Plasmepsin II, an acidic hemoglobinase from the Plasmodium falciparum foodvacuole, is active at neutral pH on the host erythrocyte membrane skeleton

Plasmepsin II, an aspartic protease from the human intraerythrocytic parasi te Plasmodium falciparum, is involved in degradation of the host cell hemog lobin within the acidic food vacuole of the parasite. Previous characteriza tion of enzymatic activities from Plasmodium soluble extracts, responsible for in vitro hydrolysis of erythrocyte spectrin, had shown that the hydroly sis process occurred at pH 5.0 and involved aspartic protease(s) cleaving m ainly within the SH3 motif of the spectrin alpha-subunit. Therefore, we use d a recombinant construct of the erythroid SH3 motif as substrate to invest igate the involvement of plasmepsins in spectrin hydrolysis, Using specific anti-plasmepsin II antibodies in Western blotting experiments, plasmepsin II was detected in chromatographic fractions enriched in the parasite SH3 h ydrolase activity. Involvement of plasmepsin II in hydrolysis was demonstra ted by mass spectrometry identification of cleavage sites in the SH3 motif, upon hydrolysis by Plasmodium extract enzymatic activity, and by recombina nt plasmepsin II, Furthermore, recombinant plasmepsin II digested native sp ectrin at pH 6.8, either purified or situated in erythrocyte ghosts, Additi onal degradation of actin and protein 4.1 from ghosts was observed. Specifi c antibodies were used in confocal imaging of schizont-infected erythrocyte s to localize plasmepsin II in mature stages of the parasite development cy cle; antibodies clearly labeled the periphery of the parasites. Taken toget her, these results strongly suggest that, in addition to hemoglobin degrada tion, plasmepsin II might be involved in cytoskeleton cleavage of infected erythrocytes.