One stop mycology

Sphingosines, or sphingoids, are a family of naturally occurring long-chain hydrocarbon derivatives sharing a common 1,3-dihydroxy-2-amino-backbone motif The majority of sphingolipids, as their derivatives are collectively known, can be found in cell membranes in the form of amphiphilic conjugates, each composed of a polar head group attached to an N-acylated sphingoid, or ceramide Glycosphingolipids (GSLs), which are the glycosides of either ceramide or myo-inositol-(1-O)-phosphoryl-(O-1)-ceramide, are a structurally and functionally diverse sphingolipid subclass; GSLs are ubiquitously distributed among all eukaryotic species and are found in some bacteria Since GSLs are secondary metabolites, direct and comprehensive analysis (metabolomics) must be considered an essential complement to genomic and proteomic approaches for establishing the structural repertoire within an organism and deducing its possible functional roles The glycosphingolipidome clearly comprises an important and extensive subset of both the glycome and the lipidome, but the complexities of GSL structure, biosynthesis, and function form the outlines of a considerable analytical problem, especially since their structural diversity confers by extension an enormous variability with respect to physicochemical properties This chapter covers selected developments and applications of techniques in mass spectrometric (MS) that have contributed to GSL structural analysis and glycosphingolipidomics since 1990 Sections are included on basic characteristics of ionization and fragmentation of permethylated GSLs and of lithium-adducted nonderivatized GSLs under positive-ion electrospray ionization mass spectrometry (ESI-MS) and collision-induced mass spectrometry (CID-MS) conditions; on the analysis of sulfatides, mainly using negative-ion techniques; and on selected applications of ESI-MS and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to emerging GSL structural, functional, and analytical issues The latter section includes a particular focus on evolving techniques for analysis of gangliosides, GSLs containing sialic acid, as well as on characterizations of GSLs from selected nonmammalian eukaryotes, such as dipterans, nematodes, cestodes, and fungi Additional sections focus on the issue of whether it is better to leave GSLs intact or remove the ceramide; on development and uses of thin-layer chromatography (TLC) blotting and TLC-MS techniques; and on emerging issues of high-throughput analysis, including the use of flow injection, liquid chromatography mass spectrometry (LC-MS), and capillary electrophoresis mass spectrometry (CE-MS)

Glycosphingolipid Structural Analysis and Glycosphingolipidomics

A mouse monoclonal antibody, MEST-1, was produced against Band 1 glycolipid antigen of Paracoccidioides brasiliensis. The glycan structure of Band 1 antigen was recently elucidated and the monosaccharides sequence was defined as: Galf beta 1-->6(Manp alpha 1-->3)Manp beta 1-->2Ins. The reactivity of MEST-1 MAb was determined by solid-phase radioimmunoassay and high performance thin layer chromatography immunostaining. Selective oxidation of galactofuranose residues and inhibition assays with different methyl-glycosides, revealed that MAb MEST-1 is directed against the terminal residue of beta-D-galactofuranose of Band 1, a phosphoglyceroglycolipid antigen of P. brasiliensis. By indirect immunofluorescence, it was observed that the epitope recognized by MEST-1 is accessible to the antibody in yeast forms of this fungus. Reactivity of MEST-1 with parasites known to express galactofuranose containing glycoconjugates was also analyzed by indirect immunofluorescence. A positive fluorescence was observed with promastigotes of Leishmania major and epimastigotes of Trypanosoma cruzi. GIPL-1 was identified as the antigen recognized by MEST-1 in Leishmania major, indicating that the MAb MEST-1 recognizes terminal galactofuranose residue in either beta 1-->6 or beta 1-->3 linkage to the mannose.

A monoclonal antibody directed to terminal residue of β-galactofuranose of a glycolipid antigen isolated from Paracoccidioides brasiliensis: cross-reactivity with Leishmania major and Trypanosoma cruzi

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus.

The role of glycosylinositol phospholipid 1 (GIPL-1) of Leishmania (Leishmania) major in the interaction of promastigotes and amastigotes with macrophages was analyzed. Monoclonal antibody MEST-1, which recognizes glycolipids containing terminal galactofuranose (Galf) residues (E. Suzuki, M. S. Toledo, H. K. Takahashi, and A. H. Straus, Glycobiology 7:463-468, 1997), was used to detect GIPL-1 in Leishmania by indirect immunofluorescence and to analyze its role in macrophage infectivity. L. major promastigotes showed intense fluorescence with MEST-1, and GIPL-1 was detected in both amastigote and promastigote forms by high-performance thin-layer chromatography immunostaining by using MEST-1. Delipidation of L. major promastigotes with isopropanol-hexane-water eliminated the MEST-1 reactivity, confirming that only GIPL-1 is recognized in either amastigotes or promastigotes of this species. The biological role of GIPL-1 in the ability of L. major to invade macrophages was studied by using either Fab fragments of MEST-1 or methylglycosides. Preincubation of parasites with Fab fragments reduced macrophage infectivity in about 80% of the promastigotes and 30% of the amastigotes. Preincubation of peritoneal macrophages with p-nitrophenyl-β-galactofuranoside (10 mM) led to significant (∼80%) inhibition of promastigote infectivity. These data suggest that a putative new receptor recognizing β-d-Galf is associated with L. major macrophage infectivity and that GIPL-1 containing a terminal Galf residue is involved in the L. major-macrophage interaction.

Role of β-d-Galactofuranose in Leishmania major Macrophage Invasion

Cell surface carbohydrates constitute the major antigenic determinants of fungi and protozoa. Glycoconjugates also represent a large variety of antigens or markers present in mammals such as histo-blood groups ABO, differentiation and heterophile antigens, among others. This article focuses on the general properties of glycoconjugate antigens and production and characterization of the anti-carbohydrate monoclonal antibodies (MoAbs). It describes the specificity and some properties of monoclonal antibodies directed against carbohydrate epitopes present in tumor-associated glycoproteins, in glycosaminoglycans of higher eukaryotes and in glycolipid antigens of protozoa and fungi. The epitopes recognized by the anti-carbohydrate MoAbs range from one sugar unit up to ten sugar units. Although most anti-carbohydrate MoAbs are directed predominantly toward terminal sugar residues, a few MoAbs are also reactive with internal sugar residues. The fine structure of the carbohydrate epitopes has been chemically defined by [1H] NMR, GC/MS of alditol acetates of partially permethylated compounds, -FAB/MS, degradation with exoglycosidases and inhibition with different methyl-glycosides and oligosaccharides.

Toledo Ms

Papers

Immunochemical characterization of carbohydrate antigens from fungi, protozoa and mammals by monoclonal antibodies directed to glycan epitopes.