Plasmepsin II, an Acidic Hemoglobinase from thePlasmodium 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 α-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.

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 ␣-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.
Malaria is a tropical disease caused by the protozoan parasite Plasmodium. In 1996, the World Health Organization estimated the annual mortality at 1.7-2.5 million and persons living in risk areas at ϳ2 billion. Resistance of Plasmodium to antimalarial drugs and vector resistance to insecticides are increasing problems in fighting the parasite. New approaches to chemotherapy based on blocking essential metabolic pathways of the parasite are necessary. Toward this end, the parasite proteases that are involved in crucial steps of Plasmodium development appear to be good targets. Several have been described that are involved in hemoglobin hydrolysis (the so-called hemoglobinases), antigen maturation, merozoite release, or invasion of new red blood cells by merozoites (1).
Among the Plasmodium proteases, the hemoglobinases have been extensively studied (2). In the food vacuole (pH 5-5.4), the 37-kDa aspartic proteases plasmepsin I and plasmepsin II initiate cleavage of the native hemoglobin tetramers, whereas the 28-kDa cysteine protease falcipain appears to digest the denatured or fragmented substrate (3). These three hemoglobinases have been cloned and sequenced (4 -6). A 43-kDa recombinant precursor of plasmepsin II lacking the first 76 residues of full-length proplasmepsin II has been expressed in Escherichia coli, and its acidification results in the production of a 38-kDa protein by autocatalytic cleavage (7). The 38-kDa enzyme showed kinetic properties similar to those of native plasmepsin II (8). Thus far, no clear difference in structure or function has emerged between plasmepsins I and II, and the reason why the parasite synthesizes both enzymes remains obscure. Unexpectedly, our work on the involvement of proteases in the invasion process yields new insights into the substrate selectivity of plasmepsin II compared with plasmepsin I.
Many years ago, the depletion of spectrin and protein 4.1, two major proteins of the erythrocyte membrane cytoskeleton, was described for infected red blood cells during the schizogonic stage of Plasmodium berghei (9). A Plasmodium lophurae 37-kDa cathepsin D was reported to digest spectrin and bands 2.1, 2.6, and 3 from duckling and human red blood cells at both pH 3.5 and 7.4 (10). Also, a 37-kDa acidic proteinase, able to cleave spectrin and protein 4.1 from human erythrocytes at both pH 5.0 and 7.2, was purified from soluble extracts of P. falciparum and P. berghei (11). In 1996, another P. falciparum proteolytic activity, clearly inhibited by 10 -20 M pepstatin A and involving protease(s) in the 35-40-kDa range, was described to act at acidic pH on human spectrin (12). This "spectrinase activity" cleaved the ␣-spectrin at a site located within the SH3 motif of the molecule.
Owing to the fact that plasmepsins I and II are pepstatin A-inhibitable acidic proteinases with a molecular mass of 37 kDa, we investigated the presence of these hemoglobinases in P. falciparum chromatographic fractions enriched for the SH3 hydrolase activity. Plasmepsin II was found in the most active * This work was supported in part by CNRS Grant GDR 1077; by Ministère de l'Enseignement Supérieur et de la Recherche Grant DSPT N 5 and Ministère de l'Education Nacionale, de l'Enseignement Supérieur et de la Recherche, Délégation Générale pour Armement; and by the European Community Life Sciences for Developing Countries Program III Program, supported by Grant TS3-*CT92-0145. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. fractions. Mass spectrometry identification of cleavage sites in the SH3 motif upon digestion by recombinant plasmepsin II or by spectrinase activity unambiguously established involvement of plasmepsin II in in vitro hydrolysis of the SH3 motif. The recombinant enzyme proved to be able to digest native spectrin at pH 6.8, either purified or in erythrocyte ghosts. Confocal microscopy analysis of infected erythrocytes with anti-plasmepsin II antibodies showed that the enzyme can be visualized outside the parasite in mature stages of Plasmodium. These results strengthen the idea of plasmepsin II being devoted to function(s) other than hemoglobin degradation in infected red blood cells and, more generally, address the question of the redistribution of some specific proteases during the intraerythrocytic development of P. falciparum.
P. falciparum in Vitro Culture and Synchronization-The FcB1 strain from Colombia was used throughout the experiments. Culture was performed according to Trager and Jensen (13). Basic culture medium contained RPMI 1640 medium (Life Technologies, Inc.), 25 mM Hepes, 27.5 mM NaHCO 3 , and 11 mM glucose (pH 7.4) supplemented with penicillin (100 IU/ml), streptomycin (100 g/ml), and 7% (v/v) heat-treated human plasma. Red blood cells from the same donor were added at a hematocrit of 2-3%, and the culture was maintained under an atmosphere of 3% CO 2 , 6% O 2 , and 91% N 2 at 37°C, with daily medium changes. Synchronization of cultures was achieved by Plasmagel treatment (14), followed, 4 -5 h later, by 5% (w/v) sorbitol treatment (15). Under our culture conditions, the in vitro life cycle of the FcB1 strain was 48 h.
Enzymatic Extracts and Enrichment of P. falciparum Spectrinase Activity-100,000 ϫ g extracts from the schizont stage of P. falciparum were prepared as described by Le Bonniec et al. (12). For enrichment of acidic spectrinase activity, gel filtration of 100,000 ϫ g extracts on Superose 12 and chromatography on alkyl-Sepharose were performed using a fast protein liquid chromatography system (Amersham Pharmacia Biotech). The gel filtration procedure has been described (12). For spectrinase enrichment through hydrophobic interactions, pooled active fractions from Superose 12 were applied to an alkyl-Superose HR 5/5 column equilibrated in 100 mM phosphate buffer (pH 7.2) containing 2 M ammonium sulfate. Elution was performed with a gradient of ammonium sulfate from 2 to 0 M in 100 mM phosphate buffer at a flow rate of 0.5 ml/min. Usually, 1-ml fractions were collected and tested for their SH3-hydrolyzing activity at pH 5.0 using the recombinant GST-SH3 peptide as substrate. Conditions for high performance electrophoresis chromatography analysis (system from Applied Biosystems, Foster City, CA) have been described previously (12).
Purification of Spectrin-Hemoglobin-free erythrocyte membranes were prepared according to Dodge et al. (16). Human spectrin was purified by incubating erythrocytes at 37°C for 30 min in 0.3 mM sodium phosphate and 0.1 mM EDTA. After centrifugation at 300,000 ϫ g for 1 h, the supernatant containing primarily spectrin and actin (17) was concentrated and enriched for spectrin by ultrafiltration using Centriprep-100 units (Amicon, Inc., Epernon, France).
Enzymatic Tests-All enzymatic reactions were performed at 37°C in 25 mM Tris adjusted to pH 6.8 or 5.0 with citrate. Usually, tests of the spectrinase-enriched fraction against GST-SH3 were performed at a ratio of 1 volume of pelleted GST-SH3-Sepharose beads (12) to 4 volumes of the enzymatic fraction. Tests of purified recombinant plasmepsin II (8) against GST-SH3 were performed by incubating 10 l of pelleted beads with 0.5 g of enzyme. Tests of recombinant plasmepsin II against native spectrin was performed by incubating 0.5 g of en-zyme with 50 g of spectrin. When indicated, recombinant plasmepsin was incubated prior to tests at pH 5.0 and 37°C for 2 h to induce maturation of the 43-kDa precursor form to the 38-kDa form by autoactivation.
Prior to mass spectrometry analysis of SH3 hydrolysis, the GST-SH3 peptide linked to Sepharose beads was incubated for 2 h at pH 5.0 and 37°C with the spectrinase-enriched fraction recovered by gel filtration or was incubated for 3 h at pH 5 and 37°C with recombinant plasmepsin II. The presence of released fragments was checked by electrophoresis on Tricine gel, which allows visualization of small peptides (18). For kinetic analysis of SH3 cleavage by the spectrinase activity, aliquots were taken after 1, 2, and 3 min of incubation of the recombinant peptide with the spectrinase-enriched fraction. The spectrinase-enriched fraction, recombinant plasmepsin II, and GST-SH3, alone and under the same experimental conditions, were used as controls in mass spectrometry analyses.
Mass Spectrometry Analysis-After digestion of the SH3 motif with either recombinant plasmepsin II or the spectrinase-enriched fraction, the released peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After digestion, the samples were acidified by performing a dilution (1:2 to 1:10) in a 0.1% aqueous trifluoroacetic acid solution. The MALDI matrix used was 2,5-dihydroxybenzoic acid (Aldrich). The acidified samples were then mixed 1:1 (v/v) with a saturated solution of 2,5-dihydroxybenzoic acid in 0.1% aqueous trifluoroacetic acid and analyzed. After determination of their molecular masses, cleavage sites in the SH3 motif were identified.
MALDI-TOF-MS measurements were performed on a Voyager Elite mass spectrometer (Perseptive Biosystems, Inc., Framingham, MA) in positive ion reflectron mode using a delayed extraction. For laser desorption, a nitrogen laser beam ( ϭ 337 nm) was used. Typically, 150 -256 shots were averaged for each acquired spectrum. External calibration was performed using a mixture of neurotensin, ACTH clip-(18 -39), and ACTH-(7-38) with m/z ratios corresponding to monoisotopic [M ϩ H] ϩ of 1672.92 and 2465.20 and average [M ϩ H] ϩ of 3660.19, respectively. Only the average m/z ratios of the protonated peptides are given under "Results." The difference between the calculated average mass and the experimental mass determination was Ͻ0.5 atomic mass unit.
SDS-PAGE and Western Blot Analysis-Electrophoresis was performed on 10 and 15% acrylamide gels according to Laemmli (19). Gels were stained with Coomassie Brilliant Blue R-250. Proteins were electrotransferred onto nitrocellulose membranes according to Towbin et al. (20). Rabbit anti-plasmepsin I and anti-plasmepsin II antibodies (21) were used at a 1:2500 dilution.

Plasmepsin II, an Acidic Endoprotease from the Food Vacuole of Plasmodium, Co-purifies with the Spectrinase Activity-
Owing to the acidic pH activity of the parasite SH3 hydrolase, we looked for the presence of the three hemoglobinases plasmepsin I, plasmepsin II, and falcipain in the fractions recovered by gel filtration of a 100,000 ϫ g extract from the schizont stage of the FcB1 strain of P. falciparum. The spectrinase activity was localized in the eluted fractions via hydrolysis of the recombinant GST-SH3 peptide at pH 5.0 and 37°C and analyzed by SDS-PAGE and Coomassie Blue staining of the gel. In controls for each fraction omitting GST-SH3, the amount of protein in the 30 -50-kDa range was too low to be 1 The abbreviations used are: GST, glutathione S-transferase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; ACTH, adrenocorticotropic hormone; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.
detected (data not shown). Plasmepsins I and II were visualized by Western blotting of the fractions using specific antiplasmepsin I (serum 574) and anti-plasmepsin II (serum 737) antibodies. The presence of falcipain was checked by hydrolysis of the fluorogenic substrate N ␣ -benzyloxycarbonyl-Phe-Arg 7-amido-4-methylcoumarin (23). Distribution of the spectrinase activity (Fig. 1A) showed that fractions 8 and 9 contained the highest SH3-hydrolyzing activity, and plasmepsin II was clearly visualized in these fractions (Fig. 1C). In contrast, plasmepsin I, although also a 37-kDa protein, was visualized in fractions 4 -9, peaking in fraction 4 (Fig. 1B), which corresponds to eluted proteins with ϳ100-kDa molecular masses. As chromatography was performed under nondenaturing conditions, early elution of plasmepsin I might be due to proteinprotein interactions. Low SH3-hydrolyzing activity was detected in plasmepsin I-containing fractions compared with plasmepsin II-containing fractions. With respect to falcipain, degradation of the Phe-Arg 7-amido-4-methylcoumarin substrate was observed with fractions 9 and 10 ( Fig. 1D) and was minimal in fraction 8, which had high proteolytic activity against the SH3 motif. Also, falcipain is a cysteine protease, hence is not inhibited by pepstatin A, in contrast to what was observed with the spectrinase activity.
To test the hypothesis that the enzyme(s) responsible for SH3 hydrolysis might be different from plasmepsin II, various biochemical procedures were tried to separate plasmepsin II from the SH3 hydrolase activity. 100,000 ϫ g schizont extracts from different cultures of the FcB1 strain were fractionated by gel filtration on a Superose 12 column, and active fractions were further enriched either by chromatography on alkyl-Sepharose or by high performance electrophoresis chromatography. As done previously, the eluted fractions were analyzed for the presence of plasmepsin II and SH3 hydrolase. In every experiment, plasmepsin II was found to coelute with the SH3hydrolyzing activity (data not shown).
Recombinant Plasmepsin II Is Able to Hydrolyze the SH3 Domain in the GST-SH3 Peptide-The presence of plasmepsin II in the SH3-hydrolyzing fractions prompted us to investigate the involvement of this enzyme in SH3 degradation. The recombinant enzyme was used in enzymatic assays with the recombinant GST-SH3 peptide as substrate. Recombinant plasmepsin II was incubated with substrate at pH 5.0 and 37°C for different times, and GST-SH3 proteolysis was analyzed by SDS-PAGE and Coomassie Blue staining of the pro-teins (Fig. 2). Under these conditions, the recombinant enzyme proved to be able to cleave GST-SH3, generating three fragments at 34, 32, and 30 kDa, a similar pattern to the one observed with the spectrinase-enriched fraction from gel filtration of a 100,000 ϫ g schizont extract (12).
Localization of Cleavage Sites in the SH3 Motif by Mass Spectrometry Analysis of the Hydrolysis Products-To unambiguously clarify the involvement of plasmepsin II in the SH3 hydrolysis, the identification of SH3 cleavage sites upon spectrinase action and upon recombinant plasmepsin II action was undertaken by MALDI-TOF-MS analysis of the released fragments. The recombinant GST-SH3 peptide linked to Sepharose beads was incubated at pH 5.0 and 37°C with recombinant plasmepsin II or with a spectrinase-enriched fraction recovered by gel filtration of a 100,000 ϫ g schizont extract. Hydrolysis products were analyzed by SDS-PAGE on a Tricine gel, allowing the visualization of peptides in the 1-5-kDa range (data not shown). Sepharose beads were pelleted by centrifugation, and the supernatant was analyzed by MALDI-TOF-MS. The identified cleavage sites along the amino acid sequence of the SH3 motif are reported in Fig. 3.
Six sites were identified due to cleavage by the spectrinaseenriched fraction (Fig. 3, sequence 2). They are distributed within three "regions" of cleavage, showing one site (F2Q), two sites (Y2V, R2R) and three contiguous sites (L2L, L2S, S2S) of cleavage, respectively. Location of the cleavage regions along the amino acid sequence corresponds to the expected generation of the 34-, 32-, and 30-kDa proteolytic fragments.
Five sites were identified due to cleavage by recombinant plasmepsin II (Fig. 3, sequence 1). Out of the five, four corresponded to the F2Q, Y2V, L2L, and L2S sites identified upon digestion of the SH3 motif by the spectrinase fraction. The fifth site (F2I) was located outside the SH3 amino acid sequence, in the recombinant plasmid sequence.
Although mass quantitation of the hydrolysis fragments is difficult by MALDI-TOF-MS, the intensity of some peaks was highly reproducible from one experiment to the other, suggesting that the corresponding sites were highly susceptible to cleavage. From this, the Y2V and S2S sites appear as major sites of cleavage with the spectrinase fraction, and Y2V and L2L are major sites with plasmepsin II. Minor sites with the spectrinase fraction and plasmepsin II are R2R, L2L, and L2S and F2I and L2S, respectively. A very minor site at F2Q was revealed for both enzymatic activities.
Two main conclusions must be drawn from these results. First, plasmepsin II is unambiguously and predominantly involved in the in vitro hydrolysis of the SH3 motif observed with the parasite spectrinase fraction; and second, enzyme(s) other than plasmepsin II are involved in this hydrolysis, cleaving at the R2R and S2S sites. To detail the sequence of events due to respective enzymes in the spectrinase fraction, kinetic analysis of the cleavage processes was performed by MALDI-TOF-MS. The identity of the peptides released upon hydrolysis of Sepharose bead-linked GST-SH3 was assessed after 1, 2, and 3

FIG. 1. Identification of the hemoglobinases in Superose 12
fractions of a 100,000 ؋ g extract from schizonts of the FcB1 strain. Elution fractions 4 -12 (approximately corresponding to the elution of proteins in the range of 100 to 20 kDa) were split into four aliquots. One aliquot was tested for its SH3-hydrolyzing capacity. The recombinant GST-SH3 peptide was incubated with the fraction for 2 h at pH 5.0 and 37°C. SH3 hydrolysis was analyzed by SDS-PAGE and Coomassie Blue staining of the gel (A). The recombinant GST-SH3 peptide was also incubated at pH 5.0 in the elution buffer alone (control (co)). The presence of plasmepsins I and II was checked by Western blot analysis of two other aliquots from each fraction using anti-plasmepsin I antibodies (1:2500; B) and anti-plasmepsin II antibodies (1:2500; C). The remaining aliquot was used to test the presence of falcipain. Enzymatic activity was checked by cleavage of the fluorogenic dipeptidyl substrate N ␣ -benzyloxycarbonyl-Phe-Arg 7-amido-4-methylcoumarin, a specific substrate for falcipain (D). Emitted fluorescent light was collected at 440 nm upon excitation at 380 nm (arbitrary units). Molecular masses are on the right. min of enzymatic incubation (Fig. 3, sequence 3). The Y2V site (attributed to cleavage by plasmepsin II) was first cleaved at 1 min, and then the S2S site at 2 min. Next, the L2L and L2S sited (both due to plasmepsin II) and the R2R site were cleaved off within 3 min. From this, we can infer that cleavage of the S2S site is due to an additional endopeptidase cleaving the fragment -SSINK . . . VPAVY.
Recombinant Plasmepsin II Is Able to Hydrolyze Native Spectrin at pH 6.8 -Hydrolysis of purified human erythroid spectrin by the Plasmodium acidic spectrinase activity generates three main fragments at 170, 150, and 125 kDa (12). The generation of the 170-and 125-kDa fragments was explained by enzyme(s) cutting within the SH3 motif of the spectrin ␣-subunit. Because plasmepsin II was found to be part of the acting proteinases in the so-called spectrinase activity, we tested the ability of recombinant plasmepsin II to digest purified native spectrin in order to compare the proteolysis pattern with the one generated by the spectrinase activity. Spectrin from normal erythrocytes was incubated with recombinant plasmepsin II for 1 h, 2 h, and overnight at pH 6.8 and 37°C. The recombinant enzyme had been preincubated or not for 2 h at pH 5.0 since such conditions induce autoactivation and shortening of the molecule from 43 to 38 kDa (7). Spectrin hydrolysis was analyzed by SDS-PAGE and Coomassie Blue staining of the proteins. Results from the overnight incubation are presented in Fig. 4A.
At pH 6.8, hydrolysis of spectrin by the 43-kDa form of the enzyme occurred, generating four major fragments at 170, 155, 148, and 125 kDa (Fig. 4A, lane 2). These fragment sizes are very close to the ones described upon spectrin degradation by the spectrinase activity. Interestingly, greater degradation of spectrin was observed at pH 6.8 in the presence of the 38-kDa form of recombinant plasmepsin II, with an additional proteolytic fragment at 112 kDa (Fig. 4A, lane 3).
Degradation of the GST-SH3 peptide by 43-kDa recombinant plasmepsin II was analyzed at pH 6.8 as well. Hydrolysis was slower than that observed at pH 5.0 (under conditions in which 43-kDa plasmepsin II is activated to 38 kDa), but it generated the expected pattern of 34-, 32-, and 30-kDa fragments (Fig. 4B).
Recombinant Plasmepsin II Selectively Digests Spectrin, Actin, and Protein 4.1 from Erythrocyte Ghosts-The recombinant plasmepsin II activity was tested against ghosts from normal erythrocytes to check if native spectrin, situated within the protein network of the membrane skeleton, would be a substrate for the enzyme and if other proteins from the ghosts would be substrates as well. Erythrocytes were lysed in 5 mM PO 4 buffer (pH 6.8), washed with the same buffer, and incubated overnight at pH 6.8 and 37°C with the 43-or 38-kDa form of recombinant plasmepsin II. Degradation of the proteins from ghosts was analyzed by SDS-PAGE and Coomassie Blue staining of the gel. As shown in Fig. 5 (lane 3), the pattern of the ghosts was dramatically altered in the presence of 38-kDa plasmepsin II. Densitometric analysis of the gel gave a 84% disappearance of the ␣-spectrin and a 52% disappearance of the ␤-spectrin with the 38-kDa form of the enzyme versus almost unchanged ␣and ␤-spectrin amounts in the presence of the 43-kDa form (Fig. 5, lane 2). Also, the amount of protein 4.1 decreased in the presence of 38-kDa plasmepsin II, and actin clearly disappeared.
Immunolabeled Plasmepsin II Can Be Visualized outside the Parasite in Schizont-infected Red Blood Cells-To address the question of the erythrocyte membrane skeleton being a physiologically relevant substrate of plasmepsin II in mature parasite-infected cells, we investigated (by confocal microscopy) the cellular location of the enzyme during the schizont stage. Previous immunoelectron microscopy experiments (5) had shown that plasmepsins are present at the surface of the parasite in trophozoites, but no analysis of plasmepsin location later during parasite intraerythrocytic development was available thus far.
Smears of FcB1-infected red blood cells were fixed in acetone/ methanol, and double labeling of cells was performed using rabbit anti-plasmepsin II polyclonal antibodies (serum 737) and mouse anti-MSP1 monoclonal antibody (ascites 22-2). Anti-MSP1 antibody was used to visualize mature intraerythrocytic parasites via labeling of the parasite membrane. Preservation of red blood cells upon fixation was checked by phasecontrast microscopy. No labeling was observed with non- immune rabbit serum (data not shown) or with secondary antibodies alone (data not shown).
Images of immunolabeled cells, summed from three 10-nm spaced out sections each, are presented in Fig. 6. Fig. 6A shows an ϳ40-h-old schizont; although MSP1 labeling is confined to the parasite (panel 3), fleecy labeling with anti-plasmepsin II antibodies can be observed inside and outside the parasite (panel 2). The presence of anti-plasmepsin II antibody labeling in the erythrocyte cytoplasm, although not found in all schizonts, was frequently observed. In Fig. 6B is presented an early segmenter (44 -46 h old). Remarkably, plasmepsin II labeling appears to delineate individualizing merozoites, in a comparable way to MSP1 labeling, although MSP1 and plasmepsin II labeling does not totally merge; labeled plasmepsin II appears as a green rim external to the yellow MSP1-delineated membrane.

DISCUSSION
Most of the current efforts in understanding the biology of Plasmodium aim at elaborating new antimalarial strategies. Protease inhibitors have been successfully employed in well known examples such as AIDS therapy, and identification of target proteases in Plasmodium is today part of the antimalarial fight. In this respect, the hemoglobinases, as participants in an essential metabolic pathway, appear to be choice targets. Plasmepsins I and II are both responsible for the initial cleavage of hemoglobin in the digestive vacuole of the intraerythrocytic parasite, but when a specific inhibitor of plasmepsin I (SC-50083) is added to cultures at the trophozoite stage, when hemoglobin is degraded, the parasites have markedly diminished hemozoin production and are killed (IC 50 ϭ 2 ϫ 10 Ϫ6 M) (5). This indicates that plasmepsin I activity is essential, but also that plasmepsins I and II have distinct hemoglobin-degrading capacities. Moreover, the inability of plasmepsin II to supplant plasmepsin I in hemoglobin degradation raises the question of the exact role of plasmepsin II in this process.
Plasmepsin II Exhibits Different Substrate Selectivity Com-pared with Plasmepsin I-We show here that proteins other than hemoglobin can be substrates of plasmepsin II, namely spectrin, protein 4.1, and actin, all components of the erythrocyte membrane skeleton. Since plasmepsin I was shown to be inactive against a variety of non-globin substrates (2) and was also found to be poorly active on the GST-SH3 substrate, in contrast to plasmepsin II, these findings reinforce the idea that the two enzymes may be devoted to different roles in the parasitized cell. The identification of the five sites cleaved at pH 5.0 by recombinant plasmepsin II in the SH3 motif of the spectrin ␣-subunit is in general agreement with the known preference of plasmepsin II for hydrophobic residues on both sides of the scissile bond (3). Only two sites, L2S and F2Q, show a hydrophobic residue at the P1 position and a hydrophilic one at the P1Ј position, extending the repertoire of the potential sites of cleavage for plasmepsin II. For plasmepsin I, if one considers amino acids at the P1 and P1Ј positions, two putative cleavage sites can be identified  6. Immunolocalization of plasmepsin II in schizont-infected erythrocytes by confocal microscopy. Blood smears of the FcB1 strain were incubated with mouse anti-MSP1 monoclonal antibody (1:50) and rabbit anti-plasmepsin II polyclonal antibodies (1:200) using tetramethylrhodamine B isothiocyanate-conjugated anti-mouse Ig antibodies (1:50) and fluorescein isothiocyanate-conjugated anti-rabbit Ig antibodies (1:100) as secondary antibodies. A, an ϳ40-h-old schizont; B, an early segmenter. Panels 1, plasmepsin II and MSP1 labeling; panels 2, plasmepsin II labeling alone; panels 3, MSP1 labeling alone. In each case, the presence of the surrounding erythrocyte cytoplasm was checked by phase-contrast microscopy. Scale bars ϭ 3 m.
within the SH3 motif: the F2Q site, deduced from the plasmepsin I preference for the phenylalanine at the P1 position, and the L2L site, listed as a secondary site on hemoglobin (3). The presence of 32-30-kDa fragment(s) upon hydrolysis of GST-SH3 by fraction 4 and adjacent functions in Fig. 1 could thus be attributed to plasmepsin I action. However, fractions in which plasmepsin II was readily detected compared with plasmepsin I (fractions 8 -10) displayed higher degradative activity. This observation suggests that, most probably, recognition of potential cleavage sites in the SH3 motif by plasmepsins I and II involves additional parameters such as identity of the surrounding amino acids and/or conformation of the polypeptide chain. In the same way, the number of spectrin sites actually cleaved by plasmepsin II appears limited since a 20-h hydrolysis of purified native spectrin at pH 6.8 by recombinant plasmepsin II produces high molecular mass fragments, although many potential sites are found scattered along the ␣and ␤-subunit amino acid sequences, predicting the generation of multiple short sized fragments. Conformational requirements might explain why the SH3 motif, whose secondary structure is different from the repetitive structure of the rest of the ␣-chain, is a privileged target for the enzyme. This interpretation would go along with what has been written about the enzyme specificity depending on the tertiary structure of its substrate hemoglobin (24).
pH Activity and Maturation of the Recombinant Enzyme-Recombinant plasmepsin II can digest the SH3 motif of ␣-spectrin, as well as native spectrin, at pH 6.8. In terms of activity of the enzyme on the GST-SH3 substrate, hydrolysis of the substrate at pH 5.0 is completed within 75 min, whereas at pH 6.8, primary degradation fragments are visible. Thus, the pH optimum of the enzymatic activity does not seem to differ from hemoglobin to the GST-SH3 substrate. The activities of purified native plasmepsins II and I on hemoglobin are known to be dramatically reduced at pH 6.5. In both cases, ϳ35% residual activity is detected (24). Further experiments are needed to quantify the activity of the recombinant enzyme against the SH3 motif at pH 6.8 and to compare it with its activity against hemoglobin at the same pH. An interesting observation is the enhanced efficiency of recombinant plasmepsin II in degrading spectrin after it has been shortened to 38 kDa by autoactivation at pH 5.0. Previous studies had demonstrated that the structural and kinetic properties of mature recombinant plasmepsin II and native plasmepsin II were similar (8), but nothing is known about the activity of the precursor form of recombinant plasmepsin II since all experiments were performed at pH 5.0. We observed that within 20 h at pH 6.8, the hydrolysis pattern of native spectrin, either purified or from ghosts, was greater when due to the mature form of the enzyme than to the precursor form. One explanation might be that the enzyme has slowly autoactivated to the 38-kDa form at pH 6.8. If not, this might indicate that some of the properties of the enzyme differ between immature and mature forms in terms of structural and/or kinetic properties.
Is the in Vitro Degradation of the Host Cell Skeleton by Plasmepsin II Physiologically Relevant?-Our results raise the question of the physiological relevance of observations made in vitro. Indeed, two points must be considered. First, the red blood cell skeleton and plasmepsin II were believed to belong to distinct cellular compartments; and second, to cleave skeleton proteins, plasmepsin II would need to act at a pH near neutrality in the cytoplasm of the red blood cell. Now, the enzyme has been found to be active at pH 6.8 against spectrin, actin, and protein 4.1, which suggests that it could be active in the red blood cell cytoplasm. Confocal imaging using anti-plasmepsin II antibodies brought clear evidence of a possible proximity between the enzyme and membrane skeleton during late parasite maturation. What has been inferred about plasmepsin I and plasmepsin II trafficking, largely deduced from the study of plasmepsin I trafficking, is that plasmepsins are secretory molecules that are synthesized as type II integral membrane proteins (21). The proplasmepsins have been detected by immunoelectron microscopy at the parasitophorous vacuolar membrane (5), and visualization of plasmepsin II outside the parasite by immunoconfocal microscopy might be due to parasitophorous membrane-originated structures extending into the red blood cell cytoplasm. Plasmepsin II in early segmenters was found located at the periphery of the parasite, and labeling appeared higher at this stage than in younger schizonts, even though massive degradation of hemoglobin is not required anymore.
Considering the broad substrate selectivity and cleavage site specificity of plasmepsin II and the large amount as well as the location of the enzyme in late schizonts, the overall results presented here strongly suggest that plasmepsin II function in the parasite intraerythrocytic cycle is not restricted to hemoglobin degradation. Its involvement in spectrin depletion in vivo remains to be established, but it is clear that trafficking of plasmepsins and their respective roles in the infected cell must be further explored.