Trypanosoma cruzi, the agent of Chagas disease contains a major cysteine proteinase, cruzipain (Cz), with an unusual carboxyl-terminal extension (C-T). We have previously reported the presence of sulfate groups in the N-linked oligosaccharide chains of this domain. In order to evaluate the immune responses to sulfated moieties on Cz, 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. Interestingly, the abolishment of IgG2b reactivity when desulfated antigens were used as immunogens demonstrates that esterified sulfate groups are absolutely required for eliciting IgG2b response to Cz. Sera from chronically T. cruzi-infected subjects with mild disease displayed higher levels of total IgG and IgG2 antibodies specific for sulfated epitopes compared with those in more severe forms of the disease. 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 in the absence of infection elicited ultrastructural abnormalities in heart tissue. Surprisingly, hearts from sulfate-depleted C-T-immunized mice did not present pathological alterations. This is the first report showing that sulfate-bearing glycoproteins from trypanosomatids are able to elicit specific humoral and cellular immune responses and appeared to be involved in the generation of heart tissue damage. These results represent a further step in the understanding of the role of Cz in the course of T. cruzi infection.

Composition-gradient multi-angle static light scattering (CG-MALS) is an emerging technique for the determination of intermolecular interactions via the second virial coefficient B22. With CG-MALS, detailed studies of the second virial coefficient can be carried out more accurately and effectively than with traditional methods. In addition, automated mixing, delivery and measurement enable high speed, continuous, fluctuation-free sample delivery and accurate results. Using CG-MALS we measure the second virial coefficient of bovine serum albumin (BSA) in aqueous solutions at various values of pH and ionic strength of a univalent salt (NaCl). The systematic variation of the second virial coefficient as a function of pH and NaCl strength reveals the net charge change and the isoelectric point of BSA under different solution conditions. The magnitude of the second virial coefficient decreases to 1.13 x 10(-5) ml*mol/g(2) near the isoelectric point of pH 4.6 and 25 mM NaCl. These results illuminate the role of fundamental long-range electrostatic and van der Waals forces in protein-protein interactions, specifically their dependence on pH and ionic strength.

Summary Sulphoglycosphingolipids, present on the surface of diverse cells, participate in the regulation of various cellular events. However, little is known about the structure and the role of sulphoglycosphingolipids in trypanosomatids. Herein, sulphated dihexosylceramide structures – composed mainly of sphingosine as the long chain base acylated with stearic acid – have been determined for the first time in Trypanosoma cruzi epimastigotes by UV‐MALDI‐TOF‐MS analysis. Interestingly, inhibition ELISA assays using cruzipain as antigen and polyclonal rabbit antibodies specific for cruzipain, the major cysteine proteinase of T. cruzi, or for its C‐terminal domain, have demonstrated (i) that sulphate epitopes are shared between cruzipain and sulphatides of T. cruzi, (ii) that cross‐reactivity maps to the C‐terminal domain and (iii) the existence of other antigenic determinants in the glycolipidic structures. These features provide evidence that sulphate groups are antigenic in sulphate‐containing parasite glycoconjugates. Furthermore, IgG2 antibody levels inversely correlate with disease severity in chronic Chagas disease patients, suggesting that IgG2 antibodies specific for sulphated epitopes might be associated with protective immunity and might be considered as potential surrogates of the course of chronic Chagas disease.