Inhibitor of Cysteine Protease of Plasmodium malariae Regulates Malapains, Endogenous Cysteine Proteases of the Parasite

Abstract

Cysteine proteases of malaria parasites have been recognized as potential targets in antimalarial drug development as they play pivotal roles in the biology of these parasites. However, strict regulation of their activities is also necessary to minimize or prevent deleterious damage to the parasite and the host. Previously, we have characterized falcipain family cysteine proteases of Plasmodium malariae, named as malapains (MPs). MPs are active hemoglobinases. They also may participate in the release of merozoites from mature schizonts by facilitating remodeling of erythrocyte skeleton proteins. In this study, we identified and characterized an endogenous inhibitor of cysteine protease of P. malariae (PmICP). PmICP shared similar structural and biochemical properties with ICPs from other Plasmodium species. Recombinant PmICP showed a broad range of inhibitory activities against diverse cysteine proteases such as falcipain family enzymes (MP-2, MP-4, VX-3, VX-4, and FP-3), papain, and human cathepsins B and L, with stronger inhibitory activities against falcipain family enzymes. The inhibitory activity of PmICP was not affected by pH. PmICP was thermo-labile, resulting in rapid loss of its inhibitory activity at a high temperature. PmICP effectively inhibited hemoglobin hydrolysis by MPs and regulated maturation of MPs, suggesting its role as a functional regulator of MPs.

Keywords: Plasmodium malariae; cathepsins; hemoglobin hydrolysis; inhibitor of cysteine protease; malapains.

Figure 1

Multiple sequence alignment. Deduced amino acid sequences of ICPs from Plasmodium species (PmICP, ICP of P. malariae; PvICP, ICP of P. vivax; PoICP, ICP of P. ovale; PbICP, ICP of P. berghei; PfICP, falstatin), cryptostatin (XP_627553.1), and chagasin (Q966X9) were aligned. Dashes represent gaps introduced to maximize alignment. The predicted N-terminal signal sequence is underlined by a black bar. Amino acid residues corresponding to L0, L2, L4, and L6 loops, which are conserved in plasmodial ICPs [17], are shaded with different colors, respectively. The NPTTG motif that is involved in the interaction with catalytic cysteines of target enzymes in the chagasin family proteins is marked by a red dotted box. Percentage of identity among sequences is represented by shading: black (>88%), dark grey (75–88%), light grey (37–75%), and no shading (<37%).

Figure 2

Phylogenetic analysis. The phylogenetic tree was constructed based on amino acid sequences of each protein by MEGA7 (http://www.megasoftware.net; accessed on 7 March 2022) using Maximum Likelihood Estimation (MLE) via Jones-Taylor-Thornton model with 1000 bootstrap replications. Plasmodial ICPs are clustered into a distinct clade of falstatin family separated from cystatin, chagasin, and toxostatin family proteins.

Figure 3

Expression and purification of recombinant PmICP. Recombinant PmICP was expressed in Escherichia coli, purified via glutathione agarose chromatography, and analyzed by 12% SDS–PAGE. Lane 1, non-induced E. coli lysate; lane 2, IPTG-induced E. coli lysate; lane 3, purified recombinant PmICP.

Figure 4

Inhibitory activity of PmICP. (a) Inhibition profiles of PmICP against diverse cysteine proteases. (b) Effect of pH. PmICP was incubated with each enzyme in different pH buffers for 20 min, after which the residual enzyme activity was assayed. (c) Thermal stability. PmICP was pre-incubated in 50 mM sodium acetate (pH 6.0) at 37 °C, 55 °C or 70 °C for the indicated time periods. The residual inhibitory activity of PmICP for each enzyme was assayed. All experiments were performed in triplicate and the mean and SD values were calculated.

Figure 5

Inhibition of hemoglobin hydrolysis and maturation of MPs. (a) Inhibition of hemoglobin hydrolysis by MPs, VXs, and FP-3. Each fully activated enzyme (50 nM) was incubated either with or without PmICP (50 nM) at room temperature for 30 min. Human hemoglobin (1 mg/mL) was added to the mixture and incubated at 37 °C for 3 h. Samples were analyzed by 15% SDS–PAGE. (b) Regulation of maturation of MPs. Each MP (10 μg) was pre-incubated with PmICP (10 μg) or E-64 (10 μM), activated, and analyzed by 12% SDS–PAGE. NC, negative control before processing; PC, positive control after processing without pre-incubation neither PmICP nor E-64. (c) Regulation of maturation process of MPs. Each MP was processed to mature enzyme in the presence or absence of PmICP (1:1 or 3:1 ratio for MP) or E-64 (10 μM). Aliquots were taken at indicated time points and assayed for enzyme activity. Enzyme activity was assayed with Z-RR-MCA and Z-LR-MCA for MP-2 and MP-4, respectively.