title: Plasmepsin II, an acidic hemoglobinase from the Plasmodium falciparum food vacuole, is active at neutral pH on the host erythrocyte membrane skeleton.
abstract: Plasmepsin II, an aspartic protease from the human intraerythrocytic parasite Plasmodium falciparum, is involved in degradation of the host cell hemoglobin within the acidic food vacuole of the parasite. Previous characterization of enzymatic activities from Plasmodium soluble extracts, responsible for in vitro hydrolysis of erythrocyte spectrin, had shown that the hydrolysis process occurred at pH 5.0 and involved aspartic protease(s) cleaving mainly within the SH3 motif of the spectrin alpha-subunit. Therefore, we used a recombinant construct of the erythroid SH3 motif as substrate to investigate 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 hydrolase activity. Involvement of plasmepsin II in hydrolysis was demonstrated by mass spectrometry identification of cleavage sites in the SH3 motif, upon hydrolysis by Plasmodium extract enzymatic activity, and by recombinant plasmepsin II. Furthermore, recombinant plasmepsin II digested native spectrin at pH 6.8, either purified or situated in erythrocyte ghosts. Additional degradation of actin and protein 4.1 from ghosts was observed. Specific antibodies were used in confocal imaging of schizont-infected erythrocytes to localize plasmepsin II in mature stages of the parasite development cycle; antibodies clearly labeled the periphery of the parasites. Taken together, these results strongly suggest that, in addition to hemoglobin degradation, plasmepsin II might be involved in cytoskeleton cleavage of infected erythrocytes.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000469:parasitology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014617:Vacuoles / Q000201:enzymology