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Prepublished online as a Blood First Edition Paper on April 30, 2002; DOI 10.1182/blood-2002-01-0101.
Submitted January 16, 2002
Accepted March 27, 2002
Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development
Manjit Hanspal*, Meenakshi Dua, Yuichi Takakuwa, Athar H Chishti, and Akiko Mizuno
Biomedical Research, St. Elizabeth's Medical Center, Boston, MA, USA
Biochemistry, Tokyo Women's Medical University, Tokyo, Japan
* Corresponding author; email: manjit_hanspal{at}cchcs.org.
Plasmodium falciparum derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH, and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these two proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and it declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2-mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin-actin binding domain that is critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2-induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite/schizont stage. It is the proteolysis of skeletal proteins that causes membrane instability, which in turn, facilitates parasite release in vivo.
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