Biology of the Cell, 64 (1988) 233-244 © Elsevier, Paris

233

T r y p a n o s o m e s and P lasmodium

Neutral proteases involved in the reinvasion of erythrocytes by Plasmodium merozoites

Joseph SCHREVEL l*, Philippe GRELLIER 1, Roger MAYER 2 and Michel MONSIGNY 2

l Laboratoire de Biologie CeUulaire, lIRA CNRS no. 80, Universitd de Poitiers, 40 avenue du Recteur Pineau, F-86022 Poitiers Cede.r; and

2 Ddpartement de Biochimie des Glycoconjuguds, Centre de Biophysique Moldculaire, CNRS et Universitd d'Oridans, I rue Haute, F-45071 Oridans Cedex 2, France

Neutral proteases of Plasmodium sp erythrocytic stages were studied by means of a sensitive fiuorogenic method and gelatin-SDS-PAGE. The substrates gluconoyl-VaI-Leu-Gly-Lys(or Arg)-3-amido-9.ethylcarbazole were selectively hydro- iyzed 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 P f 68 endopeptidases were localized in sehizonts and also in merozoites as shown by indirect im- munofluoreseence on Pb merozoites and by the Identification of the Pf68 endopeptidase activity in free viable merozoites. The Pb 68 and Pf68 endopeptidases belong to the class of eysteine proteases. Analysis by gelatin-SDS-PAGE of a Pb 68 endopeptidase-endched fraction showed a reproducible 95,000 proteolytie band. The initial extracts showed a similar 95,000 proteolytle band, and also 2 other 90,000 and 8$,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. Hydrophille peptide derivatives related to the substrate of Pf 68 endopeptidase are shown to be potential in- hibitors of the P/relnvasion process in vitro.

neutra l pro teases - - m a l a f l a - - m e r o z o i t e - - ery throeyte invas ion - - Plasmodlum falciparum - - Plasmodlum berghei - - Plasmodlum chabaudi

INTRODUCTION

The endoerythrocytic phase of the malaria parasite life- cycle begins with the invasion of erythrocytes by merozoites released from mature schizont-infected erythrocytes. Each merozoite undergoes a sequential development starting from a ring form, evolving to trophozoite and finally to schizont. The initial step, also called the reinvasion process, includes: (t.) rupture of the host cell; (it') release of merozoites into the plasma;

" Correspondence and reprints Abbreviations: AEC, 3-amino-9-ethylcarbazole; AMC, 7-amino- 4-metylcoumarin; DIPF, di-isopropyl phosphofluoridate; EDTA, ethylene-diaminetetraacetate; GIcA, gluconoyl; NEM, N-ethyl- maleimide ; Pb, Plasmodium berghei ; Pc, Plasmodium chabaudi ; Pf, Plasmodiurn falciparum ; pHMB, o-hydroxymercuribenzoate; PI-PLC, phosphatidylinositol-specific phospholipase C; PMSF, benzylsulfonyl fluoride; pNA, p-nitroaniline; Pyn, Plasmodium yoelii nigeriensis; STI, soybean trypsin inhibitor; TLCK, N-=-p- tosyl-L-lysine chloromethylketone; TPCK, N-tosyl-L-phenyl- alanine chloromethylketone; SDS-PAGE, sodium dodecyl sul- phate-polyacrylamide gel electrophoresis.

flit.) a subsequent motile Dhave; (iv) specific interactions between merozoite and erythrocyte membrane com- ponents; and (v) finally, internalization of the parasite inside the erythrocyte. Drugs effective in blocking one or several specific steps of this crucial phase could help to control the disease. Such a strategy requires a detailed analysis of the major molecules acting at each step. The synthesis of compounds structurally analogous to key molecules pertaining to this process could open new ap- proaches to antimalarial chemotherapy.

The recognition of an erythrocyte by a merozoite is mediated by receptors and/or sugar-binding proteins [26, 66]. Neither the receptors nor the ligands are clearly identified. The 2 major types of erythrocyte transmem- brane glycoproteins, glycophorins and band III, were con- sidered as potent receptors by Pasvol et al. [57] and Okoye and Bennett [55], respectively. The putative glycophorin- binding protein, initially localized at the surface of the merozoite [58], is in fact secreted from Piasmodium falciparum schizonts [7]. Furthermore, recent studies in- dicate strain differences in Pf receptors, with at least 2 types of receptors: a neuraminidas~-sensitive one, and a neuramim'dase'insensitive but trypsin-sensitive one [27, 50, 60]. After the initial recognition between the merozoite and the erythrocyte, an apical reorientation of the merozoite

234 J. SchrOvel et al.

occurs. A junction between the merozoite and the erythrocyte membrane moves from the apical to the posterior end of the merozoite [1] and the parasite is gradually isolated into a parasitophorous vacuole. Dur- ing these events, proteases are involved, as suggested by the effects of protease inhibitors on the reinvasion of erythrocytes by Plasmodium knowlesi [4, 25] and Plasmodium falciparum merozoites [17, 21, 46].

Piasmodium intraerythrocytic development takes place in the acidic parasitophorous vacuole and requires the digestion of host erythrocyte hemoglobin: depending upon species, 25%-75% of hemoglobin may be degraded [23]. By using native or denatured hemoglobin, cathepsin D- like proteases were identified in 1). berghei [43, 44], P. fal- ciparum [24, 45], P. knowlesi [32, 45], and P. yoelii [2], and an acid low molecular weight protease from P. fal- ciparum was also characterized [72].

The direct evidence of proteases acting at physiological pH during the reinvasion process is still poorly illustrated for several reasons: (0 proteases acting outside the parasitophorous vacuole complex are lost during parasite isolation; (ii) proteases act only during a short, specific step of the reinvasion; (lit.) the parasite extracts contain low amounts of active proteases which are at the lower limit of the usual enzyme detection methods using syn- thetic chromogenic substrates (e.g., p.nitroanilide, p-naphtylamides, etc). Difficulties in the identification of neutral proteases acting during the endoerythrocytic stages of the parasite arise from possible interferences of the host proteases [67]. The ratios of both reticulocytes and leukocytes to erythrocytes increase during the blood phase of malaria infection; therefore, clear separation of Plasmodium.infected erythrocytes from other blood cells is necessary. Moreover, identification of the enzymes from uninfected healthy erythrocytes is required for discrimina- tion between host and parasite enzymes,

The problems are now being addressed by using new and/or more sensitive methods for enzyme detection and by high pressure liquid chromatography. A fluorogenic assay using peptidyl-3-amido-9-ethylcarbazole (AEC) allowed the detection of very low amounts of a P, berghei neutral endopeptidase specific for the sequence GIcA-VaI. Leu-GIy-Arg.AEC [65]. This method detects trypsin at the level of a few nanograms per millilitre, which corresponds to a dramatic increase in sensitivity in comparison with methods using chromogenic substrates [51]. Other advan- tages of the method are that the released AEC can be selec- tively extracted by ethyl acetate, which allows the use of such substrates in the presence of cell fractions and cell lysates as well as supernates, and that the overlapping of fluorescence spectra of free AEC and acyl-AEC derivatives at the excitation and emission wavelengths used is negligi- ble [54]. A second strategy in the study of t'lasmodium neutral proteases was recently developed by using gelatin substrate polyacrylamide electrophoresis (gelatin-SDS- PAGE) according to Heussen and Dowdle [33]. By means of this procedure, several neutral proteases were identified in P. falcioarum trophozoites [63] as well as in schizonts and merozoites [13, 63]. In this paper, we will review the properties and characteristics of the known Plasmodium neutral proteases, especially the Pb 68 and Pf68 endopep- tidases, and present original data o n : (i) Pb 68 endopep- tidase activity on gelatin-substrate polyacrylamide gel electrophoresis; (it') the recovery of neutral proteases from the culture medium during the in vitro reinvasion process; and flit.) the possible use of synthetic peptide analogues of the substrates of Pf68 endopeptidase to block reinvasion.

NEUTRAL PROTEASES FROM PLASMODIUM EN- DOERYTHROCYTIC STAGES

The neutral proteases of different Plasmodium en- doerythrocytic stages are listed in Table I. The 6esigna- tion of the proteases was based on their Mr. The protease extracts are usually the 100,000-g cytosoluble parasite frac- tions obtained from a lysate of free parasites, except for the proteases characterized by gelatin SDS-PAGE which were obtained from pellets of free parasites.

Plasmodium neutral aminopeptidases

Several neutral aminopeptidases were detected in rodent malarial parasites and in P. falciparum by using Ala-or Leu-p-nitroanilide substrates. The localization and func- tions of these aminopeptidases are still unknown. Neutral t). falciparum aminopeptidases were reported to be pres- ent in the cytosolic fraction of trophozoites rather than in the acid food vacuole [24, 71].

Plasmodium proteases acting on gelatin substrates

Analysis of proteases pertaining to synchronized popula- tions of specific life-cycle stages by use of the gelatin SDS- PAGE method showed the presence of 3 neutral proteases in the intraerythrocytic stages of P. falciparum: a P f 28 protease in trophozoite preparations, a Pf35-40 protease in late schizont preparations, and a Pf 75 protease in merozoites [63]. Sensitivity to inhibitors of the Pf28 and Pf35-40 proteases is in agreement with the characteristics of cysteine proteases, and that of the ,of 75 protease to those of serine proteases [63]. By using the same gelatin SDS.PAGE technique, Braun-Breton et al, [13] demonstrated that treatment of intact merozoites or isolated schizont membranes with a phosphatidylinositol- specific phospholipase C (PI-PLC) induces the activation of the ,of 76 protease. As shown in this issue, Pf 76 pro- tease activation is related to merozoite maturation and/or erythrocyte invasion [12]. Stage-specific activity and the different sensitivity to inhibitors of the parasite proteases, as well as the rapid and specific release of an anchored protease from the membrane by a PI-PLC mechanism, clearly indicate that several proteases are important molecules both in the reinvasion process of erythrocytes by merozoites and in the erythrocytic parasite development.

Plasmodium neutral endopeptidases specific for GIcA-VaI-Leu-Gly-Lys (or Arg)-AEC substrates

The fluorogenic method based on the use of peptidyl-AEC substrates appears to be quite useful for the characteriza- tion of 1). berghei and P. chabaudi neutral endopeptidases [9], and in monitoring the purification of P. berghei [8] and P. falciparum [22] neutral endopeptidases. These results will be reviewed in the next chapter. Furthermore, substrate analogues of P. faiciparum endopeptidase decrease erythrocytic parasitemia in vitro by inhibiting the reinvasion process.

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236 J. Schr(~vel et al.

THE 68,000 PLASMODIUM ENDOPEPTIDASES

P. berghei, P. chabaudi, and P. falciparum-infected erythrocytes contain a neutral parasite endopep- tidase specific for the GIcA-VaI-Leu-Gly-Lys (or Arg) substrates

Selection o f the GlcA- VaI-Leu-Gly-L ys (or Arg)-AEC sequences

In a first series of experiments, P. berghei cytosoluble ex- tracts (100,000 g, 1 h, 4°C) were incubated with different GlcA-peptidyl-AEC substrates [65]. GlcA-Val-Leu-Gly- Arg-AEC, a substrate for the bacterial endopeptidase Limulus lysate test [29], was the most efficient (Kin=0.032 mM), whereas specific substrates for pan- creatic elastase (Ala-Ala-Pro-Ala), for human leukocyte elastase (Ala-Ala-Pro-Val), or for plasmin (Gly-Pro-Arg) were weakly hydrolyzed. The released AEC was related to the endopeptidase activity because the N-terminus of the substrates was acylated by a hydrosolubilizing gluconoyl group (GlcA) [52], which prevented aminopep- tidase degradation of the substrate.

Discrimination between host and parasite enzymes

Two neutral endopeptidase activities specific for GlcA- Val-Leu-Gly.Arg.AEC were identified in 2 distinct frac- tions upon Sephacryl S-200 gel filtration from soluble P. bershei and P. chabaudi extracts by Bernard et al. [9]. These 2 endopeptidase activities were distinguished by comparing Sephacryl S-200 gel filtrations run in parallel, one with extracts obtained from erythrocytes with P. bershei at 50% parasitemia, and the other with an equivalent amount of normal mouse erythrocytes. En- dopeptidase activity in the high molecular mass fraction • 200,000 was present in both cases. In contrast, endopeptidase activity in the 130,000.Mr fraction was pres- ent only in the P. berghei extract. The host origin of the activity in the • 200,000 fraction was attested (i) by per- forming extraction after lysis of samples containing leu- kocytes and 60% enriched reticulocytes and (it') by using different washing procedures during the preparation of soluble P. bere, hei extracts. The endopeptidase activity of the •200,000 fraction decreased after several washes, whereas that of the 130,000 fraction was not significantly modified. The distinction between the host and the parasite enzyme activities was confirmed by (0 their differences in optimal pH activity (pH 7.4 for the parasite enzyme and pH 8.2 for the host protease), and (it') differences in their substrate specificities. The parasite endopeptidase showed strong activity on GlcA-Val-Leu-Gly-Lys-AEC and on GlcA-Ser-Gly-Lys-AEC substrates; in contrast, the mouse- host endopeptidase poorly cleaved OlcA-Val-Leu-Oly-Lys- AEC and did not cleave GlcA-Ser-Oly-Arg-AEC [9]. Similar results were obtained with P. falciparum extracts and human erythrocytes.

Purification of the Pb 68 and Pf 68 endopeptidases and their localization in schizonts and in merozoites

In a preliminary experiment with P. chabaudi synchro- nized in mice according to David et al. [15], it was possi-

ble to show that Pc 68 endopeptidase activity increased with schizont maturation [9]. This increase in activity during schizogony was easier to demonstrate by using highly synchronized P. falciparum in vitro cultures [22]. Pf68 endopeptidase activity was 3 times higher in schizont than in trophozoite extracts.

The Pb 68 endopeptidase was purified by high pressure liquid chromatography in a 2-step procedure, using first a chromatofocusing Mono P column (Pharmacia), then a Superose 12 gel filtration column (Pharmacia) [8]. The 100,000-g P. berghei extract was subjected to a linear 6.3-4.0 pH gradient, and the enzyme activity was detected in the acidic fraction (pH 4.35) as a single major peak. The final step of enzyme purification was performed by a gel filtration step, in which endopeptidase was eluted as a 70,000-Mr fraction. The purified enzyme fraction of P. berghei showed a 68,000-Mr polypeptide in SDS-PAGE under reducing conditions [8].

A similar Mr was observed with Pf 68 endopeptidase [22]. A single polypeptide is obtained only when the most active fractions from chromatofocusing and gel filtration steps are used; when all the active fractions are analyzed by SDS-PAGE under reducing conditions, several polypeptides (96,000/76,000/68,000) are always recovered [22]. This 2-step procedure allows an 800-fold purifica- tion of the Pf68 endopeptidase with a yield of about 30O/o, as calculated from the enzyme activity of the crude ex- tract [22].

A mouse antiserum against P. berghei endopeptidase reacted by immunoblotting specifically with the P. berghei 68,000-Mr protease both in concentrated parasite crude extract and in purified enzyme; in contrast, no labelling was observed with similar fractions prepared from P. faiciparum FcR3 Gambia parasites [8]. The high solubili- ty of the Pb 68 endopeptidase was demonstrated by the very weak labelling of the pellet of a 100,000-8 extract.

Indirect immunofiuorescence assay using Pb 68 endopeptidase.specific antiserum demonstrated strong labelling of P. berghei merozoites in mature segmented schizonts and after their release from schizonts [8]. This labelling was associated mainly with the merozoite apex, and the Pb 68 antiserum did not react with P. falciparum segmented schizonts.

Grellier et aL [22] demonstrated that ,of 68 endopep- tidase was present in merozoites. Enzyme extracts were prepared from merozoites purified according to Mrema et aL [53], and the elution on the chromatofocusing Mono P column led to the detection of a single peak of ,of 68 endopeptidase similar to that observed with the schizont stage extract.

Pb 68 and Pf 68 endopeptidases belong to the cysteine protease class

The Pb 68 and Pf 68 endopeptidases could also be designated as proteinases [6]. The 4 major classes of pro- teases (serine, cysteine, aspartic, and metallo-proteases) were established according to essential catalytic residues in their active sites, and the class of a protease is usually determined according to the effects of protease inhibitors on enzyme activity.

Protease inhibitor assays showed that Pb 68 and Pf 68 endopeptidases exhibit a similar pattern of inhibition (Table II). ZnCI 2 and HgCI z at l-raM concentrations significantly reduced both enzyme activities. The usual in- hibitors of the cysteine proteases (formerly called thiol pro-

Plasmodium neutral proteases in erythrocytic stages 237

teases) such as pHMB, the alkylating reagents iodoacetamide, and N-ethylmaleimide (NEM), strongly inhibit the Pb 68 and Pf 68 endopeptidases. Pepstatin, an inhibitor of aspartic proteases, EDTA and phosphoramidon (rha-phosphoryl-Leu-Trp), 2 inhibitors of metallo-proteases, have no or little effect on these Piasmodium endopeptidases. Peptide aldehydes isolated from Actinomycetes culture filtrates (leupeptin, antipaln, chymostatin, [70]) can inhibit proteases by forming a hemiacetal or hemithioacetal with an active-site serine or cysteine residue [3, 10]. These 3 peptide aldehydes are po- tent inhibitors of the Pb 68 and Pf 68 endopeptidases at 1.0-0.1 mM concentration. As all serine proteases are in- hibited by diisopropyl phosphofluoridate (DIPF), and most by PMSF, these inhibitors were assayed on the 2 en- dopeptidases. Activity was reduced to about 10% with 1 mM DIPF; in contrast, no effect was observed at 0.1 mM. PMSF had no effect at all. Trypsin inhibitors, such as chloromethylketone (TLCK), soybean trypsin inhibitor (181 amino acids [38]), and pancreatic basic trypsin in- hibitor aprotinin (58 amino acids [37]) produced low-to- moderate inhibitory effects (Table If).

From these data, the Pb 68 and Pf 68 endopeptidases could be classified in the EC 3.4.22 class (cysteine pro- teases) according to the Enzyme Commission of the In- ternational Union of Biochemistry [73]. As the enzymes

TABLE III. -- Substrate specificity of Pf 68 endopeptidase.

No. Substrates

Ps

GIcA

GIcA GIcA GIcA

P4 P3 P2 P, Pl'

Val Leu Gly Lys AEC OlcA Ser Gly Lys AEC Val Leu Gly Arg AEC Val Thr Gly Lys AEC Ser Leu Gly Lys AEC GlcA Ser (Bzl Gly Lys AEC GIcA Ser Gly Arg AEC

Relative activity (%)

I00 95 80 42 20 5 5

Relative activity (%) was expressed in comparison with the most active substrate GIcA-VaI-Leu-GIy-Lys-AEC.

are not inhibited by EDTA, they do not belong to calpains, which are a group of cysteine endopeptidases that require calcium ions for activity [62].

Substrate specificity of the Pb 68 and Pf 68 en- dopeptidases

TABLE il. - Effects of inhibitors on Pb and Pf 68 endopep- tidases

Inhibitor

Control ZnCl2 HgCl2

pHMB lodoacetamide N-ethylmaleimide Pepstatin EDTA CaCl2 Phosphoramidon DIPF

PMSF TLCK Leupeptin

Antipain

Chymostatin

STI Aprotinin

Final Concentration

(raM)

1.0 1.0

1.0 3.0 1.0

1.0 5.0 1.0 1.0

1.0 0.I0 1.0 1.0

1.0 0.I 0.5 0.I 1,0 0.I

1.0 1.0

Activity if/0 of control)

P. falciparum (22)

I00 2 0 7 2 8

I00 72

120 99 6

IO0 98 61 6 8 7 7 2 3

36 50

P. bershei (9)

100 0 0 0

ND ND 96

100 109 ND ND ND

96 50 38

ND 10

ND 3

ND 100 96

ND, not determined. Purified endopeptidase was incubated with protease inhibitor for 30 min at 37°C. Then 1 mM GIcA-Val- Leu-GIy-Arg-AEC was added, and released AEC was measured after a 30-min incubation at 37°C.

Conventionally the amino acid sequence of protease cleav- age sites is denoted as HzN.. P4-P3-P2-PI-P{-P2'.. COOH, in which the cleavable bond is between amino acids P~ and P~' [64]. As Pb 68 and Pf68 endopeptidases display a high specificity for the substrates having a C-terminal Gly-Lys or Gly-Arg sequence, the activity of Pf 68 en- dopeptidases was tested with various fluorogenic sub- strates having a glycyl residue in P2 position, as shown in Table III. Comparison of compounds I and 3 or com- pounds 2 and 7 indicates that the enzyme favors a lysine residue at PI. Comparison of substrates 1 and 4 shows that a hydrophobic residue such as leucine is preferable to the hydrophilic threonine residue at P3. However, the presence of an aromatic ring in P3, as in compound 6, drastically lowers the affinity of such substrates. From comparison of compounds I and 5 it appears that the role of the P4 residue is also important. Other specific substrates for the Pb 68 and Pf68 endopeptidases will be synthesized according to the specific sequences of the biological targets when they are determined.

NEUTRAL PROTEASES AND THE REINVASION PROCESS

The involvement of proteases during the release of merozoites and the erythrocytic invasion by merozoites is based mainly on indirect demonstrations using protease inhibitors. The lack of information about such proteases may be related to the short time-lapse of the invasion step (about 30 s), the loss of activity of the enzymes due to their external release in the plasma, and their restricted substrate specificity. However, the isolation of Pb 68 and Pf68 en- dopeptidases in schizonts and merozoites [8, 22] and the identification of Pf 76 protease activated by PI-PLC treat- ment [12, 13] may help to clarify previously reported data on the effects of protease inhibitors either on reinvasion experiments [4, 17, 21,25] or on the processing of antigens

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Plasmodium neutral proteases in erythrocytic stages 239

[16, 18, 19, 47]. As a 68,000 protease was observed by Braun-Breton et al. [13] during the identification pro- cedure of the Pf76,000 protease, and because Rosenthal et al. [63] reported a 75,000 protease activity in schizont preparations, it was of interest to examine the properties of the Pb 68 and Pf68 endopeptidase fractions on gelatin- SDS-PAGE. Another problem is the effect of plasma pro- tease inhibitors, which are the third largest group of func- tional proteins in human plasma after albumin and immunoglobulins [69], on Plasmodium proteases during erythrocyte invasion by merozoites.

Detection of proteases in Pb 68 and Pf 68 frac- tions by the gelatin SDS-PAGE method

A Pb 100,000-g cytosoluble extract was submitted to HPLC anionic exchange chromatography (Mono Q col- umn, Pharmacia), and the active hydrolyzing fraction on the GlcA-Val-Leu-Gly-Lys-AEC substrate was analysed by gelatin-SDS-PAGE, according to Heussen and Dow- die [33] (Fig. 1). Two proteolytic bands (Mr 95,000 and 68,000) were detected in the most active fraction (hatched area Fig. IA, and Fig. 1B, lane 1). The 95,000 band is always present in our experiments, but the presence of the 68,000 band is occasional. In gelatin-SDS-PAGE, the in- itial 100,000-g soluble extracts from P. berghei and P. faiciparum schizonts showed 95,000-90,000 major pro- tease bands and 2 other minor bands, at about Mr 140,000 and> 240,000 (Fig. 1B, lanes 2 and 3). An 85,000 protease band was not always present and might correspond to the P f 7 6 protease activated by PI-PLC treatment [13]. The irregular detection of the Pb 68 and Pf68 endopeptidase activities by gelatin-SDS-PAGE could be related either to: 0 processing of a proenzyme and/or to degradation of a higher molecular mass protease; or (i0 to the relatively low sensitivity of the gelatin-SDS.PAGE method in com- parison with the high sensitivity of the fiuorogenic peptidyl-AEC method. It could be noticed that the Pb 95,000 proteolytic activity on gelatin-SDS-PAGE from the cytosoluble 100,000-8 extracts was inhibited totally by NEM (Fig. IB, lane 5) and partially by PMSF (Fig. IB, lane 4), indicating that thus protease belongs to the cys- teine protease family.

Recovery of neutral endopeptidases in culture media with and without serum during the P. falciparum in vitro reinvasion process

The effect of plasma protease inhibitors on Plasmodium proteases is difficult to determine. Delplace et al. [19]

reported that processing of the 56,000 fragment, a sub- product of the p126 parasitophorous vacuole antigen, could depend on trypsin-like activity and did not occur upon the release of merozoites, because protease inhibitors contained in the serum impaired their activity. The mechanism by which 0q-trypsin inhibitor forms com- plexes with proteases is not yet precisely known; some serine proteases such as pancreatic or neutrophile elastases, as well as cathepsin G [69], are not inhibited. In order to determine the major effects of plasma protease inhibitors on Plasmoditz.-~ proteases, the supernate of highly syn- chronized P. falciparum cultures, performed in normal medium with 5% serum and in serum-free defined medium, were collected during the reinvasion process and submitted to gelatin-SDS-PAGE.

Segmented schizont-infected erythrocytes were obtain- ed from in vitro cultures according to Trager and Jensen [68], separated by the Plasmagel procedure [56], and diluted with new erythrocytes in order to obtain 3-5% final parasitemia. The medium [74] with 5% serum and the semi-defined medium without the major serum proteins allowing a normal in vitro P. faiciparum reinvasion pro- cess (unpublished data) were collected after a 6-hr incuba- tion, centrifuged, filtered (0.22/~m), and concentrated on B.15 Amicon. The standard parasite media on gelatin- SDS-PAGE always exhibit a very strong proteolytic ac- tivity in the range of Mr 90,000-95,000, and another ac- tivity detected above the serum albumin area. However, the origin of these activities is not clear since similar ac- tivities are present in normal culture medium (data not shown). The serum-free medium shows a specific parasite proteolytic activity on gelatin-SDS-PAGE corresponding to Mr 95,000, and sometimes another of lower Mr (40,000) area (Fig. 2, lane 2). This 95,000 protease is sensitive to reducing conditions since the effect of~-mercaptoethanol allows visualization of an active proteolytic band of about 40,000 (Fig. 2, lane 3). This 95,000 proteolytic activity in the parasite serum-free medium may correspond to that observed in the P f 100,000-g soluble extracts. No pro- teolytic activity was observed in gelatin.SDS.PAGE from normal erythrocyte lysates (data not shown).

From these in vitro experiments, the presence of human protease inhibitors is not sufficient to inactivate all the pro- teases present in normal medium. In rico, the problem is more complex. As was shown by Bernard et ai. [8], 2 endopeptidases specific for the GIcA-VaI-Leu-GIy-Arg- AEC substrates were detected in P. berghei-parasitized mouse serum. After Sephacryl-S-200 gel filtration, the ac- tivity in the > 200,000 fraction was similar to that of nor- mal mouse serum, and the 130,000 fraction was of parasite origin. These activities could be the result of the associa- tion of serum and/or parasite proteases with plasma high

FIGURE 1. - (A) Anion exchange chromatography on a Mono Q column (Pharmacia) of Pb 100,000-g extract. The column was equilibrated with 50 mM Tris buffer pH 7.5. Elution was performed with a linear gradient from 0 to 0.5 M NaCI in 50 mM Tris buffer pH 7.5, at 0.5 ml/min flow rate. Pb 68 end opeptidase activity was measured by incubating 50/~11 mM GIcA-V~I-leu-Gly-Lys- AEC with 50/zl of collected fractions for 30 min at 37°C. Released AEC was extracted by ethyl acetate. Fluorescence intensity was read at 430 nm upon excitation at 370 nm. The hatched peak corresponds to the most active fraction. (B) Gelatin-SDS-PAGE. Lane 1, most active fraction against GlcA-VaI-Leu-GIy-Lys-AEC from Mono Q. (A, hatched peak) ; lane 2, P. falciparum 100,000-g ex- tract; lane 3, P. berghei 100,000-g extract; lane 4 P. berghei 100,000-g extract treated with 1 mM PMSF during all steps; lane 5, P. berghei 100,000-g extract treated with 1 mM NEM as in lane 4.

240 J. Schr6vel et al.

97400 66200

000

,q31 0 0 0

ordered cascade of reactions and, although many data on the action of protease inhibitors are becoming available, several questions have not yet been answered. For instance, the proteases responsible for the processing of the merozoite antigens and the biological targets of the known proteases have not yet been identified. To date, the main results may be summarized as follows.

500

1 2 3 4 5

FIGURE 2. -- Gelatin-SDS-PAGE of proteins released during reinvasion. Lane 1, control serum-free defined medium; lanes 2 and 3, serum-free defined medium from the reinvasion step in the Laemmli [40] dissociation buffer without and with fl-mercap- toethanol, respectively; lanes 4 and 5, SDS-PAGE standards (low molecular weight, Bio-Rad) without and with fl-mercap- toethanol, respectively.

molecular mass protease inhibitors, such as 0t2-macro- globulin and, to a lesser extent ~n-antitrypsin. As shown by Barret [5], proteases bound to ~2-macroglobulin (725,000) are unable to act on protein substrates, but they remain active against low molecular mass substrates (< 10,000). Thus, further studies on interactions of plasma protease inhibitors with Plasmodium proteases are re- quired to determine the possible escape of some highly specific parasite proteases from association with plasma protease inhibitors.

Effects of protease inhibitors on the P. falciparum in vitro reinvasion process

During the reinvasion process, proteases could act on the release of merozoites from mature schizont-infected erythrocytes, during reorientation of the apical end of the merozoite and/or during internalization of the merozoite. However, such proteases have not yet been identified. Several possibilities may occur: (a) different proteases might control the processing of precursor major surface antigens (PMMSA: 190,000 in P. falciparum, 230,000 in P. knowlest), of parasitophorous (p126, glycophorin- binding protein or GBP), of rhoptry proteins [12, 19, 30, this issue] in order to create or destroy biologically active molecules for reinvasion; (il) other proteases could act locally on erythrocyte membrane components in order to assure the internalization of the merozoite inside the erythrocyte; flit) activation of proteases could occur at very precise periods, such as in the case of the Pf 76 protease [12, 13]. All these events could correspond to a highly

Effects o f protease inhibitors on the processing o f major parasite antigens

In order to determine whether the proteolytic activity pre- sent in 1). knowlesi mature schizonts and in merozoites could be involved either in the processing of the 230,000 PMMSA antigen or in the degradation of the antigen after extraction, David et aL [16] tested protease inhibitors per- taining to the major protease classes. Only partial inhibi- tion was obtained with DIPF at concentrations higher than 10 mM.

The PMMSA 190,000 from P. falciparum is processed into a series of subfragments which show a considerable polymorphism and possess a surface merozoite localiza- tion [28, 31, 34, 35, 47-49]. As shown by Lyon and Haynes [46], a mixture at 10/~g.ml- n each of leupeptin, chymostatin, antipain and pepstatin did not noticeably decrease the processing of 190,000 PMMSA antigen. However, they reported that leupeptin increased the recovery of 83,000 and 73,000 subfragments, and chymostatin that of a 66,000 subfragment, whereas pepstatin and antipain had milder effects. In another study, Pirson and Perkins [61] showed that the cleavage of the P. falciparum 200,000 merozoite surface antigen was partially inhibited when merozoites were incubated with 1 mM Zn + + and 5 mM iodoacetamide.

The processing of the p126 parasi~ophorous vacuole an- tigen into the major 50,000, 47,000 and 18,000 fragments was investigated by Delplace et aL [18, 19] in the presence of protease inhibitors. Chymostatin, antipain, and pep. statin had no effect on p126 processing, Leupeptin in- hibited the processing of a 56,000 intermediate product to the 50,000 fragment. In the presence of TLCK and TPCK, no. p126 processing was observed, but the in- hibitors were reported to have a deleterious effect on the parasites. The proteases involved in the processing of the GBP 130 into 100,000 and 80,000 subfragments [11, 59], or of the rhoptry antigens have not yet been characteris- ed [12].

Effects o f protease inhibitors on the release o f merozoites

The mixture of I0 ~g.ml - i leupeptin, ehymostatin, anti- pain, and chymostatin inhibits the release of P. falciparum merozoites and leads to accumulation of well preserved merozoites clustered around pigment granules [46]. The clustered merozoites are surrounded by a permeable erythrocyte membrane [46]. A similar observation was previously made by Hadley et al. [25] on P. knowlesi schizonts incubated in the presence of 50 tzg.ml- i leupep- tin or chymostatin. However, Dluzewski et al. [21] showed that with 50 ~g.ml-i leupeptin alone, about 15% of P. falciparum schizonts failed to rupture, and with 50 /~g..ml -I chymostatin that proportion was reduced to 5%.

Plasmodium neutral proteases in erythrocytic stages 241

In electron microscopy P. falciparum schizonts in- cubated with 50/~g.ml-1 leupeptin appeared free in the erythroeyte cytoplasm and were not surrounded by the parasitophorous membrane [19].

Effects o f protease inhibitors on the invasion o f erythrocytes by merozoites

Banyal et al. [4] showed that leupeptin or chymostatin, at 50 ~g.ml -I each, inhibits the reinvasion of erythrocytes by P. knowlesi merozoites. By using invasive P. knowlesi merozoites and rhesus erythrocytes, Hadley et ai. [25] were able to distinguish the effects of protease inhibitors either on schizont maturation or on merozoite invasion. Chymostatin, TLCK, and TPCK caused a marked inhibition of invasion. In contrast, leupeptin, antipain, pepstatin, and PMSF had little or no effect. Furthermore, chymostatin did not inhibit the attachment of merozoite to erythroeyte, and Hadley et ai. [25] suggested that chymostatin might be acting during or after junction for- mation. In P. falciparum in vitro experiments, Dluzewski et al. [21] showed that leupeptin also had an inhibitory effect on invasion; in addition, the inhibitory effect of leupeptin or chymostatin on P. falciparum invasion was also observed when the protease inhibitors were introduced into erythrocytes by lysis and resealing. The efficiency of the inhibitors acting on the cytoplasmic side of the mem- brane was interpreted by Dluzewski et al. [21] as a conse- quence of local action of secreted proteases on proteins accessible from the outside, such as band Ill and glycophorins which are believed to be ligands of the parasite.

The effects of protease inhibitors during reinvasion, as reported in the experiments described above, are highly variable. This variability could be related to species dif-

TABLB IV. - Inhibition of ring formation by analogues of Pf68 endoprotease synthetic substrates*.

Peptides

GIcA-VaI-Leu-GIy-Lys-NH-C2H 5

Ac-Asp-Pro-Arg-Gly-NH-C2H 5

GlcA-Val-Leu-Gly-Arg-H

GlcA.Vai.Leu.GIy-Lys.[CH2NH]-Et

Concentration (mM)

5 1 0.2 0.1

95 65 0 ND

0 0 ND ND

10 0 ND ND

100 100 ND 50

A synchronized culture (FcB-I strain) containing mature parasites with 6.3% schizonts (35-42 h), 2.6% segmenters (43-48 h) was aliquoted in a 24-well plate and cultured with various concen- trations of peptide analogues, at 10% hematocrit, for 6 h. 1000 cells were counted on Giemsa-stained thin smears, and the percentage of inhibition was calculated by means of the

R formula % = 100- "~o × 100 where R stands for the number

of ring-infected erythrocytes in the treated plates and Ro stands for the number of ring-infected erythrocytes in the control plates. ND, not determined. * ([42] and unpublished data).

ferences, but also to the specific nature of the parasite pro- teases. As shown in Table II, Pb 68 and Pf 68 endopep- tidases are inhibited by leupeptin and antipain, but also by chymostatin, whereas the more efficient inhibitors of these endopeptidases are characteristic of the cysteine rather than of the serine proteinase class. Such a situa- tion indicates that the nature of enzymes in terms of tryp- sin and/or chymotrypsin-like properties is not easy to determine from in vitro reinvasion experiments using on- ly protease inhibitors as competitors.

As Pb 68 and Pf 68 endopeptidases are present in free merozoites, enzyme-specific peptide derivatives were tested in vitro in reinvasion inhibition experiments with P. falciparum.

Possible use of synthetic peptide derivatives analogous to the substrates of Pf 68 endopep- tidase for blocking the P. fa lc iparum reinvasion process

Hydrophilic derivatives of peptides specifically recognized by the Pf 68 endopeptidase were tested on different P. falciparum strains such as FcR-3 Gambia, Uganda/Palo Alto, NF 54, and FeB. 1. To avoid the deleterious effect of AEC on erythrocytes, peptidyl-alkylamide derivatives were synthesized and added to well synchronized P. falciparum cultures according to Lambros and Vanderbeg [41] and/or Inselburg and Banyal [36]. Schizonts and segmenters were incubated for 6 hr with fresh erythrocytes. In the presence of 1-5 mM peptidyl-ethylamide derivatives, the number of ring forms was drastically reduced (Table IV, and unpublished data). When free 10 mM ethylamine was added under the same conditions, the reinvasion was similar to that of control experiments. Fur- thermore, ethylamide derivatives of peptides having se- quences unrelated to those of the specific substrates, such Ac.Asp.Pro-Arg.GIy-GIu-NH-C2H s or Ac-Asp-His-Arg- GIy-NH.C2H s, were totally inactive. These results in- dicate that the in vitro inhibition of P. falciparum reinva- sion is due to the Val-Leu-Gly-Lys sequence. Looking for more powerful inhibitors, aldehyde inhibitors (such as OlcA-Val-Leu-Gly-Lysinal) which form reversible com- plexes with the enzyme, and pseudopeptides (such as GlcA- Val.Leu.GIy-Lys.c~[CH2NH].C2Hs) were synthesized. Higher inhibitory effects of P. falciparum reinvasion were obtained with the pseudopeptides (Table IV). Other new, potent, irreversible inhibitors are under investigation.

CONCLUSION

The purification of Pb 68 and Pf68 endopeptidases, loca- tion of the enzyme in the P. berghei and P. falciparum merozoites, as well as the inhibitory effects of hydrophilic peptide derivatives related to the Val-Leu-Gly-Lys or Val- Leu-Gly-Arg sequences indicate a key role for these pro- teases in the malarial erythrocytic cycle. As similar enzyme specificity is present in different P. falciparum strains and in the rodent malarial parasites P. berghei and P. chabaudi, it is likely that the active site of these endopep- tidases is well conserved among different Piasmodium species. The actual biological target of the P. falciparum endopeptidase is not yet known. However, because the Pf68 endopeptidase acts optimally at neutral pH, and because highly hydrophilic analogues are effective in

242 J. Schr6vel et al.

blocking the reinvasion process, it is likely that the en- dopeptidase is involved in at least one of the steps occur- ring during the short time between the release of merozoites from mature schizonts and the entry of a merozoite into an uninfected erythrocyte. Purified P f 68 endopeptidase offers the advantage that its action on the processing of the major merozoite antigens, as well as on the erythrocyte components, can be looked for. Interestingly the erythrocytic band III, suspected of being involved in the invasion process [55], displays highly conserved sequences in both mouse and human erythrocytes and in the human non-erythrocyte band Ill-like protein (pHKB3) [20, 39]. Some sequences are exposed on the outside surface of the erythrocyte and, among them, sequences such as Ile-Val- Gly-Arg (506-509) and Val-Met-Gly-Lys (758-751) in mouse band Ill are closely related to our substrate analogues for P f 68 and Pb 68 endopeptidases.

The use of the gelatin-SDS-PAGE method provides the possibility of detecting other neutral proteases, both in schizont and merozoite as well as in in vitro culture media. This method was efficient in the identification of the Pf 76 protease which is activated by PI-PLC treatment and could act during invasion, as suggested by Braun-Breton et al. [12]. This new example of a protein anchored to the mem- brane via glycosylphosphatidylinositol opens up an ex- citing new field of investigation into the complex mechanism of erythrocyte invasion by Plasmodium. However, the efficiency of the gelatin-SDS-PAGE method depends on the affinity of the proteases for gelatin and on the amount of proteases. In contrast, the major ad- vantages of the fluorogenic peptidyl-AEC method are the detection of very low amounts of proteases, which great- ly facilitates enzyme purification and provides indications of the nature of protease specificity.

In contrast to previous studies in which only inhibition tests were used, the purification of neutral parasitic en. zymes will allow the identification of their biological targets by a direct approach,

ACKNOWLEDGEMENTS

We thank Professor Marcel Hommel (Liverpool School of Tropical Medicine, UK), and Dr Hans Heidrich (Max Planck Institut, Martinsried, FRG) for fruitful discussions. The help of Miss Christine Barrault for the gelatin-SDS-PAGE, and of Misses Dominique Decourt and Annick Gringault for the preparation of the manuscript was particularly appreciated. This work was partly supported by grants from the Commission des Com- munaut6s Europ~ennes (TSD-147-F), the lnstitut National de la Sant~ et de la Recherche M~dicale, nos 81-2014 (M.M.), 83-1023 (M.M.), 83-847 (J.S.), the Minist~re de la Recherche et de l'Enseignement Sup6rieur no, 86-C-0325; the Fondation pour la Recherche M~dicale (J.S.), the Association pour la Recher- che sur le Cancer (M.M.), and the Fondation Jean Langlois (J.s.).

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