(Received for publication, January 21, 1997, and in revised form, March 18, 1997)
From the Howard Hughes Medical Institute, Departments of Molecular Microbiology and Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
During the intraerythrocytic stage of infection, the malaria parasite Plasmodium falciparum digests most of the host cell hemoglobin. Hemoglobin degradation occurs in the acidic digestive vacuole and is essential for the survival of the parasite. Two aspartic proteases, plasmepsins I and II, have been isolated from the vacuole and shown to make the initial cleavages in the hemoglobin molecule. We have studied the biosynthesis of these two enzymes. Plasmepsin I is synthesized and processed to the mature form soon after the parasite invades the red blood cell, while plasmepsin II synthesis is delayed until later in development. Otherwise, biosynthesis of the plasmepsins is identical. The proplasmepsins are type II integral membrane proteins that are transported through the secretory pathway before cleavage to the soluble form. They are not glycosylated in vivo, despite the presence of several potential glycosylation sites. Proplasmepsin maturation appears to require acidic conditions and is reversibly inhibited by the tripeptide aldehydes N-acetyl-L-leucyl-L-leucyl-norleucinal and N-acetyl-L-leucyl-L-leucyl-methional. These compounds are known to inhibit cysteine proteases and the chymotryptic activity of proteasomes but not aspartic proteases. However, proplasmepsin processing is not blocked by other cysteine protease inhibitors, nor by the proteasome inhibitor lactacystin. Processing is also not blocked by aspartic protease inhibitors. This inhibitor profile suggests that unlike most other aspartic proteases, proplasmepsin maturation may not be autocatalytic in vivo, but instead could require the action of an unusual processing enzyme. Compounds that block processing are expected to be potent antimalarials.
Nearly half the world's population lives in malaria endemic areas. Every year several hundred million people are afflicted and over two million people die from this disease, mainly children (1). Malaria is increasing as a global problem because parasites have developed resistance to drugs used for prophylaxis and treatment at an alarming rate. Thus, there is an urgent need to identify new drug targets (2). Plasmodium falciparum, the organism responsible for the most lethal form of malaria, is an obligate intracellular parasite that resides for most of the course of infection in the host erythrocyte. Hemoglobin degradation is essential for the survival of the parasite and agents that disrupt the hemoglobin catabolic pathway are considered good prospects for antimalarial chemotherapy (3).
During the organism's trophozoite stage most of the host cell hemoglobin is degraded (4-6), supplying the parasite with amino acids for protein synthesis and energy metabolism (7). Plasmodium ingests hemoglobin by means of the cytostome, an invagination that spans the parasite plasma membrane and the parasitophorous vacuolar membrane (a red cell-derived structure with which the parasite surrounds itself as it invades). The cytostome fills with host cell cytoplasm and pinches off to form transport vesicles. These double membrane-delimited vesicles loaded with hemoglobin fuse with the digestive vacuole and empty their contents. The digestive vacuole, an acidic proteolytic compartment with a pH between 5.0 and 5.4 (8, 9), is the site of hemoglobin degradation and heme detoxification (10). Two aspartic proteases, plasmepsins I and II and one cysteine protease, falcipain, have been purified from isolated digestive vacuoles and characterized (11, 12). A pepstatin inhibitable activity associated with vacuolar membranes has also been partially characterized and may result from a third aspartic protease (13). Studies with purified plasmepsins and falcipain suggest that hemoglobin degradation is an ordered process. Plasmepsins I and II initiate cleavage of the native hemoglobin tetramer, while falcipain digests denatured substrate (12, 14). A peptidomimetic inhibitor of plasmepsin I blocks hemoglobin degradation in vitro and kills parasites in culture (15). Also, cysteine protease inhibitors block hemoglobin degradation, kill cultured parasites, and eradicate parasitemia in a murine malaria model (16, 17). These data suggest that targeting hemoglobin digestion could be an effective anti-malarial strategy.
The genes for plasmepsins I and II were cloned (18, 15) and predicted
to encode 51-kDa proteins that are 73% identical and are about 35%
homologous to mammalian renin and cathepsin D (19). Typically aspartic
proteases are zymogens that are synthesized as pre-proproteins. The
pro-piece, characteristically less than 50 amino acids, is removed
during protein maturation (20). In contrast, plasmepsin I and II
pro-pieces are predicted to be 123 and 124 amino acids, respectively
(Fig. 1). Cleavage of the putative pro-piece would yield
mature proteins of 37 kDa, in agreement with the size of the isolated
proteins. The long pro-pieces of the plasmepsins are distinguished by
the absence of a signal sequence. Instead, 65 amino acids upstream of
the cleavage site of both plasmepsins is a hydrophobic stretch of 21 amino acids which was proposed to serve as a signal anchor sequence
(15). The hydropathy plots of the proplasmepsins are characteristic of
membrane proteins (15, 18).
We report the results of biosynthesis studies which indicate that proplasmepsins are integral membrane proteins in the secretory pathway. Instead of the typical aspartic protease autoactivation, an acid pH-dependent ALLN/ALLM inhibitable activity appears to be responsible for cleavage of both proplasmepsins. Inhibition of proplasmepsin maturation is a new target in the hemoglobin proteolysis pathway that may provide a selective and efficient means to starve the malaria parasite.
RPMI 1640 used for culturing parasites was from
Life Technologies, Inc. Enzymes for DNA modification, proteinase K, and
phenylmethanesulfonyl fluoride (PMSF),1
were from Boehringer Mannheim.
N-Acetyl-L-leucyl-L-leucyl-norleucinal (ALLN) and
N-acetyl-L-leucyl-L-leucyl-methional
(ALLM) were obtained from both Boehringer Mannheim and Sigma.
Leupeptin, trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E-64),
pepstatin, bafilomycin A1, protein A-Sepharose, apyrase, and ATPS were from Sigma. Benzoxycarbonyl
(Z)-Phe-Ala-CHN2 was from Enzyme Systems Products (Dublin,
CA). Reticulocyte lysate and canine pancreas microsomes were from
Promega. Endoglycosidase H and N-gycosidase F were from New
England Biolabs. [35S]Methionine/cysteine (Protein
Express, 1175 Ci/mmol) was from DuPont NEN.
The P. falciparum clone HB3 (a gift of Dr. W. Trager, Rockefeller University) was grown by the method of Trager and Jensen (21) using human plasma (22). The parasites were grown in A+ erythrocytes at 2-5% hematocrit and 5-15% parasitemia. Synchrony was maintained by sorbitol treatment (23).
Recombinant PlasmidsRecombinant plasmids were made using
polymerase chain reaction performed in a TC480 thermal cycler with
Ampli-Taq (Perkin-Elmer). All reactions were preceded by a
single 5-min 94 °C denaturation step and followed by a 5-min
72 °C final extension step. Proplasmepsin I was amplified by
polymerase chain reaction using plasmid (4.4) containing the
full-length plasmepsin I cDNA (15) as template and oligonucleotide
primers (sense) 5-TCGAGGATCCATAAAGATGGCTTTATCAATTAAAGAAGATTT-3
with
(antisense) 5
-TCGAGGATCCTTACAATTTTTTTTTGGCAAGGGC-3
for 30 cycles
(94 °C, 1 min, 50 °C, 1 min, and extension 72 °C, 1 min). The
product was ligated into a TA-Cloning vector following the
manufacturer's instructions (Invitrogen). Proplasmepsin II was
amplified using 12 ng of P. falciparum genomic DNA as
template and oligonucleotides designed using published plasmepsin II
sequence (18). Polymerase chain reaction of (sense)
5
-GCATAAGCTTACAAAATGGATATTACAGTAAGAGAACATGAT-3
and antisense
5
-TCGAGAATTCTCTTATATAATTATGTTTTTCCTTTG-3
generated a product that was
digested with EcoRI and HindIII and ligated into
pBSSKII+ (Stratagene). Plasmepsins I and II used in competition experiments to determine antibody specificity were purified from Escherichia coli transformed with the PET vector expression
plasmids described previously (24).
Capped PM I and PM II RNA were prepared using linearized plasmid DNA and T7 polymerase Message Machine kit (Ambion) following the manufacturer's instructions. Transcript was added to nuclease-free rabbit reticulocyte lysate (Promega) in the presence of [35S]methionine/cysteine and 10 µg of brewer's yeast tRNA per 25 µl of translation reaction. Canine pancreas microsomes (1.8 µl) were added to certain in vitro translation reactions as specified in the text.
Antibody Production and ImmunoprecipitationAntibody to plasmepsin I (Ab 574) was generated as described previously (15). Rabbit antiserum against plasmepsin II (Ab 737) was made using a multiple antigen peptide, HSHSSNDNIELNDFQNIMFYGDAEV, derived from the mature NH2 terminus of native plasmepsin II (12) prepared by Research Genetics.
Labeled trophozoite stage parasites (routinely 5 × 108) were immunoprecipitated using denaturing conditions. Trophozoites were lysed by the addition of 1% SDS (100 µl) at 100 °C for 10 min. The solution was adjusted to 150 mM NaCl, 40 mM Tris (pH 7.5), 6 mM EDTA, 0.5% deoxycholate, 1% Triton X-100, and 0.1% SDS; insoluble material was removed by centrifugation. Ab 574 or Ab 737 was added at a 1:250 dilution (determined to be saturating) along with 70 µl of protein A-Sepharose (1:1 bead slurry to immunoprecipitation buffer). Samples were mixed by nutation at 4 °C for 1-2 h, immunocomplexes collected, added to nonreducing sample buffer, boiled, and analyzed by 12% SDS-PAGE with fluorography.
Pulse-Chase ExperimentsPulse labeling experiments were
performed with synchronized cultures of trophozoite stage parasites
(between 24 and 36 h after invasion). The cultures were washed one
time in RPMI prepared without methionine and cysteine
(MetCys
), resuspended in the same medium at
a density of 2.0 × 108/ml with
[35S]methionine/cysteine (350 µCi/ml) and incubated at
37 °C for 10 min (3% O2, 3% CO2). The
cultures were chased following the addition of complete RPMI and 100 µg/ml cycloheximide, by incubation for different times depending on
the experiment but typically between 0 and 3 h. Inhibitor
experiments were performed in the same manner except that cultures were
preincubated with inhibitors for 45-60 min before metabolic labeling
commenced (except for ALLN and ALLM which were first added during
labeling). The inhibitor being tested was added to both labeling and
chase media. Free parasites were harvested by saponin lysis (11) and
stored at
70° until analyzed.
ATP requirements for processing in vivo were determined using a variation on these culture conditions. Trophozoites were incubated in glucose-free RPMI medium for 2 h, washed once in RPMI minus glucose, methionine and cysteine, collected by centrifugation, and labeled in the same medium which included 350 µCi/ml [35S]methionine/cysteine for 10 min at 37 °C. Cycloheximide (100 µg/ml) was added to stop translation and the medium was adjusted to 20 mM deoxyglucose and 0.5 mM 2,4-dinitrophenol. Cells were incubated for 2 h, harvested, and stored frozen.
Glycosidase TreatmentProplasmepsins I and II were prepared
by in vitro translation in the presence of microsomes
described above or by pulse labeling P. falciparum
trophozoites for 10 min in RPMI (MetCys
)
with [35S]methionine/cysteine (350 µCi/ml).
Immunocomplexes were collected as described above and resuspended in
denaturing buffer (0.5% SDS, 1%
-mercaptoethanol), then heated for
10 min at 100 °C. The samples were split into four equal portions.
One portion was digested with 500 units of endoglycosidase H (Endo H)
in 50 mM sodium citrate (pH 5.5) for 3 h at 37 °C.
A second portion was treated identically except that Endo H was not
added (mock). The third portion was digested with 500 units of
N-glycosidase F (PNGase F) in 50 mM sodium
phosphate (pH 7.5), 0.1% Nonidet P-40. The final portion was
untreated. Proplasmepsins I and II were also translated without
microsomes to generate unglycosylated products which were used both for
size comparison and as controls for glycosidase digestions. Endo H and
PNGase F have no activity on unglycosylated substrates (not shown). The
products were analyzed by 12% SDS-PAGE with fluorography.
Trophozoites (5 × 108) were labeled with 150 µCi/ml [35S]methionine/cysteine for 3 h at 37 °C, BFA (5 µg/ml) was added and labeling continued for 2 h. This method generated roughly equal amounts of labeled proplasmepsins and mature plasmepsins. Free parasites were harvested as above, lysed in 10 mM phosphate buffer (pH 7.1), sonicated, and split into three parts. One part served as a control for the total amount of processed and unprocessed plasmepsins I and II in the sample, the other two parts were centrifuged for 214,000 × g-h, 4 °C. The resulting membrane fractions were extracted with either phosphate-buffered saline or sequentially with 0.5 M KCl and 100 mM Na2CO3 (pH 11.0). All extractions were for 30 min on ice followed by centrifugation for 214,000 × g-h. Membrane fractions were solubilized with 1% SDS and immunoprecipitated along with supernatants and the unfractionated control. Immunoprecipitated complexes were analyzed by SDS-PAGE with fluorography.
Membrane orientation of proplasmepsins I and II was determined by
labeling 5 × 108 trophozoites in 150 µCi/ml RPMI
(MetCys
) in the presence of BFA (5 µg/ml). Cells were placed on ice for 15 min, collected by
centrifugation at 2,000 × g for 15 min, washed twice
in cold phosphate-buffered saline, and stored at
70 °C. The pellet
was resuspended gently in cold phosphate-buffered saline in the
presence of protease inhibitors (10 µM pepstatin, 10 µM E-64, 1.0 mM 1,10-phenanthroline, 100 µM ALLN, 1 mg/ml aprotinin) on ice (25). The lysate that
resulted from this treatment was adjusted to 5 mM
CaCl2 and divided into three parts: 1) untreated; 2) 1 or 2 mg/ml proteinase K; 3) 1 or 2 mg/ml proteinase K plus 0.3% Triton
X-100, and the lysates were incubated on ice for 60 min. Proteinase K
activity was inhibited by the addition of 10 mg/ml PMSF. The samples
were immunoprecipitated as above after two preclearings with protein
A-Sepharose. Analysis of immunocomplexes was by 12% SDS-PAGE with
fluorography.
Parasites were carefully synchronized by sorbitol treatment (23) and cultured at 2% hematocrit to 3-5% parasitemia. The resulting cultures (approximately 108 parasites) were labeled at specified times after invasion with 50 µCi/ml [35S]methionine/cysteine for 90 min at 37 °C. Ring-stage cultures (5 h after invasion) were labeled in the same manner in the presence of ALLN (50 µM) or bafilomycin A1 (1.3 µM). Labeled parasites were harvested by saponin lysis and immunoprecipitated in series with Ab 737 and Ab 574 using the conditions described above.
Northern Blot AnalysisSynchronized HB3 cultures were
harvested at the ring stage (10 h after invasion), trophozoite stage
(30 h after invasion), and schizont stage (40 h after invasion) by
saponin lysis (11). Total RNA was prepared by guanidinium thiocyanate
extraction and CsCl centrifugation. RNA (10 µg/lane) transferred to
Magna NT (MSI) was UV cross-linked at 1.2 × 105
µJ/cm2. Radiolabeled antisense proplasmepsin probes were
prepared by in vitro transcription in reactions containing
[-32P]UTP and T3 polymerase for plasmepsin I or SP6
polymerase for plasmepsin II. Filters were hybridized in 5 × SSPE
(0.75 M NaCl, 0.75 M sodium citrate, 50 mM sodium pyrophosphate, 5 mM EDTA), 1 × Denhardt's, 50% formamide, 0.1% sodium pyrophosphate, 1% SDS, and
100 µg/ml sheared salmon sperm DNA for 12 h at 60 °C. The final wash solution was 0.1 × SSC and 0.1% SDS at 65 °C.
To address whether the proplasmepsins are made
as membrane proteins and to examine how the proteins are processed
in vivo, biosynthesis of both proteins was followed using
specific antibodies. A plasmepsin I-specific antibody (Ab 574) was
prepared against the first 224 NH2-terminal amino acids of
the mature enzyme. The PM II antibody (Ab 737) was prepared against a
22-amino acid peptide corresponding to the mature NH2
terminus of plasmepsin II. To assess the specificity of Ab 574 (PM I)
and Ab 737 (PM II), proplasmepsins I and II radiolabeled during
in vitro translation were immunoprecipitated with both
antibodies. No cross-reactivity was observed (Fig. 2). Immunoprecipitations performed in the presence of competing unlabeled plasmepsins I or II also indicate that the antibodies are specific for
their respective proteins.
To examine the maturation of the plasmepsins, intraerythrocytic
P. falciparum trophozoites were pulse-labeled with
[35S]methionine/cysteine for 10 min, followed by a chase
in unlabeled medium with cycloheximide to prevent new protein
synthesis. Processing was monitored at specific time intervals. As
predicted, proplasmepsins I and II migrate at about 51 kDa (Fig.
3). The time course of processing for both proteins is
virtually identical. A single cleavage occurs generating mature
proteins of 37 kDa with a t1/2 of about 20 min.
Processing is complete by 60 min and the proteins are stable for at
least 3 h after synthesis.
The mechanism of protein secretion by Plasmodium is not well
characterized (26-28). As is seen for other eukaryotic cells (29), the
addition of brefeldin A (BFA) to cultures impairs protein secretion by
Plasmodium, resulting in a significant reduction in protein
export (30). However, some proteins are secreted even in the presence
of BFA. When BFA (5 µg/ml) was added to trophozoite cultures during a
3-h metabolic labeling, maturation of proplasmepsins I and II was
completely blocked. In the absence of BFA, processing proceeded
normally and mature proteins accumulated (Fig.
4A). Inhibition with BFA suggests that the
plasmepsins are secretory proteins that are transported through the
Golgi apparatus. Proplasmepsin processing is likely to occur in a
downstream compartment.
Plasmodium lacks a morphologically well defined Golgi apparatus. Earlier reports indicated that in contrast to all other eukaryotes, N-glycosylation of proteins does not occur (31). Recently N-glycoproteins were found in intraerythrocytic parasites (32), so it was of interest to determine whether the plasmepsins are N-glycosylated and if this modification plays any role in delivery of proteins to the digestive vacuole. Plasmepsin I has 7 consensus N-glycosylation sites, 5 on the putative intralumenal part of the protein and plasmepsin II has 5 consensus sites, 3 on the predicted lumenal side of the protein. When proplasmepsins I and II, translated in vitro in the presence of canine pancreas microsomes, are examined by SDS-PAGE, higher molecular weight forms (3 for plasmepsin I and 2 for plasmepsin II) are seen for both proteins (Fig. 4B). Treatment of these products with Endo H or PNGase F (not shown) results in trimmed products that comigrate with proteins synthesized without microsomes. To examine native proplasmepsins, trophozoites were metabolically labeled for 10 min and harvested. Immunocomplexes containing plasmepsins I and II were isolated, denatured, and treated with either Endo H or PNGase F. Radiolabeled proplasmepsins translated in vitro without microsomes were prepared for size comparison. The native proplasmepsins comigrate with the in vitro translated controls, with or without glycosidase treatment, as is expected if they are not N-glycosylated (Fig. 4C).
Proplasmepsins Are Type II Integral Membrane ProteinsPrevious immunoelectron microscopy studies using a
plasmepsin I-specific antibody (Ab 574) suggested that the enzyme is
membrane-associated in delivery vesicles, at the parasite cell surface
and in the cytostome (15). Trophozoites were metabolically labeled for 3 h to accumulate mature plasmepsins, then BFA was added and
labeling continued for 2 h to accumulate proplasmepsins. This
treatment produced approximately equal amounts of pro and mature
plasmepsins (Fig. 5). High speed centrifugation of
trophozoite lysate produced a pellet containing trophozoite membranes
from which the proforms of plasmepsins I and II were
immunoprecipitated. An additional membrane pellet prepared in the same
manner was extracted successively with high salt and high pH solutions.
These treatments did not dissociate proplasmepsins from the membrane
fraction, suggesting that the 21-amino acid hydrophobic stretch in the
proplasmepsins serves as a signal anchor sequence. Immunoprecipitation
of the supernatant fraction indicates that mature plasmepsins are
soluble proteins produced by cleavage of the membrane-bound proforms
(Fig. 5).
To examine the membrane topology of the proplasmepsins, trophozoites
were metabolically labeled in the presence of BFA for 3 h and the
parasitized erythrocytes were collected. The cells were frozen and
thawed in cold. This treatment lyses cells and permits microsomes to
reseal with proteins in their original orientation (25). Proteinase K
was added with or without Triton X-100 and the digested products were
immunoprecipitated and examined by SDS-PAGE (Fig. 6). A
clear difference in the size of proplasmepsin I is observed after
protease digestion of intact microsomes. This 4-kDa difference is
consistent with the predicted cleavage of 35 amino acids
NH2-terminal to the signal anchor. Disruption of microsome
integrity by Triton X-100 permitted complete plasmepsin digestion by
proteinase K, indicating that 47 kDa of plasmepsin I is contained
within the microsome. Similar results were obtained for plasmepsin II
(not shown).
Proplasmepsin Processing Is Inhibited by ALLN and ALLM in Vivo
Comparison of the putative cleavage sites deduced from the
NH2 termini of isolated mature enzymes (12), suggests that
there is conservation at the 2,
1, and +4 positions (Fig. 1).
However, in vitro recombinant proplasmepsin II activates
autocatalytically by cleavage at another site, 12 amino acids upstream
from the end identified for the native enzyme (33). This difference
might be explained if autocatalytic cleavage occurs in vivo,
and is followed by an additional processing event or by
exopeptidase-mediated trimming during protein isolation (33).
Alternatively, autocatalytic processing of proplasmepsin II might be an
in vitro artifact. Cleavage of proplasmepsin I to mature
protein has not been demonstrated in vitro (24), suggesting
either that the plasmepsins are processed differently in
vivo or that expressed proplasmepsin I resists activation because
of improper folding. Interestingly, the site of proplasmepsin II
autocatalytic cleavage is Phe-Leu; cleavage of hemoglobin at
33/34
Phe-Leu by both plasmepsins initiates its breakdown (11, 12).
Proplasmepsin I has Phe-Phe at the corresponding position in the
pro-piece.
Inhibitors were added to trophozoites during metabolic labeling to
identify the class of enzyme responsible for plasmepsin processing.
Proplasmepsins I and II were affected identically by all reagents and
conditions tested. The results shown are for plasmepsin I. Trophozoite
cultures were preincubated with inhibitor for 45 min and then cells
were pulse-labeled with 35S-labeled amino acids for 10 min,
followed by a 1-2 h chase in complete medium containing inhibitor and
cycloheximide. Since autocatalytic cleavage of the pro-piece seemed
likely, cultures were tested with the aspartic protease inhibitor
pepstatin, as well as the plasmepsin I-specific inhibitor SC-50083
(15). No inhibition of processing was observed even at concentrations
greater than the IC50 for parasite growth for these
compounds (12) (Fig. 7A). A variety of other
standard protease inhibitors were tested including the cysteine
protease inhibitors leupeptin and E-64 (also
Z-Phe-Ala-CHN2, not shown) and serine protease inhibitor PMSF. No effect on plasmepsin processing was observed. The
metalloprotease inhibitor 1,10-phenanthroline (1 mM) killed
cells rapidly, so the effect on processing could not be determined (not
shown). Additional inhibitor screening revealed that the tripeptide
aldehyde inhibitors ALLN and ALLM completely block proenzyme cleavage
in a reversible manner (Fig. 7, A and B). These
inhibitors target cysteine proteases (including calpains) and the
chymotryptic activity of proteasomes (ALLNALLM) (34-36).
Titration of ALLN and ALLM indicated that both compounds inhibit
processing equally well with an IC50 of 5 µM
for each (not shown). To further define the processing activity, the
proteasome inhibitor lactacystin (37) was tested and found to be
ineffective. Most proteins degraded by proteasomes require
ATP-dependent ubiquitination (38). No evidence for higher
molecular weight, ubiquitinated forms of proplasmepsin are observed
following ALLN treatment even when cells are labeled for several hours
(not shown) in contrast to what is seen when proteasome hydrolysis of
ubiquitinated proteins is blocked (39). Incubation of cells with
deoxyglucose and dinitrophenol (DEOX, Fig. 7A) uncouple
oxidative phosphorylation and deplete cellular ATP. These agents had no
effect on processing, suggesting that ATP is not required.
To test the possibility that the tripeptide aldehyde blockade could be
inhibition of autocatalytic plasmepsin processing, recombinant
proplasmepsin II was incubated at pH 5 and analyzed by SDS-PAGE (Fig.
8). Pepstatin blocked autocleavage well, while ALLN was
ineffective. Up to 100 µM ALLN failed to block
autoprocessing. Also, in enzyme assays using globin as substrate,
mature plasmepsin II was not inhibited by ALLN, while picomolar
concentrations of pepstatin blocked catalysis (data not shown). These
results suggest that intracellularly, ALLN is not blocking an
autocatalytic event but may be inhibiting an unusual processing
enzyme.
Proplasmepsin Processing Requires Acid pH in Vivo
Lysomotropic agents were added to trophozoite cultures
during metabolic labeling to determine whether acid conditions are required for proplasmepsin processing. Maturation was inhibited at
micromolar concentrations of chloroquine known to raise the digestive
vacuole pH (40, 41) (Fig. 9). As an alternative method of increasing
digestive vacuole pH, the fungal metabolite bafilomycin A1
was used to specifically inhibit the proton pump (42, 43). When
proplasmepsin processing was assayed in the presence of bafilomycin
A1, it was completely inhibited (Fig. 9).
These results suggest that proplasmepsin processing is a
pH-dependent event, although the effect could also be
explained by a blockade of proplasmepsin transport to the compartment
where processing occurs.
Proplasmepsin I Is Synthesized and Processed Very Early in the Parasite Life Cycle
Northern blot analysis of total RNA shows
that the plasmepsin I message is most abundant at the early (ring)
stage, while plasmepsin II message peaks at the later (trophozoite)
stage (Fig. 10A). When different stage
parasites were 35S-pulse-labeled, plasmepsin I synthesis
and processing could be detected as early as 3 h after erythrocyte
invasion (Fig. 10B). Plasmepsin II was first detected in
late rings (12 h). Synthesis of both proteins peaked in the trophozoite
stage (24-32 h). By the schizont stage (36-42 h), synthesis dropped
off markedly (not shown). Early stage parasites were fully capable of
processing proplasmepsin I and the processing was inhibited by BFA as
well as ALLN (Fig. 10C), similar to the results observed
when both plasmepsins I and II are synthesized and processed later in
development (Figs. 4 and 7).
Like other aspartic proteases, the plasmepsins are secretory proteins. Remarkably little is known about the transport of secretory proteins in Plasmodium (26, 28). When brefeldin A, a fungal metabolite known to block secretion in eukaryotes, is added to Plasmodium cultures, processing of proplasmepsins to mature forms is inhibited, presumably because delivery to the processing compartment is impaired. Whether this results because BFA disrupts and redistributes the Golgi or because it blocks secretion from the endoplasmic reticulum is unclear. Both models have been proposed to explain the effect of BFA on Plasmodium (26, 28). In fact, it has been suggested that early stage parasites may lack a Golgi altogether (28) as is the case for certain stages of the protozoan parasite Giardia lamblia (44). Stacked Golgi cisternae have not been seen in Plasmodium although some Golgi markers have been identified (26, 45). Proplasmepsins have N-glycosylation sites, some of which were recognized during our in vitro translation with canine pancreas microsomes. However, despite reports of N-glycosylation of some malarial proteins (32), native proplasmepsins were not found to be N-glycosylated, so whether they transit the Golgi could not be ascertained. How the plasmepsins escape this post-translational modification is not clear.
Typically proforms of aspartic proteases are soluble, although peripheral membrane association is sometimes observed (46-49). In contrast, proplasmepsins I and II are integral membrane proteins that are processed to form soluble mature enzymes. Cleavage of a membrane-spanning precursor to generate soluble enzyme also occurs for mammalian lysosomal acid phosphatase (50). Proenzyme processing is different from the proplasmepsins in this case, since acid phosphatase is transported to the lysosome several hours after synthesis and is released from the membrane by sequential proteolytic steps (51).
Proplasmepsins have type II membrane topology. This is consistent with the positive inside rule (52). Positively charged NH2-terminal residues have been shown to predict type II membrane topology (53, 54). Proplasmepsin I has 3 lysines and 1 arginine within 8 amino acids NH2-terminal to the signal anchor (15) and proplasmepsin II has 4 lysines (18). Plasmepsin I was previously hypothesized to traverse the parasite plasma membrane to the parasitophorous vacuolar membrane and intersect with the hemoglobin ingestion pathway (15). This delivery route places plasmepsin I near the parasitophorous vacuole membrane with the carboxyl terminus of the protein (from which the active enzyme is derived) in the erythrocyte cytoplasm. The orientation is reversed from that predicted for type II proteins that traffic through the secretory pathway by successive membrane fusion and vesiculation events (55). A simplified secretory model has been proposed for Plasmodium in which local parasite plasma membrane and parasitophorous vacuole membrane fusion occurs forming joint domains with a single membrane bilayer (26, 56). Contact sites between parasite plasma membrane and parasitophorous vacuole membrane have been observed microscopically (55, 57). Fusion of delivery vesicles containing proplasmepsins with these joint domains could yield the orientation suggested by immunoelectron microscopy for plasmepsin I. However, additional targeting studies will be required to delineate the route taken by plasmepsins to the digestive vacuole.
Proplasmepsin processing appears to require acidic conditions. Two different types of lysosomotropic agents, a proton pump inhibitor and a weak base alkalinization compound were able to block proplasmepsin maturation. It is possible that this is an indirect effect, whereby these agents prevent the proenzymes from getting to their site of processing. We have recently observed proplasmepsin processing in a cell-free system and find that cleavage also requires acid conditions in vitro.2 Digestive vacuole proteins have similar pH requirements (11, 12). These data are consistent with a model for activation in which the membrane-bound proplasmepsins are trafficked to the acidic digestive vacuole where they are cleaved by a processing enzyme. This model predicts that transport through the secretory pathway would have a t1/2 for delivery less than or equal to the t1/2 for processing (20 min). Extracellular secretion of newly synthesized proteins by Plasmodium has been observed within 5 min, suggesting that transit through the secretory apparatus is fast (58). In yeast, transport of soluble proteins proteinase A (59) and carboxypeptidase Y (60), as well as the membrane protein alkaline phosphatase (61), to the acidic food vacuole, has been observed with a t1/2 of 6 min. Thus, rapid delivery of the plasmepsins to the digestive vacuole seems entirely reasonable, but this model will require further testing.
Proplasmepsin Processing Has an Unusual Inhibitor Profile and May Be Mediated by an Unusual EnzymeThe aspartic protease inhibitors pepstatin and SC-50083 did not block proplasmepsin processing. This could result from poor penetration of the inhibitors. However, SC-50083 appears to be readily taken up by cells (15) and while pepstatin membrane permeability is poor, concentrations well above the IC50 were found to be ineffective at blocking processing. These data, combined with the effectiveness of the tripeptide aldehydes ALLN and ALLM (which do not block autoprocessing of proplasmepsin II in vitro) suggest that the activating cleavage of the proplasmepsins is not autocatalytic. This is atypical for acidic aspartic proteases (20). Instead, cleavage of both proenzymes appears to be mediated by a tripeptide aldehyde-inhibitable activity. The tripeptide aldehydes ALLN and ALLM inhibit cysteine proteases including calcium-activated cysteine proteases (calpains) (34). Such enzymes may be envisioned to encounter unprocessed plasmepsins in the parasite. Falcipain has been isolated from the digestive vacuole and was shown to have substrate specificity that matches the proplasmepsins cleavage site (12). Also, calpains I and II have been purified from erythrocyte cytoplasm (62, 63) and might interact with plasmepsins in the hemoglobin ingestion pathway. However, the processing enzyme does not appear to be any known cysteine protease since the inhibitors E-64, leupeptin, and Z-Phe-Ala-CHN2 were ineffective at inhibiting processing at high concentrations.
ALLN inhibits the chymotryptic activity and to a lesser extent the peptidylglutamic peptide hydrolase and tryptic activities of proteasomes (35, 64). Most of our experimental data suggest that proteasome involvement in proplasmepsin cleavage is unlikely. We find that ALLN and ALLM inhibit processing equally well. This is not the case for the chymotryptic activity of proteasomes against which ALLN is 5-42 times more potent than ALLM (35, 65). In addition, proteasomes generally hydrolyze proteins into peptides. Although proteasomes have infrequently been implicated in protein processing (66, 67) this appears to result when the precursor is ubiquitinated in an ATP-dependent manner and then partially degraded (66). We find no evidence of proplasmepsin ubiquitination and ATP is not required for proplasmepsin cleavage. Additionally, proteasomes work optimally at neutral pH, while processing appears to involve an acid hydrolase. Finally, the Streptomyces antibiotic lactacystin inhibits the chymotryptic activity of proteasomes (37) but does not inhibit proplasmepsin processing.
Proplasmepsin I Synthesis and Processing Commences Early in DevelopmentAt the ring stage of parasite development, plasmepsin I mRNA is abundant and protein is synthesized. At the trophozoite stage even more plasmepsin I protein is made, although the quantity of message has declined substantially. This suggests that there is better translation efficiency later in development, but what accounts for this is not clear. Also unexpected is the observation that plasmepsin I is processed in very early parasites and appears to require acidic pH for this to occur. In early ring stage parasites there is no identifiable digestive vacuole (3) and little hemoglobin degradation is thought to occur. It seems possible that plasmepsin I has some function in addition to hemoglobin metabolism. Alternatively, a low level of hemoglobin digestion may be necessary for parasite survival even at the earliest stages of development. At this time, plasmepsin I alone may suffice. Digestion of a greater mass of hemoglobin required for parasite maturation could be augmented by plasmepsin II later in development.
Proplasmepsin Activation as a Potential Target for Antimalarial ChemotherapyHemoglobin breakdown by the malaria parasite
P. falciparum is a massive catabolic process that
appears to be essential for the survival of the parasite. Plasmepsins I
and II have been shown by in vitro assay to comprise 80% of
the total globin degrading activity in the digestive vacuole (12). They
have different substrate specificities except for cleavage at
Phe33-Leu34 which is thought to initiate
hemoglobin proteolysis (12). Previously, we have shown that malaria
parasites in culture are killed by a peptidomimetic inhibitor of
plasmepsin I (SC-50083) and proposed that it could serve as a good lead
compound for developing more potent anti-plasmepsin agents (15). This
goal is complicated by the degree of difficulty involved in synthesis
of SC-50083 and its derivatives. In this study, we have determined that
processing of both plasmepsins I and II is inhibited by ALLN and ALLM.
These inhibitors are nonspecific and obviously not suitable for use as
antimalarials, however, the notion that both plasmepsins could be
completely inactivated by a single compound is appealing. ALLN and ALLM
are permeable tripeptide aldehydes that are readily taken up by cells.
They are relatively easy to synthesize which should facilitate
identification of related compounds specifically tailored to inhibit
processing. The cleavage sites of proplasmepsins I and II have been
determined and shown to have conserved sequence (12). Both have glycine
at P2, leucine at P1, and aspartic acid at P4
(Fig. 1). Additional
insight into the processing event may be derived from the observations
that ALLN and ALLM inhibit equally well, but replacing norleucinal or
methional with argininal (leupeptin) results in the loss of processing
blockade. This hint of selectivity shows promise for the development of
easily synthesized plasmepsin inhibitors that efficiently prevent
hemoglobin degradation and are lethal to the parasite.
We thank A. Oksman for her expert help with parasite cultures. I. Gluzman generously provided recombinant plasmepsin proteins. We are also indebted to K. Crowley, A. Strauss, S. Kornfeld, B. Lindenbach, D. Sibley, T. Steinberg, and J. Laing for helpful suggestions.