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.

Single units of O-linked N-acetylglucosamine (GlcNAc), usually components of nuclear and cytoplasmatic proteins, are present at the C-terminal domain of cruzipain (Cz), a lysosomal major antigen from Trypanosoma cruzi. On the other hand, antibodies directed against some self-antigens like myosin are associated with Chagas heart disease. The participation of O-GlcNAc moieties in the molecular antigenicity of Cz was determined using GlcNAc linked to aprotinin by ELISA. The immune cross-reactivity between Cz and myosin is mainly focused in the C-T domain. ELISA inhibition assays using rabbit sera specific for Cz and C-T in conjunction with immune-gold electron microscopy analysis of heart tissues from mice immunized with C-T confronted with polyclonal rabbit sera specific for Cz and C-T prior and after myosin adsorption provided evidence which indicates that O-GlcNAc moieties constitute a common epitope between Cz and either myosin or other cardiac O-GlcNAc-containing proteins, showing a new insight into the molecular immune pathogenesis of Chagas heart disease.

Cruzipain (Cz), the major cysteine proteinase of Trypanosoma cruzi, is a glycoprotein that contains sulfated high‐mannose‐type oligosaccharides. We have previously determined that these sulfate groups are targets of specific immune responses. In order to evaluate the structural requirements for antibody recognition of Cz, a systematic structure–activity study of the chemical characteristics needed for antibody binding to the Cz sulfated epitope was performed by immunoassays. With this aim, different synthesized molecules were coupled to the proteins BSA and aprotinin and confronted with (a) mouse sera specific for Cz and its carboxy‐terminal (C‐T) domain, (b) antibodies raised in rabbits immunized with Cz and its C‐terminal domain and (c) IgGs purified from human Chagas disease sera. Our results indicate that a glucosamine containing an esterifying sulfate group in position O‐6 and an N‐acetyl group was the preferred epitope for the immune recognition of sera specific for Cz and its C‐T domain. Although to a minor extent, other anionic compounds bearing sulfate groups in different positions and number as well as different anionic charged groups including carboxylated or phosphorylated monosaccharides, disaccharides and oligosaccharides were recognized. In conclusion, we found that synthetic anionic sugar conjugates containing N‐acetyl d‐glucosamine‐6‐sulfate sodium salt (GlcNAc6S) competitively inhibit the binding of affinity purified rabbit anti‐C‐T IgG to the C‐T extension of Cz. Extending these findings to the context of natural infection, immune assays performed with Chagas disease serum confirmed that the structure of synthetic GlcNAc6S mimics the N‐glycan‐linked sulfated epitope displayed in the C‐T domain of Cz.