A cysteine protease activity from Plasmodium falciparum cleaves human erythrocyte ankyrin

The malaria parasite Plasmodium falciparum undergoes distinct morphologic c hanges during its 48-h life cycle inside human red blood cells. Parasite pr oteinases appear to play important roles at all stages of the erythrocytic cycle of human malaria. Proteases involved in erythrocyte rupture and invas ion are possibly required to breakdown erythrocyte membrane skeleton. To id entify such proteases, soluble cytosolic extract of isolated trophozoites/s chizonts was incubated with erythrocyte membrane ghosts or spectrin-actin d epleted inside-out vesicles, which were then analyzed by SDS-PAGE. In both cases, a new protein band of 155 kDa was detected. The N-terminal peptide s equencing established that the 155 kDa band represents truncated ankyrin. I mmunoblot analysis using defined monoclonal antibodies confirmed that ankyr in was cleaved at the C-terminus. While the enzyme preferentially cleaved a nkyrin, degradation of protein 4.1 was also observed at high concentrations of the enzyme. The optimal activity of the purified enzyme, using ankyrin as substrate, was observed at pH 7.0-7.5, and the activity was strongly inh ibited by standard inhibitors of cysteine proteinases (cystatin, NEM, leupe ptin. E-64 and MDL 28 170), but not by inhibitors of aspartic (pepstatin) o r serine (PMSF. DFP) proteinases. Furthermore, we demonstrate that protease digestion of ankyrin substantially reduces its interaction with ankyrin-de pleted membrane vesicles. Ektacytometric measurements showed a dramatic inc rease in the rate of fragmentation of ghosts after treatment with the prote ase. Although the role of ankyrin cleavage in vivo remains to be determined , based on our findings we postulate that the parasite-derived cysteine pro tease activity cleaves host ankyrin thus weakening the ankyrin-band 3 bindi ng interactions and destabilizing the erythrocyte membrane skeleton, which, in turn, facilitates parasite release. Further characterization of the enz yme may lead to the development of novel antimalarial drugs, (C) 2000 Elsev ier Science B.V. All rights reserved.