Structural characterization of novel oligosaccharides of cell-surface glycoproteins of Trypanosoma cruzi

Affinity-purified glycopeptides were prepared from Trypanosomacruzi using the carbohydrate-specific monoclonal antibody WIC29.26.These glycopeptides contain rhamnose, fucose, xylose, and galactose,in the ratio 1:1:2:3. A series of oligosaccharides was releasedfrom the glycopeptides by mild acid hydrolysis, while, in contrast,no oligosaccharides were released by either peptide N-glycosidaseF or conventional base-catalyzed ß-elimination andreduction. This suggested the presence of a phosphodiester linkagebetween the carbohydrate and peptide, which was further supportedby the detection of phosphothreonine in the glycopeptides. Themild acid liberated (MAL) fraction was resolved into two majoracidic oligosaccharides (MAL-P1 and MAL-P2), two minor neutraloligosaccharides (MAL P1b and MAL-P2b) and a neutral fraction(MAL-N1), consisting of Gal and Xyl monosaccharides. The MAL-P1and MAL-P2 oligosaccharides proved to be hexa- and heptasaccharidesthat shared a common xylose reducing terminus, but differedby one galactofuranose residue, and their negative charge wasshown to be due to the presence of cyclic-phosphate attachedto nonreducing terminal galactofuranose residues. The MAL-P1band MAL-P2b oligosaccharides appeared to be nonphosphorylatedversions of MAL-P1 and MAL-P2. Partial structures of MAL-P1and MAL-P2 are suggested, based on compositional analyses, electrospraymass spectrometry, and tandem mass spectrometry before and afterpermethylation. The origin and significance of these uniquetrypanosomatid glycoconjugates is discussed. glycoprotein monoclonal antibody oligosaccharide structure Trapanosoma cruzi