Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3

PLoS One. 2009;4(4):e5156. doi: 10.1371/journal.pone.0005156. Epub 2009 Apr 9.


The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1) - P(4) amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2) position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18)O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2) Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antimalarials / therapeutic use
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism*
  • Hemoglobins / chemistry
  • Hemoglobins / genetics*
  • Hemoglobins / metabolism*
  • Humans
  • Hydrolysis
  • Leucine / genetics
  • Leucine / metabolism
  • Malaria / drug therapy
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Library
  • Peptides / genetics
  • Peptides / metabolism
  • Plasmodium falciparum / enzymology*
  • Plasmodium falciparum / genetics
  • Protein Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Protein Structure, Tertiary
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity


  • Antimalarials
  • Hemoglobins
  • Peptide Library
  • Peptides
  • Protein Isoforms
  • Recombinant Proteins
  • Cysteine Endopeptidases
  • falcipain 2
  • falcipain 3
  • Leucine