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

• Published in: Molecular and cellular biochemistry Vol. 341; no. 1-2; pp. 17 - 31
• Main Authors: Shakarian, Alison M, McGugan, Glen C, Joshi, Manju B, Stromberg, Mary, Bowers, Lauren, Ganim, Christine, Barowski, Jessica, Dwyer, Dennis M
• Format: Journal Article
• Language: English
• Published: Boston Boston : Springer US 01-08-2010; Springer US; Springer; Springer Nature B.V
• Subjects: Animals; Biochemistry; Biomedical and Life Sciences; Cardiology; Enzymatic activity; Enzymes; Gene expression; Gene structure; Genes, Protozoan; Genomics; Human parasite; Humans; Kinetoplastid protozoan; Leishmania; Leishmania donovani; Leishmania donovani - enzymology; Life Cycle Stages; Life cycles; Life Sciences; Lipase; Lipase - chemistry; Lipase - genetics; Lipase - physiology; Lipids; Medical Biochemistry; Oncology; Parasites; Parasitic protozoa; Pathogens; Physiological aspects; Recombinant Fusion Proteins - metabolism; RNA; RNA, Messenger - biosynthesis; Transfection; triacylglycerol lipase; Trypanosomatid; Kinetoplastid protozoan; Trypanosomatid; Lipase; Human parasite; Gene structure
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1007/s11010-010-0433-6

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.