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The parasitic protozoan organism Trypanosoma cruzi is the causative agent of Chagas disease. The insect vector-dwelling epimastigote form of the organism expresses a low abundance glycoprotein associated with the flagellum adhesion zone, called gp72. The gp72 glycoprotein was first identified with an anti-carbohydrate IgG3 monoclonal antibody called WIC29.26 and has been shown to have an unusual sugar composition. Here, we describe a new way to isolate the WIC29.26 carbohydrate epitope of gp72. Using 1H NMR and mass spectrometry before and after derivatization, we provide an almost complete primary chemical structure for the epitope, which is that of a complex phosphosaccharide: Galfß1-4Rhapa1-2Fucpa1-4(Galpß1-3)(Galpa1-2)Xylpß1-4Xylpß1-3(Xylpß1-2Galpa1-4(Galpß1-3)(Rhapa1-2)Fucpa1-4)GlcNAcp, with phosphate attached to one or other of the two Galp terminal residues and in which all residues are of the d-absolute configuration, except for fucose and rhamnose which are l. Combined with previous data (Haynes, P. A., Ferguson, M. A., and Cross, G. A. (1996) Glycobiology 6, 869-878), we postulate that this complex structure and its variants lacking one or more residues are linked to Thr and Ser residues in gp72 via a phosphodiester linkage (GlcNAcpa1-P-Thr/Ser) and that these units may form phosphosaccharide repeats through GlcNAcpa1-P-Galp linkages. The gp72 glycoprotein is associated with the flagellum adhesion zone on the parasite surface, and its ligation has been implicated in inhibiting parasite differentiation from the epimastigote to the metacyclic trypomastigote stage. The detailed structure of the unique phosphosaccharide component of gp72 reported here provides a template for future biosynthetic and functional studies.
- Antibodies, Monoclonal, Murine-Derived
- Carbohydrate Conformation
- Protozoan Proteins
- Trypanosoma cruzi