1NUPEB - UFOP- MG;2Laboratório de Imunoparasitologia-NUPEB-UFOP;3IQ-USP-SP;4DBB-UVF-MG

Leishmania major has two mapped apyrase genes in its genome (NTPDase and GDPase). Apyrase function,characterized as tri and di-nucleotide hydrolysis, usually insensitive to ATPase inhibitors, were previously demonstrated in intact L. amazonensis, L. braziliensis and L. major cells. The very distinct ecto-nucleotidase capacity between Leishmania species suggests its involvement with virulence and control of host-immune responses. In order to evaluate if a molecular difference could explain these data we amplified both genes from these 3 species and cloned in TOPO amplification-vector in order to sequence. The identity of isolated genes was evaluated by automated DNA sequencing and BlastX analysis. To perform further evaluations isolated full-apyrase genes were cloned into expression vectors (pET21b and pYES-CT). In silico molecular analysis between L. major genes showed only one possible transmembrane region in amino-terminal domain of both putative proteins. Furthermore, possible GDPase has a signal peptide at amino acid position 45 (predicted by SIGNAL P program), suggesting a possible soluble excreted protein. On the other hand, the putative N-terminal transmembrane domain in NTPDase and the presence of ecto-apyrase activity on live cells could be suggestive of an ecto-membrane localization. Despite both predicted proteins have all Apyrase Conserved Regions, align between the same revealed a very low identity (19,9%). Analysis of partial genes and deduced proteins from L amazonensis and L. braziliensis showed 100% identity at both levels (DNA and protein) with L. major isoforms, suggesting that the different ecto-nucleotidase capacity could not be explained by only this molecular approach. Confirmation of these by determination of full genes sequences, analysis of expression profiles and cellular localization will be the next steps in this investigation. Furthermore, the L. major`s GDPase and NTPDase heterologous expression and purification had been performed and biochemical characterizations of these proteins could elucidate the real role of these apyrases in Leishmania ecto-nucleotidase capacity.

Supported by: UFOP, FAPEMIG. CNPq and MEC