Molecular and Cellular Biochemistry

, Volume 341, Issue 1–2, pp 17–31 | Cite as

Identification, characterization, and expression of a unique secretory lipase from the human pathogen Leishmania donovani

  • Alison M. Shakarian
  • Glen C. McGugan
  • Manju B. Joshi
  • Mary Stromberg
  • Lauren Bowers
  • Christine Ganim
  • Jessica Barowski
  • Dennis M. Dwyer
Article
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Abstract

Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a ~33 kDa protein, with a well-conserved substrate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3::HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immuno-fluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3::HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.

Keywords

Leishmania Human parasite Gene structure Trypanosomatid Kinetoplastid protozoan Lipase 

Abbreviations

aa

Amino acid

Ab

Antibody

bp

Base pair

DIG

Digoxigenin

FBS

Fetal bovine serum

gDNA

Genomic DNA

HA

Hemagglutinin

LdLip3

Gene encoding the secretory lipase of Leishmania donovani

4MU

4-Methylumbelliferone

nt

Nucleotide

oligo

Oligodeoxy-ribonucleotide

ORF

Open reading frame

PBS

Phosphate buffered saline

PCR

Polymerase chain reaction

RT

Reverse transcription

SDS-PAGE

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis

SP

Signal peptide

SL

Spliced leader

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Notes

Acknowledgments

Dr. Alison Shakarian was supported in part by RI-INBRE Grant # P20RR016457 from the NCRR, NIH. This study was supported in part by the Intramural Research Program of the Division of Intramural Research, NIAID, NIH. Dr. Glen McGugan was supported by an Intramural Research Training Award at LPD/NIAID/NIH. We thank Dr. Greg Matlashewski (McGill University) for providing the pKSNEO leishmanial expression vector. The contents of this document are solely the responsibility of the authors and do not necessarily represent the official views of NCRR, or NIH.

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Copyright information

© US Government 2010

Authors and Affiliations

  • Alison M. Shakarian
    • 1
  • Glen C. McGugan
    • 2
  • Manju B. Joshi
    • 2
  • Mary Stromberg
    • 1
  • Lauren Bowers
    • 1
  • Christine Ganim
    • 1
  • Jessica Barowski
    • 1
  • Dennis M. Dwyer
    • 2
  1. 1.The Department of Biology and Biomedical SciencesSalve Regina UniversityNewportUSA
  2. 2.The Cell Biology Section, Laboratory of Parasitic Diseases, Division of Intramural ResearchNIAID, NIHBethesdaUSA

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