Plasmepsin II, an Acidic Hemoglobinase from the
Plasmodium falciparum Food Vacuole, Is Active at Neutral pH
on the Host Erythrocyte Membrane Skeleton*
Sylvain
Le Bonniec
,
Christiane
Deregnaucourt
§,
Virginie
Redeker¶,
Ritu
Banerjee
,
Philippe
Grellier
,
Daniel
E.
Goldberg
**, and
Joseph
Schrével
From the
Laboratoire de Biologie Parasitaire,
Muséum National d'Histoire Naturelle, EP 1790 CNRS, 61 rue
Buffon, and the ¶ Laboratoire de Neurobiologie, Ecole
Supérieure de Physique et Chimie Industrielles de la Ville de
Paris, CNRS UMR 7637, 75231 Paris Cedex 05, France and the
Howard Hughes Medical Institute, Departments of Medicine and
Molecular Microbiology, Washington University School of Medicine,
St. Louis, Missouri, 63110
 |
ABSTRACT |
Plasmepsin II, an aspartic protease from the
human intraerythrocytic parasite Plasmodium falciparum, is
involved in degradation of the host cell hemoglobin within the acidic
food vacuole of the parasite. Previous characterization of enzymatic
activities from Plasmodium soluble extracts, responsible
for in vitro hydrolysis of erythrocyte spectrin, had shown
that the hydrolysis process occurred at pH 5.0 and involved aspartic
protease(s) cleaving mainly within the SH3 motif of the spectrin
-subunit. Therefore, we used a recombinant construct of the
erythroid SH3 motif as substrate to investigate the involvement of
plasmepsins in spectrin hydrolysis. Using specific anti-plasmepsin II
antibodies in Western blotting experiments, plasmepsin II was detected
in chromatographic fractions enriched in the parasite SH3 hydrolase
activity. Involvement of plasmepsin II in hydrolysis was demonstrated
by mass spectrometry identification of cleavage sites in the SH3 motif,
upon hydrolysis by Plasmodium extract enzymatic activity,
and by recombinant plasmepsin II. Furthermore, recombinant plasmepsin
II digested native spectrin at pH 6.8, either purified or situated in
erythrocyte ghosts. Additional degradation of actin and protein 4.1 from ghosts was observed. Specific antibodies were used in confocal
imaging of schizont-infected erythrocytes to localize plasmepsin II in
mature stages of the parasite development cycle; antibodies clearly
labeled the periphery of the parasites. Taken together, these results strongly suggest that, in addition to hemoglobin degradation, plasmepsin II might be involved in cytoskeleton cleavage of
infected erythrocytes.
 |
INTRODUCTION |
Malaria is a tropical disease caused by the protozoan parasite
Plasmodium. In 1996, the World Health Organization estimated the annual mortality at 1.7-2.5 million and persons living in risk
areas at ~2 billion. Resistance of Plasmodium to
antimalarial drugs and vector resistance to insecticides are increasing
problems in fighting the parasite. New approaches to chemotherapy based on blocking essential metabolic pathways of the parasite are necessary. Toward this end, the parasite proteases that are involved in crucial steps of Plasmodium development appear to be good targets.
Several have been described that are involved in hemoglobin hydrolysis (the so-called hemoglobinases), antigen maturation, merozoite release,
or invasion of new red blood cells by merozoites (1).
Among the Plasmodium proteases, the hemoglobinases have been
extensively studied (2). In the food vacuole (pH 5-5.4), the 37-kDa
aspartic proteases plasmepsin I and plasmepsin II initiate cleavage of
the native hemoglobin tetramers, whereas the 28-kDa cysteine protease
falcipain appears to digest the denatured or fragmented substrate (3).
These three hemoglobinases have been cloned and sequenced (4-6). A
43-kDa recombinant precursor of plasmepsin II lacking the first 76 residues of full-length proplasmepsin II has been expressed in
Escherichia coli, and its acidification results in the
production of a 38-kDa protein by autocatalytic cleavage (7). The
38-kDa enzyme showed kinetic properties similar to those of native
plasmepsin II (8). Thus far, no clear difference in structure or
function has emerged between plasmepsins I and II, and the reason why
the parasite synthesizes both enzymes remains obscure. Unexpectedly,
our work on the involvement of proteases in the invasion process yields
new insights into the substrate selectivity of plasmepsin II compared
with plasmepsin I.
Many years ago, the depletion of spectrin and protein 4.1, two major
proteins of the erythrocyte membrane cytoskeleton, was described for
infected red blood cells during the schizogonic stage of
Plasmodium berghei (9). A Plasmodium lophurae
37-kDa cathepsin D was reported to digest spectrin and bands 2.1, 2.6, and 3 from duckling and human red blood cells at both pH 3.5 and 7.4 (10). Also, a 37-kDa acidic proteinase, able to cleave spectrin and
protein 4.1 from human erythrocytes at both pH 5.0 and 7.2, was
purified from soluble extracts of P. falciparum and P. berghei (11). In 1996, another P. falciparum
proteolytic activity, clearly inhibited by 10-20 µM
pepstatin A and involving protease(s) in the 35-40-kDa range, was
described to act at acidic pH on human spectrin (12). This
"spectrinase activity" cleaved the
-spectrin at a site located
within the SH3 motif of the molecule.
Owing to the fact that plasmepsins I and II are pepstatin A-inhibitable
acidic proteinases with a molecular mass of 37 kDa, we investigated the
presence of these hemoglobinases in P. falciparum chromatographic fractions enriched for the SH3 hydrolase activity. Plasmepsin II was found in the most active fractions. Mass spectrometry identification of cleavage sites in the SH3 motif upon digestion by
recombinant plasmepsin II or by spectrinase activity unambiguously established involvement of plasmepsin II in in vitro
hydrolysis of the SH3 motif. The recombinant enzyme proved to be able
to digest native spectrin at pH 6.8, either purified or in erythrocyte ghosts. Confocal microscopy analysis of infected erythrocytes with
anti-plasmepsin II antibodies showed that the enzyme can be visualized
outside the parasite in mature stages of Plasmodium. These
results strengthen the idea of plasmepsin II being devoted to
function(s) other than hemoglobin degradation in infected red blood
cells and, more generally, address the question of the redistribution of some specific proteases during the intraerythrocytic development of
P. falciparum.
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EXPERIMENTAL PROCEDURES |
Tools and Chemicals--
Saponin and
N
-benzyloxycarbonyl-Phe-Arg
7-amido-4-methylcoumarin fluorogenic substrate were purchased from
Sigma-Aldrich Chimie (St. Quentin Fallavier, France). Fluorescein
isothiocyanate-labeled anti-rabbit Ig antibodies and
tetramethylrhodamine B isothiocyanate-labeled anti-mouse Ig
antibodies were purchased from Nordic Immunological Laboratories.
Construction of the recombinant
GST1-SH3 peptide and linkage
to Sepharose beads have been previously described (12).
P. falciparum in Vitro Culture and Synchronization--
The FcB1
strain from Colombia was used throughout the experiments. Culture was
performed according to Trager and Jensen (13). Basic culture medium
contained RPMI 1640 medium (Life Technologies, Inc.), 25 mM
Hepes, 27.5 mM NaHCO3, and 11 mM
glucose (pH 7.4) supplemented with penicillin (100 IU/ml), streptomycin
(100 µg/ml), and 7% (v/v) heat-treated human plasma. Red blood cells
from the same donor were added at a hematocrit of 2-3%, and the
culture was maintained under an atmosphere of 3% CO2, 6%
O2, and 91% N2 at 37 °C, with daily medium
changes. Synchronization of cultures was achieved by Plasmagel
treatment (14), followed, 4-5 h later, by 5% (w/v) sorbitol treatment
(15). Under our culture conditions, the in vitro life cycle
of the FcB1 strain was 48 h.
Enzymatic Extracts and Enrichment of P. falciparum Spectrinase
Activity--
100,000 × g extracts from the schizont
stage of P. falciparum were prepared as described by Le
Bonniec et al. (12). For enrichment of acidic spectrinase
activity, gel filtration of 100,000 × g extracts on
Superose 12 and chromatography on alkyl-Sepharose were performed using
a fast protein liquid chromatography system (Amersham Pharmacia
Biotech). The gel filtration procedure has been described (12). For
spectrinase enrichment through hydrophobic interactions, pooled active
fractions from Superose 12 were applied to an alkyl-Superose HR 5/5
column equilibrated in 100 mM phosphate buffer (pH 7.2)
containing 2 M ammonium sulfate. Elution was performed with
a gradient of ammonium sulfate from 2 to 0 M in 100 mM phosphate buffer at a flow rate of 0.5 ml/min. Usually,
1-ml fractions were collected and tested for their SH3-hydrolyzing
activity at pH 5.0 using the recombinant GST-SH3 peptide as substrate.
Conditions for high performance electrophoresis chromatography analysis
(system from Applied Biosystems, Foster City, CA) have been described previously (12).
Purification of Spectrin--
Hemoglobin-free erythrocyte
membranes were prepared according to Dodge et al. (16).
Human spectrin was purified by incubating erythrocytes at 37 °C for
30 min in 0.3 mM sodium phosphate and 0.1 mM
EDTA. After centrifugation at 300,000 × g for 1 h, the supernatant containing primarily spectrin and actin (17) was concentrated and enriched for spectrin by ultrafiltration using Centriprep-100 units (Amicon, Inc., Epernon, France).
Enzymatic Tests--
All enzymatic reactions were performed at
37 °C in 25 mM Tris adjusted to pH 6.8 or 5.0 with
citrate. Usually, tests of the spectrinase-enriched fraction against
GST-SH3 were performed at a ratio of 1 volume of pelleted
GST-SH3-Sepharose beads (12) to 4 volumes of the enzymatic fraction.
Tests of purified recombinant plasmepsin II (8) against GST-SH3 were
performed by incubating 10 µl of pelleted beads with 0.5 µg of
enzyme. Tests of recombinant plasmepsin II against native spectrin was
performed by incubating 0.5 µg of enzyme with 50 µg of spectrin.
When indicated, recombinant plasmepsin was incubated prior to tests at
pH 5.0 and 37 °C for 2 h to induce maturation of the 43-kDa
precursor form to the 38-kDa form by autoactivation.
Prior to mass spectrometry analysis of SH3 hydrolysis, the GST-SH3
peptide linked to Sepharose beads was incubated for 2 h at pH 5.0 and 37 °C with the spectrinase-enriched fraction recovered by gel
filtration or was incubated for 3 h at pH 5 and 37 °C with recombinant plasmepsin II. The presence of released fragments was
checked by electrophoresis on Tricine gel, which allows visualization of small peptides (18). For kinetic analysis of SH3 cleavage by the
spectrinase activity, aliquots were taken after 1, 2, and 3 min of
incubation of the recombinant peptide with the spectrinase-enriched fraction. The spectrinase-enriched fraction, recombinant plasmepsin II,
and GST-SH3, alone and under the same experimental conditions, were
used as controls in mass spectrometry analyses.
Mass Spectrometry Analysis--
After digestion of the SH3 motif
with either recombinant plasmepsin II or the spectrinase-enriched
fraction, the released peptides were analyzed by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
After digestion, the samples were acidified by performing a dilution
(1:2 to 1:10) in a 0.1% aqueous trifluoroacetic acid solution. The
MALDI matrix used was 2,5-dihydroxybenzoic acid (Aldrich). The
acidified samples were then mixed 1:1 (v/v) with a saturated solution
of 2,5-dihydroxybenzoic acid in 0.1% aqueous trifluoroacetic acid and
analyzed. After determination of their molecular masses, cleavage sites
in the SH3 motif were identified.
MALDI-TOF-MS measurements were performed on a Voyager Elite mass
spectrometer (Perseptive Biosystems, Inc., Framingham, MA) in positive
ion reflectron mode using a delayed extraction. For laser desorption, a
nitrogen laser beam (
= 337 nm) was used. Typically, 150-256 shots
were averaged for each acquired spectrum. External calibration was
performed using a mixture of neurotensin, ACTH clip-(18-39), and
ACTH-(7-38) with m/z ratios corresponding to monoisotopic
[M + H]+ of 1672.92 and 2465.20 and average [M + H]+ of 3660.19, respectively. Only the average
m/z ratios of the protonated peptides are given under
"Results." The difference between the calculated average mass and
the experimental mass determination was <0.5 atomic mass unit.
SDS-PAGE and Western Blot Analysis--
Electrophoresis was
performed on 10 and 15% acrylamide gels according to Laemmli (19).
Gels were stained with Coomassie Brilliant Blue R-250. Proteins were
electrotransferred onto nitrocellulose membranes according to Towbin
et al. (20). Rabbit anti-plasmepsin I and anti-plasmepsin II
antibodies (21) were used at a 1:2500 dilution.
Confocal Microscopy Analysis--
Smears of asynchronous
cultures of the FcB1 strain were fixed for 2 min at
20 °C in
acetone/methanol (7:3, v/v). Double labeling of cells with
anti-plasmepsin II polyclonal antibodies (serum 737 (21); 1:200 in PBS
and 3% nonfat milk (pH 7.5)) and anti-MSP1 (merozoite
surface protein 1) monoclonal
antibodies (ascites 22-2 (22); 1:50 in PBS and 3% nonfat milk (pH
7.5)) was performed by successive incubations (2 h, room temperature)
with the respective antibodies. After washing in PBS and 3% nonfat
milk (3 × 10 min), slides were incubated with mixed
tetramethylrhodamine B isothiocyanate-labeled anti-mouse Ig antibodies
(1:50) and fluorescein isothiocyanate-labeled anti-rabbit Ig antibodies
(1:100) in PBS and 3% nonfat milk for 2 h at room temperature.
Control slides for labeling of cells with secondary antibodies and
nonimmune rabbit serum were processed in parallel. Slides were
washed in PBS (3 × 10 min) and mounted in
FluoroGuardTM antifade reagent (Bio-Rad) under a coverslip.
Observations were made using a confocal laser scanning microscope (MRC
1024, Bio-Rad).
 |
RESULTS |
Plasmepsin II, an Acidic Endoprotease from the Food Vacuole of
Plasmodium, Co-purifies with the Spectrinase Activity--
Owing to
the acidic pH activity of the parasite SH3 hydrolase, we looked for the
presence of the three hemoglobinases plasmepsin I, plasmepsin II, and
falcipain in the fractions recovered by gel filtration of a
100,000 × g extract from the schizont stage of the
FcB1 strain of P. falciparum. The spectrinase activity was
localized in the eluted fractions via hydrolysis of the recombinant GST-SH3 peptide at pH 5.0 and 37 °C and analyzed by SDS-PAGE and Coomassie Blue staining of the gel. In controls for each fraction omitting GST-SH3, the amount of protein in the 30-50-kDa range was too
low to be detected (data not shown). Plasmepsins I and II were
visualized by Western blotting of the fractions using specific
anti-plasmepsin I (serum 574) and anti-plasmepsin II (serum 737)
antibodies. The presence of falcipain was checked by hydrolysis of
the fluorogenic substrate
N
-benzyloxycarbonyl-Phe-Arg
7-amido-4-methylcoumarin (23). Distribution of the spectrinase activity
(Fig. 1A) showed that
fractions 8 and 9 contained the highest SH3-hydrolyzing activity, and
plasmepsin II was clearly visualized in these fractions (Fig.
1C). In contrast, plasmepsin I, although also a 37-kDa
protein, was visualized in fractions 4-9, peaking in fraction 4 (Fig.
1B), which corresponds to eluted proteins with ~100-kDa
molecular masses. As chromatography was performed under nondenaturing
conditions, early elution of plasmepsin I might be due to
protein-protein interactions. Low SH3-hydrolyzing activity was detected
in plasmepsin I-containing fractions compared with plasmepsin
II-containing fractions. With respect to falcipain, degradation of the
Phe-Arg 7-amido-4-methylcoumarin substrate was observed with fractions
9 and 10 (Fig. 1D) and was minimal in fraction 8, which had
high proteolytic activity against the SH3 motif. Also, falcipain is a
cysteine protease, hence is not inhibited by pepstatin A, in contrast
to what was observed with the spectrinase activity.

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Fig. 1.
Identification of the hemoglobinases in
Superose 12 fractions of a 100,000 × g
extract from schizonts of the FcB1 strain. Elution fractions
4-12 (approximately corresponding to the elution of proteins in the
range of 100 to 20 kDa) were split into four aliquots. One aliquot was
tested for its SH3-hydrolyzing capacity. The recombinant GST-SH3
peptide was incubated with the fraction for 2 h at pH 5.0 and
37 °C. SH3 hydrolysis was analyzed by SDS-PAGE and Coomassie Blue
staining of the gel (A). The recombinant GST-SH3 peptide was
also incubated at pH 5.0 in the elution buffer alone (control
(co)). The presence of plasmepsins I and II was checked by
Western blot analysis of two other aliquots from each fraction using
anti-plasmepsin I antibodies (1:2500; B) and anti-plasmepsin
II antibodies (1:2500; C). The remaining aliquot was used to
test the presence of falcipain. Enzymatic activity was checked by
cleavage of the fluorogenic dipeptidyl substrate
N -benzyloxycarbonyl-Phe-Arg
7-amido-4-methylcoumarin, a specific substrate for falcipain
(D). Emitted fluorescent light was collected at 440 nm upon
excitation at 380 nm (arbitrary units). Molecular masses are on the
right.
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To test the hypothesis that the enzyme(s) responsible for SH3
hydrolysis might be different from plasmepsin II, various biochemical procedures were tried to separate plasmepsin II from the SH3 hydrolase activity. 100,000 × g schizont extracts from different
cultures of the FcB1 strain were fractionated by gel filtration on a
Superose 12 column, and active fractions were further enriched either
by chromatography on alkyl-Sepharose or by high performance
electrophoresis chromatography. As done previously, the eluted
fractions were analyzed for the presence of plasmepsin II and SH3
hydrolase. In every experiment, plasmepsin II was found to coelute with
the SH3-hydrolyzing activity (data not shown).
Recombinant Plasmepsin II Is Able to Hydrolyze the SH3 Domain in
the GST-SH3 Peptide--
The presence of plasmepsin II in the
SH3-hydrolyzing fractions prompted us to investigate the involvement of
this enzyme in SH3 degradation. The recombinant enzyme was used in
enzymatic assays with the recombinant GST-SH3 peptide as substrate.
Recombinant plasmepsin II was incubated with substrate at pH 5.0 and
37 °C for different times, and GST-SH3 proteolysis was analyzed by
SDS-PAGE and Coomassie Blue staining of the proteins (Fig.
2). Under these conditions, the
recombinant enzyme proved to be able to cleave GST-SH3, generating
three fragments at 34, 32, and 30 kDa, a similar pattern to the one
observed with the spectrinase-enriched fraction from gel filtration of
a 100,000 × g schizont extract (12).

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Fig. 2.
Kinetic analysis of GST-SH3 hydrolysis by
recombinant plasmepsin II. Approximately 10 µl of pelleted
GST-SH3 substrate linked to Sepharose beads was incubated with 0.5 µg
of recombinant plasmepsin II in 25 mM Tris citrate (pH 5.0)
(control (co)). After a 2-min (lane 1), 5-min
(lane 2), 15-min (lane 3), 30-min (lane
4), 1-h (lane 5), or 20-h (lane 6)
incubation, aliquots were taken, boiled in SDS reducing buffer, and
then analyzed by SDS-PAGE on a 15% acrylamide gel. The gel was stained
with Coomassie Blue. Molecular masses (in kilodaltons) are on the
right.
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Localization of Cleavage Sites in the SH3 Motif by Mass
Spectrometry Analysis of the Hydrolysis Products--
To unambiguously
clarify the involvement of plasmepsin II in the SH3 hydrolysis, the
identification of SH3 cleavage sites upon spectrinase action and upon
recombinant plasmepsin II action was undertaken by MALDI-TOF-MS
analysis of the released fragments. The recombinant GST-SH3 peptide
linked to Sepharose beads was incubated at pH 5.0 and 37 °C with
recombinant plasmepsin II or with a spectrinase-enriched fraction
recovered by gel filtration of a 100,000 × g schizont
extract. Hydrolysis products were analyzed by SDS-PAGE on a Tricine
gel, allowing the visualization of peptides in the 1-5-kDa range (data
not shown). Sepharose beads were pelleted by centrifugation, and the
supernatant was analyzed by MALDI-TOF-MS. The identified cleavage sites
along the amino acid sequence of the SH3 motif are reported in Fig.
3.

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Fig. 3.
MALDI-TOF-MS identification of cleavage sites
in the SH3 amino acid sequence. Sequences 1 and
2, SH3 hydrolysis by recombinant plasmepsin II and the
SH3-hydrolyzing activity, respectively. Cleavage sites are indicated by
arrows. Sequence 3, kinetic analysis of SH3
hydrolysis by the SH3-hydrolyzing activity. Sites cleaved within 1, 2, and 3 min of incubation are indicated by arrows. The plasmid
sequence is underlined.
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Six sites were identified due to cleavage by the spectrinase-enriched
fraction (Fig. 3, sequence 2). They are distributed within
three "regions" of cleavage, showing one site (F
Q), two sites
(Y
V, R
R) and three contiguous sites (L
L, L
S, S
S) of cleavage, respectively. Location of the cleavage regions along the
amino acid sequence corresponds to the expected generation of the 34-, 32-, and 30-kDa proteolytic fragments.
Five sites were identified due to cleavage by recombinant plasmepsin II
(Fig. 3, sequence 1). Out of the five, four corresponded to
the F
Q, Y
V, L
L, and L
S sites identified upon digestion of
the SH3 motif by the spectrinase fraction. The fifth site (F
I) was
located outside the SH3 amino acid sequence, in the recombinant plasmid sequence.
Although mass quantitation of the hydrolysis fragments is difficult by
MALDI-TOF-MS, the intensity of some peaks was highly reproducible from
one experiment to the other, suggesting that the corresponding sites
were highly susceptible to cleavage. From this, the Y
V and S
S
sites appear as major sites of cleavage with the spectrinase fraction,
and Y
V and L
L are major sites with plasmepsin II. Minor sites
with the spectrinase fraction and plasmepsin II are R
R, L
L, and
L
S and F
I and L
S, respectively. A very minor site at F
Q was
revealed for both enzymatic activities.
Two main conclusions must be drawn from these results. First,
plasmepsin II is unambiguously and predominantly involved in the
in vitro hydrolysis of the SH3 motif observed with the
parasite spectrinase fraction; and second, enzyme(s) other than
plasmepsin II are involved in this hydrolysis, cleaving at the R
R
and S
S sites. To detail the sequence of events due to respective
enzymes in the spectrinase fraction, kinetic analysis of the cleavage processes was performed by MALDI-TOF-MS. The identity of the peptides released upon hydrolysis of Sepharose bead-linked GST-SH3 was assessed
after 1, 2, and 3 min of enzymatic incubation (Fig. 3, sequence
3). The Y
V site (attributed to cleavage by plasmepsin II) was
first cleaved at 1 min, and then the S
S site at 2 min. Next, the
L
L and L
S sited (both due to plasmepsin II) and the R
R site
were cleaved off within 3 min. From this, we can infer that cleavage of
the S
S site is due to an additional endopeptidase cleaving the
fragment -SSINK ... VPAVY.
Recombinant Plasmepsin II Is Able to Hydrolyze Native Spectrin at
pH 6.8--
Hydrolysis of purified human erythroid spectrin by the
Plasmodium acidic spectrinase activity generates three main
fragments at 170, 150, and 125 kDa (12). The generation of the 170- and 125-kDa fragments was explained by enzyme(s) cutting within the SH3
motif of the spectrin
-subunit. Because plasmepsin II was found to
be part of the acting proteinases in the so-called spectrinase activity, we tested the ability of recombinant plasmepsin II to digest
purified native spectrin in order to compare the proteolysis pattern
with the one generated by the spectrinase activity. Spectrin from
normal erythrocytes was incubated with recombinant plasmepsin II for
1 h, 2 h, and overnight at pH 6.8 and 37 °C. The
recombinant enzyme had been preincubated or not for 2 h at pH 5.0 since such conditions induce autoactivation and shortening of the
molecule from 43 to 38 kDa (7). Spectrin hydrolysis was analyzed by SDS-PAGE and Coomassie Blue staining of the proteins. Results from the
overnight incubation are presented in Fig.
4A.

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Fig. 4.
A, hydrolysis of purified spectrin by
recombinant plasmepsin II. 50 µg of native spectrin was incubated
with 0.5 µg of recombinant enzyme in 25 mM Tris citrate
(pH 6.8) for 20 h at 37 °C. Before incubation, recombinant
plasmepsin II had been (lane 3) or had not been (lane
2) matured at pH 5.0 and 37 °C for 2 h. Native spectrin
was also incubated in Tris citrate alone (lane 1). The
hydrolysis pattern was analyzed by SDS-PAGE on a 10% acrylamide gel.
The gel was stained with Coomassie Blue. Molecular masses of the
hydrolysis fragments are indicated on the right. Standard molecular
masses are on the left. B, comparative analysis of GST-SH3
hydrolysis by recombinant plasmepsin II at pH 6.8 and 5.0. GST-SH3 was
incubated in Tris citrate without recombinant plasmepsin II for 75 min
(lane 1) and 20 h (lane 3) at pH 6.8 and for
75 min (lane 1') and 20 h (lane 3') at pH
5.0 or in Tris citrate with recombinant plasmepsin II for 75 min
(lane 2) and 20 h (lane 4) at pH 6.8 and for
75 min (lane 2') and 20 h (lane 4') at pH
5.0. Results were analyzed by SDS-PAGE under reducing conditions on a
15% acrylamide gel. The gel was stained with Coomassie Blue. Molecular
masses (in kilodaltons) are on the right.
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At pH 6.8, hydrolysis of spectrin by the 43-kDa form of the enzyme
occurred, generating four major fragments at 170, 155, 148, and 125 kDa
(Fig. 4A, lane 2). These fragment sizes are very close to the ones described upon spectrin degradation by the
spectrinase activity. Interestingly, greater degradation of spectrin
was observed at pH 6.8 in the presence of the 38-kDa form of
recombinant plasmepsin II, with an additional proteolytic fragment at
112 kDa (Fig. 4A, lane 3).
Degradation of the GST-SH3 peptide by 43-kDa recombinant plasmepsin II
was analyzed at pH 6.8 as well. Hydrolysis was slower than that
observed at pH 5.0 (under conditions in which 43-kDa plasmepsin II is
activated to 38 kDa), but it generated the expected pattern of 34-, 32-, and 30-kDa fragments (Fig. 4B).
Recombinant Plasmepsin II Selectively Digests Spectrin, Actin, and
Protein 4.1 from Erythrocyte Ghosts--
The recombinant plasmepsin II
activity was tested against ghosts from normal erythrocytes to check if
native spectrin, situated within the protein network of the membrane
skeleton, would be a substrate for the enzyme and if other proteins
from the ghosts would be substrates as well. Erythrocytes were lysed in
5 mM PO4 buffer (pH 6.8), washed with the same
buffer, and incubated overnight at pH 6.8 and 37 °C with the 43- or
38-kDa form of recombinant plasmepsin II. Degradation of the proteins
from ghosts was analyzed by SDS-PAGE and Coomassie Blue staining of the
gel. As shown in Fig. 5 (lane
3), the pattern of the ghosts was dramatically altered in the
presence of 38-kDa plasmepsin II. Densitometric analysis of the gel
gave a 84% disappearance of the
-spectrin and a 52% disappearance
of the
-spectrin with the 38-kDa form of the enzyme versus almost unchanged
- and
-spectrin amounts in the
presence of the 43-kDa form (Fig. 5, lane 2). Also, the
amount of protein 4.1 decreased in the presence of 38-kDa plasmepsin
II, and actin clearly disappeared.

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Fig. 5.
Hydrolysis of red blood cell membrane
proteins by recombinant plasmepsin II at pH 6.8. A 20-µl pellet
of ghosts from normal red blood cells was resuspended in 180 µl of 25 mM Tris citrate buffer (pH 6.8), and 40-µl aliquots were
incubated overnight at 37 °C without recombinant plasmepsin II
(lane 1), with 0.5 µg of recombinant plasmepsin II in the
43-kDa form (lane 2), and with 0.5 µg of recombinant
plasmepsin II in the 38-kDa form, i.e. preincubated for
2 h at pH 5.0 and 37 °C (lane 3). Results were
analyzed by SDS-PAGE on a 10% acrylamide gel, and the gel was stained
with Coomassie Blue. The -subunit (240 kDa) and -subunit (220 kDa) of spectrin, protein 4.1 (80 kDa), and actin (43 kDa) are
indicated by arrowheads. Recombinant plasmepsin II visible
in lane 2 is indicated by the arrow. (The 38-kDa
form of the enzyme was not detected in lane 3, in agreement
with a previous control experiment on plasmepsin II activation in which
the 38-kDa form appeared faintly on a silver-stained gel compared with
the 43-kDa form). Molecular masses (in kilodaltons) are on the
left.
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Immunolabeled Plasmepsin II Can Be Visualized outside the Parasite
in Schizont-infected Red Blood Cells--
To address the question of
the erythrocyte membrane skeleton being a physiologically relevant
substrate of plasmepsin II in mature parasite-infected cells, we
investigated (by confocal microscopy) the cellular location of the
enzyme during the schizont stage. Previous immunoelectron microscopy
experiments (5) had shown that plasmepsins are present at the surface
of the parasite in trophozoites, but no analysis of plasmepsin location
later during parasite intraerythrocytic development was available thus far.
Smears of FcB1-infected red blood cells were fixed in acetone/methanol,
and double labeling of cells was performed using rabbit anti-plasmepsin
II polyclonal antibodies (serum 737) and mouse anti-MSP1 monoclonal
antibody (ascites 22-2). Anti-MSP1 antibody was used to visualize
mature intraerythrocytic parasites via labeling of the parasite
membrane. Preservation of red blood cells upon fixation was checked by
phase-contrast microscopy. No labeling was observed with non-
immune rabbit serum (data not shown) or with secondary antibodies alone
(data not shown).
Images of immunolabeled cells, summed from three 10-nm spaced out
sections each, are presented in Fig. 6.
Fig. 6A shows an ~40-h-old schizont; although MSP1
labeling is confined to the parasite (panel 3), fleecy
labeling with anti-plasmepsin II antibodies can be observed inside and
outside the parasite (panel 2). The presence of
anti-plasmepsin II antibody labeling in the erythrocyte cytoplasm,
although not found in all schizonts, was frequently observed. In
Fig. 6B is presented an early segmenter (44-46 h old).
Remarkably, plasmepsin II labeling appears to delineate individualizing
merozoites, in a comparable way to MSP1 labeling, although MSP1 and
plasmepsin II labeling does not totally merge; labeled plasmepsin II
appears as a green rim external to the yellow MSP1-delineated membrane.

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Fig. 6.
Immunolocalization of plasmepsin II in
schizont-infected erythrocytes by confocal microscopy. Blood
smears of the FcB1 strain were incubated with mouse anti-MSP1
monoclonal antibody (1:50) and rabbit anti-plasmepsin II polyclonal
antibodies (1:200) using tetramethylrhodamine B
isothiocyanate-conjugated anti-mouse Ig antibodies (1:50) and
fluorescein isothiocyanate-conjugated anti-rabbit Ig antibodies (1:100)
as secondary antibodies. A, an ~40-h-old schizont;
B, an early segmenter. Panels 1, plasmepsin II
and MSP1 labeling; panels 2, plasmepsin II labeling alone;
panels 3, MSP1 labeling alone. In each case, the presence of
the surrounding erythrocyte cytoplasm was checked by phase-contrast
microscopy. Scale bars = 3 µm.
|
|
 |
DISCUSSION |
Most of the current efforts in understanding the biology of
Plasmodium aim at elaborating new antimalarial strategies.
Protease inhibitors have been successfully employed in well known
examples such as AIDS therapy, and identification of target proteases
in Plasmodium is today part of the antimalarial fight. In
this respect, the hemoglobinases, as participants in an essential
metabolic pathway, appear to be choice targets. Plasmepsins I and II
are both responsible for the initial cleavage of hemoglobin in the digestive vacuole of the intraerythrocytic parasite, but when a
specific inhibitor of plasmepsin I (SC-50083) is added to cultures at
the trophozoite stage, when hemoglobin is degraded, the parasites have
markedly diminished hemozoin production and are killed
(IC50 = 2 × 10
6 M) (5).
This indicates that plasmepsin I activity is essential, but also that
plasmepsins I and II have distinct hemoglobin-degrading capacities.
Moreover, the inability of plasmepsin II to supplant plasmepsin I in
hemoglobin degradation raises the question of the exact role of
plasmepsin II in this process.
Plasmepsin II Exhibits Different Substrate Selectivity Compared
with Plasmepsin I--
We show here that proteins other than
hemoglobin can be substrates of plasmepsin II, namely spectrin, protein
4.1, and actin, all components of the erythrocyte membrane skeleton.
Since plasmepsin I was shown to be inactive against a variety of
non-globin substrates (2) and was also found to be poorly active on the
GST-SH3 substrate, in contrast to plasmepsin II, these findings
reinforce the idea that the two enzymes may be devoted to different
roles in the parasitized cell. The identification of the five sites
cleaved at pH 5.0 by recombinant plasmepsin II in the SH3 motif of the spectrin
-subunit is in general agreement with the known preference of plasmepsin II for hydrophobic residues on both sides of the scissile
bond (3). Only two sites, L
S and F
Q, show a hydrophobic residue
at the P1 position and a hydrophilic one at the P1' position, extending
the repertoire of the potential sites of cleavage for plasmepsin II.
For plasmepsin I, if one considers amino acids at the P1 and P1'
positions, two putative cleavage sites can be identified within the SH3
motif: the F
Q site, deduced from the plasmepsin I preference for the
phenylalanine at the P1 position, and the L
L site, listed as a
secondary site on hemoglobin (3). The presence of 32-30-kDa
fragment(s) upon hydrolysis of GST-SH3 by fraction 4 and adjacent
functions in Fig. 1 could thus be attributed to plasmepsin I action.
However, fractions in which plasmepsin II was readily detected compared
with plasmepsin I (fractions 8-10) displayed higher degradative
activity. This observation suggests that, most probably, recognition of
potential cleavage sites in the SH3 motif by plasmepsins I and II
involves additional parameters such as identity of the surrounding
amino acids and/or conformation of the polypeptide chain. In the same
way, the number of spectrin sites actually cleaved by plasmepsin II
appears limited since a 20-h hydrolysis of purified native spectrin at
pH 6.8 by recombinant plasmepsin II produces high molecular mass
fragments, although many potential sites are found scattered along the
- and
-subunit amino acid sequences, predicting the generation of
multiple short sized fragments. Conformational requirements might
explain why the SH3 motif, whose secondary structure is different from
the repetitive structure of the rest of the
-chain, is a privileged
target for the enzyme. This interpretation would go along with what has
been written about the enzyme specificity depending on the
tertiary structure of its substrate hemoglobin (24).
pH Activity and Maturation of the Recombinant
Enzyme--
Recombinant plasmepsin II can digest the SH3 motif of
-spectrin, as well as native spectrin, at pH 6.8. In terms of
activity of the enzyme on the GST-SH3 substrate, hydrolysis of the
substrate at pH 5.0 is completed within 75 min, whereas at pH 6.8, primary degradation fragments are visible. Thus, the pH optimum of the enzymatic activity does not seem to differ from hemoglobin to the
GST-SH3 substrate. The activities of purified native plasmepsins II and
I on hemoglobin are known to be dramatically reduced at pH 6.5. In both
cases, ~35% residual activity is detected (24). Further experiments
are needed to quantify the activity of the recombinant enzyme against
the SH3 motif at pH 6.8 and to compare it with its activity against
hemoglobin at the same pH. An interesting observation is the enhanced
efficiency of recombinant plasmepsin II in degrading spectrin after it
has been shortened to 38 kDa by autoactivation at pH 5.0. Previous
studies had demonstrated that the structural and kinetic properties of
mature recombinant plasmepsin II and native plasmepsin II were similar
(8), but nothing is known about the activity of the precursor form of
recombinant plasmepsin II since all experiments were performed at pH
5.0. We observed that within 20 h at pH 6.8, the hydrolysis
pattern of native spectrin, either purified or from ghosts, was greater when due to the mature form of the enzyme than to the precursor form.
One explanation might be that the enzyme has slowly autoactivated to
the 38-kDa form at pH 6.8. If not, this might indicate that some of the
properties of the enzyme differ between immature and mature forms in
terms of structural and/or kinetic properties.
Is the in Vitro Degradation of the Host Cell Skeleton by Plasmepsin
II Physiologically Relevant?--
Our results raise the question of
the physiological relevance of observations made in vitro.
Indeed, two points must be considered. First, the red blood cell
skeleton and plasmepsin II were believed to belong to distinct cellular
compartments; and second, to cleave skeleton proteins, plasmepsin II
would need to act at a pH near neutrality in the cytoplasm of the red
blood cell. Now, the enzyme has been found to be active at pH 6.8 against spectrin, actin, and protein 4.1, which suggests that it could
be active in the red blood cell cytoplasm. Confocal imaging using
anti-plasmepsin II antibodies brought clear evidence of a possible
proximity between the enzyme and membrane skeleton during late parasite
maturation. What has been inferred about plasmepsin I and plasmepsin II
trafficking, largely deduced from the study of plasmepsin I
trafficking, is that plasmepsins are secretory molecules that are
synthesized as type II integral membrane proteins (21). The
proplasmepsins have been detected by immunoelectron microscopy at the
parasitophorous vacuolar membrane (5), and visualization of plasmepsin
II outside the parasite by immunoconfocal microscopy might be due to
parasitophorous membrane-originated structures extending into the red
blood cell cytoplasm. Plasmepsin II in early segmenters was found
located at the periphery of the parasite, and labeling appeared higher at this stage than in younger schizonts, even though massive
degradation of hemoglobin is not required anymore.
Considering the broad substrate selectivity and cleavage site
specificity of plasmepsin II and the large amount as well as the
location of the enzyme in late schizonts, the overall results presented
here strongly suggest that plasmepsin II function in the parasite
intraerythrocytic cycle is not restricted to hemoglobin degradation.
Its involvement in spectrin depletion in vivo remains to be
established, but it is clear that trafficking of plasmepsins and their
respective roles in the infected cell must be further explored.
 |
ACKNOWLEDGEMENTS |
We are very grateful to Marie-Christine
Lecomte and Catherine Fournier (INSERM; U.409) for providing the
recombinant GST-SH3 clone and to Prof. Jean Rossier for fruitful
collaboration between the Laboratoire de Neurobiologie at the Ecole
Supérieure de Physique et Chimie Industrielles and our laboratory.
 |
FOOTNOTES |
*
This work was supported in part by CNRS Grant GDR 1077; by
Ministère de l'Enseignement Supérieur et de la Recherche
Grant DSPT N 5 and Ministère de l'Education Nacionale, de
l'Enseignement Supérieur et de la Recherche,
Délégation Générale pour Armement; and by the
European Community Life Sciences for Developing Countries Program III
Program, supported by Grant TS3-*CT92-0145.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 33-1-40-79-35-16;
Fax: 33-1-40-79-34-99; E-mail: florent{at}mnhn.fr.
**
Supported by National Institutes of Health Grant AI31615 and
recipient of a Burroghs Wellcome Fund Scholar Award in Molecular Parasitology.
 |
ABBREVIATIONS |
The abbreviations used are:
GST, glutathione
S-transferase;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
MALDI-TOF-MS, matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry;
ACTH, adrenocorticotropic hormone;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered
saline.
 |
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.