Toshihide Mitamura

Heparin-binding EGF-like growth factor, which acts as the diphtheria toxin receptor, forms a complex with membrane protein DRAP27/CD9, which up-regulates functional receptors and diphtheria toxin sensitivity.

DRAP27, the monkey homolog of human CD9 antigen (DRAP27/CD9) and diphtheria toxin receptor (DTR) were expressed in mouse L cells. L cells transfected transiently with both DRAP27/CD9 and DTR cDNA bound approximately 10 times more diphtheria toxin (DT) than cells transfected with DTR alone. Stable L cell transfectants expressing both DTR and DRAP27/CD9 (LCH‐1 cells) had 15 times more cell surface DT‐binding sites and were 20 times more sensitive to DT than were stable L cell transfectants expressing DTR alone (LH‐1 cells). Increased DT‐binding and DT sensitivity were not due to increased DTR transcription or increased cell surface DTR protein. Co‐immunoprecipitation of DRAP27/CD9 with DTR and chemical cross‐linking suggest a tight association of these membrane‐bound proteins. In addition, the identity of DTR and a growth factor (HB‐EGF) was established. Immobilized DT specifically adsorbed HB‐EGF precursor solubilized from transfected L cells and [125I]DT bound to immobilized recombinant HB‐EGF. We conclude that DRAP27/CD9 associates tightly with DTR/HB‐EGF and up‐regulates the number of functional DTRs and DT sensitivity, and that HB‐EGF is identical to DTR.

Plasmodium falciparum Phospholipase C Hydrolyzing Sphingomyelin and Lysocholinephospholipids Is a Possible Target for Malaria Chemotherapy

Sphingomyelinase (SMase) is one of the principal enzymes in sphingomyelin (SM) metabolism. Here, we identified a Plasmodium falciparum gene (PfNSM) encoding a 46-kD protein, the amino acid sequence of which is ∼25% identical to that of bacteria SMases. Biochemical analyses of the recombinant protein GST-PfNSM, a fusion protein of the PfNSM product with glutathione-S-transferase, reveal that this enzyme retained similar characteristics in various aspects to SMase detected in P. falciparum–infected erythrocytes and isolated parasites. In addition, the recombinant protein retains hydrolyzing activity not only of SM but also of lysocholinephospholipids (LCPL) including lysophosphatidylcholine and lysoplatelet-activating factor, indicating that PfNSM encodes SM/LCPL-phospholipase C (PLC). Scyphostatin inhibited SM/LCPL-PLC activities of the PfNSM product as well as the intraerythrocytic proliferation of P. falciparum in a dose-dependent manner with ID50 values for SM/LCPL-PLC activities and the parasite growth at 3–5 μM and ∼7 μM, respectively. Morphological analysis demonstrated most severe impairment in the intraerythrocytic development with the addition of scyphostatin at trophozoite stage than at ring or schizont stages, suggesting its effect specifically on the stage progression from trophozoite to schizont, coinciding with the active transcription of PfNSM gene.

Serum factors governing intraerythrocytic development and cell cycle progression of Plasmodium falciparum

Malaria is clinically manifested only when the human malaria parasites in the genus Plasmodium enter the obligatory intraerythrocytic life cycle. Elucidation for the roles of the serum, the key nutrient, and its components is then deemed essential for thorough understanding of the proliferation of Plasmodium cells at the erythrocytic stage. Fractionation and analysis of serum and its components was performed by chromatography, solvent extraction, and subsequent reconstitution experiments. Only fractions containing serum albumin (SA) from the serum and purified intact bovine serum albumin (BSA) showed comparable growth promoting activity with human serum (HS). Delipidated BSA can only effect parasite growth after reconstitution with lipids extracted from intact BSA. Fatty acid (FA) species in the neutral lipid fraction from intact BSA proved likewise when reconstituted with delipidated BSA. Furthermore, the involved FA species have to come in a pair of one saturated and one unsaturated, with palmitic and oleic acids as the best combination. The results were further substantiated by morphological analysis as well as biochemical analysis of the DNA synthesis during the intraerythrocytic development. This study can be a basis to explore the molecular mechanism of lipid traffic within the parasitized red blood cell (RBC), which can be an important adjunct to the development of drugs for malaria therapy.

Neutral sphingomyelinase activity dependent on Mg2+ and anionic phospholipids in the intraerythrocytic malaria parasite Plasmodium falciparum

Sphingolipid metabolism and metabolites are important in various cellular events in eukaryotes. However, little is known about their function in plasmodial parasites. Here we demonstrate that neutral sphingomyelinase (SMase) involved in the sphingomyelin (SM) catabolism is retained by the intraerythrocytic parasite Plasmodium falciparum. When assayed in a neutral pH buffer supplemented with Mg(2+) and phosphatidylserine, an activity for the release of the phosphocholine group from SM was detected in parasite-infected, but not in uninfected, erythrocyte ghosts. The SMase activity in the parasite-infected erythrocyte ghosts was enhanced markedly by anionic phospholipids including unsaturated but not saturated phosphatidylserine. Mn(2+) could not substitute for Mg(2+) to activate SMase in parasite-infected erythrocyte ghosts, whereas both Mn(2+) and Mg(2+) activated mammalian neutral SMase. The specific activity level of SMase was higher in isolated parasites than in infected erythrocyte ghosts; further fractionation of lysates of the isolated parasites showed that the activity was bound largely to the membrane fraction of the parasites. The plasmodial SMase seemed not to hydrolyse phosphatidylcholine or phosphatidylinositol. The plasmodial SMase, but not SM synthase, was sensitive to scyphostatin, an inhibitor of mammalian neutral SMase, indicating that the plasmodial activities for SM hydrolysis and SM synthesis are mediated by different catalysts. Our finding that the malaria parasites possess SMase activity might explain why the parasites seem to have an SM synthase activity but no activity to synthesize ceramide de novo.

Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA)

The Plasmodium falciparum serine repeat antigen (SERA), a malaria vaccine candidate, is processed into several fragments (P73, P47, P56, P50, and P18) at the late schizont stage prior to schizont rupture in the erythrocytic cycle of the parasite. We have established an in vitro cell-free system using a baculovirus-expressed recombinant SERA (bvSERA) that mimics the SERA processing that occurs in parasitized erythrocytes. SERA processing was mediated by parasite-derived trans-acting proteases, but not an autocatalytic event. The processing activities appeared at late schizont stage. The proteases are membrane associated, correlating with the secretion and accumulation of SERA within the parasitophorous vacuole membrane (PVM). The activity responsible for the primary processing step of SERA to P47 and P73 was inhibited by serine protease inhibitor DFP. In contrast, the activity responsible for the conversion of P56 into P50 was inhibited by each of the cysteine protease inhibitors E-64, leupeptin and iodoacetoamide. Moreover, addition of DFP, E-64 or leupeptin to the cultures of schizont-stage parasites blocked schizont rupture and release of merozoites from PVM. These results indicate that SERA processing correlates to schizont rupture and the processing is mediated by at least three distinct proteases.

Intraerythrocytic Plasmodium falciparum utilize a broad range of serum-derived fatty acids with limited modification for their growth.

Plasmodium falciparum causes the most severe form of malaria. Utilization of fatty acids in serum is thought to be necessary for survival of this parasite in erythrocytes, and thus characterization of the parasite fatty acid metabolism is important in developing a new strategy for controlling malaria. Here, we examined which combinations of fatty acids present in human serum support the continuous culture of P. falciparum in serum-free medium. Metabolic labelling and gas chromatography analyses revealed that, despite the need for particular fatty acids for the growth of intraerythrocytic P. falciparum, it can metabolize a broad range of serum-derived fatty acids into the major lipid species of their membranes and lipid bodies. In addition, these analyses showed that the parasites overall fatty acid composition reflects that of the medium, although the parasite has a limited capacity to desaturate and elongate serum-derived fatty acids. These results indicate that the Plasmodium parasite is distinct from most cells, which maintain their fatty acid composition by coordinating de novo biosynthesis, scavenging, and modification (desaturation and elongation).

Developmental-stage-specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies in Plasmodium falciparum-infected erythrocytes

Triacylglycerol (TAG) serves as a major energy storage molecule in eukaryotes. In Plasmodium, however, this established function of TAG appears unlikely, despite detecting previously considerable amount of TAG associated with intraerythrocytic parasites, because plasmodial cells have very little capacity to oxidize fatty acids. Thus, it is plausible that TAG and its biosynthesis in Plasmodium have other functions. As a first step in understanding the biological significance of TAG and its biosynthesis to the intraerythrocytic proliferation of Plasmodium falciparum, we performed detailed characterization of TAG metabolism and trafficking in parasitized erythrocyte. Metabolic labeling using radiolabeled-oleic and palmitic acids in association with serum albumin, which have been shown to be among the serum essential factors for intraerythrocytic proliferation of P. falciparum, revealed that accumulation of TAG was strikingly pronounced from trophozoite to schizont, whereas TAG degradation became active from schizont to segmented schizont; the consequent products, free fatty acids, were released into the medium during schizont rupture and/or merozoite release. These results were further supported by visualization of lipid bodies through immunofluorescence and electron microscopy. At the schizont stages, there is some evidence that the lipid bodies are partly localized in the parasitophorous vacuole. Interestingly, the discrete formation and/or trafficking of lipid bodies are inhibited by brefeldin A and trifluoperazine. Inhibition by trifluoperazine hints at least that a de novo TAG biosynthetic pathway via phosphatidic acid contributes to lipid body formation. Indeed, biochemical analysis reveals a higher activity of acyl-CoA:diacylglycerol acyltransferase, the principal enzyme in the sn-glycerol-3-phosphate pathway for TAG synthesis, at trophozoite and schizont stages. Together, these results establish that TAG metabolism and trafficking in P. falciparum-infected erythrocyte occurs in a stage-specific manner during the intraerythrocytic cycle and we propose that these unique and dynamic cellular events participate during schizont rupture and/or merozoite release.

Differential localization of processed fragments of Plasmodium falciparum serine repeat antigen and further processing of its N-terminal 47 kDa fragment

The serine repeat antigen (SERA) of Plasmodium falciparum is a blood stage malaria vaccine candidate. It has been shown that 120 kDa SERA was proteolytically processed into N-terminal 47 kDa fragment (P47), central 56 kDa fragment (P56) that was further converted to 50 kDa (P50), and C-terminal 18 kDa fragment (P18). Here, we have examined the processing of SERA and the localization of its processed fragments by using mouse antibodies directed against recombinant proteins corresponding to different domains of SERA. Western blot analysis showed that all the processing events occurred inside parasitized erythrocytes at the stage just prior to the schizont rupture, that P47 was further processed into two 25 kDa fragments and that the two fragments, which were linked to P18 through disulfide bonds, were associated with the merozoite. In contrast, P50 was completely shed into culture medium and absent from the merozoite. This observation was further supported by the results of indirect immunofluorescence assay. These results could account for the findings that antibodies against P47 were inhibitory to the parasite growth in vitro but those against P50 were not. Finally, we demonstrated that the further processing of P47 is allelic type-dependent. The results of the present study would help in vaccine designing based on SERA.

Amino-terminal Processing of Cell Surface Heparin-binding Epidermal Growth Factor-like Growth Factor Up-regulates Its Juxtacrine but Not Its Paracrine Growth Factor Activity

Human heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) expressed on Chinese hamster ovary (CHO) cells is synthesized as a 19-kDa major, and 22- and 27-kDa minor, membrane-anchored precursors (proHB-EGF). In contrast, the 27-kDa species is major and the 19- and 22-kDa ones are minor in mouse proHB-EGF. The juxtacrine growth factor activities of human and mouse proHB-EGFs on CHO cells toward EP170.7 cells in co-culture are significantly different. To investigate the relationship between the juxtacrine growth factor activities and the molecular species, we prepared human-mouse chimeras. Chimeras that have the human amino-terminal sequence with a mouse EGF-like domain showed approximately 8-fold up-regulation of the juxtacrine growth factor activity and the predominance of a 19-22-kDa major species. In contrast, chimeras that have the mouse amino-terminal sequence with a human EGF-like domain showed approximately 5-fold down-regulation of the juxtacrine activity and the predominance of the 27-kDa major species. A Gly32·HB-EGF (117-amino acid form), which is amino-terminally extended, induced the same mitogenic activity as that of Arg73·HB-EGF (75-amino acid form), which is amino-terminally truncated. These results strongly suggested that amino-terminal processing of human proHB-EGF would be required for up-regulation of its juxtacrine growth factor activity, but not for its paracrine activity.

Lipid metabolism in Plasmodium falciparum-infected erythrocytes: possible new targets for malaria chemotherapy

The emergence and spread of drug-resistant parasites coupled with the absence of an effective vaccine makes malaria treatment more complicated, and thus the development of new antimalarial drugs is one of the urgent tasks in malaria research. This review highlights lipid metabolism in Plasmodium parasite cells, the study of which would lead to providing new targets for therapeutic intervention.