Molecular & Biochemical Parasitology 120 (2002) 177–186

Characterization of proteases involved in the processing ofPlasmodium falciparum serine repeat antigen (SERA)

Jie Li a, Hiroyuki Matsuoka b, Toshihide Mitamura a, Toshihiro Horii a,*a Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka Uni�ersity, Suita, Osaka 565-0871, Japan

b Department of Medical Zoology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan

Received 18 June 2001; received in revised form 26 October 2001; accepted 11 December 2001

Abstract

The Plasmodium falciparum serine repeat antigen (SERA), a malaria vaccine candidate, is processed into several fragments (P73,P47, P56, P50, and P18) at the late schizont stage prior to schizont rupture in the erythrocytic cycle of the parasite. We haveestablished an in vitro cell-free system using a baculovirus-expressed recombinant SERA (bvSERA) that mimics the SERAprocessing that occurs in parasitized erythrocytes. SERA processing was mediated by parasite-derived trans-acting proteases, butnot an autocatalytic event. The processing activities appeared at late schizont stage. The proteases are membrane associated,correlating with the secretion and accumulation of SERA within the parasitophorous vacuole membrane (PVM). The activityresponsible for the primary processing step of SERA to P47 and P73 was inhibited by serine protease inhibitor DFP. In contrast,the activity responsible for the conversion of P56 into P50 was inhibited by each of the cysteine protease inhibitors E-64, leupeptinand iodoacetoamide. Moreover, addition of DFP, E-64 or leupeptin to the cultures of schizont-stage parasites blocked schizontrupture and release of merozoites from PVM. These results indicate that SERA processing correlates to schizont rupture and theprocessing is mediated by at least three distinct proteases. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Plasmodium falciparum ; SERA; Protease; Protein processing; Schizont rupture; Baculovirus

www.parasitology-online.com.

1. Introduction

The malaria parasite Plasmodium falciparum has anasexual life cycle in the mature human erythrocyte, inwhich it is surrounded by the parasitophorous vacuolemembrane (PVM). In the erythrocyte, the parasite un-dergoes three distinct stages of development: the ring,the trophozoite and the schizont. During theschizogony, between 16 and 32 merozoites are formedwithin the parasitophorous vacuole. The merozoitesrelease from the ruptured schizont and invade new redcells and continue the cycle. During the course ofschizont rupture, merozoite release and re-invasion, anumber of proteins associated with free merozoites,

such as merozoite surface protein-1 (MSP-1) [1], MSP-3[2], MSP-6 [3], apical membrane antigen-1 [4], andrhoptry associated protein-1 [5,6], are proteolyticallyprocessed. In the case of MSP-1, the final processingevent (i.e. conversion of MSP-142 to MSP-119) has beenshown to be necessary for merozoite invasion [7]. Fur-thermore, results from studies with protease inhibitorssuggest that parasite-derived proteases are involved inthe events of schizont rupture and meroziote re-inva-sion [8]. A recent report showed that a cysteineprotease(s) could be involved in the release of mero-zoites from within the PVM [9]. Therefore, analysis ofthese processing events and characterization of theproteases involved would help to understand the molec-ular mechanisms underlying schizont rupture, mero-zoite-release, and/or merozoite-invasion.

The serine repeat antigen (SERA) of P. falciparum[10] is synthesized at trophozoite- and schizont-stagesduring the intraerythrocytic cycle and secreted as a 120kDa protein into the parasitophorous vacuole [11,12].

Abbre�iations: bvSERA, baculovirus-expressed SERA; DFP, diiso-propyl fluorophosphate; E-64, trans-epoxysuccinyl-L-leucylamindo(4-guanidino)butine; SERA, serine repeat antigen.

* Corresponding author. Tel.: +81-6-6879-8280; fax: +81-6-6879-8281.

E-mail address: horii@biken.osaka-u.ac.jp (T. Horii).

0166-6851/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0166-6851(01)00452-2

J. Li et al. / Molecular & Biochemical Parasitology 120 (2002) 177–186178

It has been demonstrated that the 120 kDa protein isproteolytically processed into 47 kDa N-terminal (P47),56 kDa central (P56), and 18 kDa C-terminal (P18)fragments [11,12]. P47 is, depending on the allelic typeof SERA gene, further processed into two 25 kDafragments (P25n and P25c) (Li and Horii, unpublisheddata), while P56 is converted into a 50 kDa fragment(P50) [11,12], which has a significant homology topapain-family proteases but has a predicted active ser-ine instead of cysteine [13,14]. SERA processing isinitiated most likely at late schizont stage and com-pleted within the parasitized erythrocytes just prior toschizont rupture (Li and Horii, unpublished data).These observations suggest that the processing ofSERA might represent the maturation of the putativeprotease and that the processed fragments of SERA aswell as the proteases involved in the SERA processingmight play important roles in schizont rupture, mero-zoite release, and/or merozoite invasion. In the presentstudy, we have established an in vitro cell-free systemthat mimics the SERA processing that occurs withinparasitized erythrocytes. With this system, we furthercharacterized the proteases involved in SERAprocessing.

2. Materials and methods

2.1. Antibodies

Recombinant proteins SE47’ and SE50A have beendescribed [15]. A synthetic gene encoding amino acidresidues 879–989 of P. falciparum Honduras-1 SERAwas constructed by a similar method to that for theexpression of SE47’ and SE50A [15] and used to expressrecombinant P18 (SE18) in Escherichia coli as a glu-tathione-S-transferase fusion protein. Polyclonal anti-bodies specific for P47, P50 and P18 (named �P47,�P50, and �P18, respectively. See Fig. 1) were raised inBALB/c mice (SLC, Japan) and total IgG was purifiedwith a HiTrap Protein A column (Pharmacia).

2.2. Construction of plasmid pMbac-SERA encodingrecombinant SERA (b�SERA) and expression ofb�SERA in insect cells

A synthetic gene encoding the almost full sequence ofP. falciparum Honduras-1SERA (amino acid residues23–989) was constructed with previously describedDNA fragments [15] and the newly synthesized joiningfragments according to the methods described [15]. Theassembled DNA fragment of the gene was introducedinto the baculovirus transfer vector pMbac (Stratagene,La Jolla, CA) by using Sma I and Bam HI sites. Theresultant plasmid, pMbac-SERA, contains a gene thatcodes for the N-terminal 26 amino acids residues ofmelittin followed by residues 23–989 of SERA.

For the generation of recombinant baculoviruses, Sf9cells were co-transfected with 2.4 �g of pMbac-SERAand 0.12 �g of DNA of wild-type baculovirus (AcM-NPV) according to the manufacturer’s protocol(PharMingen, San Diego, CA, USA). For the expres-sion of bvSERA, High Five™ cells (Invitrogen, NVLeek, Netherlands) were seeded at 2×106 cells per 75cm2 flask in EX-Cell™ 405 medium (JRH Biosciences,Lenexa, KS, USA). The cells were infected with 2×107

PFU of the recombinant baculovirus for 2 h. Theinfected cells were incubated in the same medium at27 °C for 2 to 3 days and the culture medium wascollected by centrifugation at 600×g for 15 min andstored at −80 °C until use.

2.3. Purification of b�SERA

All the procedures described below were conductedat 4 °C except where indicated. Cleared culture super-natant of insect cells producing bvSERA was concen-trated 20–25 fold with Cetriprep 30 (Amicon). Theconcentrate was subjected to gel filtration HPLC with aTSK-gel G4000SW column (Tosoh, Japan) followed bya DE 52 (Whatman) column. The appropriate fractionswere pooled and dialyzed extensively against a phos-phate-buffered saline (PBS). The dialyzed sample wasconcentrated with Centricon 30 (Amicon). A part of thepurified bvSERA was extensively dialyzed against 0.5M Tris–HCl, pH 7.5/8 M urea/1 mM EDTA, incu-bated with 50 mM DTT for 5 h at room temperatureand then treated with 100 mM iodoacetamide at roomtemperature for 30 min in the dark. The resultantdenatured and alkylated protein was dialyzed exten-sively against PBS and concentrated as above. Theprotein was stored at −80 °C until use.

2.4. Parasite culture and preparation of parasiteextracts

Honduras-1 line of P. falciparum was routinely main-tained as described [16,17]. Synchronization of parasite

Fig. 1. Scheme of the processing of P. falciparum Honduras-1 SERA.The positions of the three predicted active amino acid residues of theprotease homologue are indicated by single letters [13,14] and theamino acid residue numbers of the N-terminal ends of the majorprocessed fragments are shown in parenthesis [12]. Hatched boxrepresents the region of serine repeat. The lines show the regions ofSERA to which probe antibodies are directed.

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culture to a ring-stage was performed by 5% D-sorbitoltreatment [18]. The synchronized parasites were furthercultivated to trophozoite or schizont stages. Thetrophozoite- or schizont-infected erythrocytes were iso-lated from the parasite cultures by 63% (vol./vol.) Per-coll (Amersham Pharmacia Biotech) density gradientcentrifugation as described [19]. The isolated parasitizederythrocytes were washed twice with PBS and immedi-ately used for further experiments or stored at −80 °Cuntil use.

Parasitized erythrocytes were thawed from −80 °C,suspended in four cell-pellet volumes of PBS containing0.25% Triton X-100 (v/v) (ICN Biochemicals Inc, OH,USA), and then incubated at 4 °C for 1 h. Aftercentrifugation at 20000×g for 20 min, the supernatant(Triton extract) was collected. Alternatively, late-schizont-infected erythrocytes were suspended in fourcell-pellet volumes of PBS containing 0.1% saponin(w/v) (Sigma) immediately after Percoll purification.The suspension was incubated at room temperature for10 min and centrifuged at 2000×g for 5 min. Theresultant supernatant (Saponin extract) was recovered.The precipitate was subjected to freezing and thawingonce and extracted with Triton X-100 as describedabove for the Triton extract. The extract thus obtainedwas named Triton re-extract.

2.5. Western blot analysis

Western blot analysis was basically performed asdescribed by Towbin et al [20]. Samples were separatedby sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) on an appropriate gel [21]and electroblotted onto a PVDF membrane (Biorad).Primary antibodies were used at either 0.1 �g ml−1 (for�P47) or 0.5 �g ml−1 (for �P50 and �P18). Biotinylatedhorse IgG specific to mouse IgG (at 1:1000), avidin-conjugated peroxidase (ABC kit) and diaminobenzidinetetrahydrochloride (Vector Laboratories) were used.

2.6. In �itro reaction for SERA processing

Triton extract of parasite was used except whereindicated. A reaction mixture (total volume 10–15 �l)was assembled that consisted of parasite cell extract(0.8–1 �l), 0.3–0.5 �g of purified bvSERA and PBS.After incubation at 37 °C for the indicated times, themixture was boiled for 3 min in the presence of reduc-ing SDS sample and subjected to Western blot analysis.To examine effects of protease inhibitors on the pro-cessing of bvSERA, a parasite extract appropriatelydiluted in PBS was pre-incubated with a protease in-hibitor (added from a 10× stock) at room temperaturefor 10 min prior to the addition of bvSERA.

2.7. Analysis of glycosylation and assays fordihydrofolate reductase and lactate dehydrogenase

Denatured and alkylated bvSERA (0.2 �g) was incu-bated with 1 unit of N-glycanase (Sigma) in 20 �l of0.25 M Tris–HCl, pH 8.0 at 37 °C for 12 h, and thensubjected to SDS-PAGE and Western blot analysis.Assay for dihydrofolate reductase activity was per-formed as previously described [22]. Plasmodial lactatedehydrogenase activity was measured basically as de-scribed [23] with some modifications. A reaction mix-ture contained 100 �l of the Mulstat™ reagent (Flow,Inc.), 20 �l of the mixture of nitroblue tetrazolium andphenazine ethosulfate (Sigma) and 20 �l of 100-folddiluted parasite extract or lysate of uninfected erythro-cytes. The activity was expressed as absorbance mea-sured at 620 nm (A620 nm).

2.8. Analysis of effects of protease inhibitors on SERAprocessing within parasitized erythrocytes andintraerythrocytic proliferation of P. falciparum

Late schizonts were Percoll-purified, re-cultured for 4h in complete medium with fresh RBC. The parasiteswere then treated with 5% D-sorbitol (0 h) and re-cul-tured to establish a 4 h early ring stage culture. After 37h of incubation, new schizont-infected erythrocyteswere Percoll-purified, re-suspended in complete mediumand inoculated into wells on a 12-well plate (IWAKI,Japan) at 1.5×107 per 1 ml per well. About 10 �l ofprotease inhibitor or solvent was added to the culturesat 38 h. After further incubation at 37 °C for 12 h, theparasitized erythrocytes remaining in each culture werecollected by centrifugation at 200×g for 3 min andlysed by boiling in 80 �l of SDS sample buffer. Then 20�l of each sample was subjected to Western blotanalysis.

To examine the effects of protease inhibitors on theproliferation of the parasite, a culture that had beentightly synchronized as described above was diluted at37 h (schizont stage) to a parasitemia of 0.86%. Theculture was then dispensed into wells of a 24-wellculture plate (IWAKI, Japan) at 0.5 ml per well and 3%hematocrit. At 38 h, 5 �l of protease inhibitor orsolvent was added to each well. After incubation at37 °C for 12 h, thin blood smears were made, Giemsa-stained, and inspected under a microscope.

3. Results

3.1. Expression and purification of recombinant SERA(b�SERA)

The synthetic gene encoding whole SERA moleculewas constructed to circumvent the difficulty in express-

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Fig. 2. Western blot analysis of recombinant baculovirus-expressedSERA (bvSERA). Cell lysate corresponding to 2×106 trophozoitesand schizonts of P. falciparum Honduras-1 (lanes 1, 3, 5 and7) and0.15 �g of purified bvSERA (lanes 2, 4, 6 and 8) were separated on8% SDS-PAGE under reducing (lanes 1–4) or non-reducing condi-tions (lanes 5–8) and probed with �P47 (lanes 1, 2, 5 and 6) or �P18(lanes 3, 4, 7 and 8). M, molecular weight markers (Biorad).

change (data not shown), suggesting that bvSERAwas not autocatalytically processed. We then exam-ined whether processing of bvSERA could be medi-ated by trans-acting protease activities derived fromparasitized erythrocytes. When bvSERA was incu-bated at 37 °C with the Triton X-100-soluble fractionof a cell lysate (Triton extract) prepared from lateschizont-infected erythrocytes, the 120 kDa bvSERAwas converted into smaller fragments correspondingto those found in the cell lysate of segmentedschizonts (Fig. 3). A major 48.5 kDa protein (P47bv)and a faint 47 kDa protein (P47en) were recognizedby �P47 (Fig. 3, lane 4). The 47 kDa fragment wasderived from the endogenous SERA because it wasalso found in the extract control and co-migratedwith P47 in the lysate of late shizonts (Fig. 3, lane 2),while the 48.5 kDa fragment was the counterpart gen-erated from bvSERA. The difference in molecular sizebetween P47bv and P47en is most likely attributed tothe five additional amino acids derived from the bac-ulovirus transfer vector after the signal peptide ofmelittin has been removed. Antibody �P50 recognizedtwo major bands (50 and 73 kDa) in the incubatedmixture containing bvSERA (Fig. 3, lane 7). The in-tensity of this 50 kDa protein band was obviouslymuch stronger than the 50 kDa band in the extractcontrol (Fig. 3, lane 6 and 7), indicating that a frag-ment corresponding to P50 was generated frombvSERA. Likewise, a 73 kDa band and an 18 kDaband were detected by �P18 in the incubated mixturecontaining bvSERA (Fig. 3, lane 10). The 73 kDaband was reactive with both �P50 and �P18, suggest-ing that it is the intermediate product composed ofP56 and P18. This is further supported by a timecourse analysis of bvSERA processing in vitro show-ing that bvSERA was successively converted to P73,P56 and P50 (Fig. 4). The in vitro results are consis-tent with the observations on SERA processing in theparasite culture (Fig. 3, lanes 2, 5 and 8). Productscorresponding to P25n and P25c that appeared in theparasite cells, however, could not be observed in thecell-free system.

Incubation of denatured and alkylated bvSERAwith the extract resulted in mere random degradation(Fig. 5A, lanes 3 and 6), indicating that the process-ing was dependent on the tertiary structure ofbvSERA. The cell extract prepared from latetrophzoites only caused non-specific degradation ofbvSERA (Fig. 5B, lane 2). In contrast, the cell ex-tract prepared from schizonts showed evident process-ing activities (Fig. 5B, lane 4), and extract preparedfrom segmented schizonts had much stronger activi-ties (Fig. 5B, lane 6). Thus the protease activities forthe processing of bvSERA appears to accumulate orto be specifically activated at late schizont stage.

ing the extremely A/T-biased native gene in a het-erologous organism. To obtain correctly folded re-combinant SERA, we expressed it as a secretedprotein by using the baculovirus expression systemand purified it from culture supernatant of infectedinsect cells. The identity of the recombinant protein(bvSERA) was confirmed through Western blot anal-ysis by using mouse antibodies (�P47 and �P18) spe-cific for the N- or C-terminal domains of SERA.Under both reducing and non-reducing conditions,bvSERA and the parasite-derived SERA co-migratedin SDS-PAGE (Fig. 2, lanes 1–6). Both showed aslower mobility under non-reducing conditions (Fig.2, lanes 5–8) than under reducing conditions (Fig. 2,lanes 1–4). Moreover, �P18, which recognized the na-tive SERA under reducing but not under non-reduc-ing conditions (Fig. 2, lanes 3 and 7), also recognizedbvSERA only under reducing conditions (Fig. 2,lanes 4 and 8). These observations suggest that thepurified bvSERA retained a similar conformation tothe native SERA. Treatment of denatured and alky-lated bvSERA with an excess amount of N-glycanasedid not cause any detectable mobility shift, suggestingthat N-glycosylation of bvSERA is not significant(data not shown).

3.2. In �itro processing of b�SERA

When the purified bvSERA was incubated at 37 °Cin PBS, RPMI1640 medium, acetate buffer (pH 5) orTris–HCl buffer (pH 9), its molecular size did not

J. Li et al. / Molecular & Biochemical Parasitology 120 (2002) 177–186 181

Fig. 3. In vitro processing of bvSERA by parasite extracts. Purified bvSERA (0.3 �g) was incubated at 37 °C for 5 h without (lane 1) or withthe Triton extract of parasite cells (lanes 4, 7 and 10) prepared as described in Section 2. Cell lysate corresponding to 2×106 segmented schizonts(lanes 2, 5 and 8) and the parasite extract incubated without bvSERA (lanes 3, 6 and 9) were included as controls. The samples were subjectedto Western blot analysis with �P 47 (lanes 1–4), �P50 (lanes 5-7) or �P18 (lanes 8–10). P47en, endogenous P47 from parasite extract; P47bv, P47generated from bvSERA.

3.3. Localization of protease acti�ities responsible forthe processing of b�SERA

To localize SERA processing activities, late schizont-infected erythrocytes were treated with 0.1% saponin asdescribed in Section 2. The native SERA was almostexclusively recovered in the Saponin extract (Fig. 6,lane 3). No activity of dihydrofolate reductase, a solu-ble cytosolic enzyme, could be detected in the Saponinextract (�0.005 U ml−1), while a significant activity(0.57 U ml−1) comparable to that found in the Tritonextract was detected in Triton re-extract. Similarly, theactivity of cytosolic plasmodial lactate dehydrogenasewas not found in the Saponin extract: A620 nm=0.052versus 0.053 for the lysate of uninfected erythrocytes, incontrast to 0.411 for the Triton extract. This indicatesthat the saponin treatment disrupted the PVM butcaused little or no permealization of the parasite cellmembrane. A substantial fraction of the processingactivities was found in the Saponin extract (Fig. 6, lane4), although less than those found in theTriton extract(Fig. 6, lane 2). The majority of the activities, however,was found in the precipitate fraction (Triton re-extract)(Fig. 6, lane 6), suggesting that the responsibleproteases are associated with membranes. This interpre-tation is supported by the observation that little of theprocessing activities were detected in the supernatantfraction from the extraction with PBS of late schizontcells disrupted by freezing and thawing (data notshown).

3.4. Effects of protease inhibitors on the processing ofb�SERA

The establishment of the in vitro cell-free system forthe SERA processing enabled us to analyze theproteases involved in SERA processing. Inhibitorsknown to block serine, cysteine, aspartic, or metallo

Fig. 4. Kinetic analysis of in vitro processing of bvSERA. A mixture(total 67.5 �l) consisting of 8.6 �g of bvSERA and 11 �l of parasiteextract was incubated at 37 °C. Aliquots of 9 �l were removed at theindicated times, boiled in reducing SDS sample buffer, and subjectedto Western blot analysis with �P50. The extract incubated alone for5 h was loaded in lane Ex.

J. Li et al. / Molecular & Biochemical Parasitology 120 (2002) 177–186182

Fig. 5. Specificity of the protease activities responsible for the processing of bvSERA in vitro. (A) Structure dependency. Intact bvSERA (lanes2 and 5) or denatured and alkylated bvSERA (lanes 3 and 6) was incubated at 37 °C for 5 h with the parasite extract. Extract controls were alsoincluded (lanes 1 and 4). The incubated samples were subjected to Western blot analysis with �P47 (lanes 1–3) or �P50 (lanes 4–6). (B) Stagespecificity of the processing activities. Parasite culture synchronized by 5% D-sorbitol treatment was further incubated for around 24, 30 or 34 hto obtain parasite cells rich in late trophozoites (lanes 1 and 2), schizonts (lanes 3 and 4) and segmented schizonts (lanes 5 and 6), respectively.Triton extracts were prepared from these cells and incubated at 37 °C for 5 h without (lanes 1, 3, and 5) or with 0.5 �g of bvSERA (lanes 2, 4,and 6). The samples were then subjected to Western blot analysis with �P47.

proteases were tested for their effects on the processingof bvSERA. Diisopropyl fluorophosphate (DFP), a ser-ine protease inhibitor, significantly inhibited the conver-sion of bvSERA to P47bv at 1 mM (Fig. 7, lane 11)and this inhibition was nearly complete at 5 mM (Fig.7, lane 12). The other inhibitors tested were withoutsuch an effect. These include Diethylenediaminete-traacetic acid (EDTA), aminophenylmethyl sulfonylfluoride (APMSF), aprotinin, trans-epoxysuccinyl-L-leucylamindo (4-guanidino) butine (E-64), leupeptin,phenylmethylsulfonyl fluoride (PMSF), pepstatin A(Fig. 7, lanes 3–9), 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF; 1 mM), N-tosyl-L-phenylalaninechloromethyl ketone (TPCK; 0.1 mM), N-a-p-tosyl-L-lysine chloromethyl ketone (TLCK; 1 mM),dichloroisocoumarin (DCI; 1 mM), soybean trypsininhibitor (0.1 mg ml−1), iodoacetoamide (1 mM), o-phenanthroline (1 mM) and bestatin (0.2 mM) (datanot shown). Treatment with E-64 or leupeptin inhibitednon-specific degradation of P47bv, causing denserP47bv band (Fig. 7, lanes 6 and 7). Thus, the proteaseresponsible for the first cleavage event of SERA pro-cessing (conversion of SERA to P47+P73) was sensi-tive to DFP but insensitive to the other serine proteaseinhibitors tested (APMSF, aprotinin, PMSF, AEBSF,TPCK, TLCK, DCI, and soybean trypsin inhibitor),suggesting that it is a unique serine protease.

In contrast, cysteine protease inhibitors E-64, leu-peptin, and iodoacetamide each at 1 mM significantlyinhibited the conversion of P73 to P56 as well as the

Fig. 6. Localisation of the processing activities. Triton extract (lanes1 and 2), Saponin extract (lanes 3 and 4), or Triton re-extract (lanes5 and 6) prepared as described in Section 2 was incubated for 5 h at37 °C without (lanes 1, 3 and 5) or with 0.3 �g of bvSERA (lanes 2,4 and 6) and then subjected to Western blot analysis with �P47.

J. Li et al. / Molecular & Biochemical Parasitology 120 (2002) 177–186 183

Fig. 7. Inhibition of processing of SERA into P47 and P73. Mixtures of parasite extract and bvSERA (0.3 �g) were incubated at 37 °C for 5 hin the presence of 1% ethanol (lane 2), 10 mM EDTA (lane 3), 1 mM APMSF (lane 4), 1.5 �M aprotinin (lane 5), 1 mM E-64 (lane 6), 1 mMleupeptin (lane 7), 1 mM PMSF (lane 8), 10 �M pepstatin A (lane 9), 1% isopropanol (lane 10), 1 mM DFP (lane 11) or 5 mM DFP (lane 12).As a control, bvSERA alone (lane 1) was also incubated. The samples were then subjected to Western blot analysis with �P47.

Fig. 8. Inhibition of processing of P73 into P56 and P50. Mixtures of parasite extract and bvSERA (0.3 �g) were incubated at 37 °C for 5 h inthe presence of 1% ethanol (lane3), 10 mM EDTA (lane 4), 1 mM APMSF (lane5), 1.5 �M aprotinin (lane 6), 1 mM E-64 (lane7), 1 mM leupeptin(lane 8), 1 mM PMSF (lane 9) 10 �M pepstatin A (lane 10) or 1 mM iodoacetamide (lane 11). As controls, parasite extract (lane 1) and bvSERA(lane 2) alone were also incubated. The incubated samples were then subjected to Western blot analysis with �P50.

conversion of P56 to P50 (Fig. 8, lanes 7, 8 and 11).With either E-64 or leupeptin, the inhibition could beachieved at as low as 0.1 �M and appeared to reachplateau at 1 �M. Complete inhibition of the conversionof P56 to P50 could be achieved at 10 �M, but only

partial inhibition of the conversion of P73 to P56 wasobserved at 0.1 �M–1 mM (data not shown). Ethanol,EDTA, APMSF, aprotinin, PMSF, pepstatin A (Fig. 8,lanes 3–6, 9–10) and AEBSF (at 1 mM; data notshown) were without effect.

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3.5. Effects of protease inhibitors on SERA processingwithin the parasitized RBC and the parasite growth

To see whether SERA processing in parasitized ery-throcytes are inhibited by DFP, leupeptin, or E-64,purified middle-stage schizonts (38–42 h post invasion)were incubated in the presence of the inhibitors for 12h. The remaining schizont cells were recovered andsubjected to Western blot analysis. As shown in Fig.9A, treatment with 1 mM DFP significantly causedaccumulation of the unprocessed SERA. By contrast,both leupeptin and E-64 at 100 �M markedly inhibitedSERA processing into P50 and caused accumulation ofP56 (Fig. 9B). However, inhibition of the conversion ofP73 to P56, which was evident in the cell-free system,could not be observed.

To examine the effects of DFP, leupeptin, and E-64on the intraerythrocytic proliferation of the parasite,cultures of middle- stage schizonts (38–42 h post inva-sion) were treated with individual inhibitors for 12 h.DFP (1 mM), leupeptin and E-64 (100 �M) individuallyblocked 35–46% of the schizonts from rupturing. No-ticeably, the majority of the remaining schizonts weremorphologically similar to the structures reported byothers [9,24].

4. Discussion

We have expressed recombinant SERA as a secretedprotein in the baculovirus expression system. By usingthe protein, we have established a cell-free system thatreproduces SERA processing that occurs in the para-sitized erythrocyte. In the system, fragments corre-sponding to P47, P73, P56, P50 and P18 were allgenerated, although some of the derived P18 moleculeswere trimmed further by non-specific protease activities.Products corresponding to P25n and P25c that ap-peared in the parasite cells, however, could not beobserved in the cell-free system even after prolongedincubation. The responsible protease might be unstablein the extract.

The in vitro system enabled us to characterize theproteases responsible for SERA processing. The pro-cessing activities are restricted to late schizont stage,which is consistent with the findings that SERA pro-cessing occurs immediately prior to schizont rupture (Liand Horii, unpublished data). The results of the frac-tionation study strongly suggest that the proteases re-sponsible for SERA processing are located in theparasitophorous vacuole, which is consistent with thatSERA is located in the same vacuole [11]. In addition,

Fig. 9. Effect of DFP, leupeptin (Lp) and E-64 on SERA processing in parasitized erythrocytes. Purified parasite cells at the middle schizont stage(38–42 h post invasion) were re-cultured at 37 °C in the presence of indicated protease inhibitors or solvent as described in Section 2. After 12h, the remaining parasite cells were recovered from the cultures and subjected to Western blot analysis with �P47 (A) or �P50 (B). C, control (0.1%DMSO in A and no addition in B).

J. Li et al. / Molecular & Biochemical Parasitology 120 (2002) 177–186 185

it is noteworthy that the proteases are membrane-as-sociated although its physiological significance is notknown.

At least three distinct proteases are involved in theprocessing of SERA. The primary step of SERA pro-cessing, conversion of SERA to P47 and P73, wasspecifically inhibited by DFP, a specific inhibitor ofserine proteases. The responsible protease seems tohold a unique structure, because it is not sensitive tomany other serine protease inhibitors. The proteolyticreaction is unlikely to be an autocatalytic process ofSERA that contains structural similarity to papainfamily [13,14]. The conversion of P56 to P50, in con-trast, is mediated by a cysteine protease but not aserine protease as has been suggested by Debrabantet al. [24]. As for the conversion of P73 to P56, al-though both E-64 and leupeptin partially inhibitedthis event in the in vitro system, they did not showany inhibitory effect in vivo. Although the reason forthis inconsistency is unknown, the results may suggesta third protease acting on SERA to convert P73 toP56.

The events of schizont rupture and merozoite re-lease are poorly understood, yet evidence suggeststhat parasite-derived proteases are involved [8]. In arecent study by Salmon et al [9], when added to mid-dle-stage schizonts, E-64 caused an accumulation ofPVM-enclosed merozoite structures. A model for theprocess of rupture was subsequently proposed bythose authors: merozoites enclosed within the PVMfirst exit from the host erythrocyte and then rapidlyescape from within the PVM by a proteolysis-depen-dent mechanism. In the present study, we also ob-served that when added to cultures of middle-stageschizonts, leupeptin and E-64 each caused an accumu-lation of unruptured schizonts morphologically similarto the PVM-enclosed merozoite structures. Concomi-tantly, both inhibitors inhibited SERA processing inthese schizonts. These data suggest that SERA pro-cessing preludes the second step of schizont rupture,the release of merozoites from the PVM. P50 ofSERA, which has a structural similarity to cysteineprotease, might function as a protease in the processof PVM rupture. Further study is required to test thishypothesis.

Acknowledgements

We thank Dr R. Brobey for help in the assay ofdihydrofolate reductase activity. We are grateful toDr D. Bzik for critical reading of the manuscript.This work was supported by Grant-in-Aid for Scien-tific Research on Priority Areas (A) (08281104) andGrant-in-Aid for Scientific Research (A) (13357002)from the Japanese Ministry of Education, Science,

Sports, Culture and Technology. This study also re-ceived financial support from the UNDP/WorldBank/WHO Special Programme for Research andTraining in Tropical Diseases (TDR), WHO, Geneva(Vaccine Discovery Research Grant 980278 to T. H.).

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