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

Alain Debrabant, Elodie Ghedin, Dennis M. Dwyer

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)16366-16372
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number21
DOIs
StatePublished - May 26 2000

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3'-nucleotidase
Dissection
Leishmania
Glycosylation
Membrane Proteins
Cell Membrane
Membranes
Leishmania donovani
Enzymes
Protein Sorting Signals
Green Fluorescent Proteins
Endoplasmic Reticulum
Nucleic Acids
Parasites
Proteins
Gene encoding
Anchors
Genes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Dissection of the functional domains of the Leishmania surface membrane 3'-nucleotidase/nuclease, a unique member of the class I nuclease family. / Debrabant, Alain; Ghedin, Elodie; Dwyer, Dennis M.

In: Journal of Biological Chemistry, Vol. 275, No. 21, 26.05.2000, p. 16366-16372.

Research output: Contribution to journalArticle

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