Margaret E. Perkins

Erythrocyte receptor recognition varies in Plasmodium falciparum isolates

N-Acetylneuraminic acid (NeuNAc) is the terminal sugar residue of the O-linked tetrasaccharide linked to erythrocyte sialoglycoproteins, glycophorins. Erythrocytes lacking NeuNAc have been shown previously to be resistant to invasion by certain isolates of Plasmodium falciparum merozoites. We report here variation between different geographic isolates of P. falciparum in their dependency on NeuNAc for invasion of host erythrocytes. Seven different geographic isolates of P. falciparum were examined for their ability to invade neuraminidase treated erythrocytes. For all isolates invasion was reduced significantly, although considerable variation in NeuNAc dependency was apparent. Three isolates, FCR-3, FVO and It2, exhibited a very high dependence on NeuNAc residues for invasion (invasion reduced greater than 90%), whereas two isolates (Thai-Tn and FC-27) exhibited a moderately high dependence (invasion reduced 75%). Two other isolates (CDC-1 and 7G8) exhibited moderate dependence on NeuNAc (invasion reduced 50%). Cleavage of the complete O-linked tetrasaccharide by O-glycanase removes all carbohydrate from glycophorin A, B and C except the single N-linked oligosaccharide on glycophorin A and C. Invasion of FCR-3 and CDC-1 isolates into O-glycanase treated erythrocytes was not markedly different from that into neuraminidase treated cells indicating that NeuNAc is the important residue of the tetrasaccharide for both isolates. Invasion into endo-beta-galactosidase treated erythrocytes, in which the lactosaminoglycan side chain of band 3 and band 4.5 is cleaved, was not significantly reduced for either the CDC-1 or FCR-3 isolates. Additional results on the trypsin insensitivity of band 3 also suggest that this erythrocyte protein is not important in P. falciparum recognition. The greatest divergence in receptor specificity between FCR-3 and CDC-1 isolates was apparent in invasion into periodate-treated erythrocytes. Periodate oxidation results in cleavage of the exocyclic hydroxyl groups of the terminal NeuNAc but leaves its COOH group unaltered. These experiments also illustrated that the negatively charged COOH group of NeuNAc is not the important group in the interaction of the merozoite with the NeuNAc. Trypsin-treated erythrocytes were almost fully resistant to invasion by CDC-1 as well as the FCR-3 isolates suggesting that the CDC-1 isolate, in addition to interacting with NeuNAc, depends on a trypsin sensitive site for invasion. This site could involve the N-linked saccharide on glycophorin A and C or a protein on the erythrocyte surface unrelated to the glycophorins.

Glutathione stability and oxidative stress in P. falciparum infection in vitro: Responses of normal and G6PD deficient cells

Abstract Red cell oxidative stress in P. falciparum infection in vitro was investigated in relation to the G6PD-Malaria hypothesis. Glutathione stability was enhanced in infected red cells; glucose consumption and pentose pathway activity were not different in normal and G6PD deficient cells, although parasite growth was impaired in G6PD deficiency. Evidence for a response to oxidative stress was not found. Infected red cells have glutamate dehydrogenase activity which was not found in uninfected cells. This enzyme provides a separate pathway for the generation of NADPH independent from the pentose shunt. The data suggest that a significant oxidative stress is not present in falciparum malaria and that another mechanism may be operative in G6PD deficiency.

A tandemly repeated sequence determines the binding domain for an erythrocyte receptor binding protein of P. falciparum

Erythrocyte invasion by the malarial merozoite is a receptor-mediated process, an obligatory step in the development of the parasite. The Plasmodium falciparum protein GBP-130, which binds to the erythrocyte receptor glycophorin, is shown here to encode the binding site in a domain composed of a tandemly repeated 50 amino acid sequence. The amino acid sequence of GBP-130, deduced from the cloned and sequenced gene, reveals that the protein contains 11 highly conserved 50 amino acid repeats and a charged N-terminal region of 225 amino acids. Binding studies on recombinant proteins expressing different numbers of repeats suggest that a correlation exists between glycophorin binding and repeat number. Thus, a repeat domain, a common feature of plasmodial antigens, has been shown to have a function independent of the immune system. This conclusion is further supported by the ability of antibodies directed against the repeat sequence to inhibit the in vitro invasion of erythrocytes by merozoites.

Interaction of the 140/130/110 kDa rhoptry protein complex of plasmodium falciparum with the erythrocyte membrane and liposomes

During Plasmodium falciparum merozoite invasion into human and mouse erythrocytes, a 110-kDa rhoptry protein is secreted from the organelle into the erythrocyte membrane. In the present study our interest was to examine the interaction of rhoptry proteins of P. falciparum with the erythrocyte membrane. It was observed that the complex of rhoptry proteins of 140/130/110 kDa bind directly to a trypsin sensitive site on intact mouse erythrocytes, and not human, saimiri, or other erythrocytes. However, when erythrocytes were disrupted by hypotonic lysis, rhoptry proteins of 140/130/110 kDa were found to bind to membranes and inside-out vesicles prepared from human, mouse, saimiri, rhesus, rat, and rabbit erythrocytes. A binding site on the cytoplasmic face of the erythrocyte membrane suggests that the rhoptry proteins may be translocated across the lipid bilayer during merozoite invasion. Furthermore, pretreatment of human erythrocytes with a specific peptide derived from MSA-1, the major P. falciparum merozoite surface antigen of MW 190,000-200,000, induced binding of the 140/130/110-kDa complex. The rhoptry proteins bound equally to normal human erythrocytes and erythrocytes treated with neuraminidase, trypsin, and chymotrypsin indicating the binding site was independent of glycophorin and other major surface proteins. The rhoptry protein complex also bound specifically to liposomes prepared from different types of phospholipids. Liposomes containing PE effectively block binding of the rhoptry proteins to mouse cells, suggesting that there are two binding sites on the mouse membrane for the 140/130/110-kDa complex, one protein and a second, possibly lipid in nature. The results of this study suggest that the 140/130/110 kDa protein complex may interact directly with sites in the lipid bilayer of the erythrocyte membrane.

Isolation and characterization of rhoptries of Plasmodium falciparum.

Rhoptries have been isolated from Plasmodium falciparum schizont-infected erythrocytes by isopycnic density centrifugation. Gradient fractions were analyzed by immunoblotting with antibodies against two polypeptides of 140 and 110 kDa, known to be components of the rhoptry. The proteins were present primarily in fractions with a density of 1.16 g ml-1. Electron microscopy of these fractions indicated they were enriched in rhoptries. For the most part, the isolated organelle retained in situ morphology, although some rhoptries were distorted, indicating the structure of some of the organelles is not rigid. Electrophoretic analysis of the rhoptry fractions indicated the presence of a number of proteins, many of which have not been identified to date. Properties of proteins in the isolated rhoptry were examined using the 140 and 110 kDa proteins as representative markers. Both proteins are present in a complex with a 130-kDa protein, as all three co-immunoprecipitate. At the late schizont stage, the rhoptry proteins are present in two distinct forms; a soluble form with an Mr of 480 000 which would correspond to a single copy of the 140/130/110 kDa complex and a form that can be sedimented at 130 000 x g. Properties of the sedimentable form suggest that the proteins are included in structures that resemble membranes. Ionic detergents were required to solubilize the proteins while high concentrations of NaCl and Na2CO3 resulted in only partial solubilization. Furthermore, treatment of disrupted rhoptries with phospholipase A and C resulted in the release of proteins into the soluble form.

Phosphorylation of erythrocyte membrane and cytoskeleton proteins in cells infected with Plasmodium falciparum

Phosphorylation changes in the erythrocyte membrane and cytoskeletal proteins as a consequence of infection by the malarial parasite Plasmodium falciparum were examined. Spectrin, band 3, band 4.1, ankyrin and glycophorin are phosphorylated in normal erythrocytes. As a consequence of invasion by the merozoite, the extracellular stage of the parasite, into 32P-prelabeled normal erythrocytes, all the major 32P-labeled erythrocyte proteins are dephosphorylated. As the parasite develops intracellularly from the immature ring stage to the mature schizont stage, selective phosphorylation of certain host proteins, spectrin, ankyrin and band 3 is observed. Band 4.1 does not appear to incorporate [32P]phosphate at any stage of parasite development. These observed phosphorylation changes may be important in the regulation of the cytoskeletal organization in P. falciparum-infected cells.

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.

Surface proteins of schizont-infected erythrocytes and merozoites of Plasmodium falciparum

Schizont-infected erythrocytes and merozoites were isolated from in vitro cultures of the human parasite, Plasmodium falciparum labeled with various radioactive substrates. The isolated merozoites were viable since they were able to reinvade fresh erythrocytes. On the basis of sensitivity to specific enzymes, eleven proteins synthesised by the parasite, were localised on the surface of the schizont-infected erythrocyte. Eight of these were glycoproteins, six of which appeared to represent three doublets. Five merozoite surface proteins were identified on the basis of their sensitivity to trypsin and chymotrypsin, treatments which also rendered the merozoite incapable of erythrocyte invasion. Merozoites appeared not to contain any glycoproteins; all of the glycoproteins synthesised by the parasite were apparently transported to the surface of the schizont-infected erythrocyte.

Binding of Plasmodium falciparum rhoptry proteins to mouse erythrocytes and their possible role in invasion.

Rhoptry proteins of Plasmodium falciparum merozoites, of 140, 130, and 110 kDa, identified by co-precipitation with Mab.1B9, bind selectively to mouse erythrocytes and reticulocytes. The properties of binding are shown to correlate with invasion of P. falciparum into mouse erythrocytes. Invasion of two strains of P. falciparum 7G8 and FCR-3, into mouse erythrocytes was examined, and was found to differ significantly. The 7G8 strain invades mouse erythrocytes at a rate of 40-60% compared to invasion into human erythrocytes, whereas FCR-3 invades at a rate of 5-15%. Both strains of P. falciparum preferentially invade reticulocytes in the in vitro invasion assay. This correlated with an increase in the amount of rhoptry protein of the 7G8 strain bound to mouse erythrocytes, compared to the FCR-3 strain and an increased binding to reticulocytes compared to mature erythrocytes. Binding of the rhoptry proteins and merozoite invasion into the erythrocyte is blocked in erythrocytes treated with trypsin and chymotrypsin but not in neuraminidase-treated erythrocytes, suggesting that the putative receptor site is exposed and accessible on the erythrocyte surface. Rabbit antiserum against gp3, the major glycophorin of mouse erythrocytes, blocks binding of the rhoptry proteins to erythrocytes and reduces merozoite invasion into mouse erythrocytes by 50%. Binding of rhoptry proteins to mouse reticulocytes was not blocked by alpha gp3 indicating a receptor difference between reticulocytes and erythrocytes. Mab.1B9 reduces merozoite invasion but does not decrease binding of the rhoptry proteins to the mouse erythrocyte. The mouse erythrocyte serves as a useful model to study the receptor-ligand interaction of rhoptry proteins and host surface proteins and to define the role of the rhoptry proteins during the invasion process.

Chemical crosslinking of Plasmodium falciparum glycoprotein, Pf200 (190–205 kDa), to the S-antigen at the merozoite surface

Merozoites were isolated from Plasmodium falciparum cultures labeled with [3H]mannose and [35S]methionine and treated with a cleavable homobifunctional crosslinker, dithiobis(succinimidyl) propionate. The crosslinked complexes were immunoprecipitated with Mab.5B1 directed against the major merozoite surface glycoprotein. Pf200 (MW 190-205), and reduced with dithiothreitol. Crosslinked immunocomplexes did not contain the second major merozoite surface glycoprotein, Pf50 (MW 45-55 kDa), or other major [35S]methionine-labeled proteins, except for a weakly labeled protein of 150 kDa. Crosslinked complexes immunoprecipitated with Mab.5B1 and then reduced with DTT were immunoblotted with antibody directed against three soluble P. falciparum antigens, a serine-rich antigen known as Pf126 or SERA, the S-antigen, and GBP-130. The 150-kDa S-antigen was readily detected in crosslinked immunocomplexes with Pf200. The SERA antigen, although crosslinked under these conditions, was not detected in association with Pf200 nor was GBP-130.