Bee venom phospholipase A2 induces stage-specific growth arrest of the intraerythrocytic Plasmodium falciparum via modifications of human serum components.

Secreted phospholipases A(2) (sPLA(2)s) from snake and insect venoms and from mammalian pancreas are structurally related enzymes that have been associated with several toxic, pathological, or physiological processes. We addressed the issue of whether toxic sPLA(2)s might exert specific effects on the Plasmodium falciparum intraerythrocytic development. We showed that both toxic and non-toxic sPLA(2)s are lethal to P. falciparum grown in vitro, with large discrepancies between respective IC(50) values; IC(50) values from toxic PLA(2)s ranged from 1.1 to 200 pm, and IC(50) values from non-toxic PLA(2)s ranged from 0.14 to 1 microm. Analysis of the molecular mechanisms responsible for cytotoxicity of bee venom PLA(2) (toxic) and hog pancreas PLA(2) (non-toxic) demonstrated that, in both cases, enzymatic hydrolysis of serum phospholipids present in the culture medium was responsible for parasite growth arrest. However, bee PLA(2)-lipolyzed serum induced stage-specific inhibition of P. falciparum development, whereas hog PLA(2)-lipolyzed serum killed parasites at either stage. Sensitivity to bee PLA(2)-treated serum appeared restricted to the 19-26-h period of the 48 h parasite cycle. Analysis of the respective role of the different lipoprotein classes as substrates of bee PLA(2) showed that enzyme treatment of high density lipoproteins, low density lipoproteins, and very low density lipoproteins/chylomicrons fractions induces cytotoxicity of either fraction. In conclusion, our results demonstrate that toxic and non-toxic PLA(2)s 1) are cytotoxic to P. falciparum via hydrolysis of lipoprotein phospholipids and 2) display different killing processes presumably involving lipoprotein by-products recognizing different targets on the infected red blood cell.

Plasmepsin II, an acidic hemoglobinase from the Plasmodium falciparum food vacuole, is active at neutral pH on the host erythrocyte membrane skeleton.

Plasmepsin II, an aspartic protease from the human intraerythrocytic parasite Plasmodium falciparum, is involved in degradation of the host cell hemoglobin within the acidic food vacuole of the parasite. Previous characterization of enzymatic activities from Plasmodium soluble extracts, responsible for in vitro hydrolysis of erythrocyte spectrin, had shown that the hydrolysis process occurred at pH 5.0 and involved aspartic protease(s) cleaving mainly within the SH3 motif of the spectrin alpha-subunit. Therefore, we used a recombinant construct of the erythroid SH3 motif as substrate to investigate the involvement of plasmepsins in spectrin hydrolysis. Using specific anti-plasmepsin II antibodies in Western blotting experiments, plasmepsin II was detected in chromatographic fractions enriched in the parasite SH3 hydrolase activity. Involvement of plasmepsin II in hydrolysis was demonstrated by mass spectrometry identification of cleavage sites in the SH3 motif, upon hydrolysis by Plasmodium extract enzymatic activity, and by recombinant plasmepsin II. Furthermore, recombinant plasmepsin II digested native spectrin at pH 6.8, either purified or situated in erythrocyte ghosts. Additional degradation of actin and protein 4.1 from ghosts was observed. Specific antibodies were used in confocal imaging of schizont-infected erythrocytes to localize plasmepsin II in mature stages of the parasite development cycle; antibodies clearly labeled the periphery of the parasites. Taken together, these results strongly suggest that, in addition to hemoglobin degradation, plasmepsin II might be involved in cytoskeleton cleavage of infected erythrocytes.

Human erythroid spectrin alpha subunit and its SH3 domain are sensitive to acidic Plasmodium falciparum proteolytic activity.

Many proteases play a crucial role in the Plasmodium intraerythrocytic life cycle. Spectrin depletion, one of the major events involved in parasite release from the red blood cell, results from proteolytic activities associated with the presence of the intracellular parasite. Here, we describe a new acidic proteolytic activity from Plasmodium falciparum, whose target is the alpha-subunit of human spectrin. Immunoblotting experiments with antibodies specific for the tryptic peptides of the alpha-chain and in vitro proteolysis tests on recombinant peptides from different regions of the spectrin alpha subunit demonstrated that cleavage sites for the parasite proteolytic activity were localized within the SH3 motif of the alpha-chain sequence. Remarkably, this Plasmodium protease activity on spectrin SH3 substrate was unable to cleave the SH3 from fodrin, a non-erythroid spectrin.

Turnover of the GPI-anchored surface antigen in Paramecium: Partial release of its acylated form into the culture medium.

Surface antigens of Paramecium are high molecular weight proteins encoded by a multigene family, and their mutually exclusive expression at the cell surface is elicited by environmental conditions: changes in external factors trigger antigenic variation. The surface antigens are anchored in the plasma membrane by a glycosyl phosphatidylinositol moiety which can be removed by an endogenous phospholipase C-like hydrolase, releasing a lipid-lacking form of the molecules. In order to understand the mechanisms of the antigenic variation and the physiological involvement of the endogenous enzyme, I studied the turnover of the G antigen stably expressed at 23°C in Paramecium primaurelia. By (35)S labeling and chase experiments, I demonstrate that the turnover occurs at a slow rate (half-life beyond 45 hours), and is concomitant with a release of the molecule into the external medium: 16% of the initial cell-associated antigen is found in the medium after 45 hours. Analysis by immunolabeling and [(3)H]myristate radiolabeling of the released antigen showed that it is acylated, indicating that the release phenomenon does not involve the endogenous phospholipase C. Furthermore, the results indicate that in addition to releasing, an internal degradative pathway intervenes in the surface antigen turnover.

A new class of Paramecium surface proteins anchored in the plasma membrane by a glycosylinositol phospholipid. Membrane anchor of Paramecium cross-reacting glycoproteins.

Treatment of paramecia with ethanol or Triton X-100 solubilizes a major membrane protein, namely the surface antigen (SAg), and a set of glycopeptides in the range 40-60 kDa, which cross-react with the SAg. We demonstrate that these glycopeptides, called 'cross-reacting glycoproteins' (CRGs), are distinct molecules from the SAg. First, after purification of CRGs from ethanolic extracts of Paramecium primaurelia expressing the 156G SAg, the amino acid composition of a given CRG was found to be different from, and incompatible with, that of the 156G SAg. Secondly, we showed that the CRGs, although not immunologically detectable, are present in fractions containing the myristoylated form of the 156G SAg. The treatment of these fractions by phosphatidylinositol-specific phospholipases C enables us to reveal the CRGs through the unmasking of two distinct epitopes. One is the 'cross-reacting determinant' (CRD), initially described for the variant surface glycoproteins (VSGs) of Trypanosoma; the other determinant, called 'det-2355', is specific to the SAg and to the CRGs. Our results suggest that (1) phosphatidylinositol is covalently linked to the CRGs and (2) the CRD and the det-2355 are localized in the same region of the CRGs. We propose that the CRGs are a new set of surface proteins anchored in the cell membrane of Paramecium via a glycosylinositol phospholipid, in the same way as the SAgs.

The membrane-anchor of Paramecium temperature-specific surface antigens is a glycosylinositol phospholipid.

The temperature-specific G surface antigen of Paramecium primaurelia strain 156 was biosynthetically labeled by [3H]myristic acid in its membrane-bound form, but not in its soluble form. It could be cleaved by a phosphatidylinositol-specific phospholipase C from Trypanosoma brucei or from Bacillus cereus which released its soluble form with the unmasking of a particular glycosidic immunodeterminant called the crossreacting determinant. The Paramecium enzyme, capable of converting its membrane-bound form into the soluble one, was inhibited by a sulphydril reagent in the same way as the trypanosomal lipase. From this evidence we propose that the Paramecium temperature-specific surface antigens are anchored in the plasma membrane via a glycophospholipid, and that an endogenous phospholipase C may be involved in the antigenic variation process.

Immunological evidence of a common structure between Paramecium surface antigens and Trypanosoma variant surface glycoproteins.

The surface antigens (SAgs) of Paramecium and the variant surface antigens (VSGs) of Trypanosoma can be purified in two distinct molecular forms: a soluble form (solubilized in dilute ethanolic solution in the case of Paramecium, or in water for Trypanosoma) and a membranal form, amphiphile (solubilized in SDS). In trypanosomes, the enzymatic conversion of the membrane form into the soluble form is accompanied by the unmasking of a particular immunological determinant, called cross-reacting determinant (CRD), which is located in the COOH-terminal phospho-ethanolamine glycopeptide. We demonstrate immunological homologies between Paramecium SAgs and Trypanosoma VSGs. A determinant corresponding to the CRD of VSGs is borne by the ethanol-soluble form of the SAgs and by two cross-reacting light chains also present in ethanolic cellular extracts (together with the soluble form), and not by the membranal form of SAgs. Furthermore, we show that the membranal form of Paramecium SAgs can be converted into soluble form and that this enzymatic conversion also yields cross-reacting light chains. We also demonstrate that the membranal form is the physiological form in paramecia stably expressing a given SAg.

Surface antigens of Paramecium primaurelia. Membrane-bound and soluble forms.

The surface antigens of Paramecium constitute a family of high molecular weight (ca 300 kD) iso-proteins whose alternative expression, adjusted to environmental conditions, involves both intergenic and interallelic exclusion. Since the surface antigen molecules had previously been shown to play a key role in the control of their own expression, it seemed important to compare the structural particularities of different surface antigens: the G and D antigens of P. primaurelia expressed at different temperatures, and which are coded by two unlinked loci. Here we demonstrate that in all cases a given surface antigen presents two biochemically distinct basic forms: a soluble form recovered from ethanolic extraction of whole cells, and a membrane-bound form recovered from ciliary membranes solubilized by detergent. The membrane-bound form differs from the soluble one by its mobility on SDS gels and by an electrophoretic mobility shift in the presence of anionic or cationic detergents. Furthermore, two 40-45 kD polypeptides sharing common determinants with soluble antigens were found exclusively in ethanolic extracts but not in ciliary membranes: the cross-reactivity of these light polypeptides with ethanol-extracted antigens could be demonstrated only after beta-mercaptoethanol treatment. Immunological comparisons between allelic and non-allelic soluble antigens demonstrate that allelic antigens share a great number of surface epitopes, most of which are not accessible in vivo, while non-allelic antigens appear to share essentially sequence-antigenic determinants. The significance of these results is discussed in relation to the mechanism of antigenic variation.