Binding of glycophorins to Plasmodium falciparum merozoites

Plasmodium falciparum merozoites recognize and attach to glycophorins, the surface sialoglycoproteins of human erythrocytes. The structural requirements for a merozoite binding site were studied with the use of two methods. In the first, certain glycophorins and their tryptic fragments were added directly to isolated merozoites prior to their addition to erythrocytes. Low concentrations (50 micrograms ml-1) of glycophorin A inhibited merozoite invasion. At higher concentrations a mixture of glycophorins A, B and C (GPS) (100 micrograms ml-1) and glycophorin B (200 micrograms ml-1) also inhibited invasion. GPS from Tn erythrocytes which lack both sialic acid and galactose residues was almost as effective as normal GPS in blocking invasion. None of the monosaccharides present on glycophorin, including N-acetylneuraminic acid, inhibited merozoite invasion. Erythrocytes treated with lectins were only partially resistant to invasion. These results indicated that the oligosaccharide side chains are not the major structural determinant of the merozoite binding site. Glycophorin A was cleaved by trypsin and the separated fragments added to merozoites. Only the external N-terminal tryptic fragment T1 and the trypsin resistant hydrophobic core, T6, showed some, but considerably less, inhibitory activity than the intact molecule. In the second approach, the binding of 125I-labeled GPS to isolated merozoites was determined. 125I-GPS binding was saturated at 0.23 micrograms for 10(9) merozoites and was competitively inhibited by unlabeled GPS but not by free sugars. Desialylated GPS bound almost to the same extent as the intact molecule.     

Microtubule associated motor proteins of Plasmodium falciparum merozoites

We have studied the occurrence, stage specificity and cellular location of key molecules associated with microtubules in Plasmodium falciparum merozoites. Antibodies to gamma tubulin, conventional kinesin and cytoplasmic dynein were used to determine the polarity of merozoite microtubules (mt), the stage specificity of the motor proteins and their location during merozoite development. We conclude that the minus ends of the mts are located at their apical pole. Kinesin was present throughout the lifecycle, appearing as a distinct crescent at the apex of developing merozoites. The vast majority of cytoplasmic dynein reactivity occurred in late merogony, also appearing at the merozoite apex. Destruction of mt with dinitroanilines did not affect the cellular location of kinesin or dynein. In invasion assays, dynein inhibitors reduced the number of ring stage parasites. Our results show that both conventional kinesin and cytoplasmic dynein are abundant, located at the negative pole of the merozoite mt and, intriguingly, appear there only in very late merogony, prior to merozoite release and invasion.     

Independent translocation of two micronemal proteins in developing Plasmodium falciparum merozoites

Apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) contains an N-terminal propeptide that is removed prior to the translocation of the mature protein onto the merozoite surface. We localized unprocessed PfAMA1 to the microneme organelles of the intraerythrocytic schizont. The results have suggested that the processed form of PfAMA1 translocates from the microneme compartment independently of another microneme protein, EBA175, which is also involved in the invasion of human erythrocytes.     

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.     

Alpha-tubulin II is a male-specific protein in Plasmodium falciparum.

The tubulin gene family in Plasmodium falciparum consists of one beta-tubulin and two alpha-tubulin genes (alpha-tubulin I and II). We present here data indicating that alpha-tubulin II is expressed only in male sexual stage parasites. An IgM mAb, 5E7, specifically reacted with stage III (day 4-5) through mature (day 10-11) male gametocytes and with emerging, exflagellating, or freely moving male gametes. No reactivity was detected in female gametocytes, female gametes, sporozoites, or asexual parasites. mAb 5E7 also specifically recognized male gametes of the avian parasite, Plasmodium gallinaceum, and immunoblotted a 50 kDa protein in extracts of male gametes from both species. This 50 kDa antigen was localized by immunoelectron microscopy to axonemes of male gametes in a pattern similar to that obtained with anti-alpha- and anti-beta-tubulin antibodies. Furthermore, mAb 5E7 specifically reacted with recombinant alpha-tubulin II protein obtained using the PCR-amplified alpha-tubulin II gene from a gametocyte-specific cDNA library. The sex-specific expression of alpha-tubulin II and its localization to axoneme of the male parasite suggest a role for this molecule in the morphologic changes that occur during exflagellation and in the motility of the parasite. alpha-Tubulin II and mAb 5E7 may prove useful tools in studies of the biology of sexual stage differentiation and development in P. falciparum in addition to the general understanding of post-translational modifications of tubulin isoforms.     

Expression and characterization of catalytic domain of Plasmodium falciparum subtilisin-like protease 3

PfSUB3 is the third subtilisin-like protease annotated in Plasmodium genome database "PlasmoDB". The other two members, PfSUB1 and PfSUB2 have been implicated in merozoite egress and invasion in asexual blood stages. In this study, we recombinantly expressed a region of PfSUB3 spanning from Asn(334) to Glu(769) (PfSUB3c) which encompassed the predicted catalytic domain with all the active site residues and predicted mature region spanning from Thr(516) to Glu(769) (PfSUB3m) in E. coli. PfSUB3m showed PMSF-sensitive proteolytic activity in in vitro assays. Replacement of active site serine with alanine in PfSUB3m resulted in inactive protein. We found that PfSUB3c and PfSUB3m undergo truncation to produce a 25-kDa species which was sufficient for proteolytic activity. Quantitative real-time PCR, immnufluorescence assay and Western blot analyses revealed that PfSUB3 is expressed at late asexual blood stages. Serine protease activity of PfSUB3 and its expression in the late stages of erythrocytic schizogony are indicative of some possible role of the protease in merozoite egress and/or invasion processes.     

A C2 domain containing plasma membrane protein of Plasmodium falciparum merozoites mediates calcium-dependent binding and invasion to host erythrocytes

BackgroundInvasion of red blood cells by Plasmodium falciparum merozoites is governed by multiple receptor–ligand interactions which are critical for bridging the two cells together. The critical function of these ligands for invasion and their direct exposure to the host immune system makes them lucrative vaccine candidates. This necessitates the discovery of new adhesins with less redundancy that mediates the binding of merozoite to the red cell, and furthermore invasion into it. Here we have identified a novel membrane associated antigen (PfC2DMA) that is conserved throughout the Plasmodium species and has a membrane targeting C2 domain at its extreme N-terminal region.MethodsRecombinant C2_dom was expressed heterologously in bacteria and purified to homogeneity. Mice antisera against C2_dom was raised and used to check the expression and intraparasitic localization of the protein. RBC and Ca²⁺ ion binding activity of C2_dom was also checked.ResultsC2_dom exhibited specific binding to Ca²⁺ ions and not to Mg²⁺ ions. PfC2DMA localized to the surface of merozoite and recombinant C2_dom bound to the surface of human RBCs. RBC receptor modification by treatment with different enzymes showed that binding of C2_dom to RBC surface is neuraminidase sensitive. Mice antisera raised against C2_dom of Pf C2DMA showed invasion inhibitory effects.ConclusionOur findings suggest that C2_dom of PfC2DMA binds to surface of red cell in a Ca²⁺-dependent manner, advocating a plausible role in invasion and can serve as a potential novel blood stage vaccine candidate.     

Isolation of a Plasmodium falciparum rhoptry protein

A monoclonal antibody raised against the malaria parasite Plasmodium falciparum recognised a protein of 140000 molecular weight which was synthesized during schizogony. The protein has been purified by monoclonal antibody affinity chromatography from extracts of parasitized red cells. Antibodies against the protein have been used to determine its subcellular location. The protein is not expressed on the merozoite surface and has been located in the rhoptries, the apical organelles of the merozoite.     

Mitochondrial protein synthesis in Plasmodium falciparum

Protein synthesis in intact Plasmodium falciparum was 333 times more sensitive to cycloheximide than to chloramphenicol. The 50% inhibitory concentration (IC50) of cycloheximide in a 27-h assay in vitro was 6 X 10(-7) M but no constant cycloheximide-insensitive fraction of total protein synthesis was observed at concentrations of this inhibitor between 10(-7) and 10(-2) M. 0.24% of total protein synthesis occurred in the presence of 10(-3) M cycloheximide but the chloramphenicol sensitivity of this fraction was similar to that of overall protein synthesis (IC50 2 X 10(-4) M). The major fraction of protein synthesis by P. falciparum, therefore, is assumed to be cytoplasmic and to occur on 80S ribosomes. Cycloheximide-insensitive, chloramphenicol-sensitive (70S ribosomal) protein synthesis being undetectable by the methods employed, mitochondrial protein synthesis in P. falciparum is presumed to constitute a considerably smaller fraction of the total protein synthetic capacity than observed in other lower eukaryotes.     

Role of the carbohydrate domains of glycophorins as erythrocyte receptors for invasion by Plasmodium falciparum merozoites.

Solubilized preparations of purified glycophorins and specific domains of these molecules were assessed for their effects as inhibitors of Plasmodium falciparum invasion of human erythrocytes in vitro. The ability of newly invaded merozoites to continue developing and incorporating [3H]hypoxanthine during a 24-h period after their invasion was used as an assay for merozoite invasion. Glycophorins A, B, and C were found to be equally effective as inhibitors. Previous studies had shown N-acetylglucosamine, a sugar component of glycophorins A and C but not B, to be an effective inhibitor. Accordingly, molecular domains common to all of the glycophorins were further assessed. Sialic acid was shown to act almost as effectively as N-acetylglucosamine, presumably because of the structural similarities between these sugars. The inhibitory ability of sialic acid is considerably enhanced when presented to the parasite in a clustered form, as in an oligosaccharide. The acetyl group of these sugars does not appear to play an essential role in this inhibition. How the P. falciparum merozoite recognizes and interacts with the sugar domains of the glycophorin molecule remains to be determined.     

Identification and characterisation of proteins associated with the rhoptry organelles of Plasmodium falciparum merozoites

We describe antigens of Plasmodium falciparum recognised by murine monoclonal antibodies which by immunofluorescence react with the rhoptry organelles of the extracellular merozoite stage. Immunoblotting shows that the antibodies recognise two major parasite antigens of M_r 82 and 65 kilodaltons (kDa). Immunoprecipitations from detergent extracts of [³⁵S]-methioninelabelled parasites show that the 82-kDa and 65-kDa antigens are parasite proteins. Pulse-chase experiments on synchronous parasite cultures show that the 82-kDa protein is synthesised during early schizogony and is later processed into the 65-kDa antigen in segmenting schizonts. In Nonidet P-40, these antigens are non-covalently associated with two other proteins of 40 kDa and 42 kDa. The 40/42-kDa doublet is synthesised in parallel with the 82 kDa antigen and persists, apparently unchanged, till the end of the cell cycle.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis-(aminoethylether) tetraacetic acid - PMSF phenylmethylsulphonylfluoride - TLCK tosyl-L-lysine chloromethyl ketone - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - M _r relative molecular mass     

The 42-kilodalton rhoptry-associated protein of Plasmodium falciparum.

The gene coding for a 42-kDa rhoptry protein of Plasmodium falciparum has been cloned. On the basis of prior monkey vaccination studies, this protein is regarded as an important vaccine candidate, but its identity has been the subject of considerable uncertainty. Analysis of the cloned sequence shows that it is a basic, hydrophobic protein, without repetitive elements, unrelated to any of the previously postulated gene products and shows minimal sequence diversity. The availability of the corresponding recombinant protein will enable studies of its efficacy in human vaccine trials to be undertaken.     

Unique insertions within Plasmodium falciparum subtilisin-like protease-1 are crucial for enzyme maturation and activity

Parasite serine proteases play essential roles in the asexual erythrocytic life cycle of the malaria parasite. The timing and location of expression of Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) are consistent with a role in erythrocyte invasion. Maturation of PfSUB-1 involves two autocatalytic processing events in which an 82 kDa precursor is converted to a 54 kDa form, followed by further cleavage to produce a 47 kDa form. Here we have compared PfSUB-1 with a number of Plasmodium orthologues and the most closely related bacterial subtilase sequences and find that, like many malarial proteins, PfSUB-1 possesses both low and high complexity insertions. The latter take the form of six surface-associated strands or loops which are conserved in all SUB-1 orthologues but not present in any other subtilase. Several mutants of PfSUB-1 with deletions of all, or part, of each of the six loop insertions were produced in an insect cell expression system. Aside from loop III, which was dispensable, individual deletion of the loop insertions revealed a role in protein maturation and/or stability. Specific substitutions within loop II inhibited maturation and enzyme activity. Mutations in loops V and VI specifically inhibited the second step of autocatalytic maturation providing evidence that the two processing steps have distinct structural requirements and that conversion to p47 is not a prerequisite for proteolytic activity in trans.     

The 22 kDa component of the protein complex on the surface of Plasmodium falciparum merozoites is derived from a larger precursor, merozoite surface protein 7

The gene coding for merozoite surface protein 7 has been identified and sequenced in three lines of Plasmodium falciparum. The gene encodes a 351 amino acid polypeptide that is the precursor of a 22-kDa protein (MSP7(22)) on the merozoite surface and non-covalently associated with merozoite surface protein 1 (MSP1) complex shed from the surface at erythrocyte invasion. A second 19-kDa component of the complex (MSP7(19)) was shown to be derived from MSP7(22) and the complete primary structure of this polypeptide was confirmed by mass spectrometry. The protein sequence contains several predicted helical and two beta elements, but has no similarity with sequences outside the Plasmodium databases. Four sites of sequence variation were identified in MSP7, all within the MSP7(22) region. The MSP7 gene is expressed in mature schizonts, at the same time as other merozoite surface protein genes. It is proposed that MSP7(22) is the result of cleavage by a protease that may also cleave MSP1 and MSP6. A related gene was identified and cloned from the rodent malaria parasite, Plasmodium yoelii YM; at the amino acid level this sequence was 23% identical and 50% similar to that of P. falciparum MSP7.