EXPERIMENTAL PARASITOLOGY72, 218-284 (1991)

Plasmodium falciparum: Isolation and Characterization of a 55kDa Protease with a Cathepsin D-Like Activity from P. falciparum ERIC BAILLY,*,?

JEAN SAVEL,~

GUY MAHOUY,$

AND GINETTE

*INSERM UI3, kidpita Claude Bernard, 10 Avenue de la Porte fllniversitk Paris V Rent! Descartes, 5 Avenue de l’observatoire, d’lmmunomodulation, Centre Hayem, Hopital Saint Louis, 75010 Paris, France

d’dubetvilliers, 75006 Paris, 2 Place du.Dr.

JAIJREGUIBERRY*,’ 75019 Paris, France; France; #Laboratoire AIfred Fournier,

BAILLY, E., SAVEL, J., MAHOUY, G., AND JAUREGUIBERRY,G. 1991. Plasmodium faland characterization of a 55kDa protease with a cathepsin D-like activity Experimental Parasitology 12, 218-284. Native electrophoresis followed by imprint digest method using hemoglobin as substrate allowed the detection of parasite hemoglobinase activity at acidic pH (3.9 to 5). This protease was inhibited specifically by pep&tin A and insensitive to other protease inhibitors. The molecular weight determination using modiied SDS-PAGE followed by imprint digest method, demonstrated a single area of activity at 55-58 kDa, similar to cathepsin D characterized in eucaryotic cells. The parasitic origin has been shown by radiolabeling experiments with [‘$Imethionine. The SS-kDa protein was immunoprecipitated by a rabbit anti-cathepsin D serum. 0 1991AcademicPress,Inc. INDEX DE~CRI~ORS AND ABBREVIATIONS: Plasmodium falciparum; Cathepsin D; Electrophoresis; Western blotting; Chloroquine sensitivity; 4-(2 Hydroxyethyl)-1-piperazine ethane sulphonic acid (Hepes); Phosphate-buffer saline (PBS); Falciparum culture M’ba number 29 (FCM29); Tris(hydroxyethyl-aminoethane) (Tris); Ethylene-diamine tetracetic acid (EDTA); Red blood cell (RBC); Pepstatin A (PA); Phenyhnethylsulfonyl fluoride (PMSF); N-ethylmaleimide (NEM); N-a-Tosyl-L-lysine-chloromethyl-ketone; Cathepsin D antiserum (CDA). ciparum: Isolation from P. falciparum.

INTRODUCTION

The malarial parasite Plasmodium falciparum undergoes a complex developmental cycle inside erythrocytes of the vertebrate host. The ingestion and digestion of host cell cytosol by the parasite provides amino acids for the synthesis of proteins required for parasite growth and multiplication (Sherman 1979). The digestion of hemoglobin, the major constituent of the host cell cytosol, takes place in the parasite food vacuole that shares many characteristics with secondary lysosomes of mammalian cells (Aikawa 1977). Food vacuoles have a low pH (Yayon et al. 1984; Krogstad et al. 1985) and probably contain hydrolytic enzymes , including proteases. The presence i To whom the correspondence dressed.

should be ad-

of acid proteases able to degrade hemoglobin has been demonstrated in various malaria species, such as P. lophurae (Sherman and Tanigoshi 1983), P. berghei (Levy and Chou, 1973), P. yoelli nigeriensis (Aissi et al. 1983), and P. knowlesi (Hempelmann and Wilson 1980), and in the human parasite P. falciparum (Gyang et al. 1982). Using specific inhibitors, one protease has been characterized and partially purified (Vander Jagt et al. 1986). The role of cysteine proteases in hemoglobin degradation in food vacuole of P. falciparum has been recently reported (Rosenthal et al. 1987, 1988). We report in this study on a 55kDa asparty1 protease from P. falciparum that closely resembles cathepsin D in terms of its substrate specificity, pH dependence, and inhibitor sensitivity.

218 0014-4894/91 $3.00 Copyright Q 1991 by Academic Press. Inc. AU rights of reproduction in any form reserved.

A 55KDA MATERIALS

ASPARTYL

PROTEASE

AND METHODS

Cultivation of parasites. P. falciparum (clone C, from FCM, strain) was cultured in vitro according to Trager and Jensen (1976) with minor modifications. Cultures were maintained in petri dishes at 37°C with a gaseous phase of 5% 02, 5% CO,, and 90% N,. Cultures consisted of 5% washed human erythrocytes in RPM1 1640 medium supplemented with 25 mM hepes, 25 mM sodium bicarbonate, 2 g.liters-’ glucose and 50 mgliters-’ hypoxanthine and 10% (v/v) human serum (AB ‘). Prior to use in culture, blood was cleared from leukocytes and platelets by passage through a column containing a 1: 1 mixture of microcrystalline cellulose (Sigmacell) and alpha cellulose (Sigma). When parasitemia reached lO-15%, cultures were used for enzyme preparation. In all experiments, more than 50% of the parasitized red blood cells (PRBC) harbored middle stage parasites (late trophozoites and early schizonts having less than four nuclei). Radiolabeling of parasite proteins. Prior to labeling cultures of P. falciparum, 700 pJ of packed cells from parasite cultures were incubated for 1 hr in 6 ml of methionine-free complete RPM1 1640 medium. Proteins synthesized by parasites were labeled by adding 50 l&i/ml [35S]methionine to the medium (sp act 800 CYmmol, Amersham) for 6 hr. Preparation ofprotein extract. Parasite cultures (1.5 ml PRBC with a parasitemia of IO-15%) were centrifuged (SOOg for 10 mitt) and the pellets were washed three times with phosphate-buffered saline (PBS), pH 7.2. The final pellet was resuspended in 1.5 vol of PBS containing 0.2% w/v saponin (Merck) and incubated for 30 min at 37°C with occasional mixing. The lysate was centrifuged (2OOOg, 4”C, 10 min) and the pellet washed with PBS until the supemate was colorless. Erythrocyte ghosts were discarded with the supemate, and the brown pellet of parasites was further processed with an equal volume of lysis solution (10 mM TrislHCl, 5 mM EDTA, 100 mM NaCl, 0.5% (v/v) Triton X-100, pH 7.2) and incubated for 30 min at 4°C. After centrifugation (3O,OOOg,4°C for 1 hr) the supernate was collected and used as crude enzyme extract. Parasite culture supemate was precipitated for 30 min at 4°C once with ammonium sulfate 40% saturated and then centrifuged (25,OOOg, 4°C). The resulting supemate was further precipitated with ammonium sulfate 90% saturated, and the resulting pellet was dissolved in 0.2 M sodium acetate pH 5. The sample was dialyzed against the same buffer for 24 hr and concentrated with a P-micro Prodicon apparatus (Biomolecular Dynamics). For the proteolytic assay the concentrated sample was purified on DEAE trisacryl-M (IBF) column preequilibrated with sodium phosphate 0.1 M, pH 7.1, buffer. Proteins were eluted with increasing stepwise sodium chloride solutions ranging from 0.1 M up to 0.5

FROM P.

fakiparum

279

M and the purified fraction was further chromatographed on Pepstatin A-agarose column (PA-agarose). Analytical SDS-PAGE. Proteins from labeled or unlabeled PRBC (equivalent to 10’ parasites per well) were electrophoresed on a 11% acrylamide gel according to Laemmli (1970). Gels containing 35S-labeled material were treated with Amplify (Amersham) and autoradiographed on a Kodak x-Omat film. i4Cmethylated molecular weight standards were purchased from Amersham. Staining of unlabeled proteins was done using a Kodak vue kit and molecular weight markers from Bio-Rad. For determination of hemoglobinase activity, enzyme samples were not boiled in B-mercaptoethanol-containing buffer. After electrophoresis the proteins were renatured by two washes of the gel in 2.5% (v/v) Triton X-100 aqueous solution for 30 min each at RT. Native PAGE. The technique of Hempelmann and Wilson (1980) was used with minor modifications: samples were electrophoresed through 7.5% acrylamide stacking gel and 11% resolving gel at constant voltage (150 v) for 20 hr at 4°C with 150 mM Tris/HCl pH 8.9 as gel buffer and 5 mM T&/35 mM glycine pH 8.5 as electrode buffer. Molecular weights were determined by the method of Bryan (1977) and Davis (1964). Conditions of enzyme extracts and standard protein (Sigma) separation and hemoglobinase activity were the same as describe above. Graphic determination of molecular weight hemoglobinase activity was made according to the instructions of the manufacturers. Detection ofprotease activity. Protease activity was revealed by the imprint digest method (Roberts et al. 1977) with the following modifications: 10 ml of 0.1 M citrate-buffered solution containing 1% agarose (Type 1 Sigma) were poured into a plastic cast to obtain a 2-mm-thick gel. After cooling the gel was overlaid with 5 ml of citrate-buffered solution containing 0.750 g.liters-’ of human hemoglobin (Sigma). After 30 min equilibration at RT excess solution was discarded. Simultaneously, slices of the polyacrylamide gel obtained by analytical and native PAGE were incubated 30 min in the same citrate buffer. Slices were then laid on the hemoglobin-imbibed agarose gel and incubated in a moist chamber at 37”C, for 20 hr for slices obtained by analytical SDS-PAGE and for 6 hr for the case of native-PAGE. The agarose gel was stained with Coomassie brillant blue R (Sigma) in a methanol-acetic acid-water solution (3:1:6). Hemoglobin degradation was detected in the agarose gel as a clear band against the blue background of undigested hemoglobin. To reveal the pH dependence of enzyme activity, experiments were performed with citrate-buffered solutions adjusted to various pH’s (between 3.9 and 5.5). Proteolytic activity assay. The assay was conducted

280

BAILLY ETAL.

according to Capony et al. (Capony et al. 1986) modified as follows: the reaction mixture contained 40 pl of pepstatin A-agarose purified fraction parasite lysates or culture supemate, 10,000 cpm (7.6 nmole) of (methyl-14C)-methylated methemoglobin (NEN, sp act 20 mCi/g), 1 mg.ml-’ unlabeled hemoglobin in a fmal volume of 200 pl of 0.1 M sodium citrate buffer at pH 3.2. After 2 hr incubation at 37°C the reaction was stopped by adding trichloracetic acid (I’CA). The radioactivity of 25 ~1 TCA-soluble material was counted. Blanks were run with dialysis buffer in place of enzyme, and were subtracted. Inhibition studies were performed in the same conditions, in the presence of various concentrations of pepstatin A (PA) (0.02-20 pM). Bovine spleen cathepsin D (Sigma) was used as reference enzyme. Znhibition studies. All protease inhibitors were purchased from Sigma. Inhibitors were added to the citrate buffer (PH 3.9) at final concentrations of 5 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM Nethyhnaleimide (NEM), 100 pM PA, 20 pg.tn-’ chymostatin, 100 pM N-a-tosyl-L-lysine-chloromethylketone (TLCK), 100 )LM L-trans-epoxysuccinyl (4guanidino)-butane (E&l), or 10 PM iodoacetamide. Hemoglobinase inhibition was performed by 3 hr incubation of gel slices in citrate buffer for 3 hr before detection of the enzymatic activity. Partial purification of parasite cathepsin D on pepstatin A-agarose. Lysates prepared as described above were adjusted to 50 mM CH,COONa, pH 5,0.1 M NaCl in order to remove Triton X-100 and passed through a PA-agarose column (1 X 3.5 cm) (Sigma). Contact was maintained for 1 hr at 4°C before washing with acetate buffer, pH 5.0. Bound proteins were eluted at room temperature with 50 r&f Tris/HCl pH 8.6, 1 M NaCl buffer, at a flow rate of 10 ml/hr. Void volume was discarded and fractions eluted in a single peak were pooled, dialyzed against PBS, pH 7.2, and concentrated. Preparation of cathepsin D antiserum (CDA). One milligram commercially purchased purified bovine cathepsin D (Sigma) was suspended in 1 ml of sterile distilled water, emulsified with an equal volume of Freund’s complete adjuvant, and injected subcutaneously to New Zealand rabbits. The immunization injections were repeated every 2 weeks thereafter four times using half the amount of protein emulsified with an equal volume of Freund’s incomplete adjuvant. Blood was collected from the ear vein 1 week after the last injection, allowed to clot at room temperature, and centrifuged 3000 rpm 10 min. Serum was collected and used for IgG purification. ZgGpurification. The rabbit serum was precipitated with ammonium sulfate as described by Bogitsh and Kirschner (1987). The precipitate was chromatographed on DEAE-trisacryl M (IBF) column preequilibrated with Tris 0.025 M, pH 8.WNaCl 0.05 M. Re-

sidual hemoglobin was discarded by gel fdtration of the IgG fraction through a Sephacryl-S-200 HR column (Pharmacia) (elution buffer 0.1 M Tris/HCl NaCl 0.2 M, pH 8). After each step of purification, the IgG fraction was dialyzed against PBS, pH 7.2, concentrated, and examined by the Ouchterlony double diffusion technique for reactivity with bovine cathepsin D, and with rabbit anti-IgG serum. Zmmunoprecipitation of radioactive proteins. Extracts of parasitized cells were mixed with rabbit CDA serum in 10 mM Tris/I-ICl, pH 7.4, EDTA 10 mM NaCl 0.4 M, 1% Triton X-100 (solution A). Anti-sera were not diluted more than 25-fold and detergent solution represented one-quarter of the total volume of the immunoprecipitation mixture. The mixture was incubated for 3 hr at 4°C. Antigen-antibody complexes were precipitated by incubation (1 hr, 4°C) with 100 t~,l of 10% Staphylococcus aureus Cowan I strain inactivated by formalin treatment (ICN) in solution A + bovine serum albumin 1 mg.ml-’ (solution B). After one wash with solution B and two washes with solution A the antigen-antibody staphA+omplexes were resuspended in solution A without Triton X-100. The antigen-antibody complexes were released from protein A bacterial absorbent by incubation at lOO”C, 4 min in the SDS-PAGE loading buffer and electrophoresed on a 11% acrylamide gel. Western blot analysis. Electrotransfer blot was made on nitrocellulose paper (Towbin et al. 1979). The separated proteins were immunodetected with rabbit CDA serum, CDA IgG fraction or specific CDA IgG (purified on a cathepsin D column) at appropriate dilutions and revealed with l/250 peroxidase-linked donkey antibodies to rabbit Ig chains with diaminobenzidine as substrate.

RESULTSAND DISCUSSION

Using the technique of Hempelmann and Wilson (1980) we were able to show hemoglobinase activity in P. falciparum, using human hemoglobin as substrate, at pH 3.9 but not at 5.5. The electrophoretic pattern was complex; three bands of hemoglobinase activity could be distinguished, with Rf values of 0.54,0.65, and 0.81, respectively. Under the same conditions noninfected red blood cells showed very light activity with Rf of 0.91, but not in the range of parasite extract activity. Leukocyte extracts showed no activity, implying the parasitic origin of the hemoglobinase activity (Fig. 1). Protein with hemoglobinase activity in a nondenaturing system migrated with Rf val-

A~KDA 1

ASPARTYL

PROTEASE

234567

FROM P.falciparum 1

281

234567

Rf

-l’-

lib-

-0.65.0.54-

FIG. 1. (A) Hemoglobinase activity (white area) revealed by imprint digest method at pH 3.9. (B) Lack of hemoglobinase activity at pH 5.5. Sample control were leucocytes extracts (lane l), RBC lysate (lane 2) ghost RBC extract prepared identically to parasite enzyme extract (lane 3), various P. falciparum culture extracts (lane 4 to 7). Rf=

AHb (front of migration) - AHb (proteolitic AHb (front of migration)

ues of 0.54 and 0.65 equivalent to molecular weights of 52 and 55-58 kDa, respectively. In analytical SDS-PAGE followed by the imprint digest method, only the 55-58 kDa zone of activity was seen (Fig. 2). The loss of the activity related to the 52-kDa protein may be due to the susceptibility of the enzymatic activity to the SDS-PAGE conditions. Inhibition of the hemoglobinase activity by PA (100 l&f) suggests that it is an aspartyl protease-like cathepsin D. Inhibitors of cysteine or serine proteases were without effects at the concentration used (Table I). Considering the pH dependence of the proteolytic activity and the calculated pH in the food vacuole (Krogstad et al. 1985) it is suggested that this protease is of lysosomal origin. The partial purification of parasite lysate and culture supemate on PA-agarose column yielded the 55-kDa species as a major constituent. Using PA-agarose purified [35S]methionine-radiolabeled parasite proteins for SDS-PAGE and autoradiography, five major proteins at 55, 52, 39, 30, and 28

area)

kDa were seen (Fig. 3A). Under the same conditions, the culture medium contained 120-l 10,60, and 52-55 kDa proteins as major constituents (Fig. 4A). It remains to be 12

FIG. 2. Hemoglobinase activity (white areas) of two different extracts revealed by imprint digest method after analytical SDS-PAGE.

282

BAILLY

ET AL.

TABLE I Inhibition of Hemoglobinase Activity by Protease Inhibitors NEM (10 m

Chymostatin (20 pg . ml-‘)

Inhibitors TLCK (la0 PM

E64 (100 lw

+ Note. - , No inhibition;

+

+ , faint inhibition;

1

2

1

2

PMSF (5 mM)

Pepstatin (100 Fw

-

++

+ + , total inhibition.

established whether they are exported from the parasite or released upon spontaneous disruption of PRBCs. Immunological detection. Rabbit CDA serum immunoprecipited specifically a 55 kDa protein from total labeled parasite protein extract. This protein was also revealed by immunoblot analysis with rabbit CDA serum, total IgG, or a specific CDA-IgG fraction (Fig. 4B). In noninfected RBC rabbit CDA serum did not detect the 55kDa protein. Preimmune serum did not detect cathepsin D protein in a control reaction, and immune serum did not detect any related 55-kDa protein in control nonparasitized RBC. Protease activity. The cathepsin D-like activity of the PA-agarose fractions obtained from parasite extract was assayed using (methyl-14C)-methylated methemoA

Iodoacetamide (10 mM)

globin as substrate. The specific activity of cathepsin D was increased by purification 5- to 12-fold and 84% of the enzymatic activity (2.32 mu) was inhibited by 2 FM PA (Fig. 5). Other investigators have already reported the presence of a 148-kDa cathepsin D-like protein in P. falciparum (Gyang et al. 1982)and a 35-kDa in P. lophurae (Sherman and Tanighoshi 1983). But one of the problems in the context of intraerythrocytic parasites is to know whether the parasite uses the residual erythrocyte cathepsin D A kDa

1

2

B kDs

B

69

46

FIG. 3. Autoradiography of SDS-PAGE analysis of labeled parasite proteins. (A) [i4C] radiolabeled molecular weight markers (lane 1), [?S]methionine-labeled parasite extract after purification on PA agarose column (lane 2). (B) Immunoprecipitation of parasite extract done with rabbit preimmune serum (lane 1) with CDA serum (lane 2).

FIG. 4. (A) PA agarose column fraction of culture supemate analyzed by SDS--PAGE and autoradiographed (lane l), colored with Kodak vuee Kit (lane 2). (B) Immunoblotting analysis of PA agarose column fraction of parasite extract revealed by specific CDA W.

A 55-KDA ASPARTYL

PROTEASE

FROM P. fakiparum

283

The existence of multiple forms of this protein, as we have shown by immunoblot analysis, lead us to believe that these forms derive from a single precursor as demonstrated in other cells types (Hasilik ef al. 1982; Nishimura et al. 1987; Morisset et al. 1986).

P. falciparum.

ACKNOWLEDGMENTS FIG. 5. Inhibition activity curve of the 55kDa protein by pep&tin as described under Materials and Methods using (methyl-r4C)-methylated methemoglobin as substrate.

and activates it, or whether it synthesizes this protease de nova. Using radiolabeled extract we were able to detect by immunoprecipitation a 55kDa protein (Fig. 3B) with rabbit CDA serum. This extract simultaneously purified on a PA-agarose column clearly showed a 55kDa protein as major constituent, able to digest (methyl14C)-methylated methemoglobin. The proteolytic activity (2.32 mu) was inhibited by 2 p,M of PA. These results confirm that the 55-kDa cathepsin D-like protein is clearly distinct from contaminating host cell enzymes. Immunodetection by Western blot of nonpurified enzymatic extracts and PAagarose fraction showed thin bands at 55 and 30 kDa with the IgG CDA. Using culture supernate both 58- and 55-kDa proteins were revealed. After PA agarose the 58-kDa was enriched and a 52-kDa protein appeared. All these polypeptides were recognized by the rabbit CDA serum (data not shown). To account for this variability, we suggest that the 58-kDa polypeptide is a precursor of the 55-kDa, and the 52-kDa is its degradation product, produced during the purification process. In conclusion, we have demonstrated the presence of a 55- to 58-kDa aspartyl protease that resembles cathepsin D in terms of its substrate specificity, pH dependence, inhibitor sensitivity, and is synthesized by

We express our thanks to Dr. J. J. Pocidalo for support and laboratory facilities. We are very grateful to Dr. H. Ginsburg for helpful discussion and useful advice and comments on this manuscript. This work was supported by Centre National de la Recherche Scientifque (CNRS) and the Institut National de la Sante et de la Recherche Mddicale (INSERM). Eric Bailly was supported by a doctoral fellowship of MRES. REFERENCES AIKAWA, M. 1977. Variations in structure and function during the life cycle of malaria parasites. Bulletin of the World Health Organisation 55, 13%156. AISSI, E., CHARET, P., BOUQUELET, S., AND BIGUET. 1983. Endoprotease in Plasmodium Yoelii nigeriensis. Journal

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