Moh'cular and Btochemical Parasttologv. 23 119,q?l IQ3-201
EI,e~ier
193
MBP U07S0
Localization, biosynthesis, processing and isolation of a major 126 k D a
antigen of the parasitophorous vacuole of Plasmodium falciparum
Patrick Delplace 1, Bernard Fortier l.z, Guy. Tronchin ~, Jean-Francois Dubremetz ~ and
Alain Vernes ~.-~
LL"
42 I.VSER.~I. I'tlleneut.e d'.4scq and :Fuculte de Mddecme. Ltlle. France
(Receixed 5 August IqS6; accepted 1 December 10Sbl
Monoclonal antibodies prepared against a 50 kDa antigen found in Plasrnodium [alciparum culture supernatants identify a 126
kDa polypeptide which can be localized b.~ immunofluorescence and immunoelectronmicroscopy at the peripher:, of the schizonts.
This polypeptide is released from the infected erythrocytes by mild saponin lysis and is probably a component of the parasitophorous vacuole. Pulse chase kinetic analysis demonstrated its disappearance from the parasitized red blood cell from 6 to 10 h
after being synthesized and the concomitant appearance of the 50 kDa molecule in the culture supernatant. Purification of metabolicalb labeled, schizont infected cells demonstrated that spontaneous release of merozoites is needed for the processing of the
126 to the 50 kDa whereas reinvasion is not. Polyclonal antibodies were raised in rabbit against affinity purified 126 kDa protein.
These antibodies, together with another 126 kDa specific monoclonal antibody have enabled us to characterize two other cleavage
products of the 126 kDa antigen in culture supernatants, namely 47 and 18 kDa polypeptides. We believe that the processing of
the 126 kDa protein into Io~ molecular weight fragments reflects a proteolytic event which may participate in merozoite release.
Key words: Plasmodium falciparum; Parasitophorous vacuole; Exoantigens; Monoclonal antibody
Introduction
In a p r e v i o u s r e p o r t , we have described a 50
k D a c u l t u r e s u p e r n a t a n t a n t i g e n specific to the
m e r o z o i t e release r e i n v a s i o n stage of Plasmod i u m falciparum [1]. U s i n g a m o n o c l o n a l antibody we have identified a 126 k D a p r o t e i n as the
p r e c u r s o r of that m o l e c u l e in schizonts.
Such a b r e a k d o w n of schizont antigens has been
d e s c r i b e d for surface p r o t e i n s of P. falciparum
[2-4] or P. k n o w l e s i [5] a n d is c o n s i d e r e d as a
m a t u r a t i o n process i n v o l v e d in the triggering of
m e r o z o i t e release or host cell invasion. Protease
Correspondence address: P. Delplace, U42 INSERM, 369 rue
J. Guesde. 59650 Villeneuve d'Ascq. France.
Abbreviations: DOC, sodium deox)cholate: EDTA, ethylenedmminetetraacetic acid; IFA, indirect fluorescence assay;
McAb. monoclonal antibody; NP40, Nonidet P40; PBS,
phosphate-buffered saline; PMSF, phenylmethylsulfonyl
fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide
gel electrophoresis.
inhibitors are also k n o w n to inhibit both processes [6,7], which gives f u r t h e r strength to these
i n t e r p r e t a t i o n s . H o w e v e r , these p h e n o m e n a are
not fully u n d e r s t o o d . Since we b e l i e v e d that the
126 to 50 k D a t r a n s f o r m a t i o n could be related to
this set of e v e n t s , we have investigated this process f u r t h e r a n d the results of this study are reported h e r e i n . W e have localized the 126 k D a in
the schizont infected cell a n d a n a l y z e d the kinetics of its biosynthesis and processing to the 50 kDa
m o l e c u l e ; then we have purified the 126 k D a ,
raised polyclonal a n t i b o d i e s which have e n a b l e d
us to identif3' o t h e r cleavage p r o d u c t s of this molecule in culture s u p e r n a t a n t s .
Material and Methods
Cultivation o f P. falciparum in vitro. All the exp e r i m e n t s have b e e n p e r f o r m e d with the F C R 3
strain of P. falciparum grown in O ÷ h u m a n red
b l o o d cells, in R P M I 1640 m e d i u m s u p p l e m e n t e d
with 10% A + h u m a n s e r u m [8].
194
Biosynthetic labeling. In all cases, metabolic labeling was performed ~ith [35S]methionine (> 81)0
Ci mmol -l, Amersham) at 40 IxCi m1-1 in methionine free medium (RPMI 1640, Selectamine kit,
G I B C O , Grand Island, NY, U.S.A.) supplemented with 10% A ÷ human serum.
Parasitized cells were washed once with methionine free medium before labeling. Labeled parasites and supernatants were obtained as follows:
an asynchronous culture of P. falciparum (10%
parasitaemia, 400 p.I of packed cells) was labeled
for 5 h with [35S]methionine in a 10 ml volume.
At the end of the labeling period, one half of the
cells were washed in phosphate-buffered saline
(PBS; 50 mM sodium phosphate buffer, pH 7.5,
150 mM NaCI) and stored at -80°C. The remaining cells were resuspended in complete medium
and reincubated for 16 h. The supernatant was
then collected, centrifuged at 20000 × g for 1 h
and stored at -80°C.
5 h. At the end of this period, the cells were labeled with 400 gCi of [35S]methionine for 6 h. In
those conditions, the synchronization was within
a 4 h window and the end of labeling coincided
with the beginning of reinvasion.
Labeled schizonts were isolated by centrifugation on metrizamide [11] and resuspended into 15
ml of culture medium; over 80% parasitaemia was
obtained, containing mostly mature schizonts. The
suspension was divided into 3 culture flasks. One
of the flasks was incubated without treatment.
Another one received 200 Isl of fresh red blood
cells: the third one was frozen by exposure at
-20°C for 1 h. The flasks were then incubated at
37°C for 16 h. The contents of the flasks were then
collected, centrifuged at 12000 x g for 15 min and
the supernatant collected for immunoprecipitation analysis with immunosorbent 24C6 1F1, and
with the immunosorbent prepared with the lgG
of a P. falciparum immune donor.
Saponin lysis of labeled P. falciparum culture. An
asynchronous P. falciparurn culture (12%, paras-
Kinetics of 126 to 50 kDa processing. An asynchronous culture (160 I.d of cells, 10% parasitaemia) was labeled for 30 min with 200 ~Ci of
[35S]methionine at a hematocrit of 4% in a 50 ml
tube incubated at 37°C in a water bath. At the end
of the labeling period, the suspension was centrifuged and resuspended into 8 ml of serum free
medium. One ml was taken as time 0 sample. The
remainder was centrifuged and resuspended into
7 ml of complete medium, and distributed by 1
ml into a 24 well tissue culture plate which was
incubated at 37°C. One well was then collected
every hour between 5 and 11 h after the end of
the labeling period and immediately centrifuged
for 15 rain at 12000 x g. Pellets and supernatants
were then processed for immunosorption with
immunosorbent 24C6 1F1.
itaemia, 150 gl of packed cells) was labeled for 3
h with [35S]methionine at a hematocrit of 3% in
a 5 ml volume. After a 2 h chase in complete medium, the parasitized cells were washed once in
serum free medium. One fifth of the cells was then
pelleted and frozen. The remaining cells were resuspended in PBS to a volume of 500 I~1. An equal
volume of PBS containing 2 mg ml -L saponin
(Merck), 0.2 mg ml -l aprotinin (Boehringer),
0.02 mg ml -~ leupeptin (Boehringer), 2 mM
phenylmethylsulfonyl fluoride (PMSF), 4 mM
ethylenediaminetetraacetic acid (EDTA) was then
added and the suspension was incubated with
gentle agitation for 5 rain at room temperature.
The suspension was then centrifuged at 12 000 x
g for 10 min at 4°C. Pellet and supernatant were
collected for immunoprecipitation with immunosorbent 24C6 1F1.
Isolation and cultivation of labeled schizonts. An
FCR 3 culture (500 I,tl of cells, 10% parasitaemia)
was treated once with sorbitol [9]. The parasitized cells were resuspended 8 h later into culture
medium supplemented with 1.5 ~g ml-I of aphidicolin (Sigma) and incubated for 16 h [10]. The
culture was then returned to normal medium for
Monoclonal antibodies (McAb). The production
of hybridomas against P. falciparum exoantigens
and the characterization of antibody 24C6 1F1
specific for the 126 to 50 kDa antigen have been
described previously [1].
Further screening led to the characterization of
other 126 kDa specific monoclonal antibodies
(23D5 2H6 and 3E9 1 A l l ) which are described
in the present paper.
195
Preparation of immunosorbents. Purification of
IgG and coupling to CNBr activated Sepharose
4B have been performed as described before [1].
Immunosorbents were prepared with monoclonal
antibodies 24C6 1F1, 23D5 2H6 and 3E9 1All
and with specific anti 126 kDa rabbit IgG. The
immunosorbent prepared with the IgG of a P.
falciparum immune donor [1], was also used.
ine/HCl pH 11.5. The eluate was dialysed and
concentrated under vacuum, diluted in 100 ml of
lysis solution for a second affinity purification. The
second eluate was dialysed and concentrated as
above, Half of the purified material was used for
immunization of a rabbit according to Vaitukaitis
[14], by two series of subcutaneous injections 4
weeks apart. Immune rabbit serum was taken two
weeks after the second injection.
lmmunosorption procedures. Two immunosorption procedures were used, depending on the immunosorbent. Immunosorption on 24C6 1F1 immunosorbent was always performed according to
Erickson and BIobel [12] as described before [1].
For all other immunosorbents, parasitized cells
were solubilized for 30 min at 0°C in PBS containing 1% (v/v) Nonidet P40 (NP40, Sigma), 0.1
mg m1-1 aprotinin, 0.01 mg m1-1 leupeptin, 1 mM
PMSF, 2 mM EDTA, 20 mM methionine. The
lysate was centrifuged 15 rain at 12000 x g and
the supernatant used for immunosorption. Culture supernatants were processed without treatment,
lmmunosorbents (20 ~1) were incubated overnight at 4°C with lysed parasites or culture medium. They were then washed 5 times with a
buffer containing 50 mM Tris-HCI pH 8.3, 1 M
NaCI, 0.5% NP40 and 20 mM methionine and
then eluted with 50 i-tl of electrophoresis sample
buffer.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performed by the method of Laemmli [15] using 12%
slab gels. The gels were stained with Coomassie
blue R, processed for fluorography [16], dried and
exposed to Kodak X-Omat AR film at -80°C.
Silver staining of the affinity purified molecule was
performed according to Morrissey [17].
Indirect Fluorescence Assay (IFA). P. falciparum
infected erythrocytes (5-10% parasitaemia) were
washed 3 times in PBS and then either dried on
multispot glass slides (unfixed cells) or resuspended for 5 min in 2% formaldehyde in PBS,
washed 3 times in PBS before being dried on the
slides (fixed cells). The slides were then stored at
-20°C until use. The IFA was performed at 37°C
in a moist chamber after a 10 min fixation in cold
acetone (-20°C).
Affinity purification. The affinity purification of
Immunoelectronmicroscopy. A 15% P. falciparum culture was washed twice in 0.1 M cacodyl-
the antigen identified by McAb 23D5 2H6 was
performed as described by Kasper et ai. [13] with
little modification. 100 ml of parasitized red blood
cells (asynchronous culture, parasitemia ranging
from 5 to 10%) were lysed by addition of two
volumes of a solution containing 50 mM Tris-HCI
pH 8.3, 2 mM EDTA, 0.5% sodium deoxycholate (DOC), 0.1 mg m1-1 aprotinin, 0.01 mg ml -~
leupeptin and 1 mM PMSF. After 10 rain on ice
the iysate was centrifuged 1 h at 100000 x g and
the supernatant was passed through 15 ml of Sepharose 4B then through 15 ml of immunosorbent 23D5 2H6. The immunosorbent was washed
by 100 ml of lysis buffer (protease inhibitors
omitted) followed by 200 ml of 50 mM Tris-HCI
buffer pH 8.3, containing 1 M NaCI. Elution was
performed with 2 volumes of 0.1 M diethylam-
ate buffer and the cells were fixed in 2% paraformaldehyde 0.1% glutaraldehyde in the same
buffer for 30 min at room temperature. The cells
were then resuspended for 1 h in a 0.1% solution
of saponin in 0.1 M cacodylate buffer; all further
incubations were performed with reagents diluted in this solution. The cells were then incubated for 4 h in a 1/20 dilution of ascitic fluid 24C6
1F1, followed by 3 washes of 15 min each. Then
the cells were resuspended in a 1/100 dilution of
peroxidase conjugated goat antimouse IgG immunoglobulins (Nordic) for 1 h. After 3 washes
of 15 min in PBS, the peroxidase activity was revealed by a 5 min incubation in 0,1 M Tris HCI
buffer pH 7.4 containing 0.05% diaminobenzidine HCI and 0.006% H20~. After washing, the
cells were fixed for 1 h in 1% glutaraidehyde in
196
0.1 M cacodylate buffer, then for 1 h in 1%. osmium tetroxide in the same buffer. They were
dehydrated in ethanol and e m b e d d e d in Epon.
Thin sections were observed uncontrasted in a
J E O L 120 CX electron microscope.
Results
Localization of the 126 kDa protein. The localization of the 126 kDa antigen in parasitized red
blood cells was at first investigated by IFA using
monoclonal antibody 24C6 1F1. In all cases, only
late stages containing a large aggregate of malarial pigment were labeled. On smears made with
unfixed cells, a mottled, irregular pattern was observed (Fig. lal; when the cells had been formalin fixed before drying, a well defined, peripheral pattern was observed, which either delineated
the schizont surface or outlined the individual
merozoites in the latest stages (Fig. lb). Free
merozoites, rings stages or trophozoites were not
labeled,
Fig. 1. Indirect immunofluorescence assa) with M c A b 24C0
1FI on P. falctparum schizont infested h u m a n erythroc}tes
unfixed (a) or formalin fixed (b) before drying. ]-he periphery
of the parasites is ~ell delineated m fixed parasites. Irregular
peripheral patterns are obtained on unfixed material.
Y
~
n
~n
t
Fig. 2. Immunoperoxidase detecuon of P 126 by McAb 2-tC~
IFI in P .tah'tparum schizont The electron dense deposit larro~) is located on the parasitophorous ~acuole surrounding
the parasite In: parasite nuclei). Bar represents 1 i-tin
Using electron microscopy the reaction products of the immunoperoxidase detection of McAb
24C6 1FI s,,ere found at the peripher 3 of schizonts, which confirmed the IFA findings (Fig. 2).
However. it was not possible to decide whether
the deposits were on the parasite surface, i n / h e
parasitophorous xacuole, or on the sacuolar
m e m b r a n e . Maurer's clefts were also labeled.
C o m p l e m e n t a r 3 data concerning the localization of the molecule were obtained by mild sa-
F~g. 3 Immunoperoxidase reaction on P..t~dctpurum schlzont
using an unrelated mouse monoclonal antibody, no label h
found on the organism (same scale as Fig. 21.
197
A
t
p
B
s
t
p
s
205
.0_ ll---t,
/
a
97.._,.
45---
29~
Fig. 4. Solubilization of P 126 by mild saponin lysis. A
[x~S]methionine labeled P. falciparum asynchronous culture
~as treated by 0.1c~ saponin for 5 min at 20°C. Untreated
parasites (tl, saponin treated parasites (p) or saponin lysis supernatant (s) were then analyzed by SDS-PAGE and fluorography before (A) or after (B) immunosorption with McAb
24Cb 1F1. P 126 is entirely recovered in saponin lysis supernatant (B,s) whereas most of the parasite proteins remain in
the parasite pellet [A.p). The apparent molecular weights ( ×
10 3) of standard proteins are given,
ponin lysis of [3-SS]methionine labeled parasites
followed by immunoprecipitation analysis of the
resulting 12000 x g pellet and supernatant. In the
conditions described in the Material and Methods section, the 126 kDa antigen was entirely recovered in the saponin supernatant whereas most
of the parasite proteins remained in the pellet
(Fig. 4).
Kinetics of biosynthesis of the 126 kDa protein.
Immunoprecipitation analysis of cells and media
samples taken hourly after a 30 min pulse showed
that the 126 kDa antigen did not change during a
6 h period after which its relative amount decreased gradually up to its complete disappearance from the cells 10 h after the end of the pulse.
The appearance and increase of the 50 kDa exoantigen was conversely observed in the culture
medium during the same period of time (Fig. 5).
Correlation between proteolysis of 126 kDa protein and merozoite release (Fig. 6). When purified
labeled schizonts were incubated either alone (almost no reinvasion could occur) or with fresh red
blood cells, no difference could be found between culture supernatants when immunoprecipitated with antibody 24C6 1F1. In both cases, a
large amount of 50 kDa exoantigen was detected,
which indicated that reinvasion was not required
for the processing of 126 kDa into its 50 kDa derivative (Fig. 6A,B).
When purified schizonts were lysed by one
freezing-thawing cycle, and then incubated in
normal culture conditions, no 50 kDa antigen was
found in the supernatant whereas a large amount
of 126 kDa antigen could still be immunoprecipitated after a 16 h incubation of the lysate. This
artificial release of the 126 kDa molecule into the
medium by schizont lysis did not lead to its processing into the 50 kDa antigen (Fig. 6C).
Identification of other processing fragments of the
126 kDa protein. Systematic screening of the
monoclonal antibodies raised against P. falciparum culture supernatant antigens led to the identification of two other antibodies which identified
a 126 kDa antigen by immunoprecipitation of labeled parasites and produced the same IFA pattern as 24C6 1F1. When assayed against 16 h
chase culture supernatants, one of these antibodies (23D5 2H6) immunoprecipitated the 50 kDa
exoantigen, whereas the other one (3E9 1 A l l )
reacted with another molecule, which had the
following characteristics: when S D S - P A G E was
performed without reducing agent, a single radioactive polypeptide of 73 kDa was observed; if
reducing agent was used (0.1 M dithiothreitol),
two radioactive components were present at 47
kDa and 18 kDa, respectively (Fig. 7).
198
PARASITIZED
.,--
TIME ( h o u r s ) o
RED
5
6
BLOOD
7
CELLS
8
9
--~-~--
10
11
0
MEDIUM
5
6
7
8
_.~
9
10 11
m
m m
205~
97~
66~
"-'- " e
45~
29~
Fig. 5. Kanetics of 120 to 50 kDa processing. An asynchronous culture of P. falciparum ~as pulse labeled for 31) min anth
[~SS]methionine follov~ed by an 11 h chase (time 0 is the end of labeling). Aliquots of parasittzed cells and culture medium were
taken houri}, immunosorbed with McAb 24C6 IFI and anal}zed by SDS-PAGE and fluorography. P 126 is found unchanged in
the parasites from time 0 to 6 h. then decreasing gradually and dnsappearing at 10 h. The 50 kDa fragment appears in the meduum
"7.h after the pulse and then increases conconutantl} with the decrease of P 126 m the cells. The apparent molecular ~eights ( .,, 10-31
of standard proteins are given.
Purification of the 126 kDa protein and production of rabbit antiserum. Monoclonal antibod~
23D5 2H6 was used for affinity purification.
Monoclonal antibody 24C6 1F1 had little affinity
for the native 126 kDa protein, which is the reason why it was always used with a denaturing immunosorption procedure, which could not be used
to purif3, the native molecule. A highly purified
preparation of the 126 kDa protein was obtained
after 2 cycles of affinity purification (Fig. 8).
lgGs from a rabbit immunized with purified 126
kDa protein were used to prepare an immunosorbent. This immunosorbent bound this latter
molecule and its 50 kDa and 73 kDa breakdown
products from culture supernatants (Fig. 7A;
analysis without reducing agent); upon reducing
conditions, four polypeptides were found at 126,
50.47 and 18 kDa (Fig. 7B).
Discussion
In an earlier report [1], we demonstrated that
a 50 kDa antigen was found in the medium after
the release-reinvasion stage of P, falciparum: a
50 kDa specific monoclonal antibody identified a
126 kDa molecule as the intracellular precursor
of the exoantigen.
The present stud}' shows that the 126 kDa molecule is located at the peripher}, of schizonts. IFA
data suggest that the molecule is not exclusively
a component of the parasitophorous vacuole
membrane since the merozoite periphery, is delineated. The remaining possibilities are thus either
the schizont membrane or the vacuolar contents.
Moreover, the irregular pattern obserxed when
cells are not fixed before du,'ing on slides, con-
199
A
1
205
..4.
B
2
2
1
2
,7
|
116
1
C
97_.
N
M
6 6 ._..
45__.. a l l B m
by fixation. Contrasting with the major surface
antigen of 195 kDa [4], the 126 kDa protein is entirely removed from parasites by a mild saponin
treatment. This also suggests that this protein is
not tightly bound to the schizont surface. Detection of the molecule in Maurer's clefts, which are
extensions of the parasitophorous vacuole is an
additional reason for us to consider this molecule
as a component of the parasitophorous vacuole.
Consistent with this conclusion is the fact that it
is not found by IFA on free merozoites or on
young intracellular stages of the parasite. We
m m
A
2
--
B
3
~
1
2
3
205~
116~
97,4-~
~
~
94~
66~
67~
29
Q
....
lID
_
i
45~
43~
Fig. 6. I m m u n o s o r p t i o n and SDS-PAGE-fluorograph} analysis of the 16 h chase culture medium of purified
[-~-~S]methionine labeled P. falciparurn schizonts incubated
alone (A) or with fresh red blood cells (B) or after lysis by
freezing-thawing (C). l m m u n o s o r p t i o n was performed with
i m m u n e h u m a n lg (1) or monoclonal antibody 24C6 IF'I (2)
immunosorbents. In A and B. the 50 kDa processing fragment is obtained (reinvasion which is almost absent from A is
not needed for 126 to 50 kDa processing). In C, P 126 is found
unchanged in the medium: when artificially released, the protein is not processed to its 50 k D a derivattve. In A and B, a
small a m o u n t of P 126 is found in the m e d i u m , probably coming from parasite death and lysis due to the purification procedure. The apparent molecular weights ( × 10 -3) of standard
proteins are given.
trasting with the clear delineation found on fixed
cells leads us to suggest that the molecule is not
membrane bound. When probed in the same
conditions, the major 195 kDa surface antigen of
schizonts shows the same pattern on fixed or unfixed specimens. This can be interpreted by the
fact that membrane antigens remain morphologically rather stable upon drying whereas soluble
components with little morphological support may
dry unevenly if they are not cross linked in situ
30~
29~
20 ~
20
14,4~
14,4~
Fig 7. Identification of the processing fragments of P 126 b.~
immunosorpuon of the 16 h chase medium of a [35S]methionme
labeled P. lalciparum culture with i m m u n o s o r b e n t s 23D5
2H611), 3EO IAIIc'2) and rabbit anti P 126(31, and SDSPAGE-fluorography analys~s in non reducing (A) or reducing
(BI conditions. A small a m o u n t of P 126 is immunosorbed in
all cases. Antibody 23D5 2H6 ident~fies a 5(1 kDa fragment in
AI and BI: antibody 3E9 I A I I identifies a 73 kDa component in non reducing conditions IAI and a 47 and an 18 kDa
c o m p o n e n t when reduced (B). Specific rabbit IgG identifies a
73 and a 50 kDa fragment in the first case {AI or a 50, a 4 "v'
and an 18 kDa fragment in reduced condihons IB) The apparent molecular ~e~ghts (× 1(]-~) of standard proteins arc
given.
200
205
,.•
116
"4"
66
b
''~
Q
45
29
Ftg. 8. Purification of P 126. Silver staining of SDS-PAGE
analysis of an aliquot of P 126 immunopurified on immunosorbent 23D5 2H6 (b) and of molecular weight markers (al.
Apparent molecular weights ( × 10-3) of protein standards are
gi,~en.
propose to name this molecule P 126.
However, as described before using McAb
24C6 1F1, culture supernatants contain a 50 kDa
fragment, not the 126 k D a antigen, which suggests that some processing occurs upon or after
schizont bursting. The incubation of lysed schizonts demonstrates that P 126 is highly stable
when artificially released in the medium since it
can still be found 16 h later and, more important,
no 50 kDa is found in those conditions. Therefore, the 126 to 50 k D a transformation is probably not due to proteolysis of P 126 after release
in the culture medium. The processing of that
molecule is more likely associated with the natural release of merozoites.
Other reports on proteolytic events occurring
upon Plasmodium merozoite release have been
published: in P. falciparum, the 195 k D a schizont
surface antigen is processed to 83, 42 and 19 kDa
antigens located on the merozoite surface [2,3], a
similar maturation occurs in P. knowlesi [5].
Other authors have shown that protease inhibitors could block merozoite release [7]. We suggest that the 126 to 50 kDa transformation is another expression of a proteolytic p h e n o m e n o n
which might be responsible at least in part for
schizont bursting and merozoite release.
As a marker of the merozoite release stage, the
processing of the 126 kDa protein to its 50 kDa
derivative provided us with a very precise probe
to analyze the biosynthesis of P 126 in the schizonts. We could work with an asynchronous culture, which is optimal for parasite growth, and
were able to obtain more precise results than by
using a synchronous culture in which the parasites are distributed into a rather large window.
Taking into account that the 126 to 50 kDa processing occurs at the very end of the cycle, our results show that P 126 is synthesized during about
4 h, between the 32nd and 36th hour of a 42 h
cycle (which is the mean cycle duration of FCR 3
in our working conditions). Since P 126 is only
detected in the parasitophorous vacuole, it must
be translocated rapidly from its biosynthesis site
to its definitive location. Its intracellular traffic
and possible precursor remain to be studied.
The characterization of P 126 had followed the
discovery of a 50 kDa fragment in the medium
release-reinvasion events. By screening other
monoclonal antibodies raised against supernatant
antigens, we have identified one hybridoma which
produced the same IFA pattern, which immunoprecipitated a 126 kDa antigen in the schizonts.
and a 73 kDa (non reducing conditions), or a 47
and an 18 kDa antigen (reducing conditions) in
the culture medium. In our previous paper, we
had identified a 47 kDa antigen which followed
the same variations as the 50 kDa one, i.e. appearing after release-reinvasion in a synchronized culture and then decreasing [1]. We were
using only reducing conditions in that work and
the 18 kDa which is in all cases less strongly labeled than the others was left unnoticed. These
data, together with the ones provided by McAb
3E9 1 A l l suggested that the 73 kDa (or 47+18
201
kDa) antigen was another processing fragment of
P 126.
This was further demonstrated by purifying
P 126 and obtaining a rabbit antiserum against this
molecule: IgGs from this rabbit recognized P 126
and the 50, 73 (or 47+18) kDa antigens in culture supernatants and no other significant polypeptide. This showed that the 73 kDa protein is
another fragment of P 126 and that the processing of this later molecule gives rise to at least three
pol}jgeptides, two of which being held together bv
disulfide bridges.
This suggests that the proteolysis of P 126 occurs at least on two different sites of the native
molecule. Further studies are needed to know
whether the 50 and the 73 kDa polypeptides
match the entire sequence of P 126 or whether
there are any overlaps or missing peptides.
This identification of P 126 and of its cleavage
products opens the way towards the analysis of the
proteolytic activity which is responsible for this
processing and we believe that this work may
bring information on the mechanism of merozoite release by malaria infected red cells.
Acknowledgements
The authors are indebted to C. Ansel, I.
Briche, A. Loyens, M. Masurelle, M. Mortuaire
for expert assistance. This work ~,,as funded by
INSERM (France) and Rhrne Mrrieux.
References
I Delplace. P., Dubremetz, J F., Fortier, B. and Vernes. A.
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