Inhibitory potential of prodomain of Plasmodium falciparum protease serine repeat antigen 5 for asexual blood stages of parasite

Abstract

Plasmodium falciparum serine repeat antigen 5 (SERA5) is a target for both drug and vaccine intervention against malaria. SERA5 is secreted in the parasitophorous vacuole where it is proteolytically processed before schizont rupture. Among the processed products is a 50.8-kDa central domain of the protease, which possesses chymotrypsin-like activity and consists of a 28.9-kDa catalytic domain with a 21.9-kDa N-terminal prodomain, which remain attached together. Because SERA5 has been implicated in merozoite egress from host erythrocytes, the effect of the prodomain and a heptapeptide derived from its C-terminus spanning from D(560) to F(566) (DNSDNMF) on parasite growth was studied. When E. coli-expressed prodomain was incubated with parasite culture, a significant delay in transition from schizont to ring stages was observed up to nanomolar concentrations. The peptide, DNSDNMF also showed similar effects but at nearly 1000-fold higher concentrations. The peptide was also found to interact with the catalytic domain. These data demonstrate the crucial role of SERA5 prodomain for the egress process. Given the inhibitory potential of the prodomain for the parasite, we suggest that peptidomimetic inhibitors based on SERA5 prodomain sequences can be developed as future therapeutics against malaria.

Figure 1. Architecture of SERA5 proenzyme.

Panel A represents the general architecture of SERA5 proenzyme showing the position of prodomain and catalytic domain. Panel B shows the sequence of SERA5 proenzyme. Prodomain sequence is shown in bold and underlined. Catalytic domain sequence is shown in normal case letters. The inhibitory heptapeptide sequence is shown in red and italics.

Figure 2. Inhibitory effect of SERA5 prodomain on parasite growth.

Panel A: Schizonts were accumulated in a dose-dependent manner upon treatment of culture with SERA5 PD (referred as PD) at ring stage. Control P. falciparum protein recombinant HMGB2 had no effect on parasite growth. E-64 (10 µM) was taken as positive control, which caused significant accumulation of schizonts. Geimsa-stained smears of parasite culture were observed by light microscopy after 48 hours of treatment. Data is representative of mean of triplicate experiments. Error bars represent standard deviation of mean. Panel B: Schizonts were accumulated in a dose-dependent manner upon treatment of culture with SERA5 PD (referred as PD) at late trophozoite stage. E-64 (10 µM) was taken as positive control, which caused significant accumulation of schizonts. Geimsa-stained smears of parasite culture were observed by light microscopy after 24 hours of treatment. Data is representative of mean of triplicate experiments. Error bars represent standard deviation of mean.

Figure 3. Localization of FITC-labeled SERA5 PD in schizont stage parasites after 48 hour of treatment.

Panel A. Culture at ring stage was treated with 100 nM of FITC-labeled SERA5 PD and observed after 48 hours after counterstaining with DAPI. FITC fluorescence is seen in schizont stage parasites but absent in uninfected RBCs. Panel B. Signals corresponding to recombinant SERA5 PD and control protein HMGB2 (both with N-terminal hexahistidine tags) were detected in SERA5 PD- and HMGB2-treated parasite lysates respectively when probed with anti-hexahistidine antibody after 48 hours of incubation.

Figure 4. Interaction of heptapeptide, DNSDNMF with SERA5 catalytic domain.

Panel A shows the interaction of DNSDNMF with SERA5 C by CD spectroscopy. The peptide induced significant conformational change when added with protein in 1∶1 molar ratio. Panel B shows the interaction of DNSDNMF with SERA5 C by spectrofluorimetry. The peptide induced quenching of the tryptophan fluorescence upon incubation with protein in 1∶1 molar ratio.

Figure 5. Inhibitory effect of heptapeptide, DNSDNMF on parasite growth.

Culture was treated with peptide, DNSDNMF and control peptide, SIINFEKL at ring stage. Geimsa-stained smears were observed after 48 hours of treatment. Accumulation of schizonts was seen at 100 µM concentration of peptide, DNSDNMF. Control peptide, SIINFEKL did not have any significant effect on culture growth. Data is representative of mean of triplicate experiments. Error bars represent standard deviation of mean.