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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.

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.

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.

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2/6Ins, NMR spectroscopy of H. capsulatum GIPCs (S. B. Levery, M. S. Toledo, A. H. Straus, and H. K. Takahashi, unpublished data) shows that the linkage is Man{alpha}1-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).

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.)

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


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 GlycobiologyHome page
C. Simenel, B. Coddeville, M. Delepierre, J.-P. Latge, and T. Fontaine
Glycosylinositolphosphoceramides in Aspergillus Fumigatus
Glycobiology, January 1, 2008; 18(1): 84 - 96.
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