Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.

Two major acidic glycolipid components (Pb-1 and Pb-2) have been extracted from the mycopathogen Paracoccidioides brasiliensis, a thermally dimorphic fungus endemic to rural areas of South and Central America. Sera of all patients exhibiting paracoccidioidomycosis were found to be reactive with Pb-1, but not with Pb-2; no reactivity was observed with sera of healthy patients or those with histoplasmosis [Toledo, M. S., Suzuki, S., Straus, A. H., and Takahashi, H. K. (1995) J. Med. Vet. Mycol. 33, 247-251]. We report here the complete structure elucidation of both P. brasiliensis glycolipids using monosaccharide, fatty acid, sphingosine, and inositol component analysis by GC-MS; 1H- and 31P NMR spectroscopy; ESI-MS and -MS/CID-MS, linkage analysis, and exoglycosidase digestion. The compounds were found to be glycosylinositol phosphorylceramides (GIPCs) with the following structures: Pb-2, Manpalpha1-->3Manpalpha1-->2Ins1-P-1Cer; and Pb-1, Manpalpha1-->3[Galfbeta1-->6]Manpalpha1-->2Ins1- P-1Cer. The serologically nonreactive Pb-2 appears to be a biosynthetic intermediate between mannosylinositol phosphorylceramide (MIPC), which is widely distributed among fungi, and the antigenic Pb-1. Pb-1 is a novel glycosphingolipid, similar to a triglycosyl IPC (Hc-VI) reported from Histoplasma capsulatum [Barr, K., Laine, R.A, and Lester, R. L. (1984) Biochemistry 23, 5589-5596], but differing in the anomeric configuration of the terminal Galf1-->6 residue, which is immunodominant. The significance of these structures as serological and taxonomic markers, as well as their potential utility as targets for immunodiagnostic agents, is discussed.

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The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional signi¢cance in Aspergillus species is unknown. We here identi¢ed and characterized a GlcCer from Aspergillus nidulans, a non-pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (P4) and D-threo-3P P,4P P-ethylenedioxy-P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a signi¢cantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Cell growth control mechanisms were studied based on organization of components in glycosphingolipid-enriched microdomain (GEM) in WI38 cells versus their oncogenic transformant VA13 cells. Levels of fibroblast growth factor receptor (FGFR) and cSrc were 4 times and 2-3 times higher, respectively, in VA13 than in WI38 GEM, whereas the level of tetraspanin CD9/CD81 was 3-5 times higher in WI38 than in VA13 GEM. Csk, the physiological inhibitor of cSrc, was present in WI38 but not in VA13 GEM.

The functional interaction ("cross-talk") of integrins with growth factor receptors has become increasingly clear as a basic mechanism in cell biology, defining cell growth, adhesion, and motility. However, no studies have addressed the microdomains in which such interaction takes place nor the effect of gangliosides and tetraspanins (TSPs) on such interaction. Growth of human embryonal WI38 fibroblasts is highly dependent on fibroblast growth factor (FGF) and its receptor (FGFR), stably associated with ganglioside GM3 and TSPs CD9 and CD81 in the ganglioside-enriched microdomain. Adhesion and motility of these cells are mediated by laminin-5 ((LN5) and fibronectin (FN) through ␣3␤1 and ␣5␤1 integrin receptors, respectively. When WI38 cells or its transformant VA13 cells were adhered to LN5 or FN, ␣3␤1 or ␣5␤1 were stimulated, giving rise to signaling to activate FGFR through tyrosine phosphorylation and inducing cell proliferation under serum-free conditions without FGF addition. Types and intensity of signaling during the time course differed significantly depending on the type of integrin stimulated (␣3␤1 versus ␣5␤1), and on cell type (WI38 versus VA13). Such effect of cross-talk between integrins and FGFR was influenced strongly by the change of GM3 and TSPs. (i) GM3 depletion by P4 caused enhanced tyrosine phosphorylation of FGFR and Akt followed by MAPK activation, without significant change of ceramide level. GM3 depletion also caused enhanced co-immunoprecipitation of FGFR with ␣3/␣5/␤1 and of these integrins with CD9/CD81. (ii) LN5-or FN-dependent proliferation of both WI38 and VA13 was strongly enhanced by GM3 depletion and by CD9/ CD81 knockdown by siRNA. Thus, integrin-FGFR crosstalk is strongly influenced by GM3 and/or TSPs within the ganglioside-enriched microdomain.Cell growth and associated differentiation are controlled by growth factors, and their receptors that have tyrosine kinases at the cytoplasmic domain (1-3). Cell adhesion to extracellular matrix and motility are mediated by integrin receptors consisting of various combinations of ␣ and ␤ subunits specific for extracellular matrix components, laminin (LN), 1 fibronectin (FN), and collagen (4, 5). In contrast, some integrins on hematopoietic cells mediate cell-to-cell interaction based on their binding capability to Ig-like receptors (ICAM-1, VCAM-1) (6). Growth factor receptors (GFRs) and integrin receptors clearly differ in their domain structure, mechanism of generating signaling, and localization in the membrane microdomain. Epidermal GFR (EGFR) and platelet-derived GFR are claimed to be associated with caveolar membrane (7, 8), whereas integrin receptors are soluble in 1% Triton X-100 and are considered to be located outside of the caveolar membrane or "raft" (9). However, in recent studies, integrin receptors have often been found associated with tetraspanins (TSPs) (10, 11), and integrin⅐TSP complexes are insoluble in 0.5% Triton X-100 (12) or in 1% Brij 98 (10, 13-16). Gangliosides are capable of interacting with and modulating t...

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Cerebroside (monohexosylceramide) components were identified in neutral lipids extracted from both the yeast and mycelial forms of the thermally dimorphic mycopathogen Histoplasma capsulatum. The components were purified from both forms and their structures elucidated by 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and low energy tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Both components were characterized as beta-glucopyranosylceramides (GlcCers) containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base, attached to 18-carbon 2-hydroxy fatty N-acyl components. However, while the fatty acid of the yeast form GlcCer was virtually all N-2'-hydroxyoctadecanoate, the mycelium form GlcCer was characterized by almost exclusive expression of N-2'-hydroxy-(E)-delta(3)-octadecenoate. These results suggest that the yeast-mycelium transition is accompanied by up-regulation of an as yet uncharacterized ceramide or cerebroside 2-hydroxy fatty N-acyl (E)-delta(3)-desaturase activity. They also constitute further evidence for the existence of two distinct pathways for ceramide biosynthesis in fungi, since glycosylinositol phosphorylceramides (GIPCs), the other major class of fungal glycosphingolipids, are found with ceramides consisting of 4-hydroxysphinganine (phytosphingosine) and longer chain 2-hydroxy fatty acids. In addition to identification of the major glucocerebroside components, minor components (< 5%) detectable by molecular weight differences in the ESI-MS profiles were also characterized by tandem ESI-MS/CID-MS analysis. These minor components were identified as variants differing in fatty acyl chain length, or the absence of the sphingoid 9-methyl group or (E)-delta(8)-unsaturation, and are hypothesized to be either biosynthetic intermediates or the result of imperfect chemical transformation by the enzymes responsible for these features. Possible implications of these findings with respect to chemotaxonomy, compartmentalization of fungal glycosphingolipid biosynthetic pathways, and regulation of morphological transitions in H.capsulatum and other dimorphic fungi are discussed.