Caracterização molecular e imunológica da nucleosídeo trifosfato difosfohidrolase (NTPDASE 1) de Leishmania amazonensis e de seu domínio B

Abstract

By in silico analysis, an antigenic conserved B domain was previously identified within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. The r-potDomB, a recombinant belonging to the conserved B domain (r78- 117) from the potato apyrase, was obtained by heterologous expression, and its reactivity with polyclonal anti-potato apyrase antibodies confirmed the existence of epitopes which induce humoral immune response in mammalian. The cloning of the gene from the Leishmania amazonensisNTPDase 1 (LamNTPDase1; NCBI AFJ22627.1) and in silico analysis reinforced the hypothesis of B-domain conservation within NTPDases. The r-potDomB, synthetic peptides LbB1LJ and LbB2LJ derived from the B-domain from LeishmaniaNTPDase 1, and the antibodies raised against these biomolecules, were used as molecular tools for specific studies of the L. amazonensisNTPDase 1 and leishmaniasis. By Western blots, theNTPDase 1 was identified as bands of 48 and 63 kDa in membrane, microsomal and flagellar fractions of promastigotes. By ultrastructural immunocytochemical, the antibodies localized the NTPDase 1 at the surface of plasma membrane, nucleus, mitochondria and kinetoplast, at flagellar pocket and flagellum, confirming its widespread distribution in this parasite. In silico analysis suggested possible post-translational modifications of the NTPDase 1, including its conjugation with SUMO protein. The polypeptides of 48 and 63 kDa and one co-migrant of 12 kDa were isolated of promastigotes by non-denaturing gel electrophoresis. By Western blots and mass spectrometry, the identity of NTPDase1 in the polypeptides of 48 e 63 kDa was confirmed. By mass spectrometry, the polypeptide of 12 kDa was identified belonging to the SUMO protein family and, also, added to the polypeptide of 63 kDa. The polypeptides of 63 and 12 kDa, but not 48 kDa, were recognized in Western blots by immune serum anti-SUMO1, suggesting binding between NTPDase 1 and SUMO, a modification described for the first time among members of the NTPDase family. BALB/c mice were infected with promastigotes, and the antibodies from these animals recognized the pure NTPDase, but not SUMO, confirming its antigenicity. During disease progression, high reactivity between r-potDomB, LbB1LJ or LbB2LJ and IgG2a antibodies of the promastigote-infected mice was observed at 40 days post-infection, revealing both the B-domain antigenicity and its ability for induce the production of this subtype in early disease stages. The IgG1 reactivity was significantly higher at 90-120 days post-infection, coinciding with the most active stage of the disease, suggesting participation of the domain in immuneregulatory events. The r-potDomB induced delayed type-hypersensitivity reaction in Swiss mice, a cellular immune response, with a concomitant increase in the levels of IgG2a and IFN-y. The r-potDomB, in the absence of adjuvant, used in pre-immunization of amastigotes-infected Swiss mice, stimulated the production of IgG2a and IFN-y, and promoted partial protection against leishmaniasis progression, as observed by measuring of footpad swelling and histopathological analysis. The results stimulate biotechnological use of the r-potDomB, or derivatives of this biomolecule, in formulations and experimental protocols of immunotherapy and/or vaccination against leishmaniasis.