Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 - PubMed (original) (raw)

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

Shoba Subramanian et al. PLoS One. 2009.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Cleavage preferences of falcipain-2 and falcipain-3 against a tetrapeptide library.

P1 P2, P3 and P4 complete diverse libraries were used to determine specificities. Activities are displayed as percentage of the maximum for each position. Amino acids are represented by the single-letter code (n is for norleucine). Error bars represent standard deviations of results from three experiments.

Figure 2

Figure 2. Hemoglobin cleavage sites for falcipain-2 and falcipain-3.

A–D. Globin peptides and intact human hemoglobin. Cleavage sites for the two proteases based on analysis as described in Methods of hydrolysis of 12-mer peptides (top arrows, data from all time points) and intact hemoglobin (lower arrows, data from both 180 min and overnight time points) are shown for α and β globin. E. Intact human hemoglobin. The schematic shows α globin subunits in white and β globin subunits in yellow. P1 residues at cleavage sites within helices are in red and within loops are in blue.

Figure 3

Figure 3. Time course for cleavage of globin peptides.

Schematic representation of cleavage fragments over the indicated time points based on analysis of cleavage of 12-mer peptides spanning the sequences of α and β globin.

Figure 4

Figure 4. Cleavage site preferences for falcipain-2 and falcipain-3.

Preferences for amino acids (single letter code) at P1-P4 and P1′-P4′ are shown schematically. At each position, the height of each amino acid corresponds to its frequency at cleavage sites based on analysis of α and β globin. WebLogo (

weblogo.berkeley.edu

), a free software for plotting consensus sequences, was used to plot P4- P4′ preferences.

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