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 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.
Debrabant
A
A
Cell Biology Section, Laboratory of Parasitic Diseases, Division of Intramural Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425, USA.
Ghedin
E
E
Dwyer
D M
DM
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008164:Luminescent Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009708:Nucleotidases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D008164:Luminescent Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009708:Nucleotidases; C040470:3'-nucleotidase