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Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA)
Molecular & Biochemical Parasitology 120 (2002) 177– 186 www.parasitology-online.com.
Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA) Jie Li a, Hiroyuki Matsuoka b, Toshihide Mitamura a, Toshihiro Horii a,* a
b
Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka Uni6ersity, Suita, Osaka 565 -0871, Japan 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 have established an in vitro cell-free system using a baculovirus-expressed recombinant SERA (bvSERA) that mimics the SERA processing that occurs in parasitized erythrocytes. SERA processing was mediated by parasite-derived trans-acting proteases, but not 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 activity responsible 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, leupeptin and iodoacetoamide. Moreover, addition of DFP, E-64 or leupeptin to the cultures of schizont-stage parasites blocked schizont rupture and release of merozoites from PVM. These results indicate that SERA processing correlates to schizont rupture and the processing 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
1. Introduction The malaria parasite Plasmodium falciparum has an asexual life cycle in the mature human erythrocyte, in which it is surrounded by the parasitophorous vacuole membrane (PVM). In the erythrocyte, the parasite undergoes three distinct stages of development: the ring, the trophozoite and the schizont. During the schizogony, between 16 and 32 merozoites are formed within the parasitophorous vacuole. The merozoites release from the ruptured schizont and invade new red cells and continue the cycle. During the course of schizont rupture, merozoite release and re-invasion, a number of proteins associated with free merozoites, Abbre6iations: bvSERA, baculovirus-expressed SERA; DFP, diisopropyl fluorophosphate; E-64, trans-epoxysuccinyl-L-leucylamindo(4guanidino)butine; SERA, serine repeat antigen. * Corresponding author. Tel.: +81-6-6879-8280; fax: +81-6-68798281. E-mail address: [email protected] (T. Horii).
such as merozoite surface protein-1 (MSP-1) [1], MSP-3 [2], MSP-6 [3], apical membrane antigen-1 [4], and rhoptry associated protein-1 [5,6], are proteolytically processed. In the case of MSP-1, the final processing event (i.e. conversion of MSP-142 to MSP-119) has been shown to be necessary for merozoite invasion [7]. Furthermore, results from studies with protease inhibitors suggest that parasite-derived proteases are involved in the events of schizont rupture and meroziote re-invasion [8]. A recent report showed that a cysteine protease(s) could be involved in the release of merozoites from within the PVM [9]. Therefore, analysis of these processing events and characterization of the proteases involved would help to understand the molecular mechanisms underlying schizont rupture, merozoite-release, and/or merozoite-invasion. The serine repeat antigen (SERA) of P. falciparum [10] is synthesized at trophozoite- and schizont-stages during the intraerythrocytic cycle and secreted as a 120 kDa protein into the parasitophorous vacuole [11,12].
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It has been demonstrated that the 120 kDa protein is proteolytically 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 type of SERA gene, further processed into two 25 kDa fragments (P25n and P25c) (Li and Horii, unpublished data), while P56 is converted into a 50 kDa fragment (P50) [11,12], which has a significant homology to papain-family proteases but has a predicted active serine instead of cysteine [13,14]. SERA processing is initiated most likely at late schizont stage and completed within the parasitized erythrocytes just prior to schizont rupture (Li and Horii, unpublished data). These observations suggest that the processing of SERA might represent the maturation of the putative protease and that the processed fragments of SERA as well as the proteases involved in the SERA processing might play important roles in schizont rupture, merozoite release, and/or merozoite invasion. In the present study, we have established an in vitro cell-free system that mimics the SERA processing that occurs within parasitized erythrocytes. With this system, we further characterized the proteases involved in SERA processing. 2. Materials and methods
2.1. Antibodies Recombinant proteins SE47’ and SE50A have been described [15]. A synthetic gene encoding amino acid residues 879–989 of P. falciparum Honduras-1 SERA was constructed by a similar method to that for the expression of SE47’ and SE50A [15] and used to express recombinant P18 (SE18) in Escherichia coli as a glutathione-S-transferase fusion protein. Polyclonal antibodies specific for P47, P50 and P18 (named aP47, aP50, and aP18, respectively. See Fig. 1) were raised in BALB/c mice (SLC, Japan) and total IgG was purified with a HiTrap Protein A column (Pharmacia).
Fig. 1. Scheme of the processing of P. falciparum Honduras-1 SERA. The positions of the three predicted active amino acid residues of the protease homologue are indicated by single letters [13,14] and the amino acid residue numbers of the N-terminal ends of the major processed fragments are shown in parenthesis [12]. Hatched box represents the region of serine repeat. The lines show the regions of SERA to which probe antibodies are directed.
2.2. Construction of plasmid pMbac-SERA encoding recombinant SERA (b6SERA) and expression of b6SERA in insect cells A synthetic gene encoding the almost full sequence of P. falciparum Honduras-1SERA (amino acid residues 23–989) was constructed with previously described DNA fragments [15] and the newly synthesized joining fragments according to the methods described [15]. The assembled DNA fragment of the gene was introduced into the baculovirus transfer vector pMbac (Stratagene, La Jolla, CA) by using Sma I and Bam HI sites. The resultant plasmid, pMbac-SERA, contains a gene that codes for the N-terminal 26 amino acids residues of melittin followed by residues 23–989 of SERA. For the generation of recombinant baculoviruses, Sf9 cells were co-transfected with 2.4 mg of pMbac-SERA and 0.12 mg of DNA of wild-type baculovirus (AcMNPV) according to the manufacturer’s protocol (PharMingen, San Diego, CA, USA). For the expression of bvSERA, High Five™ cells (Invitrogen, NV Leek, Netherlands) were seeded at 2× 106 cells per 75 cm2 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. The infected cells were incubated in the same medium at 27 °C for 2 to 3 days and the culture medium was collected by centrifugation at 600× g for 15 min and stored at − 80 °C until use.
2.3. Purification of b6SERA All the procedures described below were conducted at 4 °C except where indicated. Cleared culture supernatant of insect cells producing bvSERA was concentrated 20–25 fold with Cetriprep 30 (Amicon). The concentrate was subjected to gel filtration HPLC with a TSK-gel G4000SW column (Tosoh, Japan) followed by a DE 52 (Whatman) column. The appropriate fractions were pooled and dialyzed extensively against a phosphate-buffered saline (PBS). The dialyzed sample was concentrated with Centricon 30 (Amicon). A part of the purified bvSERA was extensively dialyzed against 0.5 M Tris –HCl, pH 7.5/8 M urea/1 mM EDTA, incubated with 50 mM DTT for 5 h at room temperature and then treated with 100 mM iodoacetamide at room temperature for 30 min in the dark. The resultant denatured and alkylated protein was dialyzed extensively against PBS and concentrated as above. The protein was stored at − 80 °C until use.
2.4. Parasite culture and preparation of parasite extracts Honduras-1 line of P. falciparum was routinely maintained as described [16,17]. Synchronization of parasite
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culture to a ring-stage was performed by 5% D-sorbitol treatment [18]. The synchronized parasites were further cultivated to trophozoite or schizont stages. The trophozoite- or schizont-infected erythrocytes were isolated from the parasite cultures by 63% (vol./vol.) Percoll (Amersham Pharmacia Biotech) density gradient centrifugation as described [19]. The isolated parasitized erythrocytes were washed twice with PBS and immediately used for further experiments or stored at −80 °C until use. Parasitized erythrocytes were thawed from − 80 °C, suspended in four cell-pellet volumes of PBS containing 0.25% Triton X-100 (v/v) (ICN Biochemicals Inc, OH, USA), and then incubated at 4 °C for 1 h. After centrifugation at 20000× g for 20 min, the supernatant (Triton extract) was collected. Alternatively, lateschizont-infected erythrocytes were suspended in four cell-pellet volumes of PBS containing 0.1% saponin (w/v) (Sigma) immediately after Percoll purification. The suspension was incubated at room temperature for 10 min and centrifuged at 2000× g for 5 min. The resultant supernatant (Saponin extract) was recovered. The precipitate was subjected to freezing and thawing once and extracted with Triton X-100 as described above for the Triton extract. The extract thus obtained was named Triton re-extract.
2.5. Western blot analysis Western blot analysis was basically performed as described by Towbin et al [20]. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on an appropriate gel [21] and electroblotted onto a PVDF membrane (Biorad). Primary antibodies were used at either 0.1 mg ml − 1 (for aP47) or 0.5 mg ml − 1 (for aP50 and aP18). Biotinylated horse IgG specific to mouse IgG (at 1:1000), avidinconjugated peroxidase (ABC kit) and diaminobenzidine tetrahydrochloride (Vector Laboratories) were used.
2.6. In 6itro reaction for SERA processing Triton extract of parasite was used except where indicated. A reaction mixture (total volume 10– 15 ml) was assembled that consisted of parasite cell extract (0.8 –1 ml), 0.3–0.5 mg of purified bvSERA and PBS. After incubation at 37 °C for the indicated times, the mixture was boiled for 3 min in the presence of reducing SDS sample and subjected to Western blot analysis. To examine effects of protease inhibitors on the processing of bvSERA, a parasite extract appropriately diluted in PBS was pre-incubated with a protease inhibitor (added from a 10× stock) at room temperature for 10 min prior to the addition of bvSERA.
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2.7. Analysis of glycosylation and assays for dihydrofolate reductase and lactate dehydrogenase Denatured and alkylated bvSERA (0.2 mg) was incubated with 1 unit of N-glycanase (Sigma) in 20 ml of 0.25 M Tris–HCl, pH 8.0 at 37 °C for 12 h, and then subjected to SDS-PAGE and Western blot analysis. Assay for dihydrofolate reductase activity was performed as previously described [22]. Plasmodial lactate dehydrogenase activity was measured basically as described [23] with some modifications. A reaction mixture contained 100 ml of the Mulstat™ reagent (Flow, Inc.), 20 ml of the mixture of nitroblue tetrazolium and phenazine ethosulfate (Sigma) and 20 ml of 100-fold diluted parasite extract or lysate of uninfected erythrocytes. The activity was expressed as absorbance measured at 620 nm (A620 nm).
2.8. Analysis of effects of protease inhibitors on SERA processing within parasitized erythrocytes and intraerythrocytic proliferation of P. falciparum Late schizonts were Percoll-purified, re-cultured for 4 h in complete medium with fresh RBC. The parasites were then treated with 5% D-sorbitol (0 h) and re-cultured to establish a 4 h early ring stage culture. After 37 h of incubation, new schizont-infected erythrocytes were Percoll-purified, re-suspended in complete medium and inoculated into wells on a 12-well plate (IWAKI, Japan) at 1.5×107 per 1 ml per well. About 10 ml of protease inhibitor or solvent was added to the cultures at 38 h. After further incubation at 37 °C for 12 h, the parasitized erythrocytes remaining in each culture were collected by centrifugation at 200× g for 3 min and lysed by boiling in 80 ml of SDS sample buffer. Then 20 ml of each sample was subjected to Western blot analysis. To examine the effects of protease inhibitors on the proliferation of the parasite, a culture that had been tightly synchronized as described above was diluted at 37 h (schizont stage) to a parasitemia of 0.86%. The culture was then dispensed into wells of a 24-well culture plate (IWAKI, Japan) at 0.5 ml per well and 3% hematocrit. At 38 h, 5 ml of protease inhibitor or solvent was added to each well. After incubation at 37 °C for 12 h, thin blood smears were made, Giemsastained, and inspected under a microscope.
3. Results
3.1. Expression and purification of recombinant SERA (b6SERA) The synthetic gene encoding whole SERA molecule was constructed to circumvent the difficulty in express-
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Fig. 2. Western blot analysis of recombinant baculovirus-expressed SERA (bvSERA). Cell lysate corresponding to 2 × 106 trophozoites and schizonts of P. falciparum Honduras-1 (lanes 1, 3, 5 and7) and 0.15 mg of purified bvSERA (lanes 2, 4, 6 and 8) were separated on 8% SDS-PAGE under reducing (lanes 1 –4) or non-reducing conditions (lanes 5 – 8) and probed with aP47 (lanes 1, 2, 5 and 6) or aP18 (lanes 3, 4, 7 and 8). M, molecular weight markers (Biorad).
ing the extremely A/T-biased native gene in a heterologous organism. To obtain correctly folded recombinant SERA, we expressed it as a secreted protein by using the baculovirus expression system and purified it from culture supernatant of infected insect cells. The identity of the recombinant protein (bvSERA) was confirmed through Western blot analysis by using mouse antibodies (aP47 and aP18) specific for the N- or C-terminal domains of SERA. Under both reducing and non-reducing conditions, bvSERA and the parasite-derived SERA co-migrated in SDS-PAGE (Fig. 2, lanes 1– 6). Both showed a slower mobility under non-reducing conditions (Fig. 2, lanes 5–8) than under reducing conditions (Fig. 2, lanes 1– 4). Moreover, aP18, which recognized the native SERA under reducing but not under non-reducing conditions (Fig. 2, lanes 3 and 7), also recognized bvSERA only under reducing conditions (Fig. 2, lanes 4 and 8). These observations suggest that the purified bvSERA retained a similar conformation to the native SERA. Treatment of denatured and alkylated bvSERA with an excess amount of N-glycanase did not cause any detectable mobility shift, suggesting that N-glycosylation of bvSERA is not significant (data not shown).
3.2. In 6itro processing of b6SERA When the purified bvSERA was incubated at 37 °C in PBS, RPMI1640 medium, acetate buffer (pH 5) or Tris– HCl buffer (pH 9), its molecular size did not
change (data not shown), suggesting that bvSERA was not autocatalytically processed. We then examined whether processing of bvSERA could be mediated by trans-acting protease activities derived from parasitized erythrocytes. When bvSERA was incubated at 37 °C with the Triton X-100-soluble fraction of a cell lysate (Triton extract) prepared from late schizont-infected erythrocytes, the 120 kDa bvSERA was converted into smaller fragments corresponding to those found in the cell lysate of segmented schizonts (Fig. 3). A major 48.5 kDa protein (P47bv) and a faint 47 kDa protein (P47en) were recognized by aP47 (Fig. 3, lane 4). The 47 kDa fragment was derived from the endogenous SERA because it was also found in the extract control and co-migrated with P47 in the lysate of late shizonts (Fig. 3, lane 2), while the 48.5 kDa fragment was the counterpart generated from bvSERA. The difference in molecular size between P47bv and P47en is most likely attributed to the five additional amino acids derived from the baculovirus transfer vector after the signal peptide of melittin has been removed. Antibody aP50 recognized two major bands (50 and 73 kDa) in the incubated mixture containing bvSERA (Fig. 3, lane 7). The intensity of this 50 kDa protein band was obviously much stronger than the 50 kDa band in the extract control (Fig. 3, lane 6 and 7), indicating that a fragment corresponding to P50 was generated from bvSERA. Likewise, a 73 kDa band and an 18 kDa band were detected by aP18 in the incubated mixture containing bvSERA (Fig. 3, lane 10). The 73 kDa band was reactive with both aP50 and aP18, suggesting that it is the intermediate product composed of P56 and P18. This is further supported by a time course analysis of bvSERA processing in vitro showing that bvSERA was successively converted to P73, P56 and P50 (Fig. 4). The in vitro results are consistent with the observations on SERA processing in the parasite culture (Fig. 3, lanes 2, 5 and 8). Products corresponding to P25n and P25c that appeared in the parasite cells, however, could not be observed in the cell-free system. Incubation of denatured and alkylated bvSERA with the extract resulted in mere random degradation (Fig. 5A, lanes 3 and 6), indicating that the processing was dependent on the tertiary structure of bvSERA. The cell extract prepared from late trophzoites only caused non-specific degradation of bvSERA (Fig. 5B, lane 2). In contrast, the cell extract prepared from schizonts showed evident processing activities (Fig. 5B, lane 4), and extract prepared from segmented schizonts had much stronger activities (Fig. 5B, lane 6). Thus the protease activities for the processing of bvSERA appears to accumulate or to be specifically activated at late schizont stage.
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Fig. 3. In vitro processing of bvSERA by parasite extracts. Purified bvSERA (0.3 mg) was incubated at 37 °C for 5 h without (lane 1) or with the 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 subjected to Western blot analysis with aP 47 (lanes 1 –4), aP50 (lanes 5-7) or aP18 (lanes 8 – 10). P47en, endogenous P47 from parasite extract; P47bv, P47 generated from bvSERA.
3.3. Localization of protease acti6ities responsible for the processing of b6SERA
3.4. Effects of protease inhibitors on the processing of b6SERA
To localize SERA processing activities, late schizontinfected erythrocytes were treated with 0.1% saponin as described in Section 2. The native SERA was almost exclusively recovered in the Saponin extract (Fig. 6, lane 3). No activity of dihydrofolate reductase, a soluble cytosolic enzyme, could be detected in the Saponin extract (B 0.005 U ml − 1), while a significant activity (0.57 U ml − 1) comparable to that found in the Triton extract was detected in Triton re-extract. Similarly, the activity of cytosolic plasmodial lactate dehydrogenase was not found in the Saponin extract: A620 nm =0.052 versus 0.053 for the lysate of uninfected erythrocytes, in contrast to 0.411 for the Triton extract. This indicates that the saponin treatment disrupted the PVM but caused little or no permealization of the parasite cell membrane. A substantial fraction of the processing activities was found in the Saponin extract (Fig. 6, lane 4), 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 responsible proteases are associated with membranes. This interpretation is supported by the observation that little of the processing activities were detected in the supernatant fraction from the extraction with PBS of late schizont cells disrupted by freezing and thawing (data not shown).
The establishment of the in vitro cell-free system for the SERA processing enabled us to analyze the proteases involved in SERA processing. Inhibitors known to block serine, cysteine, aspartic, or metallo
Fig. 4. Kinetic analysis of in vitro processing of bvSERA. A mixture (total 67.5 ml) consisting of 8.6 mg of bvSERA and 11 ml of parasite extract was incubated at 37 °C. Aliquots of 9 ml were removed at the indicated times, boiled in reducing SDS sample buffer, and subjected to Western blot analysis with aP50. The extract incubated alone for 5 h was loaded in lane Ex.
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Fig. 5. Specificity of the protease activities responsible for the processing of bvSERA in vitro. (A) Structure dependency. Intact bvSERA (lanes 2 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 also included (lanes 1 and 4). The incubated samples were subjected to Western blot analysis with aP47 (lanes 1 – 3) or aP50 (lanes 4 – 6). (B) Stage specificity of the processing activities. Parasite culture synchronized by 5% D-sorbitol treatment was further incubated for around 24, 30 or 34 h to 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 mg of bvSERA (lanes 2, 4, and 6). The samples were then subjected to Western blot analysis with aP47.
proteases were tested for their effects on the processing of bvSERA. Diisopropyl fluorophosphate (DFP), a serine protease inhibitor, significantly inhibited the conversion 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 without such an effect. These include Diethylenediaminetetraacetic acid (EDTA), aminophenylmethyl sulfonyl fluoride (APMSF), aprotinin, trans-epoxysuccinyl-Lleucylamindo (4-guanidino) butine (E-64), leupeptin, phenylmethylsulfonyl fluoride (PMSF), pepstatin A (Fig. 7, lanes 3–9), 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF; 1 mM), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK; 0.1 mM), N-a-p-tosyl-Llysine chloromethyl ketone (TLCK; 1 mM), dichloroisocoumarin (DCI; 1 mM), soybean trypsin inhibitor (0.1 mg ml − 1), iodoacetoamide (1 mM), ophenanthroline (1 mM) and bestatin (0.2 mM) (data not shown). Treatment with E-64 or leupeptin inhibited non-specific degradation of P47bv, causing denser P47bv band (Fig. 7, lanes 6 and 7). Thus, the protease responsible for the first cleavage event of SERA processing (conversion of SERA to P47+ P73) was sensitive to DFP but insensitive to the other serine protease inhibitors 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, leupeptin, and iodoacetamide each at 1 mM significantly inhibited the conversion of P73 to P56 as well as the
Fig. 6. Localisation of the processing activities. Triton extract (lanes 1 and 2), Saponin extract (lanes 3 and 4), or Triton re-extract (lanes 5 and 6) prepared as described in Section 2 was incubated for 5 h at 37 °C without (lanes 1, 3 and 5) or with 0.3 mg of bvSERA (lanes 2, 4 and 6) and then subjected to Western blot analysis with aP47.
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Fig. 7. Inhibition of processing of SERA into P47 and P73. Mixtures of parasite extract and bvSERA (0.3 mg) were incubated at 37 °C for 5 h in the presence of 1% ethanol (lane 2), 10 mM EDTA (lane 3), 1 mM APMSF (lane 4), 1.5 mM aprotinin (lane 5), 1 mM E-64 (lane 6), 1 mM leupeptin (lane 7), 1 mM PMSF (lane 8), 10 mM 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 aP47.
Fig. 8. Inhibition of processing of P73 into P56 and P50. Mixtures of parasite extract and bvSERA (0.3 mg) were incubated at 37 °C for 5 h in the presence of 1% ethanol (lane3), 10 mM EDTA (lane 4), 1 mM APMSF (lane5), 1.5 mM aprotinin (lane 6), 1 mM E-64 (lane7), 1 mM leupeptin (lane 8), 1 mM PMSF (lane 9) 10 mM 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 aP50.
conversion of P56 to P50 (Fig. 8, lanes 7, 8 and 11). With either E-64 or leupeptin, the inhibition could be achieved at as low as 0.1 mM and appeared to reach plateau at 1 mM. Complete inhibition of the conversion of P56 to P50 could be achieved at 10 mM, but only
partial inhibition of the conversion of P73 to P56 was observed at 0.1 mM –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 not shown) were without effect.
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3.5. Effects of protease inhibitors on SERA processing within the parasitized RBC and the parasite growth To see whether SERA processing in parasitized erythrocytes 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 12 h. The remaining schizont cells were recovered and subjected to Western blot analysis. As shown in Fig. 9A, treatment with 1 mM DFP significantly caused accumulation of the unprocessed SERA. By contrast, both leupeptin and E-64 at 100 mM markedly inhibited SERA processing into P50 and caused accumulation of P56 (Fig. 9B). However, inhibition of the conversion of P73 to P56, which was evident in the cell-free system, could not be observed. To examine the effects of DFP, leupeptin, and E-64 on the intraerythrocytic proliferation of the parasite, cultures of middle- stage schizonts (38– 42 h post invasion) were treated with individual inhibitors for 12 h. DFP (1 mM), leupeptin and E-64 (100 mM) individually blocked 35 –46% of the schizonts from rupturing. Noticeably, the majority of the remaining schizonts were morphologically similar to the structures reported by others [9,24].
4. Discussion We have expressed recombinant SERA as a secreted protein in the baculovirus expression system. By using the protein, we have established a cell-free system that reproduces SERA processing that occurs in the parasitized erythrocyte. In the system, fragments corresponding to P47, P73, P56, P50 and P18 were all generated, although some of the derived P18 molecules were trimmed further by non-specific protease activities. Products corresponding to P25n and P25c that appeared in the parasite cells, however, could not be observed in the cell-free system even after prolonged incubation. The responsible protease might be unstable in the extract. The in vitro system enabled us to characterize the proteases responsible for SERA processing. The processing activities are restricted to late schizont stage, which is consistent with the findings that SERA processing occurs immediately prior to schizont rupture (Li and Horii, unpublished data). The results of the fractionation study strongly suggest that the proteases responsible for SERA processing are located in the parasitophorous vacuole, which is consistent with that SERA 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 12 h, the remaining parasite cells were recovered from the cultures and subjected to Western blot analysis with aP47 (A) or aP50 (B). C, control (0.1% DMSO in A and no addition in B).
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it is noteworthy that the proteases are membrane-associated although its physiological significance is not known. At least three distinct proteases are involved in the processing of SERA. The primary step of SERA processing, conversion of SERA to P47 and P73, was specifically inhibited by DFP, a specific inhibitor of serine proteases. The responsible protease seems to hold a unique structure, because it is not sensitive to many other serine protease inhibitors. The proteolytic reaction is unlikely to be an autocatalytic process of SERA that contains structural similarity to papain family [13,14]. The conversion of P56 to P50, in contrast, is mediated by a cysteine protease but not a serine protease as has been suggested by Debrabant et al. [24]. As for the conversion of P73 to P56, although both E-64 and leupeptin partially inhibited this event in the in vitro system, they did not show any inhibitory effect in vivo. Although the reason for this inconsistency is unknown, the results may suggest a third protease acting on SERA to convert P73 to P56. The events of schizont rupture and merozoite release are poorly understood, yet evidence suggests that parasite-derived proteases are involved [8]. In a recent study by Salmon et al [9], when added to middle-stage schizonts, E-64 caused an accumulation of PVM-enclosed merozoite structures. A model for the process of rupture was subsequently proposed by those authors: merozoites enclosed within the PVM first exit from the host erythrocyte and then rapidly escape from within the PVM by a proteolysis-dependent mechanism. In the present study, we also observed that when added to cultures of middle-stage schizonts, leupeptin and E-64 each caused an accumulation of unruptured schizonts morphologically similar to the PVM-enclosed merozoite structures. Concomitantly, both inhibitors inhibited SERA processing in these schizonts. These data suggest that SERA processing preludes the second step of schizont rupture, the release of merozoites from the PVM. P50 of SERA, which has a structural similarity to cysteine protease, might function as a protease in the process of PVM rupture. Further study is required to test this hypothesis.
Acknowledgements We thank Dr R. Brobey for help in the assay of dihydrofolate reductase activity. We are grateful to Dr D. Bzik for critical reading of the manuscript. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas (A) (08281104) and Grant-in-Aid for Scientific Research (A) (13357002) from the Japanese Ministry of Education, Science,
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Sports, Culture and Technology. This study also received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), WHO, Geneva (Vaccine Discovery Research Grant 980278 to T. H.).
References [1] Holder AA, Freeman RR. The three major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight precursor. J Exp Med 1984;160:624 – 9. [2] McColl DJ, et al. Molecular variation in a novel polymorphic antigen associated with Plasmodium falciparum merozoites. Mol Biochem Parasitol 1994;68:53 – 67. [3] Trucco C, et al. The merozoite surface protein 6 gene codes for a 36 kDa protein associated with the Plasmodium falciparum merozoite surface protein-1 complex. Mol Biochem Parasitol 2001;112:91 – 101. [4] Crewther PE, Culvenor JG, Silva A, Cooper JA, Anders RF. Plasmodium falciparum: two antigens of similar size are located in different compartments of the rhoptry. Exp Parasitol 1990;49:119 – 32. [5] Bushell GR, Ingram LT, Fardoulys CA, Cooper JA. An antigenic complex in the rhoptries of Plasmodium falciparum. Mol Biochem Parasitol 1988;28:105 – 12. [6] Harnyuttanakorn P, McBride JS, Donachie S, Heidrich HG, Ridley RG. Inhibitory monoclonal antibodies recognize epitopes adjacent to a proteolytic cleavage site on the RAP-1 protein of Plasmodium falciparum. Mol Biochem Parasitol 1992;55:177 – 86. [7] Patino JAG, Holder AA, McBride JS, Blackman MJ. Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies. J Exp Med 1997;186:1689 – 99. [8] McKerrow JH, Sun E, Rothenal PJ, Bouvir J. The proteases and pathogenicity of parasitic protozoa. Annu Rev Microbiol 1993;47:821 – 53. [9] Salmon BL, Oksman A, Goldberg DE. Malaria parasite exit from the host erythrocyte: a two-step process requiring extraerythrocytic proteolysis. Proc Natl Acad Sci USA 2001;98:271 –6. [10] Bzik DJ, Li WB, Horii T, Inselburg J. Amino acid sequence of the serine repeat antigen (SERA) of Plasmodium falciparum determined from cloned cDNA. Mol Biochem Parasitol 1988;30:279 – 88. [11] Delplace P, Fortier B, Tronchin G, Dubremetz JF, Vernes A. Localization, biosynthesis, processing and isolation of a major 126 kDa antigen of the parasitophorous vacuole of Plasmodium falciparum. Mol Biochem Parasitol 1987;23:193 – 201. [12] Debrabant A, Maes P, Delplace P, Dubremetz JF, Tartar A, Camus D. Intramolecular mapping of Plasmodium falciparum P126 proteolytic fragments by N-terminal amino acid sequencing. Mol Biochem Parasitol 1992;53:89 – 96. [13] Higgins DG, McConnell DJ, Sharp PM. Malarial proteinase. Nature 1989;340:604. [14] Eakin AE, Higaki JN, McKerrow JH, Craik CS. Cysteine or serine proteinase. Nature 1989;342:132. [15] Sugiyama T, Suzue K, Okamoto M, Inselburg J, Tai K, Horii T. Production of recombinant SERA proteins of Plasmodium falciparum in Escherichia coli by using synthetic genes. Vaccine 1996;14:1069 – 76. [16] Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976;193:673 – 5.
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[17] Hanada K, Mitamura T, Fukasawa M, Magistrado PA, Horii T, Nishijima M. Neutral sphingomyelinase activity dependent on M2 + and anionic phospholipids in the intraerythrocytic malaria parasite Plasmodium falciparum. Biochem J 2000;346:671 – 7. [18] Lambros C, Vandenburg JP. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 1979;65:418 – 20. [19] Tosta CE, Sedegah M, Henderson DC, Wedderburn N. Plasmodium yoelii and Plasmodium berghei: isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation. Exp Parasitol 1980;50:7 –15. [20] Towbin H, Staehelin T, Gordon J. Electrophorectic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350 – 4.
[21] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680 – 5. [22] Sano GI, Morimatsu K, Horii T. Purification and characterization of dihydrofolate reductase of Plasmodium falciparum expressed by a synthetic gene in Escherichia coli. Mol Biochem Parasitol 1994;63:265 – 73. [23] Makler MT, Hinrichs DJ. Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia. Am J Trop Med Hyg 1993;48:205 – 10. [24] Lyon JA, Haynes JD. Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface when schizonts mature in the presence of protease inhibitors. J Immunol 1986;136:2245 – 51. [25] Debrabant A, Delplace P. Leupeptin alters the proteolytic processing of P126, the major parasitophorous vacuole antigen of Plasmodium falciparum. Mol Biochem Parasitol 1989;33:151 – 8.
Characterization of proteases involved in the processing of Plasmodium falciparum serine repeat antigen (SERA) Jie Li a, Hiroyuki Matsuoka b, Toshihide Mitamura a, Toshihiro Horii a,* a
b
Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka Uni6ersity, Suita, Osaka 565 -0871, Japan 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 have established an in vitro cell-free system using a baculovirus-expressed recombinant SERA (bvSERA) that mimics the SERA processing that occurs in parasitized erythrocytes. SERA processing was mediated by parasite-derived trans-acting proteases, but not 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 activity responsible 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, leupeptin and iodoacetoamide. Moreover, addition of DFP, E-64 or leupeptin to the cultures of schizont-stage parasites blocked schizont rupture and release of merozoites from PVM. These results indicate that SERA processing correlates to schizont rupture and the processing 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
1. Introduction The malaria parasite Plasmodium falciparum has an asexual life cycle in the mature human erythrocyte, in which it is surrounded by the parasitophorous vacuole membrane (PVM). In the erythrocyte, the parasite undergoes three distinct stages of development: the ring, the trophozoite and the schizont. During the schizogony, between 16 and 32 merozoites are formed within the parasitophorous vacuole. The merozoites release from the ruptured schizont and invade new red cells and continue the cycle. During the course of schizont rupture, merozoite release and re-invasion, a number of proteins associated with free merozoites, Abbre6iations: bvSERA, baculovirus-expressed SERA; DFP, diisopropyl fluorophosphate; E-64, trans-epoxysuccinyl-L-leucylamindo(4guanidino)butine; SERA, serine repeat antigen. * Corresponding author. Tel.: +81-6-6879-8280; fax: +81-6-68798281. E-mail address: [email protected] (T. Horii).
such as merozoite surface protein-1 (MSP-1) [1], MSP-3 [2], MSP-6 [3], apical membrane antigen-1 [4], and rhoptry associated protein-1 [5,6], are proteolytically processed. In the case of MSP-1, the final processing event (i.e. conversion of MSP-142 to MSP-119) has been shown to be necessary for merozoite invasion [7]. Furthermore, results from studies with protease inhibitors suggest that parasite-derived proteases are involved in the events of schizont rupture and meroziote re-invasion [8]. A recent report showed that a cysteine protease(s) could be involved in the release of merozoites from within the PVM [9]. Therefore, analysis of these processing events and characterization of the proteases involved would help to understand the molecular mechanisms underlying schizont rupture, merozoite-release, and/or merozoite-invasion. The serine repeat antigen (SERA) of P. falciparum [10] is synthesized at trophozoite- and schizont-stages during the intraerythrocytic cycle and secreted as a 120 kDa protein into the parasitophorous vacuole [11,12].
0166-6851/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S0166-6851(01)00452-2
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It has been demonstrated that the 120 kDa protein is proteolytically 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 type of SERA gene, further processed into two 25 kDa fragments (P25n and P25c) (Li and Horii, unpublished data), while P56 is converted into a 50 kDa fragment (P50) [11,12], which has a significant homology to papain-family proteases but has a predicted active serine instead of cysteine [13,14]. SERA processing is initiated most likely at late schizont stage and completed within the parasitized erythrocytes just prior to schizont rupture (Li and Horii, unpublished data). These observations suggest that the processing of SERA might represent the maturation of the putative protease and that the processed fragments of SERA as well as the proteases involved in the SERA processing might play important roles in schizont rupture, merozoite release, and/or merozoite invasion. In the present study, we have established an in vitro cell-free system that mimics the SERA processing that occurs within parasitized erythrocytes. With this system, we further characterized the proteases involved in SERA processing. 2. Materials and methods
2.1. Antibodies Recombinant proteins SE47’ and SE50A have been described [15]. A synthetic gene encoding amino acid residues 879–989 of P. falciparum Honduras-1 SERA was constructed by a similar method to that for the expression of SE47’ and SE50A [15] and used to express recombinant P18 (SE18) in Escherichia coli as a glutathione-S-transferase fusion protein. Polyclonal antibodies specific for P47, P50 and P18 (named aP47, aP50, and aP18, respectively. See Fig. 1) were raised in BALB/c mice (SLC, Japan) and total IgG was purified with a HiTrap Protein A column (Pharmacia).
Fig. 1. Scheme of the processing of P. falciparum Honduras-1 SERA. The positions of the three predicted active amino acid residues of the protease homologue are indicated by single letters [13,14] and the amino acid residue numbers of the N-terminal ends of the major processed fragments are shown in parenthesis [12]. Hatched box represents the region of serine repeat. The lines show the regions of SERA to which probe antibodies are directed.
2.2. Construction of plasmid pMbac-SERA encoding recombinant SERA (b6SERA) and expression of b6SERA in insect cells A synthetic gene encoding the almost full sequence of P. falciparum Honduras-1SERA (amino acid residues 23–989) was constructed with previously described DNA fragments [15] and the newly synthesized joining fragments according to the methods described [15]. The assembled DNA fragment of the gene was introduced into the baculovirus transfer vector pMbac (Stratagene, La Jolla, CA) by using Sma I and Bam HI sites. The resultant plasmid, pMbac-SERA, contains a gene that codes for the N-terminal 26 amino acids residues of melittin followed by residues 23–989 of SERA. For the generation of recombinant baculoviruses, Sf9 cells were co-transfected with 2.4 mg of pMbac-SERA and 0.12 mg of DNA of wild-type baculovirus (AcMNPV) according to the manufacturer’s protocol (PharMingen, San Diego, CA, USA). For the expression of bvSERA, High Five™ cells (Invitrogen, NV Leek, Netherlands) were seeded at 2× 106 cells per 75 cm2 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. The infected cells were incubated in the same medium at 27 °C for 2 to 3 days and the culture medium was collected by centrifugation at 600× g for 15 min and stored at − 80 °C until use.
2.3. Purification of b6SERA All the procedures described below were conducted at 4 °C except where indicated. Cleared culture supernatant of insect cells producing bvSERA was concentrated 20–25 fold with Cetriprep 30 (Amicon). The concentrate was subjected to gel filtration HPLC with a TSK-gel G4000SW column (Tosoh, Japan) followed by a DE 52 (Whatman) column. The appropriate fractions were pooled and dialyzed extensively against a phosphate-buffered saline (PBS). The dialyzed sample was concentrated with Centricon 30 (Amicon). A part of the purified bvSERA was extensively dialyzed against 0.5 M Tris –HCl, pH 7.5/8 M urea/1 mM EDTA, incubated with 50 mM DTT for 5 h at room temperature and then treated with 100 mM iodoacetamide at room temperature for 30 min in the dark. The resultant denatured and alkylated protein was dialyzed extensively against PBS and concentrated as above. The protein was stored at − 80 °C until use.
2.4. Parasite culture and preparation of parasite extracts Honduras-1 line of P. falciparum was routinely maintained as described [16,17]. Synchronization of parasite
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culture to a ring-stage was performed by 5% D-sorbitol treatment [18]. The synchronized parasites were further cultivated to trophozoite or schizont stages. The trophozoite- or schizont-infected erythrocytes were isolated from the parasite cultures by 63% (vol./vol.) Percoll (Amersham Pharmacia Biotech) density gradient centrifugation as described [19]. The isolated parasitized erythrocytes were washed twice with PBS and immediately used for further experiments or stored at −80 °C until use. Parasitized erythrocytes were thawed from − 80 °C, suspended in four cell-pellet volumes of PBS containing 0.25% Triton X-100 (v/v) (ICN Biochemicals Inc, OH, USA), and then incubated at 4 °C for 1 h. After centrifugation at 20000× g for 20 min, the supernatant (Triton extract) was collected. Alternatively, lateschizont-infected erythrocytes were suspended in four cell-pellet volumes of PBS containing 0.1% saponin (w/v) (Sigma) immediately after Percoll purification. The suspension was incubated at room temperature for 10 min and centrifuged at 2000× g for 5 min. The resultant supernatant (Saponin extract) was recovered. The precipitate was subjected to freezing and thawing once and extracted with Triton X-100 as described above for the Triton extract. The extract thus obtained was named Triton re-extract.
2.5. Western blot analysis Western blot analysis was basically performed as described by Towbin et al [20]. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on an appropriate gel [21] and electroblotted onto a PVDF membrane (Biorad). Primary antibodies were used at either 0.1 mg ml − 1 (for aP47) or 0.5 mg ml − 1 (for aP50 and aP18). Biotinylated horse IgG specific to mouse IgG (at 1:1000), avidinconjugated peroxidase (ABC kit) and diaminobenzidine tetrahydrochloride (Vector Laboratories) were used.
2.6. In 6itro reaction for SERA processing Triton extract of parasite was used except where indicated. A reaction mixture (total volume 10– 15 ml) was assembled that consisted of parasite cell extract (0.8 –1 ml), 0.3–0.5 mg of purified bvSERA and PBS. After incubation at 37 °C for the indicated times, the mixture was boiled for 3 min in the presence of reducing SDS sample and subjected to Western blot analysis. To examine effects of protease inhibitors on the processing of bvSERA, a parasite extract appropriately diluted in PBS was pre-incubated with a protease inhibitor (added from a 10× stock) at room temperature for 10 min prior to the addition of bvSERA.
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2.7. Analysis of glycosylation and assays for dihydrofolate reductase and lactate dehydrogenase Denatured and alkylated bvSERA (0.2 mg) was incubated with 1 unit of N-glycanase (Sigma) in 20 ml of 0.25 M Tris–HCl, pH 8.0 at 37 °C for 12 h, and then subjected to SDS-PAGE and Western blot analysis. Assay for dihydrofolate reductase activity was performed as previously described [22]. Plasmodial lactate dehydrogenase activity was measured basically as described [23] with some modifications. A reaction mixture contained 100 ml of the Mulstat™ reagent (Flow, Inc.), 20 ml of the mixture of nitroblue tetrazolium and phenazine ethosulfate (Sigma) and 20 ml of 100-fold diluted parasite extract or lysate of uninfected erythrocytes. The activity was expressed as absorbance measured at 620 nm (A620 nm).
2.8. Analysis of effects of protease inhibitors on SERA processing within parasitized erythrocytes and intraerythrocytic proliferation of P. falciparum Late schizonts were Percoll-purified, re-cultured for 4 h in complete medium with fresh RBC. The parasites were then treated with 5% D-sorbitol (0 h) and re-cultured to establish a 4 h early ring stage culture. After 37 h of incubation, new schizont-infected erythrocytes were Percoll-purified, re-suspended in complete medium and inoculated into wells on a 12-well plate (IWAKI, Japan) at 1.5×107 per 1 ml per well. About 10 ml of protease inhibitor or solvent was added to the cultures at 38 h. After further incubation at 37 °C for 12 h, the parasitized erythrocytes remaining in each culture were collected by centrifugation at 200× g for 3 min and lysed by boiling in 80 ml of SDS sample buffer. Then 20 ml of each sample was subjected to Western blot analysis. To examine the effects of protease inhibitors on the proliferation of the parasite, a culture that had been tightly synchronized as described above was diluted at 37 h (schizont stage) to a parasitemia of 0.86%. The culture was then dispensed into wells of a 24-well culture plate (IWAKI, Japan) at 0.5 ml per well and 3% hematocrit. At 38 h, 5 ml of protease inhibitor or solvent was added to each well. After incubation at 37 °C for 12 h, thin blood smears were made, Giemsastained, and inspected under a microscope.
3. Results
3.1. Expression and purification of recombinant SERA (b6SERA) The synthetic gene encoding whole SERA molecule was constructed to circumvent the difficulty in express-
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Fig. 2. Western blot analysis of recombinant baculovirus-expressed SERA (bvSERA). Cell lysate corresponding to 2 × 106 trophozoites and schizonts of P. falciparum Honduras-1 (lanes 1, 3, 5 and7) and 0.15 mg of purified bvSERA (lanes 2, 4, 6 and 8) were separated on 8% SDS-PAGE under reducing (lanes 1 –4) or non-reducing conditions (lanes 5 – 8) and probed with aP47 (lanes 1, 2, 5 and 6) or aP18 (lanes 3, 4, 7 and 8). M, molecular weight markers (Biorad).
ing the extremely A/T-biased native gene in a heterologous organism. To obtain correctly folded recombinant SERA, we expressed it as a secreted protein by using the baculovirus expression system and purified it from culture supernatant of infected insect cells. The identity of the recombinant protein (bvSERA) was confirmed through Western blot analysis by using mouse antibodies (aP47 and aP18) specific for the N- or C-terminal domains of SERA. Under both reducing and non-reducing conditions, bvSERA and the parasite-derived SERA co-migrated in SDS-PAGE (Fig. 2, lanes 1– 6). Both showed a slower mobility under non-reducing conditions (Fig. 2, lanes 5–8) than under reducing conditions (Fig. 2, lanes 1– 4). Moreover, aP18, which recognized the native SERA under reducing but not under non-reducing conditions (Fig. 2, lanes 3 and 7), also recognized bvSERA only under reducing conditions (Fig. 2, lanes 4 and 8). These observations suggest that the purified bvSERA retained a similar conformation to the native SERA. Treatment of denatured and alkylated bvSERA with an excess amount of N-glycanase did not cause any detectable mobility shift, suggesting that N-glycosylation of bvSERA is not significant (data not shown).
3.2. In 6itro processing of b6SERA When the purified bvSERA was incubated at 37 °C in PBS, RPMI1640 medium, acetate buffer (pH 5) or Tris– HCl buffer (pH 9), its molecular size did not
change (data not shown), suggesting that bvSERA was not autocatalytically processed. We then examined whether processing of bvSERA could be mediated by trans-acting protease activities derived from parasitized erythrocytes. When bvSERA was incubated at 37 °C with the Triton X-100-soluble fraction of a cell lysate (Triton extract) prepared from late schizont-infected erythrocytes, the 120 kDa bvSERA was converted into smaller fragments corresponding to those found in the cell lysate of segmented schizonts (Fig. 3). A major 48.5 kDa protein (P47bv) and a faint 47 kDa protein (P47en) were recognized by aP47 (Fig. 3, lane 4). The 47 kDa fragment was derived from the endogenous SERA because it was also found in the extract control and co-migrated with P47 in the lysate of late shizonts (Fig. 3, lane 2), while the 48.5 kDa fragment was the counterpart generated from bvSERA. The difference in molecular size between P47bv and P47en is most likely attributed to the five additional amino acids derived from the baculovirus transfer vector after the signal peptide of melittin has been removed. Antibody aP50 recognized two major bands (50 and 73 kDa) in the incubated mixture containing bvSERA (Fig. 3, lane 7). The intensity of this 50 kDa protein band was obviously much stronger than the 50 kDa band in the extract control (Fig. 3, lane 6 and 7), indicating that a fragment corresponding to P50 was generated from bvSERA. Likewise, a 73 kDa band and an 18 kDa band were detected by aP18 in the incubated mixture containing bvSERA (Fig. 3, lane 10). The 73 kDa band was reactive with both aP50 and aP18, suggesting that it is the intermediate product composed of P56 and P18. This is further supported by a time course analysis of bvSERA processing in vitro showing that bvSERA was successively converted to P73, P56 and P50 (Fig. 4). The in vitro results are consistent with the observations on SERA processing in the parasite culture (Fig. 3, lanes 2, 5 and 8). Products corresponding to P25n and P25c that appeared in the parasite cells, however, could not be observed in the cell-free system. Incubation of denatured and alkylated bvSERA with the extract resulted in mere random degradation (Fig. 5A, lanes 3 and 6), indicating that the processing was dependent on the tertiary structure of bvSERA. The cell extract prepared from late trophzoites only caused non-specific degradation of bvSERA (Fig. 5B, lane 2). In contrast, the cell extract prepared from schizonts showed evident processing activities (Fig. 5B, lane 4), and extract prepared from segmented schizonts had much stronger activities (Fig. 5B, lane 6). Thus the protease activities for the processing of bvSERA appears to accumulate or to be specifically activated at late schizont stage.
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Fig. 3. In vitro processing of bvSERA by parasite extracts. Purified bvSERA (0.3 mg) was incubated at 37 °C for 5 h without (lane 1) or with the 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 subjected to Western blot analysis with aP 47 (lanes 1 –4), aP50 (lanes 5-7) or aP18 (lanes 8 – 10). P47en, endogenous P47 from parasite extract; P47bv, P47 generated from bvSERA.
3.3. Localization of protease acti6ities responsible for the processing of b6SERA
3.4. Effects of protease inhibitors on the processing of b6SERA
To localize SERA processing activities, late schizontinfected erythrocytes were treated with 0.1% saponin as described in Section 2. The native SERA was almost exclusively recovered in the Saponin extract (Fig. 6, lane 3). No activity of dihydrofolate reductase, a soluble cytosolic enzyme, could be detected in the Saponin extract (B 0.005 U ml − 1), while a significant activity (0.57 U ml − 1) comparable to that found in the Triton extract was detected in Triton re-extract. Similarly, the activity of cytosolic plasmodial lactate dehydrogenase was not found in the Saponin extract: A620 nm =0.052 versus 0.053 for the lysate of uninfected erythrocytes, in contrast to 0.411 for the Triton extract. This indicates that the saponin treatment disrupted the PVM but caused little or no permealization of the parasite cell membrane. A substantial fraction of the processing activities was found in the Saponin extract (Fig. 6, lane 4), 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 responsible proteases are associated with membranes. This interpretation is supported by the observation that little of the processing activities were detected in the supernatant fraction from the extraction with PBS of late schizont cells disrupted by freezing and thawing (data not shown).
The establishment of the in vitro cell-free system for the SERA processing enabled us to analyze the proteases involved in SERA processing. Inhibitors known to block serine, cysteine, aspartic, or metallo
Fig. 4. Kinetic analysis of in vitro processing of bvSERA. A mixture (total 67.5 ml) consisting of 8.6 mg of bvSERA and 11 ml of parasite extract was incubated at 37 °C. Aliquots of 9 ml were removed at the indicated times, boiled in reducing SDS sample buffer, and subjected to Western blot analysis with aP50. The extract incubated alone for 5 h was loaded in lane Ex.
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Fig. 5. Specificity of the protease activities responsible for the processing of bvSERA in vitro. (A) Structure dependency. Intact bvSERA (lanes 2 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 also included (lanes 1 and 4). The incubated samples were subjected to Western blot analysis with aP47 (lanes 1 – 3) or aP50 (lanes 4 – 6). (B) Stage specificity of the processing activities. Parasite culture synchronized by 5% D-sorbitol treatment was further incubated for around 24, 30 or 34 h to 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 mg of bvSERA (lanes 2, 4, and 6). The samples were then subjected to Western blot analysis with aP47.
proteases were tested for their effects on the processing of bvSERA. Diisopropyl fluorophosphate (DFP), a serine protease inhibitor, significantly inhibited the conversion 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 without such an effect. These include Diethylenediaminetetraacetic acid (EDTA), aminophenylmethyl sulfonyl fluoride (APMSF), aprotinin, trans-epoxysuccinyl-Lleucylamindo (4-guanidino) butine (E-64), leupeptin, phenylmethylsulfonyl fluoride (PMSF), pepstatin A (Fig. 7, lanes 3–9), 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF; 1 mM), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK; 0.1 mM), N-a-p-tosyl-Llysine chloromethyl ketone (TLCK; 1 mM), dichloroisocoumarin (DCI; 1 mM), soybean trypsin inhibitor (0.1 mg ml − 1), iodoacetoamide (1 mM), ophenanthroline (1 mM) and bestatin (0.2 mM) (data not shown). Treatment with E-64 or leupeptin inhibited non-specific degradation of P47bv, causing denser P47bv band (Fig. 7, lanes 6 and 7). Thus, the protease responsible for the first cleavage event of SERA processing (conversion of SERA to P47+ P73) was sensitive to DFP but insensitive to the other serine protease inhibitors 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, leupeptin, and iodoacetamide each at 1 mM significantly inhibited the conversion of P73 to P56 as well as the
Fig. 6. Localisation of the processing activities. Triton extract (lanes 1 and 2), Saponin extract (lanes 3 and 4), or Triton re-extract (lanes 5 and 6) prepared as described in Section 2 was incubated for 5 h at 37 °C without (lanes 1, 3 and 5) or with 0.3 mg of bvSERA (lanes 2, 4 and 6) and then subjected to Western blot analysis with aP47.
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Fig. 7. Inhibition of processing of SERA into P47 and P73. Mixtures of parasite extract and bvSERA (0.3 mg) were incubated at 37 °C for 5 h in the presence of 1% ethanol (lane 2), 10 mM EDTA (lane 3), 1 mM APMSF (lane 4), 1.5 mM aprotinin (lane 5), 1 mM E-64 (lane 6), 1 mM leupeptin (lane 7), 1 mM PMSF (lane 8), 10 mM 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 aP47.
Fig. 8. Inhibition of processing of P73 into P56 and P50. Mixtures of parasite extract and bvSERA (0.3 mg) were incubated at 37 °C for 5 h in the presence of 1% ethanol (lane3), 10 mM EDTA (lane 4), 1 mM APMSF (lane5), 1.5 mM aprotinin (lane 6), 1 mM E-64 (lane7), 1 mM leupeptin (lane 8), 1 mM PMSF (lane 9) 10 mM 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 aP50.
conversion of P56 to P50 (Fig. 8, lanes 7, 8 and 11). With either E-64 or leupeptin, the inhibition could be achieved at as low as 0.1 mM and appeared to reach plateau at 1 mM. Complete inhibition of the conversion of P56 to P50 could be achieved at 10 mM, but only
partial inhibition of the conversion of P73 to P56 was observed at 0.1 mM –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 not shown) were without effect.
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3.5. Effects of protease inhibitors on SERA processing within the parasitized RBC and the parasite growth To see whether SERA processing in parasitized erythrocytes 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 12 h. The remaining schizont cells were recovered and subjected to Western blot analysis. As shown in Fig. 9A, treatment with 1 mM DFP significantly caused accumulation of the unprocessed SERA. By contrast, both leupeptin and E-64 at 100 mM markedly inhibited SERA processing into P50 and caused accumulation of P56 (Fig. 9B). However, inhibition of the conversion of P73 to P56, which was evident in the cell-free system, could not be observed. To examine the effects of DFP, leupeptin, and E-64 on the intraerythrocytic proliferation of the parasite, cultures of middle- stage schizonts (38– 42 h post invasion) were treated with individual inhibitors for 12 h. DFP (1 mM), leupeptin and E-64 (100 mM) individually blocked 35 –46% of the schizonts from rupturing. Noticeably, the majority of the remaining schizonts were morphologically similar to the structures reported by others [9,24].
4. Discussion We have expressed recombinant SERA as a secreted protein in the baculovirus expression system. By using the protein, we have established a cell-free system that reproduces SERA processing that occurs in the parasitized erythrocyte. In the system, fragments corresponding to P47, P73, P56, P50 and P18 were all generated, although some of the derived P18 molecules were trimmed further by non-specific protease activities. Products corresponding to P25n and P25c that appeared in the parasite cells, however, could not be observed in the cell-free system even after prolonged incubation. The responsible protease might be unstable in the extract. The in vitro system enabled us to characterize the proteases responsible for SERA processing. The processing activities are restricted to late schizont stage, which is consistent with the findings that SERA processing occurs immediately prior to schizont rupture (Li and Horii, unpublished data). The results of the fractionation study strongly suggest that the proteases responsible for SERA processing are located in the parasitophorous vacuole, which is consistent with that SERA 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 12 h, the remaining parasite cells were recovered from the cultures and subjected to Western blot analysis with aP47 (A) or aP50 (B). C, control (0.1% DMSO in A and no addition in B).
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it is noteworthy that the proteases are membrane-associated although its physiological significance is not known. At least three distinct proteases are involved in the processing of SERA. The primary step of SERA processing, conversion of SERA to P47 and P73, was specifically inhibited by DFP, a specific inhibitor of serine proteases. The responsible protease seems to hold a unique structure, because it is not sensitive to many other serine protease inhibitors. The proteolytic reaction is unlikely to be an autocatalytic process of SERA that contains structural similarity to papain family [13,14]. The conversion of P56 to P50, in contrast, is mediated by a cysteine protease but not a serine protease as has been suggested by Debrabant et al. [24]. As for the conversion of P73 to P56, although both E-64 and leupeptin partially inhibited this event in the in vitro system, they did not show any inhibitory effect in vivo. Although the reason for this inconsistency is unknown, the results may suggest a third protease acting on SERA to convert P73 to P56. The events of schizont rupture and merozoite release are poorly understood, yet evidence suggests that parasite-derived proteases are involved [8]. In a recent study by Salmon et al [9], when added to middle-stage schizonts, E-64 caused an accumulation of PVM-enclosed merozoite structures. A model for the process of rupture was subsequently proposed by those authors: merozoites enclosed within the PVM first exit from the host erythrocyte and then rapidly escape from within the PVM by a proteolysis-dependent mechanism. In the present study, we also observed that when added to cultures of middle-stage schizonts, leupeptin and E-64 each caused an accumulation of unruptured schizonts morphologically similar to the PVM-enclosed merozoite structures. Concomitantly, both inhibitors inhibited SERA processing in these schizonts. These data suggest that SERA processing preludes the second step of schizont rupture, the release of merozoites from the PVM. P50 of SERA, which has a structural similarity to cysteine protease, might function as a protease in the process of PVM rupture. Further study is required to test this hypothesis.
Acknowledgements We thank Dr R. Brobey for help in the assay of dihydrofolate reductase activity. We are grateful to Dr D. Bzik for critical reading of the manuscript. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas (A) (08281104) and Grant-in-Aid for Scientific Research (A) (13357002) from the Japanese Ministry of Education, Science,
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Sports, Culture and Technology. This study also received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), WHO, Geneva (Vaccine Discovery Research Grant 980278 to T. H.).
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