Sulfated oligosaccharides as main targets in cruzipain, the major cysteine proteinase of Trypanosoma cruzi

ACOSTA DM; SOPRANO LL; ESTEVA MI; KOVENSKY J; COUTO AS; DUSCHAK VG

Nurnberg-Alemania

Congreso; 2nd World Conference on Magic Bullets, Erlich II; 2008

Erlich II

Sulfated oligosaccharides as main targets in cruzipain, the major cysteine proteinase of Trypanosoma cruzi ACOSTA DM1, SOPRANO LL1, ESTEVA MI1, KOVENSKY J2, COUTO AS3 DUSCHAK VG1 1Inst Nac Parasitol “Dr Mario Fatala Chaben”, Ministerio de Salud, Argentina; Laboratoire des glucides, Universite Joules Verne, Amiens, France; 2CHIDECAR, FCEyN, UBA, Argentina. Background: Trypanosoma cruzi, the agent of Chagas disease contains a major cysteine proteinase, cruzipain (Cz). This lysosomal enzyme bears an unusual C-terminal domain (C-T) that contains post-translational modifications and most antibodies in natural and experimental infections are directed against it. Methods: To address the structure of the N-linked oligosaccharides present in the C-T domain, UV-MALDI-TOF mass spectrometry was used in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography. In order to evaluate the immune responses to sulfated moieties on Cz, and the involvement of anionic charged structures in the immune recognition of sulfated glycoproteins, BALB/c mice were immunized with purified Cz and C-T prior and after desulfation treatment. Results: The MALDI-TOF MS analysis allowed us to identify and characterize a new striking feature in cruzipain: sulfated high-mannose type oligosaccharides. The humoral immune response to sulfates on Cz or C-T was mainly IgG2b. IgG2b 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, hearts from sulfate-depleted C-T-immunized mice did not show pathological alterations. In contrast to anti-desulfated Cz mice serum, anti-Cz serum recognized sulfated poligalacturonic acid with relation So42-/COO- = 1(+++); 0.67 (++); 0.4 (+); 0 (-) and Glucose phosphate in a lower degree (+). Conclusions: We show for the first time 1) the presence of sulfated glycoproteins in Trypanosomatids; 2) that sulfates are able to elicit specific immune responses and appeared to be involved in the generation of heart tissue damage. 3) Our findings suggest that this effect could not be specifically due to sulfates but to anionic charged structures.