The immunologically protective P-4 antigen of Leishmania amastigotes - A developmentally regulated single strand-specific nuclease associated with the endoplasmic reticulum

The purified membrane-associated Leishmania pifanoi amastigote protein P-4 has been shown to induce protective immunity against infection and to elici t preferentially a T helper 1-like response in peripheral blood mononuclear cells of patients with American cutaneous leishmaniasis. As this molecule is potentially important for future vaccine studies, the L. pifanoi gene en coding the P-4 membrane protein was cloned and sequenced. Southern blot ana lyses indicate the presence of six tandemly arrayed copies of the P-4 gene in L. pifanoi; homologues of the P-4 gene are found in all other species of the genus Leishmania examined. DNA-derived protein sequence data indicated an identity to the Pi zinc-dependent nuclease of Penicillium citrinum (20. 8%) and the C-terminal domain of the 3' nucleotidase of Leishmania donovani (33.7%), Consistent with these sequence analyses, purified L. pifanoi P-4 protein possesses single strand nuclease (DNA and RNA) and phosphomonoester ase activity, with a preference for UMP > TMP > AMP >> CMP, Double-labeling immunofluorescence microscopic analyses employing anti-binding protein ant ibodies revealed that the P-4 protein is localized in the endoplasmic retic ulum of the amastigote. Northern blot analyses indicated that the gene is s electively expressed in the intracellular amastigote stage (mammalian host) but not in the promastigote stage (insect) of the parasite. Based upon its subcellular localization and single-stranded specific nuclease activity, p ossible roles of the P-4 nuclease in the amastigote in RNA stability (gene expression) or DNA repair are discussed.