Abstract
Class I nucleases are a family of enzymes that specifically hydrolyze single-stranded nucleic acids. Recently, we characterized the gene encoding a new member of this family, the 3'-nucleotidase/nuclease (Ld3'NT/NU) of the parasitic 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. Currently, we used a homologous episomal expression system to dissect the functional domains of the Ld3'NT/NU. Our results showed that its N-terminal signal peptide targeted this protein into the endoplasmic reticulum. Using Ld3'NT/NU-green fluorescent protein chimeras, we showed that the C-terminal domain 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, deletion of its single N-linked glycosylation site showed that such glycosylation was not required for the enzymatic functions of the Ld3'NT/NU. Thus, using the fidelity of a homologous expression system, we have defined some of the functional domains of this unique member of the class I nuclease family.
Highlights
§ National Institutes of Health Visiting Fellow
To confirm that the cloned Ld3ЈNT/NU gene product was targeted to the cell surface membrane of L. donovani, the nucleotide sequence corresponding to the deduced protein shown in Fig
L. donovani wild type (WT) and C3PO promastigotes transfected with the resulting [pKS NEO 3ЈNT/NU] plasmid were grown under increasing concentrations of G418 up to 250 g/ml
Summary
Ld3ЈNT/NU, L. donovani 3Ј-nucleotidase/nuclease; PBS, phosphate-buffered saline; SP, signal peptide; PCR, polymerase chain reaction; kb, kilobase; GFP, green fluorescent protein; TM, transmembrane domain; ConA, concanavalin A; PAGE, polyof host-derived purines (1). Whereas genes for several new members of this enzyme family have recently been identified from various plants and a proteobacterium (6, 7) based on their deduced amino acid sequence homology with the fungal nucleases, the biochemical properties of these nonfungal nucleases remain to be characterized. Within this class I nuclease family, the leishmanial enzyme is the only one to have been characterized as a cell surface membrane-anchored protein. These domains included: 1) the N-terminal signal peptide for targeting this enzyme to the endoplasmic reticulum, 2) the C-terminal putative transmembrane domain and its role in anchoring/targeting this enzyme into the parasite cell surface, and 3) the N-linked glycosylation site and its role in enzyme activity
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