Scientific Publications by FDA Staff

Eukaryot Cell 2007 Oct;6(10):1745-57

Characterization of metacaspases with trypsin-like activity and their putative role in the programmed cell death in the protozoan parasite Leishmania.

Lee N, Gannavaram S, Selvapandiyan A, Debrabant A

Debrabant A (reprint author), CBER, FDA, 29 Lincoln Dr, Bldg 29 Rm 425,HFM-310, Bethesda, MD 20892 USA Ctr Biol Evaluat & Res, US FDA, Lab Bacter Parasit & Unconvent Agents, Div Emerging & Transfus Transmitted Dis, Bethesda, MD USA

In this report, we have characterized two metacaspases of Leishmania donovani 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 approximately 42 kDa protein band in both promastigote and axenic amastigote parasite whole cell lysates by western blot. Pulse-chase experiments suggest that LdMCs are not synthesized as pro-enzymes and immunofluorescence studies show that LdMCs are associated with the acidocalcisome compartments of L. donovani. Enzymatic assays of immunoprecipitated LdMCs show that native LdMCs cleave efficiently 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 TLCK. In addition, our results show that LdMC activity was induced in parasites treated with hydrogen peroxide, a known trigger of programmed cell death in Leishmania and that parasites overexpressing metacaspases and 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.