Joseph Schrével

Purification and characterization of 37-kilodalton proteases from Plasmodium falciparum and Plasmodium berghei which cleave erythrocyte cytoskeletal components

Cytosoluble 100,000 X g extracts from Plasmodium berghei or Plasmodium falciparum infected red blood cells were shown to hydrolyze erythrocyte spectrin. By Fast Protein Liquid Chromatography (FPLC), these enzymes were purified and exhibited a pI of 4.5 and Mr of 37,000 using SDS-PAGE under reducing conditions. An immunochemical enzyme assay using anti-spectrin antibodies was developed. The optimal activity using spectrin as substrate was at pH 5.0, and the enzymes were strongly inhibited by HgCl2, ZnCl2, chymostatin, leupeptin and aprotinin, and moderately by pepstatin. These properties of the Pf37 and Pb37 proteases differ from the Plasmodium lophurae and P. falciparum cathepsin D-like enzymes and from the serine or cysteine neutral proteases previously described in P. falciparum and P. berghei infected red blood cells. While the Pf37 and Pb37 enzymes cleaved spectrin preferentially, degradation of band 4.1 was also observed with high concentration of enzyme. The parasite origin of the Pf37 protease was clearly demonstrated, since purified radiolabeled enzyme was active on spectrin. A high-molecular-weight polymer (greater than 240 kDa) was often observed on incubating purified spectrin and Pf37 protease. The breakdown of erythrocyte cytoskeletal components could be of interest in the release of merozoites from segmented schizonts or during the process of invasion of erythrocytes by merozoites.

Purification of a Plasmodium berghei neutral endopeptidase and its localization in merozoite

A Plasmodium berghei neutral endopeptidase specific for the fluorogenic substrates valyl-leucyl-glycyl-arginyl/lysyl-aminoethyl-carbazole was purified by Fast Protein Liquid Chromatography. The enzyme was a Mr 68,000 polypeptide. Immunization of mice with the purified enzyme gave a specific antiserum, as demonstrated by immunoblotting. Immunofluorescence with this antiserum showed a strong labelling of P. berghei merozoites in mature segmented schizonts and of merozoites released from schizont-infected red blood cell. This labelling was mainly associated with the merozoite apex. It is possible that this endopeptidase is involved in the reinvasion.

Purification and identification of a neutral endopeptidase inPlasmodium falciparum schizonts and merozoites

An endopeptidase specific to thePlasmodium falciparum erythrocytic schizont stage and to free merozoites was detected using the fluorogenic GlcA-Val-Leu-Gly-Lys(or Arg)-AEC substrate. The enzyme was purified by high performance liquid chromatography (HPLC); its optimal activity was around pH 7.5 and its isoelectric point was 4.4. The molecular weight of the enzyme was about 68000, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The endopeptidase was strongly inhibited by thiol proteinase inhibitors, leupeptin, and antipain. The possible involvement of this neutral endopeptidase in the reinvasion process is discussed.

Actin and spectrin-like (Mr = 260-240 000) proteins in gregarines

In Gregarina blaberae a Mr = 47 000 and a Mr = 260–240 000 doublet polypeptides reacted in immunoblotting: i) with a polyclonal monospecific rabbit antibody to frog muscular actin, a monoclonal anti‐actin antibody against chicken gizzard; and ii) with polyclonal and monoclonal antibodies to human erythrocyte β‐spectrin, respectively. The Mr = 47 000 actin‐like protein is associated with the ghost and a contractille cytoplasmic extract. The presence of an actin‐like protein in Gregarina and Lecudina and its cellular distribution in the cortex indicated that the gliding movement might involve an actin‐myosin system in contrast to previous studies. Immunofluorescence showed clear differences between the anterior part of Gregarina and Lecudina which illustrated the high cell polarity of these protozoa. The Mr = 260–240 000 doublet was detected in SDS‐PAGE from G. blaberae trophozoite ghosts but not in the cytoplasmic extracts or in extracts from sexual stages, indicating that the presence of these spectrin‐like proteins is stage‐dependent. Visualization of the Mr = 260–240 000 by immunofluorescence showed clear species differences, with rings arranged perpendicular to the longitudinal narrow folds of G. blaberae, with longitudinal lines underlying the folds of L. pellucida and with lines separating the large folds of Selenidium pendula. The cellular distribution is consistent with a stabilizer function of the spectrin‐like proteins in the scaffolding of the cortex of gregarines according to the high diversity of the cell‐shape and the cell motility systems in gregarines. The presence of spectrin‐like proteins in protozoa and particularly in parasites from primitive arthropods indicated that ancestral spectrin genes could the Mr = 260–240 000 form.

Formation and growth of gap junctions in mouse myocardium during ontogenesis: Quantitative data and their implications on the development of intercellular communication

Abstract The results of a study on the formation and growth of gap junctions in the mouse heart during ontogenesis was subjected to a statistical analysis in relation to their density and junctional surface area. A mathematical method was developed to calculate the density of gap junctions (average of gap junctions per unit area) in gap junction-containing regions. The results show that there is no mathematically significant increase in gap junction density in the gap junction-containing regions. However, a significant increase during the heart development in the ratio of gap junction to plasma membrane area is demonstrated. Results are criticized in relation to the validity of the sampling process. Implications of the results in terms of the development of non-electrical and electrical communication are discussed.

Neutral proteases involved in the reinvasion of erythrocytes by Plasmodium merozoites

Neutral proteases of Plasmodium sp erythrocytic stages were studied by means of a sensitive fluorogenic method and gelatin‐SDS‐PAGE. The substrates gluconoyl‐Val‐Leu‐Gly‐Lys(or Arg)‐3‐amido‐9‐ethylcarbazole were selectively hydrolyzed by an endopeptidase from rodent Plasmodium berghei (Pb) and Plasmodium chabaudi (Pc) and from human Plasmodium falciparum (Pf) parasites. These endopeptidases were purified from 100,000‐g soluble schizont extract by high pressure liquid chromatography; they have a similar Mr of 68,000 in SDS‐PAGE, and an optimal activity at pH 7.4. The Pb 68 and Pf 68 endopeptidases were localized in shizonts and also in merozoites as shown by indirect immunofluorescence on Pb merozoites and by the identification of the Pf68 endopeptidase activity in free viable merozoites. The Pb68 and Pf68 endopeptidases belong to the class of cysteine proteases. Analysis by gelatin‐SDS‐PAGE of a Pb 68 endopeptidase‐enriched fraction showed a reproducible 95,000 proteolytic band. The initial extracts showed a similar 95,000 proteolytic band, and also 2 other 90,000 and 85,000 major bands. During reinvasion experiments, it was possible to recover a 95,000 and a 40,000 protease band from supernates of cultures grown in a semidefined medium without serum. Hydrophilic peptide derivatives related to the substrate of Pf 68 endopeptidase are shown to be potential inhibitors of the Pf reinvasion process in vitro.

Detection and characterization of a selective endopeptidase from Plasmodium berghei by using fluorogenic peptidyl substrates

By using fluorogenic peptidyl-3-amino-9-ethyl-carbazole a highly selective endopeptidase for the Val-Leu-Gly-Arg sequence was demonstrated in endoerythrocytic stages of Plasmodium berghei. Val-Leu-Gly-Arg-endopeptidase showed a maximum activity in pH range 7.0-8.0; it was completely inhibited by 1 mM leupeptin and 1 mM antipain. A complete inhibition was also obtained by 15 mM chloroquine. This trypsin-like activity was negligible in uninfected red blood cells. The high sensitive fluorogenic procedure could be performed on cell fractions, cell lysates as well as supernatants.

Plasmodium berghei and Plasmodium chabaudi: A neutral endopeptidase in parasite extracts and plasma of infected animals

By using a sensitive fluorometric method with Val-Leu-Gly-Arg-3-amino-9-ethylcarbazole (VLGR-AEC) as a substrate, two endopeptidase activities were identified in two fractions of Sephacryl S-200 gel filtration from soluble P. berghei and P. chabaudi extracts. Controls with normal mouse erythrocytes, with leukocytes, and with reticulocyte enriched blood and different washing procedures during the preparation of soluble P. berghei extracts showed that the MW greater than 200 kDa fraction was a contaminant from erythrocytes and exhibited an optimal pH activity of 8.2. In contrast, the fraction 130 kDa was related to P. berghei and P. chabaudi and exhibited an optimal pH activity of 7.4. The two enzyme activities were compared with eight different substrates. The parasite endopeptidase showed a strong activity with Val-Leu-Gly-Lys-AEC (VLGK-AEC) and Ser-Gly-Lys-AEC (SGK-AEC) as substrates; in contrast, the mouse host endopeptidase poorly cleaved the VLGK-AEC and did not cleave SGK-AEC. Presence of the hydrophobic benzyl group on serine reduced the hydrolizing properties of P. berghei endopeptidase: the reverse was observed with host endopeptidase. The hydrolysis of the N-polyhydroxyalcanoyl-VLGK-AEC substrate by the parasite neutral endopeptidase strongly increased with the schizogonic stage, as shown with synchronized P. chabaudi in mice. By its physiological pH and specificity the release of this enzyme in mouse plasma during the infection could be of interest in a peptidyl-drug strategy.

Heat shock response of Babesia divergens and identification of the hsp70 as an immunodominant early antigen during ox, gerbil and human babesiosis

Summary— Using antisera (α‐R and α‐C7Ag) directed against the conserved Gly‐Gly‐Met‐Pro‐epitope of the hsp70 family, a single antigen was identified in the human Babesia divergens Rouen 1987 isolate by Western immunoblotting and immunoprecipitation experiments. This B divergens hsp70 is highly conserved as shown by the analysis of five other geographical B divergens isolates from different hosts (human and bovine). Indirect immunofluorescence assay performed on the asexual intraerythrocytic stages showed that the hsp70 is mainly cytoplasmic and stage‐independent. Heat‐shock experiments, with 20 min incubation at 40°C followed by a 10 to 50 min shift to 37°C in the presence of [35S]‐methionine, led to an increase of two hsp of 85 and 70 kDa while protein synthesis in general decreased within 10 min. Immunoprecipitations of [35S]‐methionine radiolabelled proteins with human, ox and gerbil antisera raised against various B divergens isolates, showed the presence of a B divergens 70 kDa protein which was demonstrated to be a hsp70 by coupling immunoblotting assays with α‐C7Ag serum on the same immunoprecipitated material. During human babesiosis, the B divergens hsp70 appears as an early antigen during the acute phase. These results are in agreement with the use of the B divergens hsp70 as an essential valence antigen in an anti‐babesiosis vaccine.

Babesia divergens vaccine

A vaccine strategy against Babesia divergens bovine babesiosis was successfully developed after perfecting of an efficient in vitro culture. Crude supernatants and purified fractions were able to induce a vaccine protection in gerbils against B. divergens infection. More, supernatants induced an effective vaccine protection in cattle. The role of B. divergens exoantigens of 17, 37, 46, 70 and 90 kDa in the development of the immune response was clearly demonstrated in gerbils, cattle, and man.