Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3.
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.
Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, California, United States of America.
Shoba
S
Subramanian
Markus
M
Hardt
Youngchool
Y
Choe
Richard K
RK
Niles
Eric B
EB
Johansen
Jennifer
J
Legac
Jiri
J
Gut
Iain D
ID
Kerr
Charles S
CS
Craik
Philip J
PJ
Rosenthal
D000595:Amino Acid Sequence; D000818:Animals; D000962:Antimalarials / Q000627:therapeutic use; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008288:Malaria / Q000188:drug therapy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000962:Antimalarials; D006454:Hemoglobins; D019151:Peptide Library; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C416224:falcipain 2; C443101:falcipain 3; D007930:Leucine