α-Galactosyl residues on Trypanosoma cruzi trypomastigote glycoconjugates contribute to host-cell invasion via Galectin-3 and Annexin A2-S100A10 complex.

Linear, nonreducing terminal a-galactopyranosyl (a-Gal) epitopes are displayed on the major surface glycosylphosphatidylinositol (GPI)-anchored glycoconjugates of infective Trypanosoma cruzi trypomastigotes, the agent of Chagas disease (CD). These immunodominant epitopes elicit abundant (3-5% of circulating IgG), trypanolytic CD anti-a-Gal antibodies, yet their role on the parasite surface remains elusive. Here, affinity purification of macrophage extracts on the immobilized Galα1-3Galβ1-4GlcNAcβ (Galili's aGal epitope) identified Galectin-3 (Gal-3) and the annexin A2-S100A10 heterotetramer (A2t) as candidate host-cell receptors, whose specificities were mapped against glycoconjugates and parasite-derived molecules. Gal-3 bound a-Gal-bearing neoglycoproteins (α-Gal-NGPs), the glycosphingolipid (GSL) iGb3 (Galα1-3Galβ1-4Glcβ1-1-Cer), trypomastigote-derived GPI-mucins (tGPI-mucins), and glycoinositolphospholipids (tGIPLs).  α-Galactosidase abolished Gal-3 binding to trypomastigotes; epimastigote binding was reduced only by β-galactosidase. Conversely, A2t bound galactosylated GSLs in calcium-dependent manner, most strongly Gb4 (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1-Cer), but not α-Gal-NGPs, and only two of 442 glycans on a printed array gave signals above background, indicating lipid-dependent recognition. A2t recognized the phospholipid-enriched fractions of both developmental stages equally, the tGIPL fraction of trypomastigotes only, and not tGPI-mucins. Antibody blockade of surface Anx2, S100A10, or Gal-3, competition with the soluble proteins, and RNA interference reduced macrophage infection by 29 to 50%, about 53%, and 42 to 59%, respectively. Terminal a-Gal epitopes on major trypomastigote GPI-anchored glycoconjugates therefore contribute to host-cell invasion, chiefly through Gal-3, whereas A2t engages a broader set of lipid-associated ligands; both contribute to a redundant entry repertoire rather than acting as obligate receptors. These interactions may offer targets for vaccine and chemotherapeutic development and serve as diagnostic and prognostic biomarkers in CD.