Homologous Expression of Glycosylphosphatidylinositol-Anchored Glycoproteins in Trypanosoma cruzi.

The surface coat of Trypanosoma cruzi is covered with glycosylphosphatidylinositol (GPI)-anchored glycoproteins (GAGPs) that contribute to parasite protection and to the establishment of a persistent infection in both the insect vector and the mammalian host. Multiple GAGPs that vary by amino acid sequence and/or posttranslational modifications are co-expressed on the parasite surface coat, hence curtailing structural/functional analyses on these molecules. Studies in our lab have indicated that GAGP-tagged variants expressed by transfected parasites undergo analogous posttranslational processing than endogenous ones and therefore constitute suitable tools to overcome these limitations. In this chapter, we detail the entire methodological pipeline for the efficient homologous expression of GAGPs in T. cruzi: from a simple strategy for the simultaneously cloning and tagging of the gene of interest to the biochemical validation of the parasite-expressed product.

Synthesis and characterization of α-d-Galp-(1 → 3)-ß-d-Galp epitope-containing neoglycoconjugates for chagas disease serodiagnosis.

The immunodominant epitope α-d-Galp-(1 → 3)-ß-d-Galp-(1 → 4)-d-GlcNAc, expressed in the mucins of the infective trypomastigote stage of Trypanosoma cruzi has been proposed for multiple clinical applications, from serodiagnosis of protozoan caused diseases to xenotransplantation or cancer vaccinology. It was previously shown that the analogue trisaccharide, with glucose in the reducing end instead of GlcNAc, was as efficient as the natural trisaccharide for recognition of chagasic antibodies. Here we describe the synthesis of α-d-Galp-(1 → 3)-ß-d-Galp-(1 → 4)-d-Glcp functionalized as the 6-aminohexyl glycoside and its conjugation to BSA using the squarate method. The conjugate of 6-aminohexyl α-d-Galp-(1 → 3)-ß-d-Galp was also prepared. Both neoglycoconjugates were recognized by serum samples of Trypanosoma cruzi-infected individuals and thus, are promising tools for the improvement of Chagas disease diagnostic applications.