Eukaryotic Cell
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Eukaryotic Cell, October 2007, p. 1745-1757, Vol. 6, No. 10
1535-9778/07/$08.00+0     doi:10.1128/EC.00123-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania{triangledown}

Nancy Lee, Sreenivas Gannavaram, Angamuthu Selvapandiyan, and Alain Debrabant*

Laboratory of Bacterial, Parasitic and Unconventional Agents, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland

Received 14 April 2007/ Accepted 7 August 2007

In this report, we have characterized two metacaspases of Leishmania donovani, L. donovani metacaspase-1 (LdMC1) and LdMC2. These two proteins show 98% homology with each other, and both contain a characteristic C-terminal proline-rich domain. Both genes are transcribed in promastigotes and axenic amastigotes of L. donovani; however, LdMC1 shows increased mRNA levels in axenic amastigotes. An anti-LdMC antibody was obtained and showed reactivity with a single ~42-kDa protein band in both promastigote and axenic amastigote parasite whole-cell lysates by Western blotting. Pulse-chase experiments suggest that LdMCs are not synthesized as proenzymes, and immunofluorescence studies show that LdMCs are associated with the acidocalcisome compartments of L. donovani. Enzymatic assays of immunoprecipitated LdMCs show that native LdMCs efficiently cleave trypsin substrates and are unable to cleave caspase-specific substrates. Consistently, LdMC activity is insensitive to caspase inhibitors and is efficiently inhibited by trypsin inhibitors, such as leupeptin, antipain, and N{alpha}-tosyl-L-lysine-chloromethyl ketone (TLCK). In addition, our results show that LdMC activity was induced in parasites treated with hydrogen peroxide, a known trigger of programmed cell death (PCD) in Leishmania and that parasites overexpressing metacaspases are more sensitive to hydrogen peroxide-induced PCD. These findings suggest that Leishmania metacaspases are not responsible for the caspase-like activities reported in this organism and suggest a possible role for LdMCs as effector molecules in Leishmania PCD.

* Corresponding author. Mailing address: CBER/FDA, 29 Lincoln Drive, Bldg. 29, Rm. 425, HFM-310, Bethesda, MD 20892. Phone: (301) 496-1652. Fax: (301) 480-7928. E-mail: alain.debrabant{at}fda.hhs.gov

{triangledown} Published ahead of print on 22 August 2007.

Eukaryotic Cell, October 2007, p. 1745-1757, Vol. 6, No. 10
1535-9778/07/$08.00+0     doi:10.1128/EC.00123-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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