Ankyrin peptide blocks falcipain-2-mediated malaria parasite release from red blood cells.
Falcipain-2 (FP-2) is a dual-function protease that cleaves hemoglobin at the early trophozoite stage and erythrocyte membrane ankyrin and protein 4.1 at the late stages of parasite development. FP-2-mediated cleavage of ankyrin and protein 4.1 is postulated to cause membrane instability facilitating parasite release in vivo. To test this hypothesis, here we have determined the precise peptide sequence at the hydrolysis site of ankyrin to develop specific inhibitor(s) of FP-2. Mass spectrometric analysis of the hydrolysis products showed that FP-2-mediated cleavage of ankyrin occurred immediately after arginine 1,210. A 10-mer peptide (ankyrin peptide, AnkP) containing the cleavage site completely inhibited the FP-2 enzyme activity in vitro and abolished all of the known functions of FP-2. To determine the effect of this peptide on the growth and development of P. falciparum, the peptide was delivered into intact parasite-infected red blood cells (RBCs) via the Antennapedia homeoprotein internalization domain. Growth and maturation of trophozoites and schizonts was markedly inhibited in the presence of the fused AnkP peptide. <10% of new ring-stage parasites were detected compared with the control sample. Together, our results identify a specific peptide derived from the spectrin-binding domain of ankyrin that blocks late-stage malaria parasite development in RBCs. Confocal microscopy with FP-2-specific antibodies demonstrated the proximity of the enzyme in apposition with the RBC membrane, further corroborating the proposed function of FP-2 in the cleavage of RBC skeletal proteins.
Dhawan
Shikha
S
Department of Biomedical Research, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.
Dua
Meenakshi
M
Chishti
Athar H
AH
Hanspal
Manjit
M
D000818:Animals; D017487:Ankyrins / Q000737:chemistry / Q000502:physiology*; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000469:parasitology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry; D010963:Plasmodium falciparum / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D017487:Ankyrins; D018076:DNA, Complementary; D010455:Peptides; D011994:Recombinant Proteins; D001120:Arginine; D003546:Cysteine Endopeptidases; C416224:falcipain 2