A Plasmodium falciparum dipeptidyl aminopeptidase I participates in vacuolar hemoglobin degradation.
Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum requires the catabolism of large amounts of host cell hemoglobin. Endoproteolytic digestion of hemoglobin to short oligopeptides occurs in an acidic organelle called the food vacuole. How amino acids are generated from these peptides is not well understood. To gain insight into this process, we have studied a plasmodial ortholog of the lysosomal exopeptidase cathepsin C. The plasmodial enzyme dipeptidyl aminopeptidase 1 (DPAP1) was enriched from parasite extract by two different approaches and was shown to possess hydrolytic activity against fluorogenic dipeptide substrates. To localize DPAP1 we created a transgenic parasite line expressing a chromosomally encoded DPAP1-green fluorescent protein fusion. Green fluorescent protein fluorescence was observed in the food vacuole of live transgenic parasites, and anti-DPAP1 antibody labeled the food vacuole in parasite cryosections. Together these data implicate DPAP1 in the generation of dipeptides from hemoglobin-derived oligopeptides. To assess the significance of DPAP1, we attempted to ablate DPAP1 activity from blood stage parasites by truncating the chromosomal DPAP1-coding sequence. The inability to disrupt the coding sequence indicates that DPAP1 is important for asexual proliferation. The proenzyme form of DPAP1 was found to accumulate in the parasitophorous vacuole of mature parasites. This observation suggests a trafficking route for DPAP1 through the parasitophorous vacuole to the food vacuole.
Department of Medicine, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Michael
M
Klemba
Ilya
I
Gluzman
Daniel E
DE
Goldberg
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D020789:Cathepsin C / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002455:Cell Division; D002875:Chromosomes / Q000378:metabolism; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010963:Plasmodium falciparum / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013997:Time Factors; D014617:Vacuoles / Q000378:metabolism*
D006454:Hemoglobins; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020789:Cathepsin C