113
Molecular and Biochemical Parasitology, 46 (1991) 113-122 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100045U MOLBIO 01515
Localization and stage specific phosphorylation of Plasmodiumfalciparum phosphoproteins during the intraerythrocytic cycle Bernd W. Suetterlin, Barbara Kappes and Richard M. Franklin Biocenter, Department of Structural Biology, Basel, Switzerland (Received 24 September 1990; accepted 22 November 1990)
Fifty-ninePlasmodiumfalciparum specific phosphoproteins with molecular weights between 15 000 and 192 000 were analyzed by SDS-PAGE and two-dimensional gel electrophoresis. 40 phosphoproteins were identified by [7-32p]ATPlabeling of cell lysates, 19 by [32p]orthophosphate labeling of parasitic cultures in vivo. Changes in the phosphorylation pattern during the infectious erythrocytic cycle were determined for all proteins. In parallel, cell fractionation studies were done to follow up possible changes in the cellular distribution of these proteins. Several phosphoproteins are associated with the membrane fraction of infected erythrocytes. One pair of proteins of approx. 88 kDa and a pI of about 5.0 was further characterized. Both proteins are located in the parasitic fractions as well as in the membrane of infected erythrocytes during the entire cycle. Phosphorylation of these proteins, however, is restricted to the trophozoite and schizont stages. Peptide mapping studies demonstrated that both proteins are identical with the exception of minor modifications which are probably not the result of differences in phosphorylation. Key words: Malaria; Plasmodiumfalciparum; Phosphoprotein
Introduction
• Proteins undergo a variety of different posttranslational covalent modifications [ 1]. One of the few reversible processes, phosphorylation and dephosphorylation, plays an essential role in regulating biochemical pathways. Very little is known about the significance of such phosphorylation and dephosphorylation events during the parasitic cycle of malaria. To date, relatively little has been published concerning the phosphoproteins and protein kinases of any malaria species. A cAMP-dependent protein kinase of Plasmodium falciparum has been partially characterized [2]. This enzyme phosphorylates some cytosolic proteins (80, 54, 51, 31.5 kDa). Besides this, a Ca2+-dependent phosphorylation of Correspondence address: R.M. Franklin, Department of Structural Biology, Biocenter, Klingelbergstrasse 70, CH4056 Basel, Switzerland.
Abbreviations: Mes, 4-morpholineethanesulfonic acid; RESA, ring-infected erythrocyte surface antigen.
five proteins (195, 158, 51, 47.5, 15 kDa) has been found in the parasitic membrane fraction [2]. Also, one cytosol specific protein kinase of the murine strains Plasmodium berghei and Plasmodium chabaudi could be identified as a type I casein kinase [3]. Cytosolic phosphoproteins of these two strains [4,5] and several parasitic phosphoproteins of P.falciparum have been described [5,6]. Phosphorylation and dephosphorylation processes also seem to play an important role during the merozoite invasion of [7] and the growth cycle in [8] red blood cells (RBCs). Amongst various general changes of the RBC membrane structure [9], malarial infection is accompanied by an altered phosphorylation pattern of major components of the erythrocytic skeleton [10-12]. New proteins derived from the parasites are phosphorylated in the membrane of erythrocytes infected with P. berghei [ 13-16] or P. chabaudi [ 14,17]. Furthermore, the major surface antigens of P. falciparum are known to be phosphoproteins [ 18-20]. None of the published reports were sufficiently detailed, however, to allow a definitive listing of the phosphoproteins found in the asexual erythro-
114 cytic cycle of P.falciparum. Therefore, we decided to investigate this problem. In this report we present a number of new P. falciparum specific phosphoproteins. Cellular fractions were analysed in order to determine the cellular localization of these proteins. Several new proteins were found to be associated with the infected host erythrocyte membrane. The stage specific phosphorylation of all proteins was studied. Materials and Methods
Parasite culture and synchronization. The P.falciparum K 1 strain, adapted to grow in horse serum, was kindly provided by H. Matile (F. Hoffmann-La Roche). The parasites were cultured in RPMI-1640 medium (Gibco) supplemented with 25 mM Hepes/0.37 mM hypoxanthine/100 mg 1-1 neomycin base/10% heat-inactivated horse serum (Amimed). The cultures were set up with a 5% hematocrit and incubated at 37°C with 4% CO2/3% 02/93% N: and 94% relative humidity. Human A + erythrocytes were obtained from the Basel Blood Donation Center. For stage-specific examinations, the parasites were synchronized 3 times according to Lambros and Vandenberg [21]. The sorbitol treatment resulted in a parasitic age difference of less than 4 h. The parasitemia and the stage of parasitic development was monitored by microscopic observation of Giemsa-stained blood smears.
quots (-80°C) in TB (20 mM Tris buffer, pH 7.0) containing a mixture of protease inhibitors (30/z~ ml 1 soybean trypsin inhibitor, Merck; 20/zg m l leupeptin, Boehringer; 50 /xg ml -I aprotinin, Sigma; 50 /~M phenylmethylsulfonyl fluoride, Sigma; 1 mM benzamidine, Sigma; 5 mM iodoacetamide, Merck). A membrane fraction of uninfected erythrocytes (control) was purified according to the same procedure. The isolated parasites were washed twice in SSC and resuspended in a 10- to 100-fold volume of TB. After homogenization for 10 min in a Potter-E1vehjem tissue grinder, the parasites were centrifuged (10 min, 13 000 x g). The pellet (parasitic membrane plus organelle fraction) was washed three times with SSC and frozen as mentioned above. The parasitic cytosol was clarified from the 13 000 x g supernatant by a second centrifugation (60 min, 100 000 x g). The protein concentrations were determined according to the procedure of Heinzel et al. [22] using BSA (fraction V, Boehringer) as a standard protein. In both parasitic fractions the activity of acetylcholinesterase (EC 3.1.1.7), a marker enzyme of the erythrocytic membrane, was measured [23]. The parasitic membrane and organelle fraction had a negligible contamination with erythrocytic membrane ghosts (less than 3%), and no acetylcholinesterase activity could be detected in the parasitic cytosol.
Protein Parasite isolation and preparation of parasitic proteins and proteins of the erythrocytic membrane. Parasite cultures were grown up to a parasitemia of about 10-15%. Infected and uninfected erythrocytes were harvested by centrifugation (10 min, 700 x g, 4°C) and washed twice with PBS (phosphate-buffered saline, pH 7.2). The culture was lysed by incubation in a 10-fold volume of 0 × 0.015% saponin in SSC (150 mM NaC1/15 mM sodium citrate, pH 7.0) for 20 min at 4°C. Released parasites were concentrated by centrifugation (15 min, 1 500 x g, 4°C). Host cell membranes were harvested from the 1 500 x g supernatant by centrifugation (20 min, 19 500 × g, 4°C) and washed 4-5 times in 30-ml aliquots of S S C until a clear white pellet was obtained. The membranes were finally stored as frozen ali-
phosphorylation. Parasitic cultures (about 10% parasitemia) were preincubated in phosphate-free medium (Amimed) for 4 h. The erythrocytes were adjusted to a 10% hematocrit and phosphorylated for 1.5 h with 0.5 mCi ml -~ [32p]orthophosphate. The parasites were subsequently isolated and the protein fractions prepared as described above. Control studies demonstrated no changes in parasite development after growing in phosphate-free medium for about 6 h. For the [y-32p]ATP phosphorylation of cell lysates we tested a variety of different labeling conditions (see Results). Optimal labeling was obtained by incubating in the presence of 10/zM [732p]ATP (5-10 Ci mmol -~) and 15 mM MgCI2 for 15 min at 30°C. The reaction was stopped by the addition of 1 vol. of double strength gel electrophoresis sample buffer (see below).
115
Protein analysis. Phosphoproteins were analyzed by SDS-PAGE [24]. Electrophoresis was carried out with 0.8 m m slabs of 7.5, 10 and 12.5% acrylamide for 3 h (max. 35 mA, 220 V). Standard marker proteins (Bio-Rad) were used to determine the individual molecular weights. Two-dimensional gel electrophoresis was carfled out according to the procedure of O'Farrell [25], with minor modifications. Gels were cast using an ampholine mixture composed of 2 parts ampholine 3-10 (LKB) and one part 4-6 (LKB). 20 mg solid urea was dissolved per 30/.d of protein sample, and finally 1 vol. oflEF sample buffer was added prior to the electrophoretic separation. PI calibration proteins (LKB) as well as several individual proteins of known pI (soybean trypsin inhibitor, Merck, pI = 4.55; carbonic anhydrase II/bovine, Sigma, pI = 5.9; carbonic anhydrase I/human, Sigma, pI = 6.6) were used as markers. 7.5 % polyacrylamide gels were employed in the second dimension. Following electrophoretic separation, the gels were stained either with silver [26] or Coomassie R-250 [27], dried, and exposed to X-ray autoradiographic film (Kodak X-OMAT AR).
Peptide mapping. The proteins of interest were
excised from 7.5% preparative polyacrylamide gels (1.5 mm) and electroeluted into 100 mM (NH4)HCO3, pH 8.3/0.1% SDS for 18 h (max. 40 mA, 20 V). The eluate was lyophilized, washed three times with acetone/H20 (9:1,4°C) to remove SDS, and dried. The pellet was resuspended in IEF sample buffer, separated by 2-dimensional gel electrophoresis, and stained with Coomassie R250. Specific proteins were excised and transferred to a 0.8 mm, 15% SDS-polyacrylamide gel. 10/xl of a given protease - Staphylococcus aureus V8 (ICN Blochemlcals, 2/xg ml ), papaln (Sigma, 20/xg -1 • -1 ml ), or t~-chymotrypsln (Merck, 100/~g ml ) was overlaid into each slot. Peptide mapping by limited proteolysis was then carded out [28]. •
•
--1
•
•
Results
P.falciparum specific phosphoproteins were analyzed by ~3(-32p]ATP labeling of cell lysates in vitro and [~'P]orthophosphate labeling of living parasites in culture. Parasites of 4 different stages were isolated at different times after infection in order to determine the stage specific protein distribution: ring stage parasites (age of 7 + 2 h), young trophozoites (22 + 2 h), old trophozoites (33 + 2 h), and schizonts (42 + 2 h). Four cellular fractions
kDa
97.4 66.2
42.7 m
31.0
A
B
Fig. 1. Autoradiographs of P.falciparum specific phosphoproteins labeled with [7-32p]ATP (A) or [32p]orthophosphate (B) and separated on 10% SDS gels. Shown is the cellular localization (C, uninfected erythrocytic membrane as control; I, membrane of parasitized erythrocytes, II, parasitic 13000 x g pellet; III, parasitic cytosol) of the proteins from different stages (R, ring; YT, young trophozoite; OT, old trophozoite; S, schizont stage). The same amount of protein lysate (10 I.tg) was applied to each lane. Arrow heads indicate the approx. 88-kDa proteins as described in the text.
116
TABLEI
SummaryofPlasmodium~lc~arumspecificphosphoproteinslabeledwith[~32p]ATP Size (kDa)
Infected
Parasitic fractions
RBC membrane R
YT
OT
13000 z g pellet S
R
YT
OT
Cytosol S
R
YT
OT
S
192.0 184.0 178.0 169.0
160.5 156.0 154.0 144.0 131.0 123.0 108.5 102.5 88.0 80.5 77.5 74.0 72.5 65.5 63.0 56.5 54.5 48.0 45.0 40.0 38.5 37.5 36.0 34.5 32.5 32.0 31.0 29.0 26.5 26.0 24.0 21.5 20.0 18.0 16.5 15.0
*
*
*
*
*
* * *
*
*
*
The phosphoproteins were analyzed from autoradiographs of SDS gels of acrylamide concentrations which varied from 7.5 to 12.5%. The cellular fractions were prepared from the following stages of the erythrocytic growth cycle: R, ring stage; YT, young trophozoites; OT, old trophozoites; S, schizonts. The star (*) indicates the specific phosphorylation pattern of each protein. This data is compiled from 3 separate experiments and all the results were consistent within these experiments.
were prepared from parasites of each stage in order to localize the proteins and to detect possible changes in the cellular distribution during the parasitic life cycle.
To establish the optimal conditions for protein kinase activity in our [~/-3zp]ATP labeling experiments, we tested a variety of buffer systems at 2 concentrations (20, 50 mM) and with different pH
117 TABLEII
SummaryofP.~lc~arums~cificphosphoproteinslabeledwith[32p]o~hophosphate Size (kDa)
Infected RBC membrane R
YT
OT
Parasitic fractions 13000 × g pellet S
R
YT
OT
Cytosol S
R
YT
OT
S
88.0 64.0 53.5 51.5 41.5 40.5 36.5 34.0 32.5 30.0 27.5 26.5 25.0 24.0 22.0 19.5 17.5 16.5 Legend: see Table I. optima (phthalate/NaOH, pH 5.0; Mes/NaOH, pH 6.0; KH2PO4/NaOH, pH 7.0; Tris/HC1, pH 7.0; Tris/HC1, pH 8.0). The addition of several divalent • 2+ 2+ 2+ • cations (Mg , Mn , or Zn ) was studied at concentrations between 10 and 60 mM. Furthermore, in some experiments we tested detergents (Triton X-100, Triton X-114, and NP-40, all at 1.0%) to enhance the solubility of some proteins. In some experiments we also used phosphatase-inhibitors ([3glycerolphosphate at 10, 40, 80 mM; (ortho)vanadate at 50, 100 ~tM; NaF at 40 mM; and EDTA at 10 mM). All labeling reactions were carried out for 15 min, incubating 10 ~tg of parasitic cell lysate, 10 ~tM [7-32p]ATP (5-10 Ci mmo1-1) at 0°C or 30°C, plus various combinations of the above-mentioned reagents. With our definitive [T-32p]ATP labeling conditions (see Materials and Methods) we were able to distinguish 40 P. falciparum specific phosphoproteins on autoradiographs from SDS gels in which the acrylamide concentration varied from 7.5 to 12.5%. With all other labeling conditions only some of the proteins were phosphorylated and no other bands appeared. Fig. 1A shows an autoradiograph of a 10% acrylamide gel used for the
analysis of the cellular and stage specific phosphorylation of proteins in the molecular weight range between 40 and 100 kDa. The complete list of all proteins identified by this means is presented in Table I. Only those protein bands which were clearly distinguishable by SDS-PAGE are summarized. Most of these [T-32p]ATP-labeled phosphoproteins were found exclusively in the parasitic fractions: 18 in the parasitic cytosol, 7 in the parasitic 13 000 x g pellet, and another 7 in both fractions. Chiefly in the parasitic cytosol the degree of phosphorylation increased rapidly and several new bands appeared during the transition from young to old trophozoites (Fig. 1A). 8 new proteins were phosphorylated in the RBC membrane fraction of the parasitized cells, as compared with that of the uninfected RBC membrane. 7 of these proteins could, however, also be detected in other parasitic fractions. Only one, with a molecular weight of about 156 kDa, was exclusively labeled in the erythrocytic membrane of ring stage infected erythrocytes. 19 phosphoproteins could be characterized after 32 . . . . [ P]orthophosphate labehng in VlVO (Fig. 1B,
118
clR
YTOT
s
kDa
97.4 m 66.2 42.7
31.0
m
21.5 m
14.4
....
Fig. 2. Stage-specific distribution of [32p]orthophosphate- labeled proteins associated with the membrane of infected erythrocytes: membrane fraction of erythrocytes infected with ring (R), young trophozoite (YT), old trophozoite (OT), and schizont (S) stage parasites (C, uninfected erythrocytic membrane as control)• The same amount of protein (20 ~tg) was applied to each lane. Shown is the autoradiograph of a 12.5 % acrylamide gel. The arrowhead indicates the approx. 41.5-kDa protein specifically labeled in the ring and schizont stages.
Table II). 15 of these proteins were exclusively located in the parasitic fractions (cytosol and/or 13 000 × g pellet). Whereas about 10 of these proteins were phosphorylated throughout the entire cycle, 5 were only phosphorylated during the late stages (old trophozoites and schizonts), i.e., at a time when mitosis and the condensation of the newly formed merozoites takes place. 4 proteins could be detected in the parasitic fractions as well as in the infected erythrocytic membrane and had a different stage specificity from the phosphoproteins mentioned above• One protein, of approx• 41.5 kDa, was only phosphorylated in schizonts and the early ring stage (Fig. 2). The phosphorylation of the other 3 proteins was first detectable in young trophozoites and remained detectable until the end of the cycle.
Only one prominent double band, of approx• 88 kDa, had an identical phosphorylation pattern using both labeling methods (Fig. 1A, B). Both proteins (pf 88-1; pf 88-2) could be identified on silverstained 2D-gels and corresponding autoradiographs as 2 neighboring protein spots (Fig. 3A-C). Their pI is about 5.0. Due to the higher resolution of 2D gels and the different solubilities of some proteins in urea, 10 additional proteins were found to be labeled by both [y-32p]ATP and [32p]orthophosphate (Fig. 3, Table III). So far, we were not able to make a precise correlation of these labeled proteins with the bands identified by SDS-PAGE. The approx• 88-kDa proteins are located in the infected erythrocytic membrane as well as in both parasitic fractions. The phosphorylation is restricted to the young and old trophozoite and the schizont stages (Fig. 1), whereas the proteins (unphosphorylated) are already present during the ring stage (Fig. 4). According to peptide mapping studies with several proteases, both proteins had identical peptide patterns, with the exception of a few small fragments (Fig. 5). The same peptides are ~2hosphorylated in the two proteins, both with [yP]ATP (not shown) or [32p]orthophosphate (Fig. 5). Discussion
The stage-dependent phosphorylation of P. falciparum specific phosphoproteins was studied during the asexual erythrocytic cycle• More phospho• protems were found by [T- P]ATP labeling of cell 32 • . lysates than by [ P]orthophosphate labelmg of intact cells. As a result of our [y-32p]ATP labeling conditions, all these phosphoproteins should be substrates of Ca 2+- or cAMP-independent protein kinases. Whether some of the proteins are themselves kinases has not yet been determined. The addition of phosphatase inhibitors prior to or during the labeling reaction had only slight effects. Only a few proteins were phosphorylated to a higher degree and no new labeled proteins could be detected• Under our in vitro labeling conditions the balance between phosphorylated and dephosphorylated forms has probably shifted to the phosphorylated form, even without using any phosphatase inhibitors. These distinct [T-32p]ATP labeling conditions may help to explain the different protein 32
•
119 pI 3.75
4.55
5.2
6.0
3.75
4.55
5.2
6.0
3.75
I
I
I
I
I
t
i
I
i
ko~
~
~ S
C
4.55
5.2
6.0
i
i
41!
200.0--
116.3
4
--
97.4--
66.2--
42.7--
Fig. 3. Protein phosphorylation patterns on 2D gels of [7-32p]ATP labeled parasitic lysates and [32P]orthophosphate labeled malarial cultures. (A) Silver-stained 2D-gel; (B) corresponding autoradiograph of the [TJ2p]ATP labeled extracts; (C) autoradiograph of [3Zp]orthophosphate labeled cultures (the silver-stained gel of this autoradiograph is not shown). 7.5% acrylamide gels were used for the second dimension. The approx. 88-kDa phosphoprotein spots (pf 88-1, pf 88-2) corresponding to the 88-kDa double band seen on SDS-PAGE (Fig. 1) are marked with arrowheads. The 10 additional phosphoproteins common to both labeling methods are indicated by arrows. TABLE III
P.falciparum specific phosphoproteins labeled by both [~/-32p]ATPand [32P]orthophosphate Protein
Size (kDa)
pI
1 2/3 4 5 6 7 8 9 10 11 12
97.5 88.0 84.0 77.5 76.0 71.0 56.0 45.5 41.5 32.5 31.5
5.0 5.0 5.0 5.4 5.1 5.0 5.0 4.9 5.0 4.9 4.6
The proteins were analyzed on 2D-gels as shown in Fig. 3.
patterns obtained in lysates ([7•P]ATP) h o s pandh ointact r yparasites l a t i o([32p]orthophosn phate). On the other hand, the rupture of intracellular barriers, such as intracellular membranes, during the preparation of in vitro lysates could lead to unnatural contacts between kinases and substrates, probably resulting in additional, artifactual phosphorylation of some proteins. Thus a nonspecific phosphorylation of non-target proteins cannot be excluded under these conditions. A further explanation for the fact that fewer proteins were phospho-
rylated during in vivo labeling could be the relatively short labeling period used there. This was done in order to examine the stage-specific activity of the kinases as accurately as possible. Some phosphoproteins of our parasitic fractions are very similar in their molecular weights to P.falciparum phosphoproteins already described. Unfortunately, the published labeling conditions are usually different from ours. Furthermore, the similar molecular weights, invariably the only information available, do not enable us to draw any firm conclusions concerning the possible identity of phosphoproteins described by us and by other workers. Nevertheless there are 2 proteins which we shall mention. One protein (approx. 156 kDa) detected in the membrane of ring infected erythrocytes corresponds in molecular weight and stage specificity to RESA [20]. The approx. 88-kDa proteins (pf 88-1, pf 88-2) show similarities to a 85kDa protein which is in the phosphorylated state in the membrane of trophozoite/schizont-infected erythrocytes [11]. The present investigation of plasmodial phosphoproteins clearly supports the assumption that phosphorylation and dephosphorylation processes play a major role in parasitic development as well as in malaria-specific alterations of the erythrocytic
120
pI
5.2
4.55
5.2
I
I
1
I
A
kDa
97.4
4.55
--
_
B
-,,-t
66.2 --
~
•
z! Fig. 4. Two-dimensional distribution of parasitic, ring-stage specific proteins. (A) Segment of a silver-stained 2D-gel; (B) corresponding autoradiograph of the [32p]onhophosphate labeled proteins. 7.5% acrylamide gels were used for the second dimension. In the silver-stained gel both approx. 88-kDa proteins (pf 88-1, pf 88-2) are indicated by arrow heads (A); the autoradiograph (B) demonstrates that these proteins are not phosphorylated. For orientation, two other phosphoproteins are marked by arrows.
membrane during infection. At the transition from young to old trophozoites, a time when DNA replication and mitosis takes place, the degree of phosphorylation increased considerably and several new proteins were phosphorylated, chiefly in the parasitic cytosol. As in other eukaryotes, protein kinases are probably involved in the regulation of plasmodial proliferation and differentiation processes, especially in the above-mentioned DNA replication and mitosis. Several phosphoproteins were associated with the membrane fraction of infected erythrocytes. With the exception of the approx. 156-kDa protein, all these proteins had corresponding bands in parasitic fractions, suggesting that these proteins are synthesized by the parasite and transported to the erythrocytic membrane. The approx. 156-kDa protein, if identical with RESA, would also be of parasitic origin. The stage specific phosphorylation pattern of the individual membrane associated proteins during the entire erythrocytic cycle demonstrates the permanent parasitic influence on the surrounding erythrocyte. Some proteins, especially the approx. 41.5-kDa protein phosphorylated only in the membrane fraction of schizont and ring stage infected erythrocytes, could, furthermore, be involved in the merozoite invasion. Although the func-
tional role of the phosphoproteins remains unknown, they seem to be important members in the connection between erythrocytes and parasites. Therefore, from a medical point of view, they might be interesting targets in the development of antimalarial agents. Let us now turn to our analysis of the approx. 88 kDa proteins (pf 88-1, pf 88-2). They have identical cellular distributions and the same stage specific phosphorylation. The addition of ~-glycerolphosphate (80 mM) during our [7-32p]ATP labeling assay resulted in an increased phosphorylation of this pair, probably underlining the involvement in a regulatory process which may start with their phosphorylation at the transition from the ring to the young trophozoite stage. Only minor differences were found in the peptide maps of these two proteins. None of the divergent fragments were phosphorylated, suggesting that other modifications (e.g., glycosylation, acetylation) of these otherwise identical proteins might be taking place. The precise biochemical nature of these modifications is now under investigation. Many proteins described in this paper must be important during the malarial growth cycle since they are only phosphorylated during the period of biosynthesis of macromolecules (see Tables I and
121
I kDa 97.4
11
1
2
66.2 w
V8
2
1
2
ch
1
2
w
42.7
|'=
ii
iiii~ili!i¸
i~ .....
!i~i ¸¸
~il ~
~
31.0 w v
•
~
0
I
•
e
•
o
21.5
14.4 m
f
°
i~
.... ~ ~
O
i¸ S
A
S
A
Fig. 5. Peptide maps of the [32 P]orthophosphate-labeled approx. 88-kDa proteins (1: pf 88-1,2: pf 88-2). Both proteins, isolated from 2D gels, were digested with S. aureus V8 protease (I) or ~-chymotrypsin (II). A third digest with papain, supporting the same conclusions, is not shown. S, silver-stained digest; A, corresponding autoradiograph; V8, protease control digest; ch, ¢~-chymotrypsin control digest. Small differences in the peptide pattern are indicated by arrows. II). F u r t h e r m o r e , a discrete n u m b e r o f proteins can be identified by both labeling m e t h o d s (Table III). W e are n o w cloning selected genes w h i c h code for s o m e o f the p h o s p h o p r o t e i n s in the hope o f learning m o r e about the function of these proteins.
Acknowledgements W e thank Dr. G e o r g e T h o m a s for helpful discussions and, together with Heidi Lane, the critical reading o f the manuscript. This w o r k was supported in part by funds f r o m the Canton Basel-Stadt and the Fonds National Suisse (Grant N u m b e r 319098.87/2).
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122 larial parasite Plasmodiumfalciparum. Biochim. Biophys. Acta 1053, 118-124. 7 Rangachari, K., Dluzewski, A., Wilson, R.J.M. and Gratzer, W.B. (1986) Control of malarial invasion by phosphorylation of the host cell membrane cytoskeleton. Nature 324, 364-365. 8 Yuthavong, Y. and Limpaiboon, T. (1987) The relationship of phosphorylation of membrane proteins with the osmotic fragility and filterability of Plasmodium berghei-infected mouse erythrocytes. Biochim. Biophys. Acta 929,278-287. 9 Howard, R.J. (1982) Alterations in the surface membrane of red blood cells during malaria. Immunol. Rev. 61, 67107. 10 Chaimanee, P. and Yuthavong, Y. (1986) Characteristics of membrane protein phosphorylation in Plasmodium berghei-infected mouse erythrocytes. J. Protozool. 33,446454. 11 Murray, M.C. and Perkins, M.E. (1989) Phosphorylation of erythrocyte membrane and cytoskeleton proteins in cells infected with Plasmodium falciparum. Mol. Biochem. Parasitol. 34,229-236. 12 Lustigman, S., Anders, R.F., Brown, G.V. and Coppel, R.L. (1990) The mature-parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum associates with the erythrocyte membrane skeletal protein, band 4.1. Mol. Biochem. Parasitol. 38,261-270. 13 Wiser, M.F., Wood, P.A., Eaton, J.W. and Sheppard, J.R. (1983) Membrane-associated phosphoproteins in Plasmodium berghei-infected murine erythrocytes. J. Cell Biol. 97,196-201. 14 Wiser, M.F., Leible, M.B. and Plitt, B. (1988) Acidic phosphoproteins associated with the host erythrocyte membrane of erythrocytes infected with Plasmodium berghei and P. chabaudi. Mol. Biochem. Parasitol. 27, 11-22. 15 Wiser, M.F. (1988) Phosphorylation of Plasmodium berghei-derived phosphoproteins associated with the host erythrocyte membrane by a spectrin kinase. Mol. Cell. Biochem. 84, 51-57. 16 Wiser, M.F., Sartorelli, A.C. and Patton, C.L. (1990) Association of Plasmodium berghei proteins with the host erythrocyte membrane: binding to inside-out vesicles. Mol. Biochem. Parasitol. 38,121-134. 17 Wiser, M.F. (1987) Phosphoproteins associated with the
18
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27
28
host erythrocyte membrane during Plasmodium chabaudi infection. In: Host-parasite Cellular and Molecular Interactions in Protozoal Infections (Chang, K.P. and Snary, D., eds.), pp. 315-320, NATO ASI Series, Springer-Verlag, Heidelberg. Howard, R.F., Stanley, H.A. and Reese, R.T. (1988) Characterization of a high-molecular-weight phosphoprotein synthesized by the human malarial parasite Plasmodiumfalciparum. Gene 64, 65-75. Coppel, R.L., Lustigman, S., Murray, L. and Anders, R.F. (1988) MESA is a Plasmodiumfalciparum phosphoprotein associated with the erythrocyte membrane skeleton. Mol. Biochem. Parasito|. 31,223-232. Foley, M., Murray, L.J. and Anders, R.F. (1990) The ringinfected erythrocyte surface antigen protein of Plasmodium falciparum is phosphorylated upon association with the host cell membrane. Mo|. Biochem. Parasitol. 38, 6976. Lambros, C. and Vandenberg, J.P. (1979) Synchronization ofPlasmodiumfalciparum erythrocytic stages in culture. J. Parasitol. 65, 418-420. Heinzel, W., Vogt, A., Kallee, E. and Faller, W. (1965) A new method for the quantitative determination of antibody and antigen protein, with a sensitivity of five micrograms. J. Lab. Clin. Med. 66,334-343. Whittaker, M. (1984) Cholinesterases. In: Methods of Enzymatic Analysis, 3rd ed. (Bergmeyer, H.U., ed.), Vol. 4, pp. 52-74, Verlag Chemie, Weinheim. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685. O'Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 40074021. Ansorge, W. (1985) Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J. Biochem. Biophys. Methods 11, 13-20. Harlow, E. and Lane, D. (1988) Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Cleveland, D.W., Fischer, S.G., Kirschner, M.W. and Laemmli, U.K. (1977) Peptide mapping by 'limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102-1106.
Molecular and Biochemical Parasitology, 46 (1991) 113-122 © 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100045U MOLBIO 01515
Localization and stage specific phosphorylation of Plasmodiumfalciparum phosphoproteins during the intraerythrocytic cycle Bernd W. Suetterlin, Barbara Kappes and Richard M. Franklin Biocenter, Department of Structural Biology, Basel, Switzerland (Received 24 September 1990; accepted 22 November 1990)
Fifty-ninePlasmodiumfalciparum specific phosphoproteins with molecular weights between 15 000 and 192 000 were analyzed by SDS-PAGE and two-dimensional gel electrophoresis. 40 phosphoproteins were identified by [7-32p]ATPlabeling of cell lysates, 19 by [32p]orthophosphate labeling of parasitic cultures in vivo. Changes in the phosphorylation pattern during the infectious erythrocytic cycle were determined for all proteins. In parallel, cell fractionation studies were done to follow up possible changes in the cellular distribution of these proteins. Several phosphoproteins are associated with the membrane fraction of infected erythrocytes. One pair of proteins of approx. 88 kDa and a pI of about 5.0 was further characterized. Both proteins are located in the parasitic fractions as well as in the membrane of infected erythrocytes during the entire cycle. Phosphorylation of these proteins, however, is restricted to the trophozoite and schizont stages. Peptide mapping studies demonstrated that both proteins are identical with the exception of minor modifications which are probably not the result of differences in phosphorylation. Key words: Malaria; Plasmodiumfalciparum; Phosphoprotein
Introduction
• Proteins undergo a variety of different posttranslational covalent modifications [ 1]. One of the few reversible processes, phosphorylation and dephosphorylation, plays an essential role in regulating biochemical pathways. Very little is known about the significance of such phosphorylation and dephosphorylation events during the parasitic cycle of malaria. To date, relatively little has been published concerning the phosphoproteins and protein kinases of any malaria species. A cAMP-dependent protein kinase of Plasmodium falciparum has been partially characterized [2]. This enzyme phosphorylates some cytosolic proteins (80, 54, 51, 31.5 kDa). Besides this, a Ca2+-dependent phosphorylation of Correspondence address: R.M. Franklin, Department of Structural Biology, Biocenter, Klingelbergstrasse 70, CH4056 Basel, Switzerland.
Abbreviations: Mes, 4-morpholineethanesulfonic acid; RESA, ring-infected erythrocyte surface antigen.
five proteins (195, 158, 51, 47.5, 15 kDa) has been found in the parasitic membrane fraction [2]. Also, one cytosol specific protein kinase of the murine strains Plasmodium berghei and Plasmodium chabaudi could be identified as a type I casein kinase [3]. Cytosolic phosphoproteins of these two strains [4,5] and several parasitic phosphoproteins of P.falciparum have been described [5,6]. Phosphorylation and dephosphorylation processes also seem to play an important role during the merozoite invasion of [7] and the growth cycle in [8] red blood cells (RBCs). Amongst various general changes of the RBC membrane structure [9], malarial infection is accompanied by an altered phosphorylation pattern of major components of the erythrocytic skeleton [10-12]. New proteins derived from the parasites are phosphorylated in the membrane of erythrocytes infected with P. berghei [ 13-16] or P. chabaudi [ 14,17]. Furthermore, the major surface antigens of P. falciparum are known to be phosphoproteins [ 18-20]. None of the published reports were sufficiently detailed, however, to allow a definitive listing of the phosphoproteins found in the asexual erythro-
114 cytic cycle of P.falciparum. Therefore, we decided to investigate this problem. In this report we present a number of new P. falciparum specific phosphoproteins. Cellular fractions were analysed in order to determine the cellular localization of these proteins. Several new proteins were found to be associated with the infected host erythrocyte membrane. The stage specific phosphorylation of all proteins was studied. Materials and Methods
Parasite culture and synchronization. The P.falciparum K 1 strain, adapted to grow in horse serum, was kindly provided by H. Matile (F. Hoffmann-La Roche). The parasites were cultured in RPMI-1640 medium (Gibco) supplemented with 25 mM Hepes/0.37 mM hypoxanthine/100 mg 1-1 neomycin base/10% heat-inactivated horse serum (Amimed). The cultures were set up with a 5% hematocrit and incubated at 37°C with 4% CO2/3% 02/93% N: and 94% relative humidity. Human A + erythrocytes were obtained from the Basel Blood Donation Center. For stage-specific examinations, the parasites were synchronized 3 times according to Lambros and Vandenberg [21]. The sorbitol treatment resulted in a parasitic age difference of less than 4 h. The parasitemia and the stage of parasitic development was monitored by microscopic observation of Giemsa-stained blood smears.
quots (-80°C) in TB (20 mM Tris buffer, pH 7.0) containing a mixture of protease inhibitors (30/z~ ml 1 soybean trypsin inhibitor, Merck; 20/zg m l leupeptin, Boehringer; 50 /xg ml -I aprotinin, Sigma; 50 /~M phenylmethylsulfonyl fluoride, Sigma; 1 mM benzamidine, Sigma; 5 mM iodoacetamide, Merck). A membrane fraction of uninfected erythrocytes (control) was purified according to the same procedure. The isolated parasites were washed twice in SSC and resuspended in a 10- to 100-fold volume of TB. After homogenization for 10 min in a Potter-E1vehjem tissue grinder, the parasites were centrifuged (10 min, 13 000 x g). The pellet (parasitic membrane plus organelle fraction) was washed three times with SSC and frozen as mentioned above. The parasitic cytosol was clarified from the 13 000 x g supernatant by a second centrifugation (60 min, 100 000 x g). The protein concentrations were determined according to the procedure of Heinzel et al. [22] using BSA (fraction V, Boehringer) as a standard protein. In both parasitic fractions the activity of acetylcholinesterase (EC 3.1.1.7), a marker enzyme of the erythrocytic membrane, was measured [23]. The parasitic membrane and organelle fraction had a negligible contamination with erythrocytic membrane ghosts (less than 3%), and no acetylcholinesterase activity could be detected in the parasitic cytosol.
Protein Parasite isolation and preparation of parasitic proteins and proteins of the erythrocytic membrane. Parasite cultures were grown up to a parasitemia of about 10-15%. Infected and uninfected erythrocytes were harvested by centrifugation (10 min, 700 x g, 4°C) and washed twice with PBS (phosphate-buffered saline, pH 7.2). The culture was lysed by incubation in a 10-fold volume of 0 × 0.015% saponin in SSC (150 mM NaC1/15 mM sodium citrate, pH 7.0) for 20 min at 4°C. Released parasites were concentrated by centrifugation (15 min, 1 500 x g, 4°C). Host cell membranes were harvested from the 1 500 x g supernatant by centrifugation (20 min, 19 500 × g, 4°C) and washed 4-5 times in 30-ml aliquots of S S C until a clear white pellet was obtained. The membranes were finally stored as frozen ali-
phosphorylation. Parasitic cultures (about 10% parasitemia) were preincubated in phosphate-free medium (Amimed) for 4 h. The erythrocytes were adjusted to a 10% hematocrit and phosphorylated for 1.5 h with 0.5 mCi ml -~ [32p]orthophosphate. The parasites were subsequently isolated and the protein fractions prepared as described above. Control studies demonstrated no changes in parasite development after growing in phosphate-free medium for about 6 h. For the [y-32p]ATP phosphorylation of cell lysates we tested a variety of different labeling conditions (see Results). Optimal labeling was obtained by incubating in the presence of 10/zM [732p]ATP (5-10 Ci mmol -~) and 15 mM MgCI2 for 15 min at 30°C. The reaction was stopped by the addition of 1 vol. of double strength gel electrophoresis sample buffer (see below).
115
Protein analysis. Phosphoproteins were analyzed by SDS-PAGE [24]. Electrophoresis was carried out with 0.8 m m slabs of 7.5, 10 and 12.5% acrylamide for 3 h (max. 35 mA, 220 V). Standard marker proteins (Bio-Rad) were used to determine the individual molecular weights. Two-dimensional gel electrophoresis was carfled out according to the procedure of O'Farrell [25], with minor modifications. Gels were cast using an ampholine mixture composed of 2 parts ampholine 3-10 (LKB) and one part 4-6 (LKB). 20 mg solid urea was dissolved per 30/.d of protein sample, and finally 1 vol. oflEF sample buffer was added prior to the electrophoretic separation. PI calibration proteins (LKB) as well as several individual proteins of known pI (soybean trypsin inhibitor, Merck, pI = 4.55; carbonic anhydrase II/bovine, Sigma, pI = 5.9; carbonic anhydrase I/human, Sigma, pI = 6.6) were used as markers. 7.5 % polyacrylamide gels were employed in the second dimension. Following electrophoretic separation, the gels were stained either with silver [26] or Coomassie R-250 [27], dried, and exposed to X-ray autoradiographic film (Kodak X-OMAT AR).
Peptide mapping. The proteins of interest were
excised from 7.5% preparative polyacrylamide gels (1.5 mm) and electroeluted into 100 mM (NH4)HCO3, pH 8.3/0.1% SDS for 18 h (max. 40 mA, 20 V). The eluate was lyophilized, washed three times with acetone/H20 (9:1,4°C) to remove SDS, and dried. The pellet was resuspended in IEF sample buffer, separated by 2-dimensional gel electrophoresis, and stained with Coomassie R250. Specific proteins were excised and transferred to a 0.8 mm, 15% SDS-polyacrylamide gel. 10/xl of a given protease - Staphylococcus aureus V8 (ICN Blochemlcals, 2/xg ml ), papaln (Sigma, 20/xg -1 • -1 ml ), or t~-chymotrypsln (Merck, 100/~g ml ) was overlaid into each slot. Peptide mapping by limited proteolysis was then carded out [28]. •
•
--1
•
•
Results
P.falciparum specific phosphoproteins were analyzed by ~3(-32p]ATP labeling of cell lysates in vitro and [~'P]orthophosphate labeling of living parasites in culture. Parasites of 4 different stages were isolated at different times after infection in order to determine the stage specific protein distribution: ring stage parasites (age of 7 + 2 h), young trophozoites (22 + 2 h), old trophozoites (33 + 2 h), and schizonts (42 + 2 h). Four cellular fractions
kDa
97.4 66.2
42.7 m
31.0
A
B
Fig. 1. Autoradiographs of P.falciparum specific phosphoproteins labeled with [7-32p]ATP (A) or [32p]orthophosphate (B) and separated on 10% SDS gels. Shown is the cellular localization (C, uninfected erythrocytic membrane as control; I, membrane of parasitized erythrocytes, II, parasitic 13000 x g pellet; III, parasitic cytosol) of the proteins from different stages (R, ring; YT, young trophozoite; OT, old trophozoite; S, schizont stage). The same amount of protein lysate (10 I.tg) was applied to each lane. Arrow heads indicate the approx. 88-kDa proteins as described in the text.
116
TABLEI
SummaryofPlasmodium~lc~arumspecificphosphoproteinslabeledwith[~32p]ATP Size (kDa)
Infected
Parasitic fractions
RBC membrane R
YT
OT
13000 z g pellet S
R
YT
OT
Cytosol S
R
YT
OT
S
192.0 184.0 178.0 169.0
160.5 156.0 154.0 144.0 131.0 123.0 108.5 102.5 88.0 80.5 77.5 74.0 72.5 65.5 63.0 56.5 54.5 48.0 45.0 40.0 38.5 37.5 36.0 34.5 32.5 32.0 31.0 29.0 26.5 26.0 24.0 21.5 20.0 18.0 16.5 15.0
*
*
*
*
*
* * *
*
*
*
The phosphoproteins were analyzed from autoradiographs of SDS gels of acrylamide concentrations which varied from 7.5 to 12.5%. The cellular fractions were prepared from the following stages of the erythrocytic growth cycle: R, ring stage; YT, young trophozoites; OT, old trophozoites; S, schizonts. The star (*) indicates the specific phosphorylation pattern of each protein. This data is compiled from 3 separate experiments and all the results were consistent within these experiments.
were prepared from parasites of each stage in order to localize the proteins and to detect possible changes in the cellular distribution during the parasitic life cycle.
To establish the optimal conditions for protein kinase activity in our [~/-3zp]ATP labeling experiments, we tested a variety of buffer systems at 2 concentrations (20, 50 mM) and with different pH
117 TABLEII
SummaryofP.~lc~arums~cificphosphoproteinslabeledwith[32p]o~hophosphate Size (kDa)
Infected RBC membrane R
YT
OT
Parasitic fractions 13000 × g pellet S
R
YT
OT
Cytosol S
R
YT
OT
S
88.0 64.0 53.5 51.5 41.5 40.5 36.5 34.0 32.5 30.0 27.5 26.5 25.0 24.0 22.0 19.5 17.5 16.5 Legend: see Table I. optima (phthalate/NaOH, pH 5.0; Mes/NaOH, pH 6.0; KH2PO4/NaOH, pH 7.0; Tris/HC1, pH 7.0; Tris/HC1, pH 8.0). The addition of several divalent • 2+ 2+ 2+ • cations (Mg , Mn , or Zn ) was studied at concentrations between 10 and 60 mM. Furthermore, in some experiments we tested detergents (Triton X-100, Triton X-114, and NP-40, all at 1.0%) to enhance the solubility of some proteins. In some experiments we also used phosphatase-inhibitors ([3glycerolphosphate at 10, 40, 80 mM; (ortho)vanadate at 50, 100 ~tM; NaF at 40 mM; and EDTA at 10 mM). All labeling reactions were carried out for 15 min, incubating 10 ~tg of parasitic cell lysate, 10 ~tM [7-32p]ATP (5-10 Ci mmo1-1) at 0°C or 30°C, plus various combinations of the above-mentioned reagents. With our definitive [T-32p]ATP labeling conditions (see Materials and Methods) we were able to distinguish 40 P. falciparum specific phosphoproteins on autoradiographs from SDS gels in which the acrylamide concentration varied from 7.5 to 12.5%. With all other labeling conditions only some of the proteins were phosphorylated and no other bands appeared. Fig. 1A shows an autoradiograph of a 10% acrylamide gel used for the
analysis of the cellular and stage specific phosphorylation of proteins in the molecular weight range between 40 and 100 kDa. The complete list of all proteins identified by this means is presented in Table I. Only those protein bands which were clearly distinguishable by SDS-PAGE are summarized. Most of these [T-32p]ATP-labeled phosphoproteins were found exclusively in the parasitic fractions: 18 in the parasitic cytosol, 7 in the parasitic 13 000 x g pellet, and another 7 in both fractions. Chiefly in the parasitic cytosol the degree of phosphorylation increased rapidly and several new bands appeared during the transition from young to old trophozoites (Fig. 1A). 8 new proteins were phosphorylated in the RBC membrane fraction of the parasitized cells, as compared with that of the uninfected RBC membrane. 7 of these proteins could, however, also be detected in other parasitic fractions. Only one, with a molecular weight of about 156 kDa, was exclusively labeled in the erythrocytic membrane of ring stage infected erythrocytes. 19 phosphoproteins could be characterized after 32 . . . . [ P]orthophosphate labehng in VlVO (Fig. 1B,
118
clR
YTOT
s
kDa
97.4 m 66.2 42.7
31.0
m
21.5 m
14.4
....
Fig. 2. Stage-specific distribution of [32p]orthophosphate- labeled proteins associated with the membrane of infected erythrocytes: membrane fraction of erythrocytes infected with ring (R), young trophozoite (YT), old trophozoite (OT), and schizont (S) stage parasites (C, uninfected erythrocytic membrane as control)• The same amount of protein (20 ~tg) was applied to each lane. Shown is the autoradiograph of a 12.5 % acrylamide gel. The arrowhead indicates the approx. 41.5-kDa protein specifically labeled in the ring and schizont stages.
Table II). 15 of these proteins were exclusively located in the parasitic fractions (cytosol and/or 13 000 × g pellet). Whereas about 10 of these proteins were phosphorylated throughout the entire cycle, 5 were only phosphorylated during the late stages (old trophozoites and schizonts), i.e., at a time when mitosis and the condensation of the newly formed merozoites takes place. 4 proteins could be detected in the parasitic fractions as well as in the infected erythrocytic membrane and had a different stage specificity from the phosphoproteins mentioned above• One protein, of approx• 41.5 kDa, was only phosphorylated in schizonts and the early ring stage (Fig. 2). The phosphorylation of the other 3 proteins was first detectable in young trophozoites and remained detectable until the end of the cycle.
Only one prominent double band, of approx• 88 kDa, had an identical phosphorylation pattern using both labeling methods (Fig. 1A, B). Both proteins (pf 88-1; pf 88-2) could be identified on silverstained 2D-gels and corresponding autoradiographs as 2 neighboring protein spots (Fig. 3A-C). Their pI is about 5.0. Due to the higher resolution of 2D gels and the different solubilities of some proteins in urea, 10 additional proteins were found to be labeled by both [y-32p]ATP and [32p]orthophosphate (Fig. 3, Table III). So far, we were not able to make a precise correlation of these labeled proteins with the bands identified by SDS-PAGE. The approx• 88-kDa proteins are located in the infected erythrocytic membrane as well as in both parasitic fractions. The phosphorylation is restricted to the young and old trophozoite and the schizont stages (Fig. 1), whereas the proteins (unphosphorylated) are already present during the ring stage (Fig. 4). According to peptide mapping studies with several proteases, both proteins had identical peptide patterns, with the exception of a few small fragments (Fig. 5). The same peptides are ~2hosphorylated in the two proteins, both with [yP]ATP (not shown) or [32p]orthophosphate (Fig. 5). Discussion
The stage-dependent phosphorylation of P. falciparum specific phosphoproteins was studied during the asexual erythrocytic cycle• More phospho• protems were found by [T- P]ATP labeling of cell 32 • . lysates than by [ P]orthophosphate labelmg of intact cells. As a result of our [y-32p]ATP labeling conditions, all these phosphoproteins should be substrates of Ca 2+- or cAMP-independent protein kinases. Whether some of the proteins are themselves kinases has not yet been determined. The addition of phosphatase inhibitors prior to or during the labeling reaction had only slight effects. Only a few proteins were phosphorylated to a higher degree and no new labeled proteins could be detected• Under our in vitro labeling conditions the balance between phosphorylated and dephosphorylated forms has probably shifted to the phosphorylated form, even without using any phosphatase inhibitors. These distinct [T-32p]ATP labeling conditions may help to explain the different protein 32
•
119 pI 3.75
4.55
5.2
6.0
3.75
4.55
5.2
6.0
3.75
I
I
I
I
I
t
i
I
i
ko~
~
~ S
C
4.55
5.2
6.0
i
i
41!
200.0--
116.3
4
--
97.4--
66.2--
42.7--
Fig. 3. Protein phosphorylation patterns on 2D gels of [7-32p]ATP labeled parasitic lysates and [32P]orthophosphate labeled malarial cultures. (A) Silver-stained 2D-gel; (B) corresponding autoradiograph of the [TJ2p]ATP labeled extracts; (C) autoradiograph of [3Zp]orthophosphate labeled cultures (the silver-stained gel of this autoradiograph is not shown). 7.5% acrylamide gels were used for the second dimension. The approx. 88-kDa phosphoprotein spots (pf 88-1, pf 88-2) corresponding to the 88-kDa double band seen on SDS-PAGE (Fig. 1) are marked with arrowheads. The 10 additional phosphoproteins common to both labeling methods are indicated by arrows. TABLE III
P.falciparum specific phosphoproteins labeled by both [~/-32p]ATPand [32P]orthophosphate Protein
Size (kDa)
pI
1 2/3 4 5 6 7 8 9 10 11 12
97.5 88.0 84.0 77.5 76.0 71.0 56.0 45.5 41.5 32.5 31.5
5.0 5.0 5.0 5.4 5.1 5.0 5.0 4.9 5.0 4.9 4.6
The proteins were analyzed on 2D-gels as shown in Fig. 3.
patterns obtained in lysates ([7•P]ATP) h o s pandh ointact r yparasites l a t i o([32p]orthophosn phate). On the other hand, the rupture of intracellular barriers, such as intracellular membranes, during the preparation of in vitro lysates could lead to unnatural contacts between kinases and substrates, probably resulting in additional, artifactual phosphorylation of some proteins. Thus a nonspecific phosphorylation of non-target proteins cannot be excluded under these conditions. A further explanation for the fact that fewer proteins were phospho-
rylated during in vivo labeling could be the relatively short labeling period used there. This was done in order to examine the stage-specific activity of the kinases as accurately as possible. Some phosphoproteins of our parasitic fractions are very similar in their molecular weights to P.falciparum phosphoproteins already described. Unfortunately, the published labeling conditions are usually different from ours. Furthermore, the similar molecular weights, invariably the only information available, do not enable us to draw any firm conclusions concerning the possible identity of phosphoproteins described by us and by other workers. Nevertheless there are 2 proteins which we shall mention. One protein (approx. 156 kDa) detected in the membrane of ring infected erythrocytes corresponds in molecular weight and stage specificity to RESA [20]. The approx. 88-kDa proteins (pf 88-1, pf 88-2) show similarities to a 85kDa protein which is in the phosphorylated state in the membrane of trophozoite/schizont-infected erythrocytes [11]. The present investigation of plasmodial phosphoproteins clearly supports the assumption that phosphorylation and dephosphorylation processes play a major role in parasitic development as well as in malaria-specific alterations of the erythrocytic
120
pI
5.2
4.55
5.2
I
I
1
I
A
kDa
97.4
4.55
--
_
B
-,,-t
66.2 --
~
•
z! Fig. 4. Two-dimensional distribution of parasitic, ring-stage specific proteins. (A) Segment of a silver-stained 2D-gel; (B) corresponding autoradiograph of the [32p]onhophosphate labeled proteins. 7.5% acrylamide gels were used for the second dimension. In the silver-stained gel both approx. 88-kDa proteins (pf 88-1, pf 88-2) are indicated by arrow heads (A); the autoradiograph (B) demonstrates that these proteins are not phosphorylated. For orientation, two other phosphoproteins are marked by arrows.
membrane during infection. At the transition from young to old trophozoites, a time when DNA replication and mitosis takes place, the degree of phosphorylation increased considerably and several new proteins were phosphorylated, chiefly in the parasitic cytosol. As in other eukaryotes, protein kinases are probably involved in the regulation of plasmodial proliferation and differentiation processes, especially in the above-mentioned DNA replication and mitosis. Several phosphoproteins were associated with the membrane fraction of infected erythrocytes. With the exception of the approx. 156-kDa protein, all these proteins had corresponding bands in parasitic fractions, suggesting that these proteins are synthesized by the parasite and transported to the erythrocytic membrane. The approx. 156-kDa protein, if identical with RESA, would also be of parasitic origin. The stage specific phosphorylation pattern of the individual membrane associated proteins during the entire erythrocytic cycle demonstrates the permanent parasitic influence on the surrounding erythrocyte. Some proteins, especially the approx. 41.5-kDa protein phosphorylated only in the membrane fraction of schizont and ring stage infected erythrocytes, could, furthermore, be involved in the merozoite invasion. Although the func-
tional role of the phosphoproteins remains unknown, they seem to be important members in the connection between erythrocytes and parasites. Therefore, from a medical point of view, they might be interesting targets in the development of antimalarial agents. Let us now turn to our analysis of the approx. 88 kDa proteins (pf 88-1, pf 88-2). They have identical cellular distributions and the same stage specific phosphorylation. The addition of ~-glycerolphosphate (80 mM) during our [7-32p]ATP labeling assay resulted in an increased phosphorylation of this pair, probably underlining the involvement in a regulatory process which may start with their phosphorylation at the transition from the ring to the young trophozoite stage. Only minor differences were found in the peptide maps of these two proteins. None of the divergent fragments were phosphorylated, suggesting that other modifications (e.g., glycosylation, acetylation) of these otherwise identical proteins might be taking place. The precise biochemical nature of these modifications is now under investigation. Many proteins described in this paper must be important during the malarial growth cycle since they are only phosphorylated during the period of biosynthesis of macromolecules (see Tables I and
121
I kDa 97.4
11
1
2
66.2 w
V8
2
1
2
ch
1
2
w
42.7
|'=
ii
iiii~ili!i¸
i~ .....
!i~i ¸¸
~il ~
~
31.0 w v
•
~
0
I
•
e
•
o
21.5
14.4 m
f
°
i~
.... ~ ~
O
i¸ S
A
S
A
Fig. 5. Peptide maps of the [32 P]orthophosphate-labeled approx. 88-kDa proteins (1: pf 88-1,2: pf 88-2). Both proteins, isolated from 2D gels, were digested with S. aureus V8 protease (I) or ~-chymotrypsin (II). A third digest with papain, supporting the same conclusions, is not shown. S, silver-stained digest; A, corresponding autoradiograph; V8, protease control digest; ch, ¢~-chymotrypsin control digest. Small differences in the peptide pattern are indicated by arrows. II). F u r t h e r m o r e , a discrete n u m b e r o f proteins can be identified by both labeling m e t h o d s (Table III). W e are n o w cloning selected genes w h i c h code for s o m e o f the p h o s p h o p r o t e i n s in the hope o f learning m o r e about the function of these proteins.
Acknowledgements W e thank Dr. G e o r g e T h o m a s for helpful discussions and, together with Heidi Lane, the critical reading o f the manuscript. This w o r k was supported in part by funds f r o m the Canton Basel-Stadt and the Fonds National Suisse (Grant N u m b e r 319098.87/2).
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