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

• Published in: PloS one Vol. 4; no. 4; p. e5156
• Main Authors: Subramanian, Shoba, Hardt, Markus, Choe, Youngchool, Niles, Richard K, Johansen, Eric B, Legac, Jennifer, Gut, Jiri, Kerr, Iain D, Craik, Charles S, Rosenthal, Philip J
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 09.04.2009; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Amino acids; Animals; Antimalarial agents; Antimalarials - therapeutic use; Biochemistry/Chemical Biology of the Cell; Biochemistry/Drug Discovery; Biology; Chemical Biology/Protein Chemistry and Proteomics; Cleavage; Cysteine; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Cysteine proteinase; Cystine; Enzymes; Erythrocytes; Food; Gene expression; Glycerol; Glycosylated hemoglobin; Hemoglobin; Hemoglobins - chemistry; Hemoglobins - genetics; Hemoglobins - metabolism; Humans; Hydrolysis; Infectious diseases; Infectious Diseases/Neglected Tropical Diseases; Leucine - genetics; Leucine - metabolism; Libraries; Malaria; Malaria - drug therapy; Medicine; Models, Molecular; Molecular Sequence Data; Mortality; Parasites; Peptide Library; Peptides; Peptides - genetics; Peptides - metabolism; Pharmaceuticals; Plasmodium falciparum; Plasmodium falciparum - enzymology; Plasmodium falciparum - genetics; Protease inhibitors; Proteases; Protein biosynthesis; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Structure, Tertiary; Protein synthesis; Proteinase inhibitors; Proteins; Proteolysis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Substrate Specificity; Substrates; Thiols; Vector-borne diseases; United States > US; San Francisco California; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0005156

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.