Relevance of sulfated oligosaccharides as main components of cruzipain, the major cysteine proteinase of Trypanosoma cruzi

D.M. ACOSTA; L.L. SOPRANO; M LANDONI,; M. POURCELOT; ESTEVA M. I.; J. KOVENSKY; A. S. COUTO; DUSCHAK, V.G.;

Buzios

Congreso; XIII International Congress of Protistology; XXV Annual Meeting of the Brazilian Society of Protozoology; XXXVI Annual Meeting on Basic Research in Chagas’Disease; 2009

Trypanosoma cruzi contains a major cysteine proteinase, cruzipain (Cz). This lysosomal enzyme bears an unusual C-terminal domain (C-T) that contains a number of post-translational modifications and is responsible for most antibodies in natural and experimental infections. UV-MALDI-TOF MS analysis, allowed us to identify the presence of sulfated high-mannose type oligosaccharides in the C-T as a new striking feature of this molecule. In order to evaluate the immune responses to sulfated moieties on Cz,  the involvement of anionic charged structures in the immune recognition of sulfated glycoproteins, and /or in the crossreactivity between myosin and cruzipain, BALB/c mice were immunized with purified Cz and C-T prior and after desulfation treatment. The humoral immune response to sulfates on Cz or C-T was mainly IgG2b. This reactivity was abolished when desulfated antigens were used as immunogens showing that sulfates are absolutely required for eliciting IgG2b response to Cz. A significant reduction of C-T-specific delayed-type hypersensitivity reaction in C-T-immunized mice was observed when desulfated C-T was challenged, suggesting the involvement of sulfate groups in the generation of memory T-cell responses. Moreover, immunization with C-T elicited ultrastructural abnormalities in heart tissue. Surprisingly, pathological alterations were not observed in hearts from sulfate depleted-C-T-immunized mice and a lower labelling was observed by immunoelectron microscopy using myosin-adsorbed specific policlonal anti-Cz serum. In order  to study the involvement of sulfates in immunerecognition, different sulfated/desulfated molecules were coupled to BSA and preliminar dot assays showed a growing recognition to sulfated structures with specific anti-Cz serum: SO42-/COO- = 1(+++), 0.67 (++), 0.4 (+), 0 (-). Our results highlight the relevance of sulfated groups as main components of this molecule. Ongoing assays using specific anti-sulfate antibodies will help to elucidate the involvement of sulfates in the antigenicity and crossreactivity of the molecule and/or in the immunopathology of Chagas disease.