E) and once with chloroform-methanol-water [30:18:4 (v/v/v), containing 0.020% (w/v) CaCl2; solvent F). After HPTLC development, plates were dried, soaked in 0.5% polymethacrylate in hexane, dried again, and blocked for 2 h with 1% BSA in 0.01 M PBS, pH 7.2. Plates were then incubated with monoclonal antibody MEST-1 or aspergillosis serum overnight; this was followed by sequential incubation with either rabbit anti-mouse IgG or rabbit anti-human IgG, respectively, and 125I-labeled protein A (4 x 105 cpm/ml) (35).

Extraction and purification of glycosphingolipids
Extraction and purification of glycosphingolipids were carried out as described previously (27, 36, 37), but with some modifications and additional steps introduced for the purpose of reducing the amounts of irrelevant substances to be dealt with earlier in the protocol. Briefly, glycosphingolipids were extracted by homogenizing mycelia (25–35 g wet weight) in an Omni-mixer (Sorvall, Inc., Wilmington, DE) three times with 200 ml of solvent A and three times with 200 ml of solvent B. The six extracts were pooled, dried on a rotary evaporator, dialyzed against water, and lyophilized. The dried residue was partitioned between water and 1-butanol presaturated with water (200 ml each) with vigorous shaking in a separatory funnel. The lower (water) layer was removed and similarly extracted three more times with equal volumes of water-saturated 1-butanol. The four 1-butanol extracts were combined in a round-bottom flask, evaporated to dryness, resuspended in a minimal volume of solvent C, and applied to a column of DEAE-Sephadex A-25 (Ac– form). Neutral glycosphingolipids were eluted with 5 volumes of solvent C. Acidic glycosphingolipids were eluted with 5 volumes of 0.5 M sodium acetate in methanol. The acidic fraction was dried, dialyzed exhaustively against deionized water, redried, and treated with 20 ml of methanol-water-1-butanol (4:3:1, v/v/v) containing 25–30% methylamine at 55°C for 4 h (flask tightly stoppered), with occasional agitation. After removal of the reagent solution by rotary evaporation, the acidic lipids were further fractionated by repetitive preparative-scale HPLC, using columns of either 60 cm x 4.6 mm Iatrobeads (Iatron Chemical Co., Tokyo, Japan) 6RS-8010 or 50 cm x 4.6 mm Sphereclone (Phenomenex, Torrance, CA) 10 µm porous spherical silica. The mobile phase was a 2-propanol-hexane-water gradient programmed from 55:40:5 to 55:25:20 over 120 min, followed by isocratic elution for 40 min, with a flow rate of 0.5 ml/min. Generally, 40 x 2 ml fractions were collected for first-stage purifications and 80 x 1 ml fractions were collected for second-stage purifications, where required. The identity and purity of each fraction were assessed by analytical HPTLC as described above, and by one-dimensional (1-D) 1H-NMR spectroscopy, before further characterization by the full range of NMR and MS techniques described below.

1-D 1H- and 2-D 1H-1H and 1H-13C-nuclear magnetic resonance spectroscopy
Samples of underivatized (G)IPCs (~0.5–1.0 mg) were deuterium-exchanged by repeated lyophilization from deuterium oxide (D2O) and then dissolved in 0.5 ml of DMSO-d6/2% D2O (30, 38) for NMR analysis. 1-D 1H-NMR, 2-D 1H-1H- gradient enhanced correlation spectroscopy (gCOSY), 1H-1H total correlation spectroscopy (TOCSY), and proton-detected 1H-13C- gradient enhanced heteronuclear single-quantum correlation (gHSQC) experiments were performed at 35°C on Varian (Palo Alto, CA) Unity Inova 500 MHz (Department of Chemistry, University of New Hampshire) and 600 and 800 MHz (Complex Carbohydrate Research Center, University of Georgia) spectrometers using standard acquisition software available in the Varian VNMR software package. Proton chemical shifts are referenced to internal tetramethylsilane ({delta} = 0.000 ppm). Carbon chemical shifts are referenced to solvent DMSO ({delta} = 40.0 ppm); in some cases, these were not obtained from directly detected 1-D spectra but measured in F1 of the gHSQC experiment; therefore, the values are given to single decimal places only.

Positive ion mode electrospray ionization mass spectrometry
Mass spectrometry of the IPC fraction (Af-0) was performed via electrospray ionization (ESI) in the positive ion mode (+ESI) on a linear ion trap instrument (LTQ; Thermo-Finnigan, San Jose, CA). Mass spectrometry of all other fractions containing GIPCs was performed in the positive ion mode on a Micromass Global (Manchester, UK) hybrid ESI-Q/q-oa-TOF-MS instrument (Q-TOF). All (G)IPC samples were introduced into the ion source via direct infusion in 100% methanol (~100 ng/µl). The flow rate was usually 0.5 µl/min, but for analysis of samples available in limited amounts, a nanospray capillary tip was used, from which the flow rate was estimated to be ~200 nl/min. To generate [M(Li)+Li]+ adducts of (G)IPC molecular species, LiI (10 mM) in methanol was added to the analyte solution until the observed ratio of [M(Li)+Li]+ adducts to mixed Na+/Li+ adducts in MS profile mode was >5:1; the necessary LiI concentration was generally in the range 3–5 mM (39, 40). In general, spectra represent summations of 50–350 scans (30 scans/min) for MS [M(Li)+Li]+ profiles and MS/collision-induced decomposition (CID)-MS (or LTQ MSn) experiments. Resolution on the Q-TOF was generally 4,000 (5% valley) for MS profile spectra and 3,000 for MS/CID-MS experiments. Extraction cone voltage (analogous to orifice-to-skimmer potential in Sciex API series instruments) was 35 V for MS profile spectra and MS/CID-MS experiments and 80 V for pseudo-+ESI-(CID-MS)2 experiments. Additional matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry was performed on an Axima CFR (Shimadzu Biotech, Columbia, MD) instrument. Lithiation was accomplished by doping 2,5-dihydroxybenzoate matrix with LiI (41).

Fragment ion naming conventions and interpretation
Nominal, monoisotopic m/z values are used in the labeling and description of +ESI-MS results. Fragment naming conventions and interpretation of spectra derived from [M(Li)+Li]+ adducts of GIPC molecular species are based on those of Adams and Ann (42) and Singh, Costello, and Beach (43), as described previously (39–41); additional naming conventions for ceramide fragments are derived from Hsu and Turk (44). Essential characteristics of these spectra are summarized below with reference to Scheme 2 (see also Schemes 3–7 below accompanying the mass spectra). In general, +ESI-MS/CID-MS spectra of GIPCs acquired [via +ESI-Q/q(CID)-oa-TOF-MS or other tandem MS/CID-MS configurations (30, 39–41)] from either disodiated or dilithiated molecular adducts exhibit abundant pairs of fragment ions representing the complete glycosylinositol ([Bn+Cat]+ and [Cn+Cat]+) and glycosylinositol phosphate ([BnPO3(Cat)+Cat]+ and [CnPO3(Cat)+Cat]+) moieties, along with ceramide ([Y0+Cat]+ and [Z0+Cat]+) and ceramide phosphate ([Y0PO3(Cat)+Cat]+ and [Z0PO3(Cat)+Cat]+) ions. Ions in parallel series, representing glycosidic fragmentations occurring singly or in combination with other cleavages, are also observed ([Cn+Cat]+, [Bn+Cat]+, [Yn+Cat]+, [Zn+Cat]+, [Ym/Cn+Cat]+, [Ym/Bn+Cat]+, [Ym/CnPO3(Cat)+Cat]+, and [Ym/BnPO3(Cat)+Cat]+). In many cases, these are accompanied by related [Zm/BnPO3(Cat)+Cat]+ and [Zm/Bn+Cat]+ ions. It has been found that the [Ym/CnPO3(Cat)+Cat]+ and [Ym/BnPO3(Cat)+Cat]+ series are the most useful fragments for deducing glycosidic sequences, because the presence of the phosphate moiety clearly labels the inositol-containing reducing end of the glycan, so that monosaccharide cleavages must yield losses from the nonreducing end. In general, +ESI-MS/CID-MS or +ESI-MSn of [M(Li)+Li]+ adducts of GIPCs appear to yield better overall S/N than [M(Na)+Na]+ adducts (39–41).


Figure 16
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  		Scheme 2. Characteristic fragmentations of glycosylated IPCs, exemplified by M{alpha}3M{alpha}2IPC. A: Nomenclature based on that of Adams and Ann (42) for fragmentation of ceramide moiety and of Singh, Costello, and Beach (43) for glycosyl-myo-inositol phosphoryl group. B, C: Hydrated analogs of sphingoid d3b and c1b ions [nomenclature of Hsu and Turk (44)], proposed as products of t18:0 or t20:0 phytosphingosine-containing ceramides (39–41).

 

Figure 17
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  		Scheme 3. Fragmentation of Af-3a in positive ion mode ESI-MS and ESI-MS/CID-MS.

 

Figure 18
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  		Scheme 4. Fragmentation of Af-3b in positive ion mode ESI-MS and ESI-MS/CID-MS.

 

Figure 19
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  		Scheme 5. Fragmentation of Af-4 in positive ion mode ESI-MS and ESI-MS/CID-MS.

 

Figure 20
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  		Scheme 6. Molecular ions and fragmentation of Af-3c and N-acetylated Af-3c in positive ion mode ESI-MS and ESI-MS/CID-MS. Phosphorylated fragments are labeled as Li+ salt adducts, but Na+ salt adducts can be named by substitution of Li with Na. Nominal, monoisotopic m/z values for both disodiated and dilithiated molecular species are given below the structure.

 

Figure 21
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  		Scheme 7. Divergent structures and hypothetical pathways for the biosynthesis of GIPCs from the common mannosyl-IPC intermediate Man{alpha}1->2InsPCer (M{alpha}2IPC). These include InsP1->6Man{alpha}1->2InsPCer, a known product of the IPT1-encoded enzyme that transfers a second mole of myo-inositol-1-O-phosphate from phosphatidylinositol to M{alpha}2IPC (left arrow) in S. cerevisiae. In basidiomycetes, a commonly observed intermediate GIPC is Galß1->6Man{alpha}1->2InsPCer (Galß6M{alpha}2IPC; Ba-2), synthesized from M{alpha}2IPC by an as yet unknown Galß6-T (right arrow). In euascomycetes, a commonly observed intermediate GIPC is Man{alpha}1->3Man{alpha}1->2InsPCer (Man{alpha}3M{alpha}2IPC; Eu-2a), synthesized from M{alpha}2IPC by an as yet unknown Man{alpha}3-T (lower left). In A. nidulans, the major GIPC is a nonantigenic trimannosyl-IPC, Man{alpha}1->3(Man{alpha}1->6)Man{alpha}1->2InsPCer (Man{alpha}3[Man{alpha}6]M{alpha}2IPC; An-3), derived from Eu-2a by the addition of a Man{alpha}1->6 residue.

 
Monosaccharide, inositol, fatty acid, sphingoid, and linkage analysis by GC-MS
Monosaccharides were analyzed as their per-O-trimethylsilyl methyl glycosides. A re-N-acetylation step was used after methanolytic depolymerization to ensure that hexosamines, if present, would be detected as their acetamido derivatives. Ceramide-derived sphingoid bases and 2-hydroxy fatty acids were detected as their N-acetyl-per-O-trimethylsilyl and 2-O-trimethylsilyl methyl ester derivatives, respectively. Myo-inositol (Ins) was detected in the monosaccharide analysis as its per-O-trimethylsilyl derivative, although optimal conditions for its release (20) were not used. All derivatives were prepared according to protocols described previously (30). Linkage analysis was performed by the method of partially methylated alditol acetates. Permethylation was performed by microscale adaptation of the method of Ciucanu and Kerek (45); hydrolysis, reduction, and acetylation to produce partially methylated alditol acetates were performed as described by Levery and Hakomori (46). Instruments used for GC-MS were a GCQ (Thermo-Finnigan) and a GC3800-MS1200 (Varian), operated in electron ionization mode. All component analyses were performed on a 30 m DB-5 (or equivalent) bonded-phase fused silica capillary column, temperature programmed from 160°C to 200°C at 2°/min and from 200°C to 260°C at 10°/min (for per-O-trimethylsilyl methyl glycosides) or from 140°C to 320°C at 4°/min (for fatty acid methyl esters and N-acetyl-per-O-trimethylsilyl sphingosines). Partially methylated alditol acetates were analyzed with the same system, temperature programmed from 160°C to 260°C at 2°/min. All derivatives were identified by comparison of retention times and mass spectra compared with authentic standards and published data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Preliminary comparison of the immunoreactivity of A. fumigatus and A. nidulans GIPCs with monoclonal antibody MEST-1
In Fig. 1 are reproduced HPTLC profiles of crude fungal acidic lipid fractions stained with orcinol (panel A), which reacts with hexose residues characteristic of GIPCs, and the monoclonal antibody MEST-1 (panel B), which specifically recognizes terminal Galfß residues (25).2 These compare the reactivity profiles of GIPCs from A. fumigatus strains 9197 and 237 (cultured at 37°C; lanes 1 and 2, respectively) with those from P. brasiliensis strain 18 (lane 3) and A. nidulans strain A28 (lane 4). The characterizations of the components from P. brasiliensis and A. nidulans have been described previously (30, 40), except that the component formerly named Pb-1 is here named Pb-3.3 The structural basis for naming the component bands from A. fumigatus will be described below. The P. brasiliensis antigen Pb-3 [Manp({alpha}1->3)[Galf(ß1->6)]Manp({alpha}1->2)InsPCer], but not Pb-2 [Manp({alpha}1->3)Manp({alpha}1->2)InsPCer], was previously observed to react with MEST-1 (25). The branching Galf(ß1->6) residue is known to be essential for the MEST-1 reactivity of Pb-3. Here, comigrating bands corresponding to Af-3b and Pb-3 from A. fumigatus and P. brasiliensis, respectively, are both clearly stained with MEST-1 (Fig. 1B, lanes 1–3), suggesting that Af-3b may have a structure similar or identical to that of Pb-3. Interestingly, bands corresponding to Af-4 from both strains of A. fumigatus also reacted, along with at least two other components with lower Rf in each strain. This suggests that Af-4 contains the common branching Galf(ß1->6) residue responsible for MEST-1 reactivity. No other components of P. brasiliensis appeared to be reactive with MEST-1 (Fig. 1B, lane 3). No GIPC components from A. nidulans reacted (Fig. 1B, lane 4), as expected, because they have been shown to have divergent oligo-{alpha}-mannosyl structures without Galfß (40). Af-3a appears to be unstained by MEST-1; although this is not completely clear because of the small difference in Rf with respect to Af-3b, the result would be consistent with absence, or unreactive presentation, of the Galfß residue.


Figure 1
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  		Fig. 1. High-performance thin-layer chromatography (HPTLC) immunostaining of crude fungal glycosylinositol phosphorylceramide (GIPC) fractions by monoclonal antibody MEST-1. A: Orcinol staining. B: MEST-1 immunostaining. Crude acidic fractions are as follows: lane 1, A. fumigatus (A.f.) strain 9197; lane 2, A. fumigatus strain 237; lane 3, P. brasiliensis (P.b.) strain 18; lane 4, A. nidulans strain A28.

 
Fractionation of major A. fumigatus GIPCs
The HPTLC results in Fig. 2A compare crude acidic lipid fractions from A. nidulans strain A28 (lane 1) and A. fumigatus strain 237 (lane 2), cultured at 30°C, with a reference standard containing Man{alpha}3Man{alpha}2IPC (M2IPC = Pb-2) and Galfß6(Man{alpha}3)Man{alpha}2IPC (GfM2IPC = Pb-3) from P. brasiliensis (lane 3). Note that the component with the highest Rf value (Af-2) comigrated with authentic M2IPC components previously characterized from P. brasiliensis (Pb-2) (30) and A. nidulans (An-2) (40), but the most abundant component (second band, Af-3a) had a slightly higher Rf than the other major GIPC from P. brasiliensis (Pb-3). A third, less abundant component (Af-3b) migrated at the same Rf as Pb-3, and the fourth band (Af-4) had a slightly lower Rf, consistent with three to four monosaccharide units attached to IPC. Af-3b and Af-4 appeared to migrate, respectively, slightly above and slightly below the major GIPC component of A. nidulans, Man{alpha}6(Man{alpha}3)Man{alpha}2IPC (M3IPC = An-3) (40). A fifth component, designated Af-3c, exhibiting a much lower Rf value by HPTLC, appeared in some preparations with a higher Rf and, as was discovered later, sometimes appeared in the neutral (DEAE-Sephadex pass-through) fraction. The reason for this, that it is a zwitterionic GIPC component, will be discussed below. Acidic lipids from strain 9197 exhibited a qualitatively similar profile, but with less Af-3a and more Af-3b and Af-4 (data not shown). This A. fumigatus acidic fraction appeared to lack some of the additional low Rf components that were observed when the fungus was cultured at 37°C (see above).


Figure 2
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  		Fig. 2. A: HPTLC analysis comparing GIPC profiles (as orcinol-stained acidic lipid components) of A. nidulans strain A28 (lane 1) and A. fumigatus strain 237 (lane 2) alongside a mixture of GIPCs (Pb-2 and Pb-3) previously isolated and characterized from P. brasiliensis (lane 3). Note that P. brasiliensis component Pb-3 was previously referred to as Pb-1 (30) but is here redesignated according to the number of monosaccharide residues in its structure. A. nidulans components An-2, An-3, and An-5 were characterized previously by Bennion et al. (40). B: HPTLC analysis showing four major GIPCs in a profile of A. fumigatus strain 9197 (lane C), which were subsequently isolated as individual components (lanes 1–4) by repetitive HPLC fractionation. Designations of bands are given beside the panels, corresponding to relative Rf values and the number of monosaccharide residues found in each component. Mobile phases are as follow: panel A, chloroform-methanol-water [50:47:14 (v/v/v), containing 0.038% (w/v) CaCl2]; panel B, chloroform-methanol-water [50:55:19 (v/v/v), containing 0.046% (w/v) CaCl2].

 
Crude acidic lipids from both strains 237 and 9197 were fractionated by preparative-scale HPLC, with each of the first four major GIPC components obtained in at least one fraction as a single orcinol-stained band (Fig. 2B, lanes 1–4); these components, designated Af-2, Af-3a, Af-3b, and Af-4, respectively, were subjected to characterization by 1H-NMR, MS, and GC-MS techniques as described below. An amount of Af-3c sufficient for extensive 2-D NMR analysis was accumulated from three HPLC fractionations of crude neutral lipids. An additional component, eluting before Af-2 in HPLC but not stained by orcinol, was also collected and analyzed (it will be referred to as Af-0). Some intermediate fractions were found to be mixtures; analysis of such mixtures also produced useful results, as reported below.

Monosaccharide, inositol, fatty acid, and sphingoid component analysis of A. fumigatus GIPCs
Mannose was essentially the only monosaccharide identified by GC-MS analysis of the trimethylsilylated methyl glycosides produced after methanolysis of Af-2 components from strains 237 or 9197. The Af-3a (237) fraction was observed to consist mainly of mannose, although a trace of galactose was also detected. Af-3b (9197) contained both galactose and mannose in an ~1:2 ratio. In Af-3c, 2-deoxy-2-amino-glucose was detected (as N-acetyl-glucosamine), along with mannose, in a ratio of ~1:2. Ins was detected in all four fractions. The major long-chain fatty acid detected was h24:0, identified by its characteristic retention time and fragments observed in its electron ionization mass spectrum (47). Small amounts of nonhydroxylated 16:0, 18:0, and 24:0 fatty acids were also detected, along with traces of h25:0, h26:0, and the dihydroxy fatty acid 2h24:0. Major sphingoid components detected were t18:0 and t20:0 4-hydroxysphinganines (phytosphingosines), in ratios varying between the different fractions. Under conditions of ion trap detection, electron ionization spectra of the 4-hydroxysphinganine-derived N-acetyl-1,3,4-tri-O-trimethylsilyl-4-hydroxysphinganines were qualitatively similar in most respects than those described previously by Thorpe and Sweeley (48), but with more abundant representation of high m/z ions, including some additional molecular mass-related ions as observed by Levery et al. (30). Thus, [M–15]+, [M–15–90]+, [M–59–90]+, [M–174]+, and [M–174–90]+ ions were observed in high abundance at m/z 560, 470, 426, 401, and 311 for the t18:0 base derivative and at m/z 588, 498, 454, 429, and 329 for the later-eluting t20:0 base derivative (data not shown; for detailed interpretations, including identifications of lower m/z ions, see Refs. 48–50).

Structural analysis of A. fumigatus IPC (Af-0)
A partial 1-D 1H-NMR spectrum of fraction Af-0, reproduced in Fig. 2, exhibited resonances characteristic for a Ins spin system as well as downfield resonances characteristic of the highly hydroxylated portion of phytoceramide, which we previously observed in GIPCs, but no resonances were observed characteristic for monosaccharide residues. Upfield resonances (data not shown) included a composite multiproton alkyl/acyl CH2 signal at ~1.23 ppm and a characteristic 6H triplet for both the alkyl and acyl CH3 at 0.853 ppm. Therefore, this fraction appears to contain IPC, the obligate intermediate in GIPC synthesis. The resonances were assigned as shown in Fig. 3 and Table 1 . The resonances assigned to Ins H-1 and Sph-1a/1b all exhibited additional three-bond coupling to the phosphorus atom of the phosphodiester (Table 1).


Figure 3
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  		Fig. 3. Downfield expansion (4.20–2.80 ppm) of one-dimensional (1-D) 1H-NMR spectra (500 MHz; DMSO-d6/2% D2O; 35°C) of the inositol phosphorylceramide (IPC) fraction (Af-0) isolated from A. fumigatus strain 9197.

 

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  		TABLE 1. 1H chemical shifts (ppm) for monosaccharide, inositol, ceramide sphingoid, and fatty-N-acyl (in parentheses) residues of IPC (myo-Ins1<-P->1Cer = Af-0) and Man{alpha}3Man{alpha}2IPC (Af-2) from A. fumigatus in DMSO-d6/2% D2O at 35°C

 
Because NMR analysis is less useful for determining ceramide size and relative sphingoid/fatty-N-acyl chain length distributions, the Af-0 fraction was further analyzed by +ESI-MSn in a linear ion trap (Fig. 4 ). Two major lipoforms were detected as a pair of [M(Na)+Na]+ salt-adduct ions observed at m/z 970 and 998, consistent with IPC having ceramides consisting of t18:0 and t20:0 4-hydroxysphinganines in combination with h24:0 fatty-N-acylation (Fig. 4A). To confirm that the m/z 28 (C2H4) increment is in the sphingoid and not the fatty-N-acyl moiety, further MSn steps were carried out to isolate and fragment the ceramide ions; because we have observed that phytoceramide ions are somewhat difficult to generate and fragment under low-energy conditions but that fragmentation is facilitated by lithium adduction (39–41), the fraction was first treated with excess LiI and reinfused into the ESI source. As shown in Fig. 4B, this resulted in essentially complete conversion to a pair of predominant [M(Li)+Li]+ adducts at m/z 938 and 966. MS2 product ion spectra (data not shown) of these two molecular adducts yielded highly abundant ceramide primary fragments [Y0+Li]+ at m/z 690 and 718, respectively, from losses of [InsOPO2(Li)–H] (with back transfer of H, as shown in Scheme 2). MS3 spectra of the [Y0+Li]+ ions at m/z 690 (Fig. 4C, m/z 938->690->) and 718 (Fig. 4D, m/z 966->718->) yielded products consistent with t18:0 and t20:0 4-hydroxysphinganines, respectively, as the major sphingoid components in the two lipoforms. Aside from the loss of H2O from the [Y0+Li]+ ions (m/z 672 and 700 in Fig. 4C, D, respectively), the most abundant fragments were those corresponding to loss of the acyl chain ([N+Li]+ {equiv} [O/Y0+Li]+) and a sphingoid rearrangement including additional loss of the amino group (d3b), as observed previously in other low-energy fragmentation modes (Scheme 2, structure B) (39–41). These ions are observed at m/z 324 and 291 in the spectrum from m/z 690 (Fig. 4C) and at m/z 352 and 319 in the spectrum of products from m/z 718 (Fig. 4D), confirming that the m/z 28 differences in the ceramide moieties reside entirely in the sphingoid chain lengths, whereas the N-acyl group is h24:0 in both ceramide species. Minor products included ions derived from these by dehydration or further fragmentation. Ions derived from the loss of all or most of the sphingoid alkyl chain, while retaining the fatty-N-acyl chain (e.g., m/z 432 [S+Li]+, 416 [T+Li]+, and 390 [U+Li]+), as well as an aldehyde ion derived from the fatty-N-acyl C2–C{omega} (m/z 345 [W+Li]+), are observed at the same m/z in both spectra (Scheme 2).


Figure 4
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  		Fig. 4. Positive ion mode electrospray ionization (+ESI-MSn) spectra of Af-0. A: Profile of molecular ions as [M(Na)+Na]+ adducts. B: Profile of molecular ions as [M(Li)+Li]+ adducts. C: MS3 products of the [Y0+Li]+ primary fragment at m/z 690 (m/z 938->690->). D: MS3 products of [Y0+Li]+ at m/z 718 (m/z 966->718->). The adduct designation "+Li" and the charge form have been omitted from the fragment labels for clarity.

 
Structural analysis of A. fumigatus GIPCs
Af-2 from strains 237 and 9197 A 1-D 1H-NMR spectrum of an Af-2 fraction from strain 237 is reproduced in Fig. 5A . Aside from the presence of minor impurities, it is essentially identical to a spectrum previously published for Man{alpha}3Man{alpha}2IPC (Pb-2) from P. brasiliensis and for which most of the resonances were assigned by 2-D homonuclear COSY and TOCSY experiments (30). For confirmation, Af-2 was subjected to a similar sequence of 2-D 1H-NMR analyses; the resonance assignments are listed for reference in Table 1. Key points are the {alpha}-Man anomeric resonances observable at 5.062 ppm (Man{alpha}2 H-1) and 4.896 ppm (Man{alpha}3 H-1); the corresponding {alpha}-Man H-2 resonances at 3.919 and 3.732 ppm, respectively; Ins H-2, H-3, and H-5 resonances at 3.968, 2.945, and 3.230 ppm, respectively; sphingoid H-1b and H-2 resonances at 4.044 and 3.837, respectively; and the fatty-N-acyl H-2 resonance at 3.845 ppm. Note that the {alpha}-mannosylation at Ins O-2 correlates with downfield shift increments for the Ins H-1, H-2, and H-3 resonances with respect to their values in InsPCer ({Delta}{delta} = 0.13, 0.18, and 0.10 ppm, respectively), consistent with increments found by comparison with data previously reported for Man{alpha}2InsPCer (22). The minor anomeric resonances at 5.030 and 4.905 ppm are consistent with a small contamination by a triglycosyl-IPC component (see below) incompletely separated from Af-2 in this fraction. A 1-D 1H-NMR spectrum of the Af-2 component from strain 9197 was of somewhat lower quality, but it lacked contamination from the triglycosyl-IPC component (data not shown). All of the salient features discussed above were visible in this spectrum, confirming its identity to the Man{alpha}3Man{alpha}2IPC isolated from strain 237.


Figure 5
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  		Fig. 5. Downfield expansion (5.15–2.85 ppm) of 1-D 1H-NMR spectra (800 MHz; DMSO-d6/2% D2O; 35°C) of GIPC fractions isolated from A. fumigatus strain 237. Spectra were acquired on fractions corresponding to lanes 1 and 2 of Fig. 1B. A: Af-2. B: Af-3a. Resonances denoted by parentheses are assigned to protons of residual Af-2 in the sample.

 
For further characterization, particularly of the ceramide moiety, which could differ from those of other species and is not well defined by NMR methods, a molecular profile of Af-2 from strain 9197 was acquired via +ESI-Q/oa-TOF-MS. These data are described in detail in the supplementary data (supplementary Figs. I, II and Scheme I). These results were consistent with an M2IPC having ceramides consisting of t18:0 and t20:0 4-hydroxysphinganines with h24:0 fatty-N-acylation and minor lipoforms with compositions h25:0/t18:0 and h24:0/t19:0.

Af-3a from strain 237 The 1-D 1H-NMR spectrum of a fraction of Af-3a (Fig. 5B) showed it to be not a homogeneous GIPC component but a mixture of Af-2 and an additional triglycosyl-IPC having a set of three anomeric protons at 4.905, 5.029, and 5.064 ppm. The anomeric protons from Af-2 are observed as lower intensity signals at 4.897 and 5.064 ppm, the latter coincident with one of the signals from the major triglycosyl-IPC component, as indicated by the greater relative intensity of that signal. Conversely, resonances from the major Af-3a component are observable as minor signals in the spectrum of Af-2 from strain 237 (cf. Fig. 5A). Unfortunately, because of the insufficient amount and the presence of two closely related structures in the mixture, we were not able to obtain high-quality 2-D NMR data specific for the Af-3a component from this fraction, resulting in only fragmentary resonance assignments. Signals assignable by apparent analogy were Ins H-3 and H-5 resonances at 2.948 and 3.231 ppm, respectively; sphingoid H-1b and H-2 resonances at 4.047 and 3.847, respectively; and a fatty-N-acyl H-2 resonance at 3.827 ppm. The similarity in chemical shift of two of the H-1 signals to those of Af-2 suggested that it could be a derivative of Man{alpha}3Man{alpha}2IPC, although potential glycosylation-induced changes in chemical shifts preclude reliable assignments based purely on analogy. A triglycosyl structure consisting solely of {alpha}-Manp residues would be consistent with the similar 3J1,2 values (1.5–2.0 Hz) of all H-1 resonances observed in the spectrum.

A linkage analysis by GC-MS detected derivatives corresponding to T-Man, ->3Man, and ->2Man. The first two derivatives would be produced from the Af-2 known to be present, but the latter, assuming that Af-3a is a mannosylated product of Af-2, as the NMR data suggest, would only be consistent with addition of the third Man residue in 1->2 linkage. Such a structure would also produce the same T-Man and ->3Man derivatives. Therefore, the structure Man{alpha}2Man{alpha}3Man{alpha}2IPC is proposed for Af-3a. This structure could be consistent with the NMR spectrum, provided that the signal at 5.029 ppm is assigned to H-1 of the penultimate Man{alpha}3 residue, because it is known that glycosylation of an {alpha}-Man residue by Man{alpha}2 induces a substantial downfield shift of H-1 of the substituted residue (51). The resonance at 4.905 ppm, therefore, is assigned to H-1 of the nonreducing terminal Man{alpha}2 residue, with H-1 of the reducing end Man{alpha}2 residue remaining at 5.064 ppm, apparently unaffected by the substitution (Table 2 ).


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  		TABLE 2. Comparison of 1H chemical shifts (ppm) for H-1 of monosaccharide residues of Man{alpha}3Man{alpha}2IPC (Af-2), Man{alpha}2Man{alpha}3Man{alpha}2IPC (Af-3a), Man{alpha}3(Galfß6)Man{alpha}2IPC (Af-3b), and Man{alpha}2Man{alpha}3(Galfß6)Man{alpha}2IPC (Af-4) from A. fumigatus in DMSO-d6/2% D2O at 35°C

 
A molecular profile of Af-3a was acquired via +ESI-Q/oa-TOF-MS (Fig. 6A ); a pair of [M(Li)+Li]+ salt-adduct ions observed at m/z 1,424 and 1,452 is consistent with a triglycosyl-IPC having ceramides consisting of t18:0 and t20:0 4-hydroxysphinganines and h24:0 fatty-N-acylation. A +ESI-MS/CID-MS spectrum acquired from the dilithiated molecular adduct at m/z 1,424 is reproduced in Fig. 6B, C, showing the predominant [B3PO3(Li)+Li]+/[C3PO3(Li)+Li]+ pair (m/z 741/759) and other abundant fragments from glycosidic cleavages (Scheme 3 ). In addition to glycosidic fragment series derived from the glycosylinositol phosphate and glycosylinositol moieties, fragments from sequential loss of monosaccharide residues from [M(Li)+Li]+ ([Ym(Li)+Li]+) appear at lower abundance in the spectrum. In the same region of the spectra and at similar abundance, ions from loss of the acyl chain and loss of acyl C2–C{omega} ([O(Li)+Li]+, [J(Li)+Li]+, and [J'(Li)+Li]+) are also observed (Scheme 2). Within this group, the latter ion appears to be predominant. Similar to previous results, the ceramide and ceramide phosphate ions ([Y0+Li]+, [Z0+Li]+, [Y0PO3(Li)+Li]+, and [Z0PO3(Li)+Li]+) appear at intermediate abundances. The [O(Li)+Li]+, [J(Li)+Li]+, [J'(Li)+Li]+, and [O/Z0PO3(Li)+Li]+ ions all provide information about the carbon number of the sphingoid and, by difference, the N-acyl chain, all or part of which is lost in the process of their formation. A similar spectrum was acquired from the [M(Li)+Li]+ adduct at m/z 1,452 (data not shown), differing only in the m/z of sphingoid-containing fragments. Thus, in agreement with the sphingoid and fatty acid analysis, these spectra indicate that the m/z 28 increment between the two predominant molecular species is attributable primarily to a corresponding C2H4 difference in the sphingoid rather than the N-acyl chain. Therefore, the [M(Li)+Li]+ adducts at m/z 1,424 and 1,452 correspond to lipoforms containing t18:0 and t20:0 4-hydroxysphinganines, respectively, with h24:0 fatty-N-acylation.


Figure 6
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  		Fig. 6. +ESI-Qq/oa-TOF spectra of Af-3a. A: Profile of molecular ions as [M(Li)+Li]+ adducts. B: MS/collision-induced decomposition (CID)-MS of [M(Li)+Li]+ at m/z 1,424, low m/z region. C: MS/CID-MS of [M(Li)+Li]+ at m/z 1,424, high m/z region. The y axis expansion in C relative to B is 27x. Ion designations correspond to Scheme 4. The designations "+Li" and "(Li)+Li" and the charge form have been omitted from the fragment labels for clarity.

 
Af-3b from strain 9197 The 1-D 1H-NMR spectrum of fraction Af-3b showed it to be a nearly homogeneous GIPC component, although a few major peaks from non-GIPC impurities could be observed (data not shown). Three anomeric proton resonances are clearly observed in the downfield region of the spectrum (Fig. 7A ) at 5.005 ppm (3J1,2 < 2.0 Hz), 4.889 ppm (3J1,2 < 2.0 Hz), and 4.828 ppm (3J1,2 = 2.0 Hz). These values are almost identical to those previously observed for the branched Galfß6-containing triglycosyl-IPC isolated from P. brasiliensis, Man{alpha}3(Galfß6)Man{alpha}2IPC (30), referred to here as Pb-3. This structure would be consistent with its HPTLC Rf value and immunoreactivity toward MEST-1 (see below). There were some differences in the spectra, however. These could be attributable to impurities, but to be more certain of the resonance and structure assignments, a series of 2-D 1H-1H and 13C-1H spectra were acquired, because off-diagonal correlations originating from H-1 resonances in 2-D 1H-1H spectra of glycoconjugates can generally be assigned in the presence of obscuring impurity peaks, and from these monosaccharide ring 1H spin system assignments most 13C-1H correlations can then be interpreted.


Figure 7
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  		Fig. 7. Downfield expansion of the anomeric proton region (5.2–4.4 ppm) of 1-D 1H-NMR spectra (500 MHz; DMSO-d6/2% D2O; 35°C) of GIPC fractions isolated from A. fumigatus strain 9197. A: Af-3b. B: Af-4. Spectra were acquired on fractions corresponding to lanes 3 and 4 of Fig. 1B.

 
Thus, almost all monosaccharide, inositol, and ceramide resonances were unambiguously assignable from 2-D 1H-1H gCOSY and TOCSY experiments (data not shown; Table 3 ). Analysis of approximate 3Ji,j proton-coupling constants around the three monosaccharide spin systems confirmed the presence of two {alpha}-Man residues, particularly recognizable by their signature small values for 3J1,2 and 3J2,3. Definitive analysis of the ß-Galf spin system was more problematic, but it was characterized by its essentially identical appearance to that observed previously for this residue in Pb-3 (30); acquisition of 13C resonance NMR data (which had not been obtained previously) proved helpful in eliminating any doubt about the identity of this residue in particular (see below). Analysis of approximate 3Ji,j coupling constants around the cyclic Ins spin system confirmed it as Ins (all hydroxyl groups equatorial except that at C-2), whereas the chemical shift pattern, compared with previously acquired data, was consistent with glycosylation of Ins at O-2 (22, 23, 30, 52). An interesting characterizing feature of the Af-3b 1H-NMR spectral data is the downfield shift of the Man{alpha}2 H-5 (4.053 ppm), which was observed previously for Pb-3 (30), as well as another GIPC having the Man{alpha}2 residue substituted at O-3 and O-6 [i.e., Man{alpha}3(Man{alpha}6)Man{alpha}2IPC (An-3) from Aspergillus nidulans (40)].


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  		TABLE 3. 1H and 13C chemical shifts (ppm) for monosaccharide, inositol, ceramide sphingoid, and fatty-N-acyl (in parentheses) residues of Man{alpha}3(Galfß6)Man{alpha}2IPC (Af-3b) from A. fumigatus in DMSO-d6/2% D2O at 35°C

 
Finally, 13C resonance assignments (Table 3), made from a 2-D 1H-detected, 13C-1H gHSQC spectrum (data not shown), were also consistent with the linkage of two glycosyl {alpha}-Manp residues to the core Man{alpha}2 residue at O-3 and O-6; this is supported by a pattern of substantial downfield shift increments ({alpha}-effects) for Man{alpha}2 C-3 and C-6, along with upfield shift decrements (ß-effects) (53) for Man{alpha}2 C-2, C-4, and C-5, compared with 13C spectral data for the parent compound, Man{alpha}2IPC (22). A striking feature of the 13C-NMR spectral data is the extreme downfield position of the C-1 resonance correlated with H-1 at 4.828 ppm; its chemical shift of 108.3 ppm essentially confirms the identification of a ß-Galf residue (53). All other 13C chemical shifts are consistent with the proposed Man{alpha}2IPC core structure, including those characteristic for the Ins residue substituted at O-2 (22, 40). Based on all of these data, the structure of Af-3b was proposed with high confidence to be Man{alpha}3(Galfß6)Man{alpha}2IPC.

A molecular profile of Af-3b was acquired via +ESI-Q/oa-TOF-MS (Fig. 8A ); a pair of [M(Li)+Li]+ salt-adduct ions observed at m/z 1,424 and 1,452 is consistent with a triglycosyl-IPC having ceramides consisting of t18:0 and t20:0 4-hydroxysphinganines with h24:0 fatty-N-acylation. Lower m/z expansions of +ESI-MS/CID-MS spectra acquired from the dilithiated molecular adducts at m/z 1,424 and 1,452 are reproduced in Fig. 8B, C, showing the predominant [B3PO3(Li)+Li]+/[C3PO3(Li)+Li]+ pair (m/z 741/759) and other abundant fragments. Similar to other GIPC components discussed, fragments from sequential loss of monosaccharide residues from [M(Li)+Li]+ ([Ym(Li)+Li]+), as well as ions from loss of the acyl chain and loss of acyl C2–C{omega} ([O(Li)+Li]+, [J(Li)+Li]+, and [J'(Li)+Li]+), were also observed at lower abundance in higher m/z regions of the spectra (data not shown). As described above, the ceramide and ceramide phosphate ions ([Y0+Li]+, [Z0+Li]+, [Y0PO3(Li)+Li]+, and [Z0PO3(Li)+Li]+) appear at intermediate abundances. The [O(Li)+Li]+, [J(Li)+Li]+, [J'(Li)+Li]+, and [O/Z0PO3(Li)+Li]+ ions (data not shown) all agreed with the sphingoid and fatty acid analysis, which indicates that the m/z 28 increment between the two predominant molecular species is primarily attributable to a corresponding C2H4 difference in the sphingoid rather than the N-acyl chain. Thus, the [M(Li)+Li]+ adducts at m/z 1,424 and 1,452 again correspond to lipoforms containing t18:0 and t20:0 4-hydroxysphinganines, respectively, with h24:0 fatty-N-acylation.


Figure 8
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  		Fig. 8. +ESI-Qq/oa-TOF spectra of Af-3b. A: Profile of molecular ions as [M(Li)+Li]+ adducts. B: MS/CID-MS of [M(Li)+Li]+ at m/z 1,424, low m/z region. C: MS/CID-MS of [M(Li)+Li]+ at m/z 1,452, low m/z region. Ion designations correspond to Scheme 5. The designations "+Li" and "(Li)+Li" and the charge form have been omitted from the fragment labels for clarity.

 
Inspection of the series of fragments derived by glycosidic bond cleavages from the [B3PO3(Li)+Li]+/[C3PO3(Li)+Li]+ pair showed that, consistent with previous findings (39–41), a somewhat lower abundance is observed for glycosylinositol phosphate fragment ions requiring at least two glycosidic cleavages for their appearance ([Y2{alpha}/Y2ß/B3PO3(Li)+Li]+ and [Y2{alpha}/Y2ß/C3PO3(Li)+Li]+, at m/z 417 and 435, respectively), compared with other ions in the [Ym/BnPO3(Li)+Li]+ and [Ym/CnPO3(Li)+Li]+ series, indicating the presence of a branch point in the glycan (Scheme 4 ). In particular, the ratios of the relative abundances of m/z 435 versus m/z 273 (0.14), and m/z 435 versus m/z 597 (0.25), are attenuated by factors of 5 and 2, respectively, in Af-3b compared with the same ratios in the isomeric unbranched Af-3a (0.70 and 0.52, respectively). Reductions in these abundance ratios have been observed previously for branched GIPCs under these