Biochemical Properties of a Novel Cysteine Protease of Plasmodium vivax, Vivapain-4

Abstract

BackgroundMultiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins) in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX)-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive.FindingsWe characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA was highest at acidic pH (5.5), whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5–7.5). VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5). Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180). Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively.ConclusionVX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular environments. VX-4 might function as a hemoglobinase in the acidic parasite food vacuole, a maturase of P. vivax plasmepsin 4 at neutral or acidic pH, and a cytoskeleton-degrading protease in the neutral erythrocyte cytosol.

Centenary celebrations article

“…Recently, vivapain-4 has been shown to express in all intraerythrocytic stages of P. vivax (Na et al 2010). The majority of vivapain-4 localization appeared to be limited to the food vacuole with dark hemozoin pigment, while some protein was also labelled diffusely in the parasite cytoplasm (Na et al 2010). As mentioned earlier, vivapains has hydrolytic activity against cytosketal proteins, and their cytoplasmic distributions may suggest their cytoplasmic roles such as cytosketal remodelling, erythrocytes rupture, and hemoglobin transport.…”

“…7). Homology modelling of vivapain-4 suggests that overall topology is similar to falcipain-2, falcipain-3, vivapain-2 and vivapain-3 (Na et al 2010). However, numbers of substitutions are recognized between vivapain-4 and other vivapains, including Ala 90, Gly154, Glu180 at the S2 pocket.…”

“…Recently, vivapain-4 has been shown to express in all intraerythrocytic stages of P. vivax (Na et al 2010). The majority of vivapain-4 localization appeared to be limited to the food vacuole with dark hemozoin pigment, while some protein was also labelled diffusely in the parasite cytoplasm (Na et al 2010).…”

“…Biochemical analysis of falcipain-2, falcipain-3 as well as vivapain-2, vivapain-3, vivapian-4 showed that they are efficient hemoglobinases. These enzymes efficiently hydrolyze human hemoglobin in acidic food vacuole of parasite (Shenai et al 2000;Sijwali et al 2001;Na et al 2004;Na et al 2010). Recent study indicated that plasmepsin-4, an aspartic protease of P. vivax, acted synergistically with vivapain-2 and vivapain-3, in the hydrolysis of hemoglobin (Moon et al 2011).…”

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

“…However, these drug discovery projects should ideally be extended to the homologous proteases of P. vivax and P. knowlesi to develop common inhibitors of these proteases as a broadly effective antimalarial therapy. Homologous P. vivax cysteine proteases, known as vivapains, have been characterized [13], [14], and homologs in P. knowlesi need to be characterized to augment ongoing falcipain-based drug development projects.…”