Glycolipids from<i>Paracoccidioides brasiliensis</i>. Isolation of a galactofuranose-containing glycolipid reactive with sera of patients with paracoccidioidomycosis

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 (, and Manp(a1Y3)Manp(a1Y6)GlcpN(a1Y2)Ins-PCer (Af-3c) (where Ins 5 myo-inositol and P 5 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 GlcNa1Y2Ins linkage may proceed by a two-step process, similar to the GlcNa1Y6Ins 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 (b1Y6) 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 thinlayer 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 (b1Y6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.-Toledo, M. S., S. B. Levery, B. Bennion, L. L.

Section: Extraction and Purification Of Glycosphingolipidsmentioning

confidence: 99%

mentioning

confidence: 99%

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus

Toledo

Levery

Bennion

et al. 2007

“…Phospholipids were purified by preparative HPTLC in solvent C. Separated phospholipids were visualized under ultraviolet light after spraying with 0.01% primulin in 90% aqueous acetone. Individual phospholipids were isolated from HPTLC plates by scraping and sonication in solvent A (22) …”

Section: Lipid Analysismentioning

confidence: 99%

Characterization of Leishmania (Viannia) braziliensis membrane microdomains, and their role in macrophage infectivity

Yoneyama

Tanaka

Silveira

et al. 2006

Detergent-resistant membranes (DRMs) from Leishmania (Viannia) braziliensis promastigotes, insoluble in 1% Triton X-100 at 4°C, were fractionated by sucrose density gradient ultracentrifugation. They were composed of glycoinositolphospholipids (GIPLs), inositol phosphorylceramide (IPC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and sterols. In contrast, 1% Triton X-100-soluble fraction was composed of PE, phosphatidylcholine, phosphatidylserine, PI, IPC, sterol, and lyso-PI. High-performance thin-layer chromatography (HPTLC) immunostaining using monoclonal antibody SST-1 showed that 85% of GIPLs are present in DRMs, and immunoelectron microscopic analysis showed that SST-1-reactive components are located in patches along the parasite surface. No difference in GIPL pattern was observed by HPTLC between Triton X-100-soluble versus -insoluble fractions at 4°C. Analysis of fatty acid composition in DRMs by GC-MS showed the presence of GIPLs containing an alkylacylglycerol, presenting mainly saturated acyl and alkyl chains. DRMs also contained sterol, IPC with saturated fatty acids, PI with at least one saturated acyl chain, and PE with predominantly oleic acid. Promastigotes treated with methyl-b-cyclodextrin to disrupt lipid microdomains showed significantly lower macrophage infectivity, suggesting a relationship between lipid microdomains and the infectivity of these parasites. In the fluid bilayer of eukaryotic plasma membranes, various lipid species are asymmetrically distributed within the exoplasmic and cytoplasmic layers, and specific subsets of proteins and glycolipids are organized as specialized microdomains (1). In mammalian cells, certain plasma membrane proteins, such as receptors and proteins anchored by glycosylphosphatidylinositol (GPI) or acyl moieties, are associated with sphingolipid/cholesterol-rich microdomains termed "lipid rafts." Such microdomains have also been termed "detergent-resistant membranes" (DRMs) because they can be isolated by density gradient ultracentrifugation on the basis of their low density and insolubility in cold, nonionic detergent (2).DRMs containing GPI-anchored proteins have also been isolated from trypanosomatids such as Leishmania (3, 4). Leishmania is a digenetic parasite whose life cycle includes motile promastigotes in the alimentary tract of its insect vector, the phlebotomine sandfly, and intracellular nonmotile amastigotes in mononuclear phagocytes of mammalian hosts. Numerous studies during the past 15 years indicate that the infection process in parasites depends on surface glycoconjugates such as lipophosphoglycans, .The purpose of this study was to characterize the lipids present in lipid raft microdomains in promastigote forms and to analyze the involvement of these microdomains in parasite-macrophage interaction in a particular Leishmania species, Leishmania (Viannia) braziliensis. This species belongs to the L. Viannia subgenus that causes human cutaneous and mucocutaneous leishmaniasis in the New World (13). Members of this subg...

“…Extraction and purification of glycosphingolipids were carried out as described previously (19)(20)(21). Briefly, glycosphingolipids were extracted by homogenizing mycelia (40-80 g wet weight) in a glasswalled blender once with 200 ml of solvent A, two times with 200 ml of solvent B, and once more with 200 ml of solvent A.…”

Section: Extraction and Purification Of Glycosphingolipidsmentioning

confidence: 99%

Neutral glycolipids of the filamentous fungus Neurospora crassa:altered expression in plant defensin-resistant mutants

Park

Bennion

François

et al. 2005

. For this study, neutral glycolipid components were extracted from wild-type and plant defensin-resistant mutant strains of N. crassa . The structures of purified components were elucidated by NMR spectroscopy and mass spectrometry. Neutral glycosphingolipids of both wild-type and mutant strains were characterized as ␤ -glucopyranosylceramides, but those of the mutants were found with structurally altered ceramides. Although the wild type expressed a preponderance of N -2 -hydroxy-( E )-⌬ 3 -octadecenoate as the fatty-N -acyl component attached to the long-chain base (4 E ,8 E )-9-methyl-4,8-sphingadienine, the mutant ceramides were found with mainly N -2 -hydroxyhexadecanoate instead. In addition, the mutant strains expressed highly increased levels of a sterol glucoside identified as ergosterol-␤ -glucoside. The potential implications of these findings with respect to defensin resistance in the N. crassa mutants are discussed.