Dissection of the functional domains of the Leishmania surface membrane 3 '-nucleotidase/nuclease, a unique member of the class I nuclease family

Class I nucleases are a family of enzymes that specifically hydrolyze singl e-stranded nucleic acids. Recently, we characterized the gene encoding a ne w member of this family, the 3'-nucleotidase/nuclease (Ld3'NT/NU) of the pa rasitic protozoan Leishmania donovani. The Ld3'NT/NU is unique as it is the only class I nuclease that is a cell surface membrane-anchored protein. Cu rrently, we used a homologous episomal expression system to dissect the fun ctional domains of the Ld3'NT/NU. Our results showed that its N-terminal si gnal peptide targeted this protein into the endoplasmic reticulum, Using Ld 3'NT/NU-green fluorescent protein chimeras, we showed that the C-terminal d omain of the Ld3'NT/NU functioned to anchor this protein into the parasite cell surface membrane. Further, removal of the Ld3'NT/NU C-terminal domain resulted in its release/secretion as a fully active enzyme. Moreover, delet ion of its single N-linked glycosylation site showed that such glycosylatio n was not required for the enzymatic functions of the Ld3'NT/NU. Thus, usin g the fidelity of a homologous expression system, we have defined some of t he functional domains of this unique member of the class I nuclease family.