Isolation and characterization of the gene encoding the surface membrane 3'-nucleotidase/nuclease of Leishmania donovani

Mol Biochem Parasitol. 1995 Apr;71(1):51-63. doi: 10.1016/0166-6851(95)00035-y.


Leishmania donovani and related trypanosomatid protozoa possess an externally oriented surface membrane enzyme capable of hydrolyzing both 3'-nucleotides and nucleic acids. By virtue of these activities, this 3'-nucleotidase/nuclease (3'-NT/Nu), previously shown to be analogous to fungal and plant class-I single-strand-specific nucleases, is thought to play a critical role in the salvage of purines, essential for the survival of these organisms. The 43-kDa 3'-NT/Nu was purified from L. donovani promastigotes and trypsin treated. Four of the released tryptic peptide fragments yielded amino-acid sequence information (Pept-1 to Pept-4) which provided the basis for the preparation of oligonucleotide primers used for PCR amplification of an approx. 300-bp DNA fragment. This fragment was cloned, sequenced and used to probe a genomic L. donovani cosmid library. Nucleotide sequence analysis of a 4.5-kb SmaI fragment, isolated from a cosmid clone, revealed an open reading frame (ORF) of 1434 nt encoding a 477-amino-acid protein. Pept-1 to Pept-4 were mapped onto the ORF-deduced protein sequence. Peptides corresponding to Pept-1 to Pept-4 were synthesized and used to immunize rabbits. The resulting anti-peptide antibodies recognized the 43-kDa protein on Western blots and immunoprecipitated the native 3'-nucleotidase activity from L. donovani membrane extracts. Further, the ORF-deduced protein shared significant sequence identity with the S1 and P1 fungal nucleases of Aspergillus oryzae and Penicillium citrinum, respectively. Cumulatively, these results demonstrated that the ORF corresponded to a gene for the L. donovani 3'-nucleotidase/nuclease. In Northern blots a nucleotide probe specific for the 3'-NT/Nu gene hybridized to a single 2.5-kb messenger RNA. Results of Southern blot analyses were consistent with the 3'-NT/Nu being encoded by a single copy gene. These data constitute the first report of the gene for this unique trypanosomatid surface membrane enzyme.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Membrane / enzymology*
  • Cloning, Molecular
  • DNA, Protozoan / analysis
  • Genes, Protozoan / genetics*
  • Leishmania donovani / enzymology
  • Leishmania donovani / genetics*
  • Molecular Sequence Data
  • Molecular Weight
  • Nucleotidases / chemistry
  • Nucleotidases / genetics*
  • Nucleotidases / isolation & purification
  • Open Reading Frames / genetics
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / immunology
  • RNA, Protozoan / analysis
  • Sequence Alignment
  • Sequence Analysis


  • DNA, Protozoan
  • Peptide Fragments
  • RNA, Protozoan
  • Nucleotidases
  • 3'-nucleotidase

Associated data

  • GENBANK/L35078