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 elicit 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 encoding the P-4 membrane protein was cloned and sequenced. Southern blot analyses 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 phosphomonoesterase activity, with a preference for UMP > TMP > AMP >> CMP, Double-labeling immunofluorescence microscopic analyses employing anti-binding protein antibodies revealed that the P-4 protein is localized in the endoplasmic reticulum of the amastigote. Northern blot analyses indicated that the gene is selectively 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, possible roles of the P-4 nuclease in the amastigote in RNA stability (gene expression) or DNA repair are discussed.