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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(α1→3)Manp(α1→2)Ins-P-Cer (Af-2), Manp(α1→2)Manp(α1→3)Manp(α1→2)Ins-P-Cer (Af-3a), Manp(α1→3)[Galf(β1→6)]Manp(α1→2)-Ins-P-Cer (Af-3b), Manp(α1→2)-Manp(α1→3)[Galf(β1→6)]Manp(α1→2)Ins-P-Cer (Af-4), and Manp(α1→3)Manp(α1→6)GlcpN(α1→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 GlcNα1→2Ins linkage may proceed by a two-step process, similar to the GlcNα1→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(β1→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(β1→6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.

monoclonal antibody, NMR spectroscopy, mass spectrometry, galactofuranose, glycolipid, Electrospray Ionization, fungus, sphingolipid, ion trap

NCBI PubMed ID: 17488996
Publication DOI: 10.1194/jlr.M700149-JLR200
Journal NLM ID: 0376606
Publisher: ASBMB
Correspondence: Levery SB cisunix.unh.edu>, Takahashi HK epm.br>
Institutions: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA, Department of Biochemistry, Universidade Federal de São Paulo/Escola Paulista de Medicina, São Paulo, Brazil, Department of Chemistry, University of New Hampshire, Durham, North Carolina, USA, Department of Plant Biology, University of Georgia, Athens, Georgia, USA
Methods: 13C NMR, 1H NMR, methylation, NMR-2D, GC-MS, ESI-MS, composition analysis, serological methods, HPLC, ion-exchange chromatography, extraction, CID-MS, HPTLC, ESI-QTOF-MS, HPTLC immunostaining