Sera of patients with chronic Chagas' disease (American trypanosomiasis) contain elevated levels of anti-alpha-galactosyl antibodies that are lytic to Trypanosoma cruzi. The T. cruzi trypomastigote F2/3 antigen complex recognized by these antibodies runs as a broad smear on SDS/PAGE [Almeida, Krautz, Krettli and Travassos (1993) J. Clin. Lab. Anal. 7, 307-316]. Treatment of T. cruzi trypomastigote cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) abolished most of their reactivity to chronic Chagas'-disease ((Chagasic, Ch) anti-alpha-galactosyl antibodies (anti-Gal). The F2/3 antigen complex, purified by solvent extraction and hydrophobic-interaction chromatography, contained 60% carbohydrate by weight and substantial amounts of Thr, Ser, Glx, Asx, Gly, Ala and Pro, but relatively few hydrophobic amino acids. The presence of myoinositol, ethanolamine and 1-O-hexadecylglycerol suggested the presence of glycosyl-phosphatidylinositol membrane anchors. This was confirmed by PI-PLC treatment, which rendered the F2/3 molecules hydrophilic and reactive to anti-(cross-reacting determinant) antibodies. The majority of the GlcNAc content of the F2/3 antigens was found at the reducing termini of oligosaccharides in O-glycosidic linkage to Thr residues. These O-linked oligosaccharides could be released by beta-elimination and by mild hydrazinolysis. The smallest released oligosaccharitol that was reactive with the Ch anti-Gal was Gal alpha 1-3Gal beta 1-4GlcNAcol (where GlcNAcol is N-acetyl-glucosaminitol). Several other Gal-containing oligosaccharitols were observed, most of which were branched and contained 4,6-di-O-substituted GlcNAcol at their reducing termini. About half of the total released oligosaccharitols could bind to immobilized Ch anti-Gal, but none of them bound to the anti-Gal isolated from normal human sera. These data suggest that the specificities of the Ch anti-Gal are quite different from the natural anti-Gal isolated from normal human sera. Therefore, these novel T. cruzi O-linked oligosaccharides are highly immunogenic under the conditions of natural infection and are the targets for lytic Ch anti-Gal.

The major acceptors of sialic acid on the surface of metacyclic trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to the parasite membrane via glycosylphosphatidylinositol anchors. Here we have compared the lipid and the carbohydrate structure of the glycosylphosphatidylinositol anchors and the O-linked oligosaccharides of the mucins isolated from metacyclic trypomastigotes and noninfective epimastigote forms obtained in culture. The single difference found was in the lipid structure. While the phosphatidylinositol moiety of the epimastigote mucins contains mainly 1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol, the phosphatidylinositol moiety of the metacyclic trypomastigote mucins contains mostly (70%) inositol phosphoceramides, consisting of a C sphinganine long chain base and mainly C and C fatty acids. The remaining 30% of the metacyclic phosphatidylinositol moieties are the same alkylacylphosphatidylinositol species found in epimastigotes. In contrast, the glycosylphosphatidylinositol glycan cores of both molecules are very similar, mainly Manα1-2Manα1-2Manα1-6Manα1-4GlcN. The glycans are substituted at the GlcN residue and at the third αMan distal to the GlcN residue by ethanolamine phosphate or 2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote mucin-like molecules, released by β-elimination with concomitant reduction, are identical to the structures reported for the epimastigote mucins (Previato, J. O., Jones, C., Gon¸alves, L. P. B., Wait, R., Travassos, L. R., and Mendo¸a-Previato, L.(1994) Biochem. J. 301, 151-159). In addition, a significant amount of nonsubstituted N-acetylglucosaminitol was released from the mucins of both forms of the parasite. Taken together, these results indicate that when epimastigotes transform into infective metacyclic trypomastigotes, the phosphatidylinositol moiety of the glycosylphosphatidylinositol anchor of the major acceptor of sialic acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged.