Biosynthesis, localization, and processing of falcipain cysteine proteases of Plasmodium falciparum.
Falcipain-2 and -3 are cysteine proteases of erythrocytic Plasmodium falciparum parasites that appear to function principally as hemoglobinases. To better understand their biological roles, we analyzed the biosynthesis, localization, and processing of these enzymes in cultured parasites. Immunoprecipitation of metabolically labeled proteins indicated that falcipain-2 was synthesized through the trophozoite stage, falcipain-3 appeared in late trophozoites/early schizonts, and both proteases persisted for at least 6 h after synthesis. Falcipain-2 and -3 were localized to the food vacuole, the site of hemoglobin hydrolysis, by immunofluorescence and immunoelectron microscopy. Subcellular fractionation experiments indicated that both enzymes were synthesized as membrane bound proforms that were processed to soluble mature forms, but falcipain-2 was processed to the mature protease much more quickly than was falcipain-3. Cysteine protease inhibitors and brefeldin A, but not aspartic or serine protease inhibitors, blocked the processing of both enzymes, suggesting that falcipain-2 and -3 process by autohydrolysis after exiting the endoplasmic reticulum/Golgi network. However, although all tested cysteine protease inhibitors blocked hemoglobinase activity in the food vacuole, only lipophilic inhibitors (E-64d, Mu-Leu-Hph-VSPh, and ALLN), blocked intracellular processing of falcipain-2 and -3. More hydrophilic inhibitors (E-64 and leupeptin) did not block processing, suggesting that autocatalytic processing of falcipain-2 and -3 occurs in a specific cellular compartment before delivery to the food vacuole. Our results support overlapping but not fully redundant roles for falcipain-2 and -3, which are targeted to the food vacuole and activated sequentially to degrade hemoglobin in erythrocytic parasites.
Dahl
Erica L
EL
Department of Medicine, San Francisco General Hospital, University of California, Box 0811, San Francisco, CA 94143-0811, USA.
Rosenthal
Philip J
PJ
D000818:Animals; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000469:parasitology; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D008018:Life Cycle Stages; D010963:Plasmodium falciparum / Q000201:enzymology / Q000254:growth & development*; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology
D015853:Cysteine Proteinase Inhibitors; D006454:Hemoglobins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C099607:falcipain