Relevance of sulfated sugars as main components of cruzipain, the major cysteine proteinase of Trypanosoma cruzi in the molecule antigenicity

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

Córdoba-Argentina

Congreso; XXIV REUNION Científica ANUAL SOC. ARG.de Protozoología; 2010

SOC. ARG.de Protozoología

Trypanosoma cruzi, causative agent of Chagas disease, contains a major cysteine proteinase, cruzipain (Cz). The presence of sulfated high-mannose type oligosaccharides in its C-terminal domain was identified as a new striking feature of this molecule [Febs J., 272, 3803-3815 (2005)], responsible for most antibodies in natural and experimental. infections. With the aim of studiung the influence of type, amount, and localization of the anionic charged group in the oligosaccharide structure involved in the immune response of cruzipain molecule mice sera specific for Cz and C-T were used as tools and anionic charged poli, oligo, di and monosacharides and/or glycopeptides with different structures using heparin as source were obtained and tested, concluding that 1) Although carbohydrates bearing carboxylate or phosphate groups cross react with Cz, our results show a clear preference for sulfated sugars. 2) The reactivity increased concomitantly with the increase of sulfate groups in the sugar antigen used. 3) Interestingly, heparin was not reactive, although fractions obtained from partially degraded heparine were weakly recognized, Maltotetraose, a neutral oligo-saccharide was used as negative control. In all cases recognition was abolished when  anti-desulfated Cz sera, was used.                 In order to determine the involvement of the different anionic charged structures obtained in the antigenic  properties of cruzipain, rabbit sera specific for Cz and C-T prior and after desulfation treatment were used as tools for ELISA inhibition assays post adsorption with different sulfated and non-sulfated molecules. In the case of direct immune recognition, sulfated and non sulfated molecules were coupled to BSA to prepare new conjugates containing GlcNAc, GlcN6S, GlcNS, GlcNAc, and uronic acids. The sugar/protein ratio in the conjugate was determined by UV-MALDI-TOF MS.  Getting deeper, rabbit serum specific for C-T was able to discriminate between positions  2 and 6 of the sulfate sustituent of GlcN obtaining the highest reactivity with GlcN6S. Structures containing phosphates and carboxylates were also recognized although in lower degree. Anti Cz sera did not allow to discriminate the position 2 or 6 of the sulfate group present in the Glucosamine unit by dot assay but preference for 6 position was observed by ELISA inhibition assays. However these assays showed the highest cross reactivity for NAcGlc 6SO4, in accordance with the putative structure present in natural Cz. These results were confirmed by using anti C-T sera. It is woth noting that sera from mice immunized with Cz with or without desulfation treatment showed absence of cross-reactivity between cruzipain and heparin and other sulfated glycoproteins (FSH/LH mix). Ongoing assays using specific anti-sulfate antibodies will help to elucidate the involvement of sulfates in the antigenicity and cross-reactivity of the molecule and/or in the immunopathology of Chagas disease.