Identificação de um domínio conservado e antigênico de ATP difosfohidrolases de parasitos, também presente em uma isoforma ativa de Leishmania (Leishmania) chagasi

Abstract

By alignment of amino acid sequences, phylogenetic analysis and peptides prediction, we demonstrated evolutionary and closer structural relationships between a particular domain from the potato apyrase and several proteins from pathogenic organisms, all of them belonging to the ATP diphosphohydrolase family. A recombinant fragment based on this domain (rDomB) was obtained as a 6xHis fusion protein. By ELISA, significant IgG seropositivity for rDomB was observed in serum samples from patients (n=30) with schistosomiasis (37%) or tegumental leishmaniasis (40%), but not from Chagas disease patients (20%). In addition, inoculation of healthy Balb/c mice (n=9) with rDomB induced significant immunostimulatory ac¬tivity, increasing the amount of IgG antibody, and IgG1 and IgG2a subtypes, as observed by ELISA, suggesting that the rDomB has distinct epitopes capable of inducing Th1 and Th2-type immune responses. Therefore, a specific domain is suggested to be potentially involved in the immune response, and it also seems to be conserved during host and parasites co-evolution. A putative protein identified as NDPase, homologous to the potato apyrase, was found in the genome of Leishmania infantum (sin. L. (L.) chagasi), the causative agent of canine visceral leishmaniasis. An ATP diphosphohydrolase activity was then characterized in Leishmania (L.) chagasi promastigotes (MHOM/BR/1972/BH46 strain) preparations. The hydrolysis of di- or triphosphate nucleosides upon bivalent metal ion activation, non-significantly affected by other phosphohydrolases, associated to the inhibition promoted by sodium azide, suggested strongly an ATP diphosphohydrolase activity. Western blots developed with both rabbit polyclonal anti-potato apyrase antibodies or mouse polyclonal anti-rDomB recognized the same band of approximately 50 kDa. Anti-potato apyrase or anti-rDomB antibodies immobilized on Protein A-Sepharose depleted about 86% and 41%, respectively, for both ATPase and ADPase activities from a C12E9-solubilized promastigotes preparation. Both immunoprecipitated resin-rabbit antibody-antigen and resin-mouse antibody-antigen complexes were separated by SDS-PAGE, and Western blots developed respectively with mouse polyclonal anti-rDomB or rabbit polyclonal anti-potato apyrase antibodies identify the same 50 kDa band. In addition, anti-rDomB antibodies inhibited about 44% of the ATPase and ADPase activities from this preparation. By Western blots and ELISA, using potato apyrase as antigen, significant IgG antibody reactivity was observed in sera from dogs with visceral leishmaniasis (INF; n=38) domiciled in endemic area, suggesting that those shared epitopes are antigenic. The IgG antibody seropositivity from INF group (38%) for rDomB was elevated, and significantly (P<0.001) higher than that found in healthy dogs also domiciled in endemic area (n=18; 12%). The IgG antibody seropositivity for rDomB was significantly higher in dogs groups clinically classified as symptomatic (n=15; 67%) when compared to the asymptomatic (n=11; 18%) or oligosymptomatic (n=12; 18%) group. Individual analysis revealed variable IgG1 and IgG2 antibody reactivity for rDomB, suggesting that different antigenic epitopes could be involved in susceptibility or resistance to canine visceral leishmaniasis. In conclusion, our results confirmed that an active ATP diphosphohydrolase isoform is expressed in L. (L.) chagasi promastigote, and it has a conserved and antigenic domain shared with potato apyrase. Furthermore, we indicate the rDomB as a new biomolecule to be tested in protocols of studies of the canine visceral leishmaniasis.