Expression, Characterization, and Cellular Localization of Knowpains, Papain-Like Cysteine Proteases of the Plasmodium knowlesi Malaria Parasite

Abstract

Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries against knowpains for developing broadly effective compounds active against multiple human malaria parasites.

The genomes of three stocks comprising the most widely utilized live sporozoite Theileria parva vaccine exhibit very different degrees and patterns of sequence divergence

“…In T. parva they are thought to facilitate lymphocyte invasion [ 16 ]. Papain family cysteine proteases have been considered as potential vaccine targets in the related apicomplexan Plasmodium falciparum [ 17 ], and the previously described T. parva antigen Tp8, in chromosome 2 (TpMuguga_02g00140), is a papain family cysteine protease. The polymorphic papain cysteine protease identified here (TpMuguga_03g00280) is located in chromosome 3, and its antigenic potential warrants further investigation.…”

Approaches for the generation of active papain-like cysteine proteases from inclusion bodies of Escherichia coli

“…DTT was found to enhance enzyme activities of knowpains (Prasad et al 2012), Falcipain-2 (Sarduy et al 2012, DerF1 and DerP1 (Takai et al 2002) in a dose-dependent manner. Different pH buffers also showed significant effects on cathepsin F-like cysteine protease (Joo et al 2007), knowpains (Prasad et al 2012), Tr-cp 14 (Asp et al 2004) and Falcipain-2 (Sarduy et al 2012) activities; the maximum activities often obtained in acidic pH. The concentration of cysteine protease used in enzyme activity assays is between 0.1 and 10 nM (Bromme et al 2004).…”

“…The buffer, DTT and EDTA concentrations vary between protocols, and their effect on the enzyme activities was in controversy. Some researchs deemed that these factors don't have a significant effect on the enzyme activities (Bromme et al 2004;Joo et al 2007;Prasad et al 2012). However, some other literatures showed different perspectives.…”

Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part III: Protozoa (2): Plasmodium spp.

“…Also, KP-4 was observed to have essential role in erythrocyte cytoskeleton breakdown facilitating merozoite egress. Accordingly, the investigators suggested using in vitro culture of P. knowlesi for searching a VP specific inhibitor in the absence of an efficient in vitro culture system for P. vivax [58] .…”