A chimeric cysteine protease of Plasmodium berghei engineered to resemble the Plasmodium falciparum protease falcipain-2.
The cysteine proteases falcipain-2 and falcipain-3 are hemoglobinases and potential targets for chemotherapy directed against Plasmodium falciparum, the most important human malaria parasite. Most in vivo evaluations of candidate antimalarials are conducted in murine malaria models, and falcipain homologs from rodent malaria parasites differ importantly from falcipain-2 and falcipain-3. We expressed berghepain-2, the single homolog of falcipain-2 and falcipain-3 of the rodent parasite P. berghei, in Escherichia coli, and characterized the refolded active enzyme. Berghepain-2 was biochemically very similar to the previously characterized rodent plasmodial protease vinckepain-2, but differed from falcipain-2 and falcipain-3 in its fine substrate and inhibitor specificity. We then used homology modeling and evolutionary trace analysis to predict key amino acids that mediate functional differences between falcipain-2 and berghepain-2. Thirteen amino acids were sequentially altered to replace berghepain-2 residues with those in falcipain-2. Mutant enzymes varied in activity and sensitivity to inhibitors. A berghepain-2 mutant with eight substitutions retained good activity and demonstrated fine substrate and inhibitor sensitivity more similar to that of falcipain-2 than berghepain-2. These results suggest that, to facilitate drug discovery, we can produce mutant animal model malaria parasites with biochemical properties more like those of the key drug target, P. falciparum.
Singh
Ajay
A
Department of Medicine, San Francisco General Hospital, University of California, CA 94143, USA.
Walker
K Jordan
KJ
Sijwali
Puran S
PS
Lau
Anthony L
AL
Rosenthal
Philip J
PJ
D019943:Amino Acid Substitution; D000818:Animals; D003546:Cysteine Endopeptidases / Q000235:genetics*; D050506:Mutant Chimeric Proteins; D010962:Plasmodium berghei / Q000201:enzymology*; D010963:Plasmodium falciparum / Q000201:enzymology*; D015202:Protein Engineering / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics
D050506:Mutant Chimeric Proteins; D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases; C416224:falcipain 2