Characterization of P0, a Ribosomal Phosphoprotein ofPlasmodium falciparum

Abstract

A cDNA expression clone of the human malarial parasite Plasmodium falciparum, Pf4, which was reactive only to the immune sera and not to the patient sera, has recently been found to be the P. falciparum homologue of the P0 ribosomal phosphoprotein gene. A Northern analysis of the P0 gene revealed the presence of two transcripts, both present in all the different intraerythrocytic stages of the parasite life cycle. A 138-base pair amino-terminal domain of this gene was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal antibodies raised against this domain immunoprecipitated the expected 38-kDa P0 protein from the 35 S-labeled as well as 32 P-labeled P. falciparum cultures. Monospecific human immune sera affinity-purified using the expression clone Pf4 also immunoprecipitated the same size protein from [35 S]methionine-labeled P. falciparum protein extract. Purified IgG from polyclonal antibodies raised against the amino-terminal domain of P0 protein completely inhibited the growth of P. falciparum in vitro. This inhibition appears to be mainly at the step of erythrocyte invasion by the parasites.

Antibodies against Ribosomal Phosphoprotein P0 ofPlasmodium falciparumProtect Mice against Challenge withPlasmodium yoelii

“…We have recently reported the characterization of the amino-terminal domain of the P. falciparum P0 ribosomal phosphoprotein PfP0N (16), which was isolated in a differential immunoscreen using malaria-immune and patient sera (22). Subsequent analysis showed that the PfP0C fusion protein also reacted extensively with the immune serum samples and that IgG from sera raised against both these domains inhibited the growth of P. falciparum in vitro in a concentration-dependent manner (8).…”

“…Mice immunized with IgG prepared from rabbit anti-GST sera also showed no difference in parasitemia compared to mice receiving no IgG (data not shown). It has been documented earlier that administration of anti-GST IgG has no effect on the growth of the malarial parasite either in vitro or in vivo (16,23). Figure 4 shows the parasitemia profile of each mouse over a period of 28 days for experiments I and II.…”

“…A glutathione S-transferase (GST) reporter-based vector was used as an expression vector as described earlier (8,16). The amino-terminal 45-amino-acid (a.a.) domain (a.a. 17 to 61) and the 256-amino-acid carboxy-terminal region (a.a. 61 to 316) of PfP0 were expressed as fusion proteins designated PfP0N and PfP0C, respectively.…”

“…The liberated merozoites were harvested at 4°C, washed, and resuspended in complete RPMI 1640 medium at a concentration of 10 7 merozoites/ml. Intracellular parasites were liberated from infected erythrocytes by saponin lysis (16). The total intracellular parasite pellet and the merozoite pellet were solubilized with 10 volumes of solubilization buffer (20 mM Tris-HCl [pH 7.8], 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetic acid, 0.1% sodium azide) at 4°C for 1 h. This was then spun at 12,000 rpm for 20 min, and the supernatant containing the solubilized protein suspension was used for Western blot analysis.…”

“…This was found to be the P. falciparum gene homologue of the ribosomal phosphoprotein P0 (PfP0). Further characterization showed that antibodies raised specifically to PfP0 inhibited the growth of P. falciparum in vitro and reacted to the surface of merozoites (8,16).…”