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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 fumigatus

Marcos S. Toledo^*, Steven B. Levery^1,, Beau Bennion, Luciana L. Guimaraes^*, Sherry A. Castle, Rebecca Lindsey, Michelle Momany, Chaeho Park^**, Anita H. Straus^* and Helio K. Takahashi^1,*

^* Department of Biochemistry, Universidade Federal de São Paulo/Escola Paulista de Medicina, 04023-900 São Paulo, Brazil
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

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

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(13)Manp(12)Ins = Man3Man2Ins]. In some cases, Man may be further abbreviated as M, Ins as I, and Cer as C.

³ We previously designated the P. brasiliensis GIPCs Man3Man2IPC and Man3(Galfß6)Man2IPC 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 (R_f) values in HPTLC.

⁴ Although Barr, Laine, and Lester (21) did not specify precisely the linkage between Man and Ins in their compounds, but characterized it as Man12/6Ins, NMR spectroscopy of H. capsulatum GIPCs (S. B. Levery, M. S. Toledo, A. H. Straus, and H. K. Takahashi, unpublished data) shows that the linkage is Man12Ins1-P-1Cer, as reported previously for GIPCs of P. brasiliensis (30) and many other fungi (40, 52, 56).

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(13)Manp(12)Ins-P-Cer (Af-2), Manp(12)Manp(13)Manp(12)Ins-P-Cer (Af-3a), Manp(13)[Galf(ß16)]Manp(12)-Ins-P-Cer (Af-3b), Manp(12)-Manp(13)[Galf(ß16)]Manp(12)Ins-P-Cer (Af-4), and Manp(13)Manp(16)GlcpN(12)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 GlcN12Ins linkage may proceed by a two-step process, similar to the GlcN16Ins 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(ß16) 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(ß16) 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; R_f, 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-demethylase, leading to an inhibition of ergosterol biosynthesis and the accumulation of 14-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 ).

View larger version (15K): [in this window] [in a new window]   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) Mana2-T (function requires two genes in Saccharomyces cerevisiae, SUR1/CSG1 and CSG2), making Man12InsPCer (M2IPC); (ii) Man6-T, making Man6InsPCer (M6IPC); (iii) GlcNAc2-T, followed by action of de-N-acetylase, making GlcN12InsPCer (GlcN2IPC).

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 ±RManp(13)[Galf(ß16)]Manp(12)Ins structural motif. Although one GIPC antigen isolated from A. fumigatus, Manp(13)[Galf(ß16)]Manp(12)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(13)Manp(16)GlcpN(12)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(12)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(12)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 ( = 0.000 ppm). Carbon chemical shifts are referenced to solvent DMSO ( = 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 ([B_n+Cat]⁺ and [C_n+Cat]⁺) and glycosylinositol phosphate ([B_nPO₃(Cat)+Cat]⁺ and [C_nPO₃(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 ([C_n+Cat]⁺, [B_n+Cat]⁺, [Y_n+Cat]⁺, [Z_n+Cat]⁺, [Y_m/C_n+Cat]⁺, [Y_m/B_n+Cat]⁺, [Y_m/C_nPO₃(Cat)+Cat]⁺, and [Y_m/B_nPO₃(Cat)+Cat]⁺). In many cases, these are accompanied by related [Z_m/B_nPO₃(Cat)+Cat]⁺ and [Z_m/B_n+Cat]⁺ ions. It has been found that the [Y_m/C_nPO₃(Cat)+Cat]⁺ and [Y_m/B_nPO₃(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).

View larger version (14K): [in this window] [in a new window]   Scheme 2. Characteristic fragmentations of glycosylated IPCs, exemplified by M3M2IPC. 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).

View larger version (12K): [in this window] [in a new window]   Scheme 3. Fragmentation of Af-3a in positive ion mode ESI-MS and ESI-MS/CID-MS.

View larger version (13K): [in this window] [in a new window]   Scheme 4. Fragmentation of Af-3b in positive ion mode ESI-MS and ESI-MS/CID-MS.

View larger version (15K): [in this window] [in a new window]   Scheme 5. Fragmentation of Af-4 in positive ion mode ESI-MS and ESI-MS/CID-MS.

View larger version (18K): [in this window] [in a new window]   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.

View larger version (20K): [in this window] [in a new window]   Scheme 7. Divergent structures and hypothetical pathways for the biosynthesis of GIPCs from the common mannosyl-IPC intermediate Man12InsPCer (M2IPC). These include InsP16Man12InsPCer, a known product of the IPT1-encoded enzyme that transfers a second mole of myo-inositol-1-O-phosphate from phosphatidylinositol to M2IPC (left arrow) in S. cerevisiae. In basidiomycetes, a commonly observed intermediate GIPC is Galß16Man12InsPCer (Galß6M2IPC; Ba-2), synthesized from M2IPC by an as yet unknown Galß6-T (right arrow). In euascomycetes, a commonly observed intermediate GIPC is Man13Man12InsPCer (Man3M2IPC; Eu-2a), synthesized from M2IPC by an as yet unknown Man3-T (lower left). In A. nidulans, the major GIPC is a nonantigenic trimannosyl-IPC, Man13(Man16)Man12InsPCer (Man3[Man6]M2IPC; An-3), derived from Eu-2a by the addition of a Man16 residue.

	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(13)[Galf(ß16)]Manp(12)InsPCer], but not Pb-2 [Manp(13)Manp(12)InsPCer], was previously observed to react with MEST-1 (25). The branching Galf(ß16) 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 R_f in each strain. This suggests that Af-4 contains the common branching Galf(ß16) 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--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 R_f with respect to Af-3b, the result would be consistent with absence, or unreactive presentation, of the Galfß residue.

View larger version (15K): [in this window] [in a new window]   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.

View larger version (17K): [in this window] [in a new window]   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].

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).

View larger version (11K): [in this window] [in a new window]   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.

View this table: [in this window] [in a new window]   TABLE 1. 1H chemical shifts (ppm) for monosaccharide, inositol, ceramide sphingoid, and fatty-N-acyl (in parentheses) residues of IPC (myo-Ins1P1Cer = Af-0) and Man3Man2IPC (Af-2) from A. fumigatus in DMSO-d6/2% D2O at 35°C

View larger version (12K): [in this window] [in a new window]   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 938690). D: MS3 products of [Y0+Li]+ at m/z 718 (m/z 966718). The adduct designation "+Li" and the charge form have been omitted from the fragment labels for clarity.

View larger version (26K): [in this window] [in a new window]   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.

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 Man3Man2IPC, although potential glycosylation-induced changes in chemical shifts preclude reliable assignments based purely on analogy. A triglycosyl structure consisting solely of -Manp residues would be consistent with the similar ³J_1,2 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 12 linkage. Such a structure would also produce the same T-Man and 3Man derivatives. Therefore, the structure Man2Man3Man2IPC 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 Man3 residue, because it is known that glycosylation of an -Man residue by Man2 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 Man2 residue, with H-1 of the reducing end Man2 residue remaining at 5.064 ppm, apparently unaffected by the substitution (Table 2 ).

View this table: [in this window] [in a new window]   TABLE 2. Comparison of 1H chemical shifts (ppm) for H-1 of monosaccharide residues of Man3Man2IPC (Af-2), Man2Man3Man2IPC (Af-3a), Man3(Galfß6)Man2IPC (Af-3b), and Man2Man3(Galfß6)Man2IPC (Af-4) from A. fumigatus in DMSO-d6/2% D2O at 35°C

View larger version (19K): [in this window] [in a new window]   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.

View larger version (14K): [in this window] [in a new window]   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.

View this table: [in this window] [in a new window]   TABLE 3. 1H and 13C chemical shifts (ppm) for monosaccharide, inositol, ceramide sphingoid, and fatty-N-acyl (in parentheses) residues of Man3(Galfß6)Man2IPC (Af-3b) from A. fumigatus in DMSO-d6/2% D2O at 35°C

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]⁺ ([Y_m(Li)+Li]⁺), as well as ions from loss of the acyl chain and loss of acyl C₂–C ([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.

View larger version (30K): [in this window] [in a new window]   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.

2

2

Af-4 from strain 9197 In the downfield 1-D ¹H-NMR spectrum of fraction Af-4 (Fig. 7B), four major anomeric proton signals can be observed at 4.826, 4.901, 5.000, and 5.023 ppm (all ³J_1,2 1.5–2.0 Hz), suggesting that it represents a tetraglycosyl-IPC. Two of these chemical shifts (4.901 and 5.023) are similar to those observed for H-1 resonances of Af-3a, whereas the other two (4.826 and 5.000) are similar to those observed for H-1 resonances of Af-3b, suggesting that it might contain features common to both triglycosyl-IPCs. In terms of glycosylation-induced chemical shift changes for the H-1 resonances, Af-4 could be thought of as having the same relationship with Af-3a as Af-3b has with Af-2 (addition of branching Galfß6 to the Man3 residue), or the same relationship with Af-3b as Af-3a has with Af-2 (addition of terminal Man2 to the Man3 residue); for this reason, the structure Man2Man3(Galfß6)Man2IPC was proposed.

A molecular profile of Af-4 was acquired via ⁺ESI-Q/oa-TOF-MS (Fig. 9A ); a pair of [M(Li)+Li]⁺ salt-adduct ions observed at m/z 1,586 and 1,614 is consistent with a tetraglycosyl-IPC having ceramides consisting of t18:0 and t20:0 4-hydroxysphinganines with h24:0 fatty-N-acylation. Other ions in the profile at m/z 1,602 and 1,630 yielded spectra consistent with mixed [M(Na)+Li]⁺ salt adducts (indicating somewhat inadequate LiI addition, but not affecting conclusions drawn from the fully lithiated molecular ions). 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. 9B, C, showing the predominant [B₄PO₃(Li)+Li]⁺/[C₄PO₃(Li)+Li]⁺ pair (m/z 903/921) and other abundant fragments. Similar to other GIPC components discussed, fragments from sequential loss of monosaccharide residues from [M(Li)+Li]⁺ ([Y_m(Li)+Li]⁺), as well as ions from loss of the acyl chain and loss of acyl C₂–C ([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 1,586 and 1,614 again correspond to lipoforms containing t18:0 and t20:0 4-hydroxysphinganines, respectively, with h24:0 fatty-N-acylation.

View larger version (22K): [in this window] [in a new window]   Fig. 9. +ESI-Qq/oa-TOF spectra of Af-4. A: Profile of molecular ions as [M(Li)+Li]+ adducts. B: MS/CID-MS of [M(Li)+Li]+ at m/z 1,586, low m/z region. C: MS/CID-MS of [M(Li)+Li]+ at m/z 1,614, 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.

Af-3c from strain 9197 A significant amount of this component was isolated from both the acidic and neutral fractions of A. fumigatus lipids. Component analysis by GC-MS indicated that the low-R_f fraction Af-3c contained mannose and glucosamine (detected as GlcNAc) in a 2:1 ratio; other components detected were Ins, t18:0 and t20:0 4-hydroxysphinganines (phytosphingosines), and h24:0 fatty acid (major). The relevant downfield section of a 1-D ¹H-NMR spectrum of fraction Af-3c (corresponding material isolated from neutral fraction lipids) is reproduced in Fig. 10A ; a section of the TOCSY spectrum corresponding to this region is reproduced in Fig. 10B. These data show a marked similarity to those obtained previously for Man3Man6GlcN2IPC (Ss-Y6) previously isolated and characterized from the yeast form of Sporothrix schenckii (23). We still consider this structure particularly unusual (see Discussion) and therefore sought to confirm its occurrence in this context with a thorough reanalysis of fraction Af-3c, supporting it with additional heteronuclear experiments not reported previously.

View larger version (28K): [in this window] [in a new window]   Fig. 10. Downfield expansion (F2, 5.20–2.30 ppm) of 1-D and 2-D 1H-NMR spectra (800 MHz; DMSO-d6/2% D2O; 35°C) of A. fumigatus GIPC fraction Af-3c. A: 1-D spectrum. B: Lower right section of 2-D 1H-1H total correlation spectroscopy (TOCSY) spectrum (200 ms mixing time; F1, 5.22–3.94 ppm). C: 2-D 1H-1H nuclear Overhauser effect spectroscopy spectrum (200 ms mixing time; F1, 5.20–4.50 ppm). In A, resonances marked with asterisks are from residual monosaccharide hydroxy protons; in B, TOCSY correlations originating from these resonances have been deleted for clarity. In C, interresidue nuclear Overhauser effect correlations directly across glycosidic linkages are boxed, weaker interresidue correlations are marked by dashed boxes, and intraresidue correlations have been left unmarked.

View this table: [in this window] [in a new window]   TABLE 4. 1H and 13C chemical shifts (ppm) for monosaccharide, inositol, ceramide sphingoid, and fatty-N-acyl (in parentheses) residues of Man3Man6GlcN2IPC (Af-3c) from A. fumigatus in DMSO-d6/2% D2O at 35°C

View larger version (10K): [in this window] [in a new window]   Fig. 11. Downfield expansion (F2, 5.20–3.50 ppm; F1, 120–60 ppm) of 2-D 1H-detected 13C-1H-g heteronuclear multiple bond correlation spectrum (800 MHz; DMSO-d6/2% D2O; 35°C) of A. fumigatus GIPC fraction Af-3c. Interglycosidic three-bond 13C-1H correlations are circled and labeled with connectivities thus established.

More detailed analysis was carried out via ⁺ESI-MS and ⁺ESI-MS/CID-MS of lithium salt adducts on the Q-TOF instrument (after treatment of the N-acetylated Af-3c sample with concentrated aqueous NH₃ to remove O-acetylated components). An ⁺ESI-MS profile now exhibited the two major [M(Li)+Li]⁺ salt adduct ions for N-acetylated Af-3c at m/z 1,465 and 1,493 (Fig. 12 ). Corresponding ⁺ESI-MS/CID-MS product ion spectra were acquired with the selection of each molecular adduct precursor; the spectrum of products from m/z 1,465 is reproduced in Fig. 12). The glycosylinositol phosphate fragments [C₄PO₃(Li)+Li]⁺ and [B₄PO₃(Li)+Li]⁺ are now observed abundantly at m/z 800 and 782, respectively; the corresponding glycosylinositol fragments [C₄+Na]⁺ and [B₄+Na]⁺ are observed at m/z 714 and 696, respectively. Fragment ions arising from additional glycosidic cleavages of all four of these primary products are also observed in abundance in these spectra, especially significant being the phosphorylated ion series including m/z 255 ([Y₁/B₄PO₃(Li)+Li]⁺) and 273 ([Y₁/C₄PO₃(Li)+Li]⁺), 458 ([Y₂/B₄PO₃(Li)+Li]⁺) and 476 ([Y₂/C₄PO₃(Li)+Li]⁺), and 620 ([Y₃/B₄PO₃(Li)+Li]⁺) and 638 ([Y₃/C₄PO₃(Li)+Li]⁺). Again, the increment of m/z 203 between [Y₁/-] and [Y₂/C₄PO₃(Li)+Li]⁺ (or between [Y₁/-] and [Y₂/B₄PO₃(Li)+Li]⁺) clearly reflects the attachment of Glc(NAc) directly to the Ins residue (Scheme 6 ). The structure of Af-3c is thus clearly confirmed by the combination of NMR and mass spectrometric analysis as Man3Man6GlcN2IPC.

View larger version (17K): [in this window] [in a new window]   Fig. 12. +ESI-Qq/oa-TOF spectra of N-acetylated Af-3c. A: Profile of molecular ions as [M(Li)+Li]+ adducts. B: MS/CID-MS of [M(Li)+Li]+ at m/z 1,465, low m/z region. Ion designations correspond to Scheme 6. The designations "+Li" and "(Li)+Li" and the charge form have been omitted from the fragment labels for clarity.

View larger version (28K): [in this window] [in a new window]   Fig. 13. +ESI-Qq/oa-TOF spectra of mixed fraction containing Af-3c*. 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 1,465, low m/z region. Ion designations for C correspond to Scheme 6; R = NAc. The designations "+Li" and "(Li)+Li" and the charge form have been omitted from the fragment labels for clarity.

Immunoreactivity of A. fumigatus GIPCs with aspergillosis serum
In Fig. 14 are reproduced HPTLC profiles of crude and purified fungal GIPCs stained with orcinol (panel A) and the serum of a patient with aspergillosis (panel B). 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) as well as the reactivity with purified A. fumigatus Af-3a (lane 4) and A. nidulans components An-2 and An-3 (lanes 5 and 6, respectively). These show more extensive staining, including most of the significant bands from both A. fumigatus strains (Fig. 14B, lanes 1 and 2) and P. brasiliensis Pb-3 (Fig. 14B, lane 3) and the purified Af-3a (Fig. 14B, lane 4). The latter staining shows unambiguously in this case that Af-3a is reactive with the serum, along with components Af-3b/Pb-3 and Af-4. A variety of lower R_f components from A. fumigatus also reacted with the serum. The M₂IPC and M₃IPC components (An-2 and An-3, respectively) from the nonpathogenic A. nidulans appeared to be nonreactive (Fig. 14B, lanes 5, 6).

View larger version (18K): [in this window] [in a new window]   Fig. 14. HPTLC immunostaining of crude and purified fungal GIPC fractions by serum from a patient with disseminated aspergillosis. A: Orcinol staining. B: Serum 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. Purified GIPC fractions are as follows: lane 4, Af-3a from A. fumigatus strain 237; lane 5, An-3 from A. nidulans strain A28; lane 6, An-5 from A. nidulans strain A28.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Af-2 represents an apparently common intermediate dimannosyl-IPC that has now been found in a number of mycelial or dimorphic euascomycete fungi (Scheme 7 , Eu-2a) (20–22, 30, 39, 40)⁴ but has not been reported in hemiascomycetous yeasts such as S. cerevisiae and Candida albicans (54), nor in any basidiomycete species (52, 55, 56), with the sole exception of Cantharellus cibarius (chantarelle) (52). In S. cerevisiae and C. albicans, GIPC biosynthesis diverges (Scheme 7, IPM2IPC) by addition of a second mole of myo-inositol-1-phosphate to the Man residue of mannosyl-IPC, a process shown in S. cerevisiae to be dependent on the IPT1 gene (57). In basidiomycetes, on the other hand, the dominant structural modification of mannosyl-IPC appears to be conversion to the alternative intermediate diglycosyl-IPC Galp(ß16)Manp(12)InsPCer (Scheme 7, Ba-2), which precedes further glycosylation steps in the formation of diverse "basidiolipids" which are found in mushrooms (52, 55), as well as in GIPCs of the yeast-like basidiomycete mycopathogen Cr. neoformans (56). The Galpß6- and Manp3-transferases responsible for the further glycosylation of Manp(12)InsPCer in euascomycetes and basidiomycetes, respectively, remain unidentified. In A. nidulans, the major GIPC was found to be the product of An-2 (= Eu-2a) with a Man6-transferase to yield a branched tri--mannosyl structure (Scheme 7, An-3) similar to that of the Man₃GlcNAc₂ core of protein N-linked glycans (except in that case the branching Man residue is ß-, rather than -).

Among the triglycosyl-IPCs found in A. fumigatus, Af-3b has been reported previously as a component of both yeast and mycelial forms of the dimorphic mycopathogen P. brasiliensis (30). A closely related structural variant, identical to Af-3b but possessing a branching -, rather than ß-, Galf residue was reported among the GIPCs of the dimorphic mycopathogen Histoplasma capsulatum (20, 21).⁴ The H. capsulatum antigen was strongly reactive with sera from patients with histoplasmosis, but, unlike the case with P. brasiliensis, it was detected only in GIPCs from the yeast form. The glycosylinositol structure of Af-3a is novel, apparently unreactive with MEST-1 but reactive with polyclonal sera from a patient with aspergillosis. The tetraglycosyl-IPC Af-4, reactive with both MEST-1 and aspergillosis serum, appears to be a product of further glycosylation of the Manp3 unit in Af-3a or Af-3b by Galfß6 or Manp2 residues, respectively; putative convergent pathways to Af-4 via these intermediate triglycosyl-IPC structures are illustrated in Scheme 8 .

View larger version (16K): [in this window] [in a new window]   Scheme 8. Structures and hypothetical pathways for the biosynthesis of A. fumigatus GIPC antigens Af-3a, Af-3b, and Af-4, starting from the common dimannosyl-IPC intermediate Af-2, Man13Man12InsPCer (Man3M2IPC).

On the other hand, it was hypothesized that, similar to the case with GlcNp16Ins of GPI anchors, the GlcNp12Ins structure might be formed by a process requiring two enzymes: transfer of GlcNAc to the Ins moiety, followed by de-N-acetylation of the GlcNAc residue (23). More recently, a series of novel GIPCs, also based on the GlcNp12Ins core structure, was isolated from mycelia of Acremonium species (31). In this case, the alternative triglycosyl-IPC structure Manp(16)Manp(16)GlcNp(12)InsPCer was identified, along with all of the presumptive intermediates. In addition, a remarkable finding was the addition of phosphocholine to the 6-hydroxyl of the terminal -Manp residues of both Manp(16)GlcNp(12)InsPCer and Manp(16)Manp(16)GlcNp(12)InsPCer. The suggestion of a two-step conversion of InsPCer to the core intermediate GlcNp(12)InsPCer was again invoked (31). We propose that the small amount of triglycosyl-IPC observed in a mixed fraction of A. fumigatus GIPCs (Af-3c*), clearly containing a HexNAc residue directly linked to Ins, is Manp(13)Manp(16)GlcNAcp(12)InsPCer: in other words, a structural analog of Af-3c derived from incompletely de-N-acetylated GlcNAcp(12)InsPCer. Although this needs further confirmation, it lends support to the suggestion that a two-step process, similar to that observed for GlcNp16Ins in the GPI anchor pathway, also applies to GlcNp12Ins in the GIPC pathway. Whether the same de-N-acetylase could be involved is an interesting question that should be answered by further in vitro and in vivo studies. A more important question, of course, is whether any special functional role(s) might be associated with this zwitterionic class of GIPCs, with or without the addition of phosphocholine. In this regard, it is significant that the Acremonium species GIPCs were found to induce cell death in suspension-cultured rice cells (31).

Thus, we consider that three core motifs have been confirmed at the monosaccharide level for fungal GIPCs (by identification in structural studies from at least two different laboratories); these are Man12Ins, Man16Ins, and GlcN12Ins (Scheme 1). Of these, the Man16Ins core has been reported only for GIPCs of S. schenckii (22, 24); remarkably, S. schenckii expresses GIPCs with all three core structures (23). As shown in the current study, A. fumigatus is sufficiently versatile at least to use two of these. Notably, the GlcN12Ins core has not been observed in GIPCs from the related model fungus A. nidulans; to date, only oligo--mannosyl-IPCs, based on the Manp(13)Manp(12)InsPCer core, have been isolated from A. nidulans, with no evidence for the expression of antigenic determinants containing ß-Galf residues (40). This interesting dichotomy suggests that wider versatility in GIPC biosynthesis may be one factor in the survival of A. fumigatus in highly diverse environments, including the entire range of mammalian host cells, tissues, and organs.

	ACKNOWLEDGMENTS

 
The authors thank James Rankin and Emma Arigi for technical assistance with growing A. fumigatus cultures and isolating some of the title compounds. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paolo and Conselho Nacional de Desenvolvimento Científìco e Technológico (Brasil) (M.S.T., A.H.S., and H.K.T.), a Glycoscience Research Award from Neose Technologies, Inc., the National Institutes of Health Resource Center for Biomedical Complex Carbohydrates (Grant P41 RR-05351), and the Biological Research Infrastructure Network-Center for Structural Biology (Grant P20 RR-16459).

Submitted on March 28, 2007

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