Characterization of Plasmodium falciparum protein kinase 2

A sustained elevation of free Ca(2+) is observed on the rupture and release of merozoites of Plasmodium falciparum from the erythrocytes. The immunoelectron micrographs demonstrate that calmodulin is localized in merozoites. To elucidate the Ca(2+) signal of P. falciparum invasion, we attempted to characterize P. falciparum protein kinase 2 (PfPK2), which is homologous to human calcium calmodulin-dependent protein kinase (CaMK). PfPK2 was purified as a fusion protein that was labeled with [gamma-(32)P]ATP; this labeling was then eliminated by phosphatase. This phosphorylation was eliminated when the putative catalytic lysine residue of PfPK2 was replaced with alanine. PfPK2 phosphorylated histone II(AS) as a representative substrate in a Ca(2+)- and calmodulin-dependent manner. Calmodulin antagonists inhibited the phosphorylation of PfPK2 in vitro and markedly decreased the parasitemia of ring forms in an invasion assay, whereas CaMKII-specific inhibitors had no effect. PfPK2 was localized in the merozoites in the culture of P. falciparum. Thus, purified PfPK2 possesses protein kinase activity in a Ca(2+)- and calmodulin-dependent manner and the catalytic lysine of this protein was determined. These data suggest that PfPK2 is the Plasmodium protein kinase expressed in the merozoites during the invasion stage.     

ATPase activity of purified plasma membranes and digestive vacuoles from Plasmodium falciparum

The ATPase activity of the human malaria parasite, Plasmodium falciparum was investigated using two experimental systems, (i) digestive vacuoles, and (ii) purified plasma membranes isolated from a chloroquine-sensitive and a chloroquine-resistant strain. No correlation between the level of ATPase activity and chloroquine sensitivity could be detected. In both systems, the ATPase activity of the chloroquine-resistant and -sensitive strain was decreased in the presence of the P-glycoprotein inhibitor vanadate. Susceptibility to inhibition by vanadate together with the lack of effect of ouabain implies a P-type ATPase activity in the plasma membrane. Furthermore, the inhibition of Fac8 ATPase activity by oligomycin both in the digestive vacuoles and the plasma membranes would be consistent with higher levels of Pgh1 in Fac8. Our data are consistent with the presence of a V-type H+-ATPase in the parasite food vacuole. Bafilomycin A1 and N-ethylmaleimide decreased the vacuolar ATPase activity in both chloroquine-resistant and -sensitive strains. Interestingly, a 30% decrease was observed between the ATPase activity of plasma membranes isolated from Fac8 and D10 in the presence of bafilomycin A1, suggesting the presence of a V-type ATPase in D10 plasma membrane that is underexpressed or altered in the plasma membrane of the chloroquine-resistant Fac8. The chemosensitisers tested had no effect on the ATPase activity of chloroquine-resistant P. falciparum in both systems suggesting that their activity is not mediated through an ATP-dependent mechanism. No effect was observed on the vacuolar ATPase activity in the presence of the antimalarials tested indicating that an ATP-dependent transport has not been activated.     

Characterization of a 225 kilodalton rhoptry protein of Plasmodium falciparum

A monoclonal antibody (24C6 4F12) raised against Plasmodium falciparum culture supernatant antigens gave a multiple dot picture on schizonts when assayed by immunofluorescence on P. falciparum erythrocytic stages. The corresponding antigen was localized in the peduncle of rhoptries by immunoelectronmicroscopy. On Western blots of P. falciparum schizonts, a major antigen of 225 kDa and a minor one of 240 kDa were recognized by this McAb. Pulse chase analysis of [35S]methionine biosynthetic labeling of P. falciparum culture demonstrated that the 240 kDa molecule was the precursor of the 225 kDa and that its processing occurred between 0 and 4 h after synthesis. Biosynthesis of the 240-225 kDa antigen occurred only during schizogony.     

Characterization of a PRL protein tyrosine phosphatase from Plasmodium falciparum

Isoprenylated proteins have important functions in cell growth and differentiation of eukaryotic cells. Inhibitors of protein prenylation in malaria have recently shown strong promise as effective antimalarials. In studying protein prenylation in the malaria protozoan parasite Plasmodium falciparum, we have shown earlier that the incubation of P. falciparum cells with (3)H-prenol precursors resulted in various size classes of labeled proteins. To understand the physiological function of prenylated proteins of malaria parasites, that are targets of prenyltransferase inhibitors, we searched the PlasmoDB database for proteins containing the C-terminus prenylation motif. We have identified, among other potentially prenylated proteins, an orthologue of a PRL (protein of regenerating liver) subgroup protein tyrosine phosphatases, termed PfPRL. Here, we show that PfPRL is expressed in the parasite's intraerythrocytic stages, where it partially associates with endoplasmic reticulum and within a subcompartment of the food vacuole. Additionally, PfPRL targeting parallels that of apical membrane antigen-1 in developing merozoites. Recombinant PfPRL shows phosphatase activity that is preferentially inhibited by a tyrosine phosphatase inhibitor suggesting that PfPRL functions as a tyrosine phosphatase. Recombinant PfPRL can also be farnesylated in vitro. Inhibition of malarial farnesyltransferase activity can be achieved with the heptapetide RKCHFM, which corresponds to the C-terminus of PfPRL. This study provides the first evidence for expression of enzymatically active PRL-related protein tyrosine phosphatases in malarial parasites, and demonstrates the potential of peptides derived from Plasmodium prenylated proteins as malarial farnesyltransferase inhibitors.     

Purification of Plasmodium falciparum digestive vacuoles and partial characterization of the vacuolar membrane ATPase

Plasmodium falciparum digestive vacuoles containing ferric oxide granules were purified from parasite homogenates by centrifugation on discontinuous sucrose gradients. Digestive vacuole membranes prepared by osmotic lysis and washed with KCl showed no detectable contamination by erythrocyte membrane proteins and only minimal contamination by non-vacuolar parasite proteins. Purified vacuolar membranes were 2.6-fold enriched in total parasite membrane ATPase activity. This ATPase was optimally active at pH 7 in the presence of at least 2 mM Mg2+. Ca2+ and Mn2+ were approximately 80-90% as effective as Mg2+, and Zn2+, Co2+ and Fe2+ also exerted some stimulatory effect. The vacuolar membrane also hydrolyzed GTP, UTP, CTP and ADP, but AMP and 3',5'-cyclic AMP were hydrolyzed only one-tenth as effectively as ATP. The ATPase was unaffected by vanadate, ouabain or oligomycin but was significantly inhibited by the proton pump inhibitors NEM and NBD-Cl. Of 6 antimalarial drugs tested, quinine and quinacrine were the most effective inhibitors and mefloquine was the least effective.     

Novel RuvB nuclear ATPase is specific to intraerythrocytic mitosis during schizogony of Plasmodium falciparum

RuvB protein belongs to AAA+ family of enzymes involved in diverse cellular activities. In addition to the annotated two RuvB proteins in Plasmodium falciparum database, we report that a third RuvB protein is also present. The amino acid sequence analysis has revealed that P. falciparum RuvB3 (PfRuvB3) possesses Walker motif A, Walker motif B, sensor I and sensor II conserved motifs similar to yeast and human RuvB like proteins. The phylogenetic analysis revealed that PfRuvB3 is closely related to yeast RuvB like proteins which are essential for the survival of yeast. The biochemical characterization of recombinant PfRuvB3 confirms its ssDNA dependent ATPase activity. Using the truncated derivatives we show that Walker motif A is essential for the enzymatic activity of PfRuvB3. Using the immunodepletion assays we further show that the ATPase activity is attributable to PfRuvB3 protein. The endogenous P. falciparum RuvB3 contains the characteristic ATPase and some DNA helicase activities. The confocal microscopy analysis showed that this protein is mainly expressed during intraerythrocytic schizont stages of the parasite and is localized to the nuclear region. Once merozoite comes out from schizont, PfRuvB3 protein distinctly relocalized to the subnuclear region. The co-localization studies with a nucleolar marker PfNop1 further suggest that in P. falciparum RuvB3 localizes into a discrete nuclear compartment. On the basis of these studies it can be speculated that P. falciparum RuvB3 is most likely required for intraerythrocytic schizogony.     

Characterization of domains of the phosphoriboprotein P0 of Plasmodium falciparum

Antibodies against the amino-terminal domain of the Plasmodium falciparum P0 phosphoriboprotein were detected extensively in immune people living in malaria endemic areas of India. It has been shown earlier that specific antibodies raised against the PfP0N domain (17-61 amino acid) of the PfP0 protein inhibit P. falciparum growth in vitro. To study the properties of the rest of the protein, the remaining 61-316 amino acids on the carboxy-side of the PfP0 protein were expressed as a glutathione-S-transferase fusion protein (PfP0C). Antibodies raised against PfP0C identified the 38 kDa P0 protein on a parasite Western blot analysis. An ELISA assay using both the PfP0N and PfP0C fusion proteins showed no reactivity with malaria patient sera samples, but showed extensive reactions with the immune sera. Antibodies against both the PfP0C and PfP0N domains were raised in rabbits and different inbred strains of mice. T-cells from immunized mice showed lymphoproliferation when presented with PfP0 protein domains. IgG from both anti-PfP0N and anti-PfP0C sera inhibited the growth of P. falciparum in vitro in a concentration dependent manner. The IgG did not show any significant effect on the growth of intraerythrocytic stages, but specifically inhibited re-invasion of red cells. Merozoites and sexual stages showed surface reactivity to both anti-PfP0N and anti-PfP0C antibodies in immunofluorescence assays. These properties strongly indicate PfP0 as a possible target for invasion-blocking antibodies.     

Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum

EBA-175 is a soluble 175-kDa Plasmodium falciparum antigen that is released into culture supernatants during rupture of schizont-infected erythrocytes. EBA-175 binds to erythrocytes and binding is sialic acid-dependent. A clone expressing the gene encoding EBA-175 was obtained previously by screening a genomic DNA expression library with antibodies that had been affinity-purified from EBA-175. Antibodies were raised against a 43-amino-acid peptide (EBA-peptide 4) synthesized according to the deduced amino acid sequence. Antibodies to peptide 4 and affinity-purified antibodies specific for EBA-175 were used to characterize further EBA-175 giving the following results: (1) EBA-175 differs biochemically and immunologically from other reported malarial antigens; (2) the EBA-175s from six geographical isolates of P. falciparum are antigenically conserved; (3) EBA-175 is expressed during schizogony as a 190-kDa protein which is larger than the culture supernatant form of the antigen. The 190-kDa form of the protein is recovered from the cell pellet in schizont-infected erythrocytes and partitions to the soluble fraction when extracted with detergent; (4) release of soluble EBA-175 into the culture supernatant coincides with schizont rupture; (5) there was no observable change in pI (pI=6.86) by isoelectric focusing between the cellular and supernatant species of the protein; and (6) release of EBA-175 into the culture supernatant is inhibited by the addition of chymostatin and leupeptin. The continued research into the role of EBA-175 during erythrocyte invasion may aid in vaccine development for malaria.     

Codon usage in Plasmodium falciparum

The codon frequencies used in 7874 codons from 17 sequences of Plasmodium falciparum have been examined. The frequency distribution is markedly biased. A and C occur with similar frequency in all positions but G is predominantly in the first base and T is predominantly in the last position. This information can be used to predict the coding strand and reading frame of P. falciparum genes.     

Characterization of a hemoglobin-degrading, low molecular weight protease from Plasmodium falciparum

A protease from Plasmodium falciparum was purified 150 fold by high performance liquid chromatography on a TSK-G-3000 SW exclusion column. The enzyme is not retained during pressure filtration with an Amicon PM10 membrane but is retained by a YM5 membrane. The molecular weight of the protease is less than 10,000, based upon mobility on a calibrated TSK column. The enzyme catalyzes the hydrolysis both of acid denatured hemoglobin and of albumin. The hydrolysis is optimal at pH4.5, but considerable activity is seen at pH 6.0. Pepstatin strongly inhibits the protease (I50 = 70 nM) while bestatin, antipain and phosphoramidon produce moderate inhibition (I50 = 30, 30 and 3 microM, respectively). The protease is inhibited by ferriprotoporphyrin IX (I50 ca. 5 microM). This inhibition is insensitive to pH between pH 4.5 and 6. Although chloroquine does not strongly inhibit the protease, chloroquine-ferriprotoporphyrin IX complex produces inhibition similar to that of ferriprotoporphyrin IX. It is suggested that the antimalarial effect of chloroquine is due to the formation of ferriprotoporphyrin IX-chloroquine complex which prevents the sequestration of ferriprotoporphyrin IX into malarial pigment, thereby providing both ferriprotoporphyrin IX and its chloroquine complex as inhibitors of one of the proteases required for the degradation of hemoglobin.     

Characterization of gp195 processed products purified from Plasmodium falciparum culture supernates

Schizonts of the malaria parasite Plasmodium falciparum synthesize a 195 kDa surface glycoprotein (gp195) that is processed into several smaller products including one of 83 kDa, which, in the case of the Camp strain, is sequentially processed into 73 and 67 kDa products. gp195 and its processing intermediates larger than 83 kDa were not precipitated from culture supernates, but the 83 and 73 kDa products were precipitated by three monoclonal antibodies (McAbs). The 83 and 73 kDa products were affinity purified from culture supernates by adsorbing to McAb 7B2 coupled to Affigel 10 and eluting either with 0.2 N acetic acid, pH 2.8, or with 3 M potassium isothiocyanate (KSCN). The epitope recognized by McAb 7B2 was denatured by acid elution but could be regenerated by treating with 8 M urea followed by dialysis. The implications of renaturing antigens to regenerate epitopes should be considered in studies on the purification, function and immunogenicity of malaria antigens.     

Plasmodium falciparum biosynthesizes sulfoglycosphingolipids

Sulfated glycosphingolipids are present on the surface of a variety of cells. They are active participants in adhesion processes in many systems and appear to be involved in the regulation of cell proliferation, differentiation and other developmental cellular events. However, the body of knowledge about synthesis, structure, and function of glycolipids in parasitic protozoa is very limited so far. In this work, we show by metabolic incorporation of [(14)C]palmitic acid, [(14)C]glucose and Na(2)(35)SO(4) that sulfoglycosphingolipids are biosynthesized in the three intraerythrocytic stages of Plasmodium falciparum. After saponification, purification of the labelled acidic components was achieved and two components named SPf1 and SPf2 were characterized. Chemical degradations and TLC analysis pointed out to sulfolipidic structures. Analysis by UV-MALDI-TOF mass spectrometry in the negative ion mode using nor-harmane as matrix showed for SPf2 a structure consisting in a disulfated hexose linked to a 20:1 sphingosine acylated with C18:0 fatty acid. Interestingly, parasite treatment with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) caused an arrest on parasite development associated to the inhibition of sulfoglycolipid biosynthesis. Taking into account that sulfoglycolipidic structures are currently involved in adhesion processes, our findings open the possibility to study the participation of this type of structures in the described aggregation phenomena in severe malaria and may contribute to clarify the pathogenesis of the disease. This report shows for the first time the synthesis of sulfoglycolipids in Apicomplexa.     

Features of mimicry in Plasmodium falciparum

In this study, we aim to show, by comparing the amino-acid sequences of several antigens of Plasmodium falciparum with those of some proteins manufactured by the host immune system, that the parasite appears to have a remarkable capacity for mimicry. This would help greatly to reduce the efficiency of the immune response during its asexual cycle. Indeed, the major sporozoite surface protein (CSP) has amino-acid sequences in common with interleukin 1; homologies between Pf 11, expressed at the trophozoite stage, and thymosin alpha 1 may be found. Lastly, RESA present at the schizont stage and protein S liberated when the parasitized erythrocyte is lysed, have sequences in common, respectively with thymosin alpha 1 and thymulin.