Glycosphingolipids of the model fungus Aspergillus nidulans

Bennion, Beau; Park, Chaeho; Fuller, Matthew D.; Lindsey, Rebecca L.; Momany, Michelle; Jennemann, Richard; Levery, Steven B.

doi:10.1194/jlr.m300184-jlr200

Cited by 24 publications

(8 citation statements)

References 75 publications

(98 reference statements)

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“…Extraction of Glycosphingolipids from Mature Flies, Larvae, and CHO Cells-glycosphingolipids were extracted and fractionated by methods similar to those described previously (16). Freeze-dried flies (ϳ5-10 g) were homogenized (Polytron, Kinematica AG, Luzern, Switzerland) in ϳ5 volumes of solvent A (2-isopropanol/hexane/water, 55:25:20, v/v/v, upper phase discarded), centrifuged, and the supernatant removed; this step was repeated with ϳ5 volumes of solvent B (chloroform/methanol, 1:1, v/v) and then sequentially again with ϳ5 volumes each of solvent A and solvent B.…”

Section: Methodsmentioning

confidence: 99%

Egghead and Brainiac Are Essential for Glycosphingolipid Biosynthesis in Vivo

Wandall

Pizette

Pedersen

et al. 2005

Journal of Biological Chemistry

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The Drosophila genes, brainiac and egghead, encode glycosyltransferases predicted to act sequentially in early steps of glycosphingolipid biosynthesis, and both genes are required for development in Drosophila. egghead encodes a ␤4-mannosyltransferase, and brainiac encodes a ␤3-N-acetylglucosaminyltransferase predicted by in vitro analysis to control synthesis of the glycosphingolipid core structure, GlcNAc␤1-3Man␤1-4Glc␤1-Cer, found widely in invertebrates but not vertebrates. In this report we present direct in vivo evidence for this hypothesis. egghead and brainiac mutants lack elongated glycosphingolipids and exhibit accumulation of the truncated precursor glycosphingolipids. Furthermore, we demonstrate that despite fundamental differences in the core structure of mammalian and Drosophila glycosphingolipids, the Drosophila egghead mutant can be rescued by introduction of the mammalian lactosylceramide glycosphingolipid biosynthetic pathway (Gal␤1-4Glc␤1-Cer) using a human ␤4-galactosyltransferase (␤4Gal-T6) transgene. Conversely, introduction of egghead in vertebrate cells (Chinese hamster ovary) resulted in near complete blockage of biosynthesis of glycosphingolipids and accumulation of Man␤1-4Glc␤1-Cer. The study demonstrates that glycosphingolipids are essential for development of complex organisms and suggests that the function of the Drosophila glycosphingolipids in development does not depend on the core structure.

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Section: Methodsmentioning

confidence: 99%

Egghead and Brainiac Are Essential for Glycosphingolipid Biosynthesis in Vivo

Wandall

Pizette

Pedersen

et al. 2005

Journal of Biological Chemistry

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“…Numerous GIPCs have been described at the cell membrane of other filamentous fungi and yeast cells but are absent from mammalian cells. The ceramide structure of these glycosphingolipids seems conserved between species with the presence of a phytosphingosine, associated to a saturated fatty acid, mainly the 2-hydroxylignoceric acid (2OH-C 24:0 ) (27)(28)(29)(30)(31)(32)(33)(34)(35). In contrast to the lipid moiety, the glycan moiety is heterogenous between fungal species with the presence of mannose, galactose in a pyranic or furanic configuration, and even fucose residue in higher mushrooms (36).…”

Section: Purification and Composition Of The Lipogalactomannan-mentioning

confidence: 99%

Glycosylphosphatidylinositol-anchored Fungal Polysaccharide in Aspergillus fumigatus

Costachel

Coddeville

Latgé

et al. 2005

Journal of Biological Chemistry

100

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Galactomannan is a characteristic polysaccharide of the human filamentous fungal pathogen Aspergillus fumigatus that can be used to diagnose invasive aspergillosis. In this study, we report the isolation of a galactomannan fraction associated to membrane preparations from A. fumigatus mycelium by a lipid anchor. Specific chemical and enzymatic degradations and mass spectrometry analysis showed that the lipid anchor is a glycosylphosphatidylinositol (GPI). The lipid part is an inositol phosphoceramide containing mainly C 18 -phytosphingosine and monohydroxylated lignoceric acid (2OH-C 24:0 fatty acid). GPI glycan is a tetramannose structure linked to a glucosamine residue: Man␣1-2Man␣1-2Man␣1-6Man␣1-4GlcN. The galactomannan polymer is linked to the GPI structure throught the mannan chain. The GPI structure is a type 1, closely related to the one previously described for the GPI-anchored proteins of A. fumigatus. This is the first time that a fungal polysaccharide is shown to be GPI-anchored.Aspergillus fumigatus has become over the past decade the most prevalent airborne fungal pathogen causing fatal invasive infections in immunocompromised patients (1). One of the characteristic components of this fungus is composed of a linear mannan chain with a tetra-␣-mannoside repeating unit and side chains of ␤1-5 galactofuranoside residues (2). The galactomannan from A. fumigatus has been described as a free polysaccharide found in the culture medium, and it is also covalently associated to the cell wall through the ␤1-3 glucan net and plays a role in the structural organization of the cell wall (2, 3). A monoclonal antibody directed against the galactofuranose side chain has been used to detect the presence of this polymer in the sera of patient with invasive aspergillosis (4, 5). Biosynthesis of the galactomannan is totally unknown in this fungus. During the search of lipid molecules that could be an anchor for the galactomannan, we isolated a membrane bound fraction that contained galactomannan. We investigated the nature of the membrane anchor of the galactomannan by GLC-MS 2 (gas liquid chromatography-mass spectrometry) and ES-MS-MS (electrospraytandem mass spectrometry) and found that the lipid anchor was a glycosylphosphatidylinositol. EXPERIMENTAL PROCEDURES Fungal Culture and Membrane PreparationA. fumigatus, strain CBS 144-89 was grown in a 15-L fermenter in 2% glucose and 1% mycopeptone (Biokar Diagnostics, Pantin, France) for 24 h at 25°C as described previously (6). Total membrane preparation from mycelium was done as described previously (7). Extraction and Purification of the LipogalactomannanLipids and polar glycolipids were extracted from membrane preparation by an organic extraction. A chloroform/methanol 1/1 (v/v) solution was added to the membrane suspension to reach a final concentration of chloroform/methanol/water (10:10:3 v/v/v). The mixture was stirred for 2 h at room temperature to extract lipids, then centrifuged at 10,000 ϫ g for 10 min. The pellet was resuspended in the same solvent, and the e...

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“…The t18:0 phytosphingosine backbone, C2 hygroxylated fatty acyls and phosphorylinositol containing polar head group are the unique feature of IPC derivatives ( Figure 2C ). IPC structures and pathway of synthesis have been characterized in several fungi like S. cereviseae ( Ejsing et al, 2009 ; Voynova et al, 2014 ), C. neoformans ( Luberto et al, 2001 ), A. fumigatus ( Kuroda et al, 1999 ; Heidler and Radding, 2000 ), A. nidulans ( Bennion et al, 2003 ), Histoplasma capsulatum ( Guimarães et al, 2014 ), and C. albicans ( Singh et al, 2010 ; Cheon et al, 2012 ).…”

Section: Introductionmentioning

confidence: 99%

Sphingolipidomics: An Important Mechanistic Tool for Studying Fungal Pathogens

Singh

Poeta

2016

Front. Microbiol.

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Sphingolipids form of a unique and complex group of bioactive lipids in fungi. Structurally, sphingolipids of fungi are quite diverse with unique differences in the sphingoid backbone, amide linked fatty acyl chain and the polar head group. Two of the most studied and conserved sphingolipid classes in fungi are the glucosyl- or galactosyl-ceramides and the phosphorylinositol containing phytoceramides. Comprehensive structural characterization and quantification of these lipids is largely based on advanced analytical mass spectrometry based lipidomic methods. While separation of complex lipid mixtures is achieved through high performance liquid chromatography, the soft – electrospray ionization tandem mass spectrometry allows a high sensitivity and selectivity of detection. Herein, we present an overview of lipid extraction, chromatographic separation and mass spectrometry employed in qualitative and quantitative sphingolipidomics in fungi.

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Glycosphingolipids of the model fungus Aspergillus nidulans

Cited by 24 publications

References 75 publications

Egghead and Brainiac Are Essential for Glycosphingolipid Biosynthesis in Vivo

Egghead and Brainiac Are Essential for Glycosphingolipid Biosynthesis in Vivo

Glycosylphosphatidylinositol-anchored Fungal Polysaccharide in Aspergillus fumigatus

Sphingolipidomics: An Important Mechanistic Tool for Studying Fungal Pathogens

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