Molecular and Biochemical Parasitology, 56 (1992) 59-68 4,) 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00
59
MOLBIO 01827
Structure of a Plasmodium chabaudi acidic phosphoprotein that is associated with the host erythrocyte membrane Willy Deleersnijder, P o r n t h i p P r a s o m s i t t i , S u m a l e e T u n g p r a d u b k u l , D i a n a H e n d r i x , C6cile H a m e r s - C a s t e r m a n a n d R a y m o n d H a m e r s Laboratorium Algemene Biologie, Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Brussels, Belgium (Received 27 January 1992; accepted 22 July 1992)
We have characterized by molecular cloning and sequencing a Plasmodium chabaudi antigen that is associated with the membrane of the infected erythrocyte throughout the entire intraerythrocytic cycle. The protein (PcEMA1) has a predicted size of 50 kDa and contains a major tandem repeat array of 16 octapeptides that constitutes almost 30% of the protein. At its amino-terminus, PcEMAI has a string of hydrophobic residues characteristic of a secreted protein, but does not contain a hydrophobic membrane-spanning segment. The antigen appears to reside on the cytoplasmic face of the erythrocytic membrane. PcEMA1 has a predicted pI of 4.4 and is a potential phosphoprotein. Key words: Malaria; Plasmodium chabaudi; Erythrocyte cytoskeleton; Tandem repeats; Phosphoprotein
Introduction
During their intraerythrocytic growth and development, Plasmodium parasites induce profound changes in the structural, functional and antigenic properties of the host red cell membrane (see refs. 1-4 for review). Among these are alterations in membrane fluidity, reduced deformability and change in shape of erythrocytes infected with mature parasites, the creation of new metabolic channels, the Correspondence address: Raymond Hamers, Laboratorium Algemene Biologie, Instituut voor Moleculaire Biologic, Paardenstraat 65, 1640 St. Genesius Rode, Belgium. Note." Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M95789. Abbreviations." PcEMAI, P. chabaudi erythrocyte membrane antigen 1; KAHRP, knob-associated histidine-rich protein; MESA, mature parasite-infected erythrocyte surface antigen; RESA, ring-infected erythrocyte surface antigen; PfEMP1 or 2, P. falciparum erythrocyte membrane protein 1 or 2; APP, acidic phosphoprotein; pI, isoelectric point.
appearance of electron-dense protrusions (knobs) on the membrane and the acquisition of cytoadherent properties. These modifications can be mediated either by direct insertion of parasite-derived proteins in the red cell plasma membrane, e.g., the PfEMP1 antigen [5] or the transferrin receptor [6,7], or indirectly via their association and interaction with components of the red cell cytoskeleton. Wellcharacterized Plasmodium antigens that are targeted to the red cell membrane and interact with the erythrocyte cytoskeleton include the knob-associated histidine-rich protein (KAHRP) [8], the ring-infected erythrocyte surface antigen (RESA) [9,10] and the mature parasite-infected erythrocyte surface antigen (MESA/PfEMP2) [11-14]. Both MESA and RESA are present on the membrane of the infected erythrocyte in a phosphorylated state. In addition, Wiser et al. have reported the appearance of new phosphoproteins on the host erythrocyte membrane during infection with P. berghei (65 and 46 kDa) and P. chabaudi (93, 90 and 76 kDa) [15,16]. These
60
parasite-derived phosphoproteins were tightly associated with the submembranous cytoskeleton of the host erythrocyte, had a pI of less than 4.0 and have been referred to as APP (acidic phosphoproteins). In this report we describe the molecular cloning and sequence analysis of a P. chabaudi gene encoding a protein that is associated with the host red cell membrane throughout the entire erythrocytic cycle. We also demonstrate that this antigen is a potential phosphoprotein.
Materials and Methods
Parasites. P. chabaudi chabaudi strain IP-PC1 clone C (IP-PC1/C) was used for all work in this report and has been described before [17]. Parasites were grown in OF1 outbred mice (Iffa Credo), kept in an inverted nycthemeral cycle for diurnal schizogony. Construction o['cDNA and genomic libraries. c D N A and genomic libraries were constructed and screened as described before [17]. Sequencing. Inserts of overlapping clones were subcloned into p U C or pBluescript (Stratagene) vectors and totally (UL20, 46A) or partially (L21, X2A, 81 and 44) sequenced. Subclones were generated that contained progressive unidirectional deletions of each insert by controlled exonuclease III digestion (Erase-a-base, Promega). Sequencing was done on double-stranded templates by the dideoxy chain-termination method of Sanger et al. [18], using modified T7 D N A polymerase (Sequenase, USB). Both strands were entirely sequenced in the coding area. We were unable to determine unambiguously nucleotide position 1721. Computer-assisted storage and analysis of sequence data was facilitated using the P C / G E N E software package (Intelligenetics). Indirect immunoJluorescence in suspension. Infected erythrocytes were either fixed by overnight incubation in phosphate buffered saline (PBS - 0.145 M NaCI/8 mM phos-
phate, pH 7.2) containing 3.7% formaldehyde, or used without fixation. After 3 washes with PBS small amounts of red cells (10/A pellets) were resuspended for 30 min in 100 /tl of the appropriate sera (diluted 1/200 in PBS). After 3 further washes with PBS, erythrocytes were incubated in 50 /~1 of FITC-conjugated goat anti-rabbit Ig antibody solution for 30 min. Finally, cells were washed 3 more times, resuspended to an appropriate dilution and viewed under the fluorescence microscope. Production ~[" rabbit sera against Jusion proteins. Y1089 bacteria (ATCC37196), lysogenic for 2gtll clones 81, 43 and 44 were grown at 32°C to A6o0 = 0.5. The culture was subsequently continued at 44°C for 20 min with good aeration. Isopropyl-/:~-thiogalactopyranoside (IPTG) was added to a final concentration of 2 mM and the culture was continued for 1 h at 37~C before harvest. Bacterial pellets were frozen at - 8 O C , thawed, and sonicated. The sonicated extract of a 50 ml culture was mixed with 1 ml SDSPAGE sample buffer, boiled for 5 min at 100~C, spun to remove insoluble debris and loaded on a preparative SDS-PAGE gel. The gel slice containing the recombinant fusion protein (identified after Coomassie blue staining) was frozen in liquid nitrogen and ground to a fine powder in a mortar. The powder was resuspended in 1 ml of PBS, emulsified with incomplete Freund's adjuvant and injected subcutaneously into a rabbit. Three injections were given at 1 month intervals. Rabbits were bled 2 weeks after the last injection. Sera were denoted L81, L43 and L44 respectively. 2gtll clones 81 and 44 express part of the PcEMA1 antigen (see Fig. 1). 2gtl 1 clone 43 expresses an unrelated P. chabaudi antigen that is also associated with the membrane of the infected red cell. It should be noted that polyclonal rabbit serum L43 is unrelated to mouse monoclonal antibody 43 (mAb 43), that is also described in the text. The (membrane associated) antigen recognized by L43 is different from the one recognized by mAb 43. The fact that both sera have been denoted '43' is entirely coincidental.
61
Preparation of erythrocyte ghosts and parasitic fractions. Uninfected or infected (30-50% parasitemia) blood was pelleted and the buffy coat removed. Ghosts were prepared by hypotonic lysis in 5 mM phosphate buffer pH 8.0 and centrifugation at 22 000 x g (10 min). The fluffy creamy layer containing membranous ghosts was removed from the underlying dark pellet of parasites and extensively washed in the same buffer until completely devoid of hemoglobin. The particulate pellet, containing the liberated parasite material was washed 3 times in the hypotonic lysis buffer. This is referred to as the parasitic fraction.
Labeling of membrane and parasitic fractions with [?-32p]ATP. One fifth of the erythrocyte ghosts or of the parasitic fraction obtained from the infected (40% parasitemia) or uninfected blood of 1 mouse was incubated for 2 min at 37°C in a final volume of 100/~1 50 m M 4-morpholineethanesulfonic acid (Mes), pH 6.0/10 mM MgC12/5 #M [7-32p]ATP (80 Ci m m o l - 1). For immunoprecipitation, the radiolabeled fractions were washed 3 times in PBS and then solubilized in 20 #1 PBS/2% SDS/5 mM phenylmethylsulfonylfluoride (PMSF) (15 rain at room temperature, followed by centrifugation at 12000 x g for 10 min to remove insoluble material). 180 /tl of Netto (10 mM Tris-HCl pH 8.3/100 mM NaCI/10 mM E D T A / I % Triton X-100/0.1% ovalbumin/1 mM PMSF/1 mM N-c~-p-tosyl-L-lysine chloromethylketone HC1 (TLCK)) was added, and 50 /~1 of rabbit antiserum. This mixture was incubated for 3 h at 4°C under gentle agitation. 10 mg of protein A-Sepharose-CL4B beads (Pharmacia) were added and incubation continued for another hour. The beads were washed 3 times in Netto followed by l wash in 10 mM Tris-HCl pH 8.3/100 mM NaCI/10 mM EDTA/1 mM PMSF/I mM TLCK. Immunocomplexes were then released by heating for 3 min at 100°C in electrophoresis sample buffer and analysed on 10% SDSPAGE slabs.
Immunoblot analysis.
One-fifth of the ery-
throcyte ghosts obtained from the blood of 1 mouse were loaded per lane (1 cm wide) of a 1.5-mm-thick 12.5% SDS-polyacrylamide gel slab. The gel was electroblotted onto a nitrocellulose filter which was saturated afterwards with TBS-T buffer (10 mM Tris-HC1, pH 8.0/150 mM NaCI/0.05% Tween 20) containing 1% BSA. Separate lanes on the filter were incubated with appropriate rabbit sera diluted 1/5000 in TBS-T. After subsequent washing affinity purified alkaline phosphatase conjugated goat anti-rabbit Ig antibodies were added to the filter strips for 1 h. After a final round of washes, substrate (0.05 mg ml ~ 5bromo-6-chloro-3-indolyl acetate/0.1 mg ml l nitro blue tetrazolium in 10 mM MgC12/100 mM Tris-HCl, pH 9.5) was added to the filters and the enzymatic reaction was stopped after 5-15 min by rinsing the membranes with water.
Results
Cloning strategy. 2gtl 1 clone 46 was initially obtained as a mosaic clone, containing part of the MSA-1 coding sequence ligated to an unrelated c D N A molecule of approximately 1 kb [17]. As sequencing of this extraneous c D N A fragment (named 46A) revealed some interesting features (long repeat area) it was further studied. Clone 46A was further
ATG
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Fig. 1. Cloning strategy for the PcEMAI gene. 46A, L21, UL20, 81 and 44 are 2gtll c D N A clones. X2A is a genomic 2GEM-2 clone of 2.3 kb. The position of the tandem repeats is indicated by chequered boxes.
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63
A contiguous stretch of 1846 nucleotides was obtained containing one major open reading frame, starting at nucleotide 324 and ending at position 1646 (Fig. 2). This encodes a protein of 441 amino acids with a calculated size of 49 707 (denoted PcEMA1 -- P. chabaudi erythrocyte membrane antigen l). Clones X2A and 46A did not differ by a single nucleotide in areas where sequence had been determined from both clones (totalling about 630 nucleotides) indicating that X2A was not derived from a pseudogene. As the last 200 nucleotides of the PcEMA1 coding sequence were determined from a genomic DNA clone the occurrence of an intron in this area could not be ruled out. We therefore verified the collinearity of c D N A and genomic DNA in this region by a PCR approach. P. chabaudi total RNA (2.5 /~g) was first treated with DNaseI to destroy any remaining traces of gDNA and poly(A) + RNA was subsequently converted to first strand cDNA. This first strand c D N A was then amplified by PCR using oligonucleotides ON19-ON20 and ON13-ON20 as a primer pair. The positions of these primers were: ON13 (nucleotide positions 1395-1411 and repeated in 1422-1438), ON19 (nucleotide Sequence analysis.
positions 452-469) and ON20 (complementary to nucleotide positions 1701-1718). Amplification was performed in a thermal cycler for 36 cycles (45 s at 94°C, 45 s at 54°C and 1 min at 72°C), using 0.l /~g of each primer. The PCR-products obtained from the ON19/ON20 and ONI 3/ON20 amplifications were analyzed by agarose gel electrophoresis and consisted of a 1.3-kb and a 0.3-kb fragment respectively (data not shown). No PCR products were obtained when these amplifications were performed on RNA that had not been reverse transcribed. These results clearly demonstrate that there is no hidden intron in the last 200 bp of the determined DNA sequence. The primary structure of PcEMAI exhibits a number of interesting features: (1) The Nterminal sequence was identified as a potential secretory signal peptide by computer analysis according to the method of Von Heijne [19]. The predicted cleavage site is between residues 15-16 or 24~25 ( - 3 , - 1 rule). The first 6 amino acids of the PcEMA1 signal peptide are almost identical to the first 6 amino acids of the P. chabaudi major merozoite surface antigen 1 (MSA 1) signal peptide (MKAISL versus M K A I G L ) [17]. (2) PcEMAI contains
A
1 2
GAA GAA
GAA GAA
GAC GAC
CCA GCA
TAC CTC
TTA TCA
TTA TTA
CAG ATG
E E D P Y L L Q E E D A L S L M
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GCA GCA GCA
GAG GAT GGC
ACA ACA ACA
CTA CTA CTA
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13 G A A T A T G A C
GCA GGT ACA
CTA AAT
E Y D A G T L N
14 G A A 15 G A A 16 G A A
GGA GAA GAA
ACA TCC GCA
E E E G S T T N E A G E G T S N E A G E G "fAN
GAA GCT GCT
GAG GGT GGC
AGC GGT GGT
ACA ACA ACA
AAT AAT AAT
C
l
Fig. 3. Structure of the major tandem repeat array of PcEMA1 at the nucleotide (left) and amino acid (right) level.
64
in its central part an 8 amino acid sequence repeated in tandem 16 times, that constitutes almost 30% of the total protein. These tandem repeats are incompletely conserved (Fig. 3). An unrelated tandem repeat structure of 2 completely conserved nonapeptides occurs a little downstream of the major repeat area. (3) Immediately following the nonapeptide repeat structure and almost continuing to the Cterminal end is an area that is extremely rich in Lysine residues (31 out of 47 residues between amino acid positions 371-417). (4) Overall PcEMA1 is rather hydrophilic. One third of all residues are either Asp (7.0%), Glu (15.9%) or Lys (12.7%). The predicted pI of PcEMA1 is 4.4 (PC/GENE C H A R G P R O software). (5) Motif searching using the P C / G E N E PROSITE version 3.0 software revealed the presence of 21 potential casein kinase II phosphorylation sites (Fig. 2). 15 of these occur in the tandem repeat oligopeptides. There are also 4 potential protein kinase C phosphorylation sites. (6) Two potential Nglycosylation sites are present in the sequence (amino acid positions 21 and 112). A search of
the SWISS-PROT release 21.0 protein data bank using the FASTA programme [20] showed that PcEMA1 was not significantly similar to any protein in the data bank.
Immunofluorescence and immunoblotting. Sera raised against expressed parts of PcEMA1 (clones 81 and 44) reacted with the membrane of formaldehyde fixed infected red cells, showing a rim-like fluorescence on the surface of the cell (Fig. 4). No fluorescence was observed on unfixed infected red cells in suspension indicating that PcEMA1 is not 1 •
2
3
4
94--
I
I
67-
43--
30-
Fig. 4. Indirect immunofluorescence using antiserum (L81) to the expressed product of clone 81. Fluorescein staining of formaldehyde fixed erythrocytes infected with P. chabaudi. The noninfected erythrocytes are indicated by arrows.
Fig. 5. lmmunoblot analysis. Infected red cell ghosts were separated on a 12.5% SDS-PAGE gel and blotted onto a nitrocellulose filter. Filter strips were p r o b e d with antiserum (L81) to the fusion product of )~gtll clone 81 (lane 1), antiserum (L44) to the clone 44 (lane 2), antiserum (L43) raised against the fusion product of 2gtl 1 clone 43, which expresses part of an unrelated P. chabaudi antigen that is also associated with the membrane of the infected red cell (lane 3, our unpublished results) and L81 preimmunization serum (lane 4).
65
accessible from the outside. The rim-like fluorescent staining was observed on the surface of erythrocytes containing parasites at all stages of parasite development (rings, trophozoites and schizonts). Sera against the recombinant fusion protein of clones 81 and 44 recognized a 85-kDa band on Western blots of total membrane preparations from infected red cells (Fig. 5). Apart from the principal 85-kDa band both sera also immunostained a minor band of slightly higher electrophoretic mobility.
could be a substrate for kinases of parasitic or erythrocytic origin. Incubation of the crude parasitic fraction with [TYP]ATP resulted in the phosphorylation of many proteins of varying size (Fig. 6, lane 1). Serum L81 specifically immunoprecipitated a 85-kDa phosphoprotein (Fig. 6, lane 3) from the [7-32P]ATP-labeled parasitic fraction. Immunoprecipitation of [7-32p]ATP labeled membrane preparations from infected red cells with L81 yielded a very weak 85-kDa band that was only apparent after 14 days' autoradiography (data not shown).
Phosphorylation of PcEMA1.
Since analysis of the PcEMA 1 protein sequence indicated the existence of numerous potential phosphorylation sites, we investigated whether PcEMA1
Discussion We have identified by molecular cloning a P.
chabaudi protein that is associated with the 2
3
4
!i~~iiiiii
94" 67.
43
Fig. 6. Overnight autoradiography of the [y-32p]ATPlabeled parasite fraction. Lane 1, total [7-32P]ATP labeled parasite fraction. Lanes 2-4, immunoprecipitations of radiolabeled parasite fraction using preimmunization serum L81 (lane 2), L81 serum against the expressed fusion product of clone 81 (lane 3) and polyclonal rabbit antiserum L43, raised against part of a P. chabaudi antigen, unrelated to PcEMA1, that is also associated with the membrane of the infected red cell (lane 4; see also Fig. 5).
membrane of infected erythrocytes and have determined its primary structure. The discrepancy between the predicted Mr of PcEMA1 (50000) and its apparent Mr on SDS-PAGE (85000) can most likely be attributed to the pronounced hydrophilic character of this protein and the extensive array of tandem repeats that it contains. This has been commonly observed for malarial antigens of this type [21]. There is considerable diversity between the individual octapeptide units of the major repeat region. Except for the first AA of each unit, which is invariably Glu, at least 3 and sometimes up to 6 different AA can be found at any given position in the octapeptide unit (Fig. 3). Units 4 and 5 are the only ones that are repeated exactly and in tandem, both at the AA and at the nucleotide level. Units 6, 12 and 13 are completely conserved at the AA level but not at the nucleotide level (there is a silent thirdbase substitution in codon 5 of unit 13). Likewise, units 3 and 10 are identical at the AA level but not at the DNA level (two silent third base substitutions). The remaining 10 octapeptide units each have a unique structure. It is however possible to subdivide the major repeat region of PcEMA1 into a number of blocks on the basis of local homology between adjacent units.
66
Units 3-6 and 10-13 show close homology and have the general structure: E(Y/G)DA(E/ D / G ) T L N (denoted here as blocks B and B' respectively). Blocks B and B' are interrupted by 3 units (7, 8, 9; = block C) which are less homogeneous and appear to be repeated in units 14-15-16 (block C') although a number of point mutations have occurred between blocks C and C'. Units 1 and 2 are also clearly related and can be considered to constitute a separate block ( = block A). The overall structure of the repeat region can thus be represented as: ABCB'C'. It is clear from the sequence data of the major repeat area that a mechanism is operating to homogenize the repeat units at the D N A level. As well as from the global homogenization throughout the entire repeat array, this can also be seen from the apparent spreading of mutations to equivalent positions in neighboring units; see, e.g., codon No. 2 in units 4 and 5; codon 7 in units 7 8 and codons 3~4 in units 8-9). Unequal crossing-over, gene conversion or slippage replication can all be invoked to explain the concerted evolution of repeat units as well as the internal duplication that presumably took place in the repeat array (ABC ~ ABCB'C'). Antisera raised against a large portion of PcEMA1 stained the membrane of formaldehyde fixed infected red cells in an indirect immunofluorescence assay in suspension. No fluorescent staining of the membrane was observed when the infected erythrocytes were not fixed. This finding is in keeping with the determined structural feature of PcEMA1. Apart from the N-terminal secretory signal peptide no hydrophobic transmembrane anchor sequences can be found in PcEMA1. This suggests that PcEMA1 is not an integral but rather a peripheral membrane protein. The pronounced hydrophilicity of PcEMA1 is also consistent with this notion. We conclude therefore that PcEMA1 resides on the cytoplasmic face of the erythrocyte membrane. We have shown that PcEMA1 is a potential substrate for kinases. PcEMA1 is readily phosphorylated in the parasitic fraction but only very faintly in an equivalent (prepared
from an equal number of infected erythrocytes) ghost membrane preparation. From these data we cannot conclude whether the kinase that is responsible for this phosphorylation is of parasitic or of erythrocytic origin. The fractionation method used to separate membranes from parasites is only crude and it is likely that the parasite fraction is contaminated with 'membrane' material and vice versa. It remains to be determined whether PcEMA1 phosphorylation also takes place in live parasites. One could indeed argue that the disruption of the intracellular structure as in our parasitic fractions will artifactually expose substrates to 'opportunistic' kinases. However since PcEMA1 is associated with the red cell membrane and since many erythrocyte membrane associated parasite antigens that have been identified to date are phosphorylated it is not unlikely that PcEMA1 is also phosphorylated under in vivo conditions. The characteristic features of P c E M A I as outlined here, resemble very closely those of a family of acidic phosphoproteins identified by Wiser et al. on the membranes of P. chabaudi [Pc(em)93; Pc(em)90 and Pc(em)76] and P. berghei [Pb(em)46 and Pb(em)65] infected red cells [15,16]. These include the acidic nature of PcEMA1, its close association with the red cell membrane possibly as a phosphoprotein, its presence throughout the entire erythrocytic cycle and the approximate Mr. The difference in apparent Mr between PcEMA1 (85 000) and any of the P. chabaudi phosphoproteins described by Wiser et al. (93 000, 90000 and 76000) might be explained by the fact that both proteins have probably been characterized from different strains. It can be expected that the large tandem repeat array is liable to rapid diversification between different strains (as is the case for most malarial tandem repeat arrays) both in terms of its encoded sequence as in terms of its length. Pb(em)65 was actually reported to exhibit Mr heterogeneity between different strains [16]. A similar phenomenon is observed for other red cell membrane associated malarial proteins such as MESA and K A H R P . These antigens also contain extensive tandem repeat areas and do exhibit a
67
variant Mr between different isolates [8,11]. We can however definitely exclude that PcEMA1 is identical to Pc(em)93 on the following grounds. Monoclonal antibody 43 (mAb 43), that was made in this laboratory and recognizes an unrelated P. chabaudi antigen that is also associated with the red cell membrane, reacts with Pc(em)93 (M. Wiser, personal communication). In our hands mab 43 detects a band of 105 kDa on Western blots of P. chabaudi IP-PC1/C extracts and does not react with the PcEMA1 85-kDa band. This 105-kDa band is most probably the murine equivalent of RESA (our unpublished results). Also, the reported amino acid composition of Pc(em)93 [14] is different from the predicted amino acid composition of PcEMA1. The occurrence of an acidic phosphoprotein (pI = 5.0; Mr = 88 000) on the membranes of P.falciparum infected erythrocytes has recently been reported [22]. It is also possible that PcEMA1 is homologous to this antigen or/and a 85-kDa P. fi~lciparum phosphoprotein detected by Murray et al. [23] on the membrane of infected red cells. The identification and characterization of PcEMA1 adds another member to the expanding family of parasite antigens that interact with the membrane of the infected red cell. Although it is probably safe to say that the function of these proteins during infection, is to modulate the structure of the red cell membrane to the advantage of the parasite, the precise role of most of these proteins and especially PcEMA1 has still to be defined. The cloning, sequencing and partial expression of PcEMAI, as reported here, will provide the molecular tools to delineate its precise biological role during future work.
Acknowledgements This work was supported by EEC contracts TSD-M-152 and TS-2-0148 and Belgian government fund 'BIO 10'. The author wish to thank Jos6e Hanegreefs for skillful technical support and Andre van Dormael for photographic work.
References 1 Sherman, l.W. (1985) Membrane structure and function of malaria parasites and the infected erythrocyte. Parasitology 91,609-645. 2 Hommel, M. and Semoff, S. (1988) Expression and function of erythrocyte-associated surface antigens in malaria. Biol. Cell 64, 183 203. 3 Aikawa, M. (1988) Morphological changes in erythrocytes induced by malarial parasites. Biol. Cell 64, 173 181. 4 Wiser, M.F. (1991) Malarial proteins that interact with the erythrocyte membrane and cytoskeleton. Exp. Parasitol. 73, 515 523. 5 Leech, J.H., Barnwell, J.W., Miller, L.H. and Howard, R.J. (1984) Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium Jalciparum-infected erythrocytes. J. Exp. Med. 159, 1567 1575. 6 Haldar, K., Henderson, C.L. and Cross, G.A.M. (1986) Identification of the parasite transferrin receptor of Plasmodium falciparum-infected erythrocytes and its acylation via 1,2-diacyl-sn-glycerol. Proc. Natl. Acad. Sci. USA 83, 8565-8569. 7 Rodriguez, M.H. and Jungery, M. (1986) A protein on Plasmodium falciparum-infected erythrocytes functions as a transferrin receptor. Nature 324, 388 391. 8 Leech, J.H., Barnwell, J.W., Miller, L.H. and Howard, R.J. (1984) Plasmodiumfalciparum malaria: association of knobs on the surface of infected erythrocytes with a histidine-rich protein and the erythrocyte skeleton. J. Cell Biol. 98, 12561264. 9 Brown, G.V., Culvenor, J.G., Crewther, P.E., Bianco, A.E., Coppel, R.L., Saint, R.B., Stahl, H.-D., Kemp, D.J. and Anders, R.F. (1985) Localization of the ringinfected erythrocyte surface antigen (RESA) of Plasmodium falciparum in merozoites and ring-infected erythrocytes. J. Exp. Med. 162, 774-779. 10 Favaloro, J.M., Coppel, R.L., Corcoran, L.M., Foote, S.J., Brown, G.V,, Anders, R.F. and Kemp, D.J. (1986) Structure of the RESA gene of Plasmodiumfalciparum. Nucleic Acids Res. 14, 8265 8277. 11 Coppel, R.L., Culvenor, J.G., Bianco, A.E., Crewther, P.E., Stahl, H.-D., Brown, G.V., Anders, R.F. and Kemp, D.J. (1986) Variable antigen associated with the surface of erythrocytes infected with mature stages of Plasmodium falciparum. Mol. Biochem. Parasitol. 20, 265-277. 12 Coppel, R.L., Lustigman, S., Murray, L. and Anders, R.F. (1988) MESA is a Plasmodium falciparum phosphoprotein associated with the erythrocyte membrane skeleton. Mol. Biochem. Parasitol. 31, 223 232. 13 Coppel, R.L. (1992) Repeat structures in a Plasmodium falciparum protein (MESA) that binds human erythrocyte protein 4.1. Mol. Biochem. Parasitol. 50, 335-348. 14 Howard, R.J., Lyon, J.A., Uni, S., Saul, A.J., Aley, S.B., Klotz, F., Panton, L.J., Sherwood, J.A., Marsh, K., Aikawa, M. and Rock, E. (1987) Transport of an Mr 300 000 Plasmodium falciparum protein (Pf EMP-2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane. J. Cell Biol. 104, 1269 1280. 15 Wiser, M.F., Wood, P.A., Eaton, J.W. and Sheppard, J.R. (1983) Membrane associated phosphoproteins in Plasmodium berghei infected murine erythrocytes. J.
68 Cell Biol. 97, 196 201. 16 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. 17 Deleersnijder, W., Hendrix, D., Bendahman, N., Hanegreefs, J., Brijs, L., Hamers-Casterman, C. and Hamers, R. (1990) Molecular cloning and sequence analysis of the gene encoding the major merozoite surface antigen of Plasmodium chabaudi chahaudi IPPCI. Mol. Biochem. Parasitol. 43, 231 244. 18 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 5467. 19 Von Heijne, G. (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14,
4683 4690. 20 Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444~-2448. 21 Anders, R.F., Coppel, R.L., Brown, G.V. and Kemp, D.J. (1988) Antigens with repeated amino acid sequence from the asexual blood stages of Plasmodium Jalciparum. Prog. Allergy 41, 148 172. 22 Suetterlin, B.W., Kappes, B. and Franklin, R.M. (1991) Localization and stage specific phosphorylation of Plasmodium Jalciparum phosphoproteins during the intraerythrocytic cycle. Mol. Biochem. Parasitol. 46, 113 122. 23 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.
59
MOLBIO 01827
Structure of a Plasmodium chabaudi acidic phosphoprotein that is associated with the host erythrocyte membrane Willy Deleersnijder, P o r n t h i p P r a s o m s i t t i , S u m a l e e T u n g p r a d u b k u l , D i a n a H e n d r i x , C6cile H a m e r s - C a s t e r m a n a n d R a y m o n d H a m e r s Laboratorium Algemene Biologie, Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, Brussels, Belgium (Received 27 January 1992; accepted 22 July 1992)
We have characterized by molecular cloning and sequencing a Plasmodium chabaudi antigen that is associated with the membrane of the infected erythrocyte throughout the entire intraerythrocytic cycle. The protein (PcEMA1) has a predicted size of 50 kDa and contains a major tandem repeat array of 16 octapeptides that constitutes almost 30% of the protein. At its amino-terminus, PcEMAI has a string of hydrophobic residues characteristic of a secreted protein, but does not contain a hydrophobic membrane-spanning segment. The antigen appears to reside on the cytoplasmic face of the erythrocytic membrane. PcEMA1 has a predicted pI of 4.4 and is a potential phosphoprotein. Key words: Malaria; Plasmodium chabaudi; Erythrocyte cytoskeleton; Tandem repeats; Phosphoprotein
Introduction
During their intraerythrocytic growth and development, Plasmodium parasites induce profound changes in the structural, functional and antigenic properties of the host red cell membrane (see refs. 1-4 for review). Among these are alterations in membrane fluidity, reduced deformability and change in shape of erythrocytes infected with mature parasites, the creation of new metabolic channels, the Correspondence address: Raymond Hamers, Laboratorium Algemene Biologie, Instituut voor Moleculaire Biologic, Paardenstraat 65, 1640 St. Genesius Rode, Belgium. Note." Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M95789. Abbreviations." PcEMAI, P. chabaudi erythrocyte membrane antigen 1; KAHRP, knob-associated histidine-rich protein; MESA, mature parasite-infected erythrocyte surface antigen; RESA, ring-infected erythrocyte surface antigen; PfEMP1 or 2, P. falciparum erythrocyte membrane protein 1 or 2; APP, acidic phosphoprotein; pI, isoelectric point.
appearance of electron-dense protrusions (knobs) on the membrane and the acquisition of cytoadherent properties. These modifications can be mediated either by direct insertion of parasite-derived proteins in the red cell plasma membrane, e.g., the PfEMP1 antigen [5] or the transferrin receptor [6,7], or indirectly via their association and interaction with components of the red cell cytoskeleton. Wellcharacterized Plasmodium antigens that are targeted to the red cell membrane and interact with the erythrocyte cytoskeleton include the knob-associated histidine-rich protein (KAHRP) [8], the ring-infected erythrocyte surface antigen (RESA) [9,10] and the mature parasite-infected erythrocyte surface antigen (MESA/PfEMP2) [11-14]. Both MESA and RESA are present on the membrane of the infected erythrocyte in a phosphorylated state. In addition, Wiser et al. have reported the appearance of new phosphoproteins on the host erythrocyte membrane during infection with P. berghei (65 and 46 kDa) and P. chabaudi (93, 90 and 76 kDa) [15,16]. These
60
parasite-derived phosphoproteins were tightly associated with the submembranous cytoskeleton of the host erythrocyte, had a pI of less than 4.0 and have been referred to as APP (acidic phosphoproteins). In this report we describe the molecular cloning and sequence analysis of a P. chabaudi gene encoding a protein that is associated with the host red cell membrane throughout the entire erythrocytic cycle. We also demonstrate that this antigen is a potential phosphoprotein.
Materials and Methods
Parasites. P. chabaudi chabaudi strain IP-PC1 clone C (IP-PC1/C) was used for all work in this report and has been described before [17]. Parasites were grown in OF1 outbred mice (Iffa Credo), kept in an inverted nycthemeral cycle for diurnal schizogony. Construction o['cDNA and genomic libraries. c D N A and genomic libraries were constructed and screened as described before [17]. Sequencing. Inserts of overlapping clones were subcloned into p U C or pBluescript (Stratagene) vectors and totally (UL20, 46A) or partially (L21, X2A, 81 and 44) sequenced. Subclones were generated that contained progressive unidirectional deletions of each insert by controlled exonuclease III digestion (Erase-a-base, Promega). Sequencing was done on double-stranded templates by the dideoxy chain-termination method of Sanger et al. [18], using modified T7 D N A polymerase (Sequenase, USB). Both strands were entirely sequenced in the coding area. We were unable to determine unambiguously nucleotide position 1721. Computer-assisted storage and analysis of sequence data was facilitated using the P C / G E N E software package (Intelligenetics). Indirect immunoJluorescence in suspension. Infected erythrocytes were either fixed by overnight incubation in phosphate buffered saline (PBS - 0.145 M NaCI/8 mM phos-
phate, pH 7.2) containing 3.7% formaldehyde, or used without fixation. After 3 washes with PBS small amounts of red cells (10/A pellets) were resuspended for 30 min in 100 /tl of the appropriate sera (diluted 1/200 in PBS). After 3 further washes with PBS, erythrocytes were incubated in 50 /~1 of FITC-conjugated goat anti-rabbit Ig antibody solution for 30 min. Finally, cells were washed 3 more times, resuspended to an appropriate dilution and viewed under the fluorescence microscope. Production ~[" rabbit sera against Jusion proteins. Y1089 bacteria (ATCC37196), lysogenic for 2gtll clones 81, 43 and 44 were grown at 32°C to A6o0 = 0.5. The culture was subsequently continued at 44°C for 20 min with good aeration. Isopropyl-/:~-thiogalactopyranoside (IPTG) was added to a final concentration of 2 mM and the culture was continued for 1 h at 37~C before harvest. Bacterial pellets were frozen at - 8 O C , thawed, and sonicated. The sonicated extract of a 50 ml culture was mixed with 1 ml SDSPAGE sample buffer, boiled for 5 min at 100~C, spun to remove insoluble debris and loaded on a preparative SDS-PAGE gel. The gel slice containing the recombinant fusion protein (identified after Coomassie blue staining) was frozen in liquid nitrogen and ground to a fine powder in a mortar. The powder was resuspended in 1 ml of PBS, emulsified with incomplete Freund's adjuvant and injected subcutaneously into a rabbit. Three injections were given at 1 month intervals. Rabbits were bled 2 weeks after the last injection. Sera were denoted L81, L43 and L44 respectively. 2gtll clones 81 and 44 express part of the PcEMA1 antigen (see Fig. 1). 2gtl 1 clone 43 expresses an unrelated P. chabaudi antigen that is also associated with the membrane of the infected red cell. It should be noted that polyclonal rabbit serum L43 is unrelated to mouse monoclonal antibody 43 (mAb 43), that is also described in the text. The (membrane associated) antigen recognized by L43 is different from the one recognized by mAb 43. The fact that both sera have been denoted '43' is entirely coincidental.
61
Preparation of erythrocyte ghosts and parasitic fractions. Uninfected or infected (30-50% parasitemia) blood was pelleted and the buffy coat removed. Ghosts were prepared by hypotonic lysis in 5 mM phosphate buffer pH 8.0 and centrifugation at 22 000 x g (10 min). The fluffy creamy layer containing membranous ghosts was removed from the underlying dark pellet of parasites and extensively washed in the same buffer until completely devoid of hemoglobin. The particulate pellet, containing the liberated parasite material was washed 3 times in the hypotonic lysis buffer. This is referred to as the parasitic fraction.
Labeling of membrane and parasitic fractions with [?-32p]ATP. One fifth of the erythrocyte ghosts or of the parasitic fraction obtained from the infected (40% parasitemia) or uninfected blood of 1 mouse was incubated for 2 min at 37°C in a final volume of 100/~1 50 m M 4-morpholineethanesulfonic acid (Mes), pH 6.0/10 mM MgC12/5 #M [7-32p]ATP (80 Ci m m o l - 1). For immunoprecipitation, the radiolabeled fractions were washed 3 times in PBS and then solubilized in 20 #1 PBS/2% SDS/5 mM phenylmethylsulfonylfluoride (PMSF) (15 rain at room temperature, followed by centrifugation at 12000 x g for 10 min to remove insoluble material). 180 /tl of Netto (10 mM Tris-HCl pH 8.3/100 mM NaCI/10 mM E D T A / I % Triton X-100/0.1% ovalbumin/1 mM PMSF/1 mM N-c~-p-tosyl-L-lysine chloromethylketone HC1 (TLCK)) was added, and 50 /~1 of rabbit antiserum. This mixture was incubated for 3 h at 4°C under gentle agitation. 10 mg of protein A-Sepharose-CL4B beads (Pharmacia) were added and incubation continued for another hour. The beads were washed 3 times in Netto followed by l wash in 10 mM Tris-HCl pH 8.3/100 mM NaCI/10 mM EDTA/1 mM PMSF/I mM TLCK. Immunocomplexes were then released by heating for 3 min at 100°C in electrophoresis sample buffer and analysed on 10% SDSPAGE slabs.
Immunoblot analysis.
One-fifth of the ery-
throcyte ghosts obtained from the blood of 1 mouse were loaded per lane (1 cm wide) of a 1.5-mm-thick 12.5% SDS-polyacrylamide gel slab. The gel was electroblotted onto a nitrocellulose filter which was saturated afterwards with TBS-T buffer (10 mM Tris-HC1, pH 8.0/150 mM NaCI/0.05% Tween 20) containing 1% BSA. Separate lanes on the filter were incubated with appropriate rabbit sera diluted 1/5000 in TBS-T. After subsequent washing affinity purified alkaline phosphatase conjugated goat anti-rabbit Ig antibodies were added to the filter strips for 1 h. After a final round of washes, substrate (0.05 mg ml ~ 5bromo-6-chloro-3-indolyl acetate/0.1 mg ml l nitro blue tetrazolium in 10 mM MgC12/100 mM Tris-HCl, pH 9.5) was added to the filters and the enzymatic reaction was stopped after 5-15 min by rinsing the membranes with water.
Results
Cloning strategy. 2gtl 1 clone 46 was initially obtained as a mosaic clone, containing part of the MSA-1 coding sequence ligated to an unrelated c D N A molecule of approximately 1 kb [17]. As sequencing of this extraneous c D N A fragment (named 46A) revealed some interesting features (long repeat area) it was further studied. Clone 46A was further
ATG
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63
A contiguous stretch of 1846 nucleotides was obtained containing one major open reading frame, starting at nucleotide 324 and ending at position 1646 (Fig. 2). This encodes a protein of 441 amino acids with a calculated size of 49 707 (denoted PcEMA1 -- P. chabaudi erythrocyte membrane antigen l). Clones X2A and 46A did not differ by a single nucleotide in areas where sequence had been determined from both clones (totalling about 630 nucleotides) indicating that X2A was not derived from a pseudogene. As the last 200 nucleotides of the PcEMA1 coding sequence were determined from a genomic DNA clone the occurrence of an intron in this area could not be ruled out. We therefore verified the collinearity of c D N A and genomic DNA in this region by a PCR approach. P. chabaudi total RNA (2.5 /~g) was first treated with DNaseI to destroy any remaining traces of gDNA and poly(A) + RNA was subsequently converted to first strand cDNA. This first strand c D N A was then amplified by PCR using oligonucleotides ON19-ON20 and ON13-ON20 as a primer pair. The positions of these primers were: ON13 (nucleotide positions 1395-1411 and repeated in 1422-1438), ON19 (nucleotide Sequence analysis.
positions 452-469) and ON20 (complementary to nucleotide positions 1701-1718). Amplification was performed in a thermal cycler for 36 cycles (45 s at 94°C, 45 s at 54°C and 1 min at 72°C), using 0.l /~g of each primer. The PCR-products obtained from the ON19/ON20 and ONI 3/ON20 amplifications were analyzed by agarose gel electrophoresis and consisted of a 1.3-kb and a 0.3-kb fragment respectively (data not shown). No PCR products were obtained when these amplifications were performed on RNA that had not been reverse transcribed. These results clearly demonstrate that there is no hidden intron in the last 200 bp of the determined DNA sequence. The primary structure of PcEMAI exhibits a number of interesting features: (1) The Nterminal sequence was identified as a potential secretory signal peptide by computer analysis according to the method of Von Heijne [19]. The predicted cleavage site is between residues 15-16 or 24~25 ( - 3 , - 1 rule). The first 6 amino acids of the PcEMA1 signal peptide are almost identical to the first 6 amino acids of the P. chabaudi major merozoite surface antigen 1 (MSA 1) signal peptide (MKAISL versus M K A I G L ) [17]. (2) PcEMAI contains
A
1 2
GAA GAA
GAA GAA
GAC GAC
CCA GCA
TAC CTC
TTA TCA
TTA TTA
CAG ATG
E E D P Y L L Q E E D A L S L M
3 4 5 6
GAA GAA GAA GAA
TAC GGA GGA TAT
GAC GAC GAC GAC
GCA GCA GCA GCA
GAA GAA GAA GGC
ACA ACA ACA ACA
CTA CTA CTA CTA
AAT AAT AAT AAT
E E E E
A E T L N A E T L N A E T L N A G T L N
B
7 8 9
GAA GAA GAA
GAA GCT GAA
GAC GGC GGC
GCA GAA GAA
GGT GGT GGT
ACA ACA GCA
ACA ACA GCC
AAT AAT AAT
E E D A G T T N E A G E G T T N E E G E G A A N
C
i0 G A A 11GAA 12 G A A
TAT TAT TAT
GAC GAC GAC
GCA GCA GCA
GAG GAT GGC
ACA ACA ACA
CTA CTA CTA
AAT AAT AAT
E Y D A E T L N E Y D A D T L N E Y D A G T L N
B ,
Y G G Y
D D D D
13 G A A T A T G A C
GCA GGT ACA
CTA AAT
E Y D A G T L N
14 G A A 15 G A A 16 G A A
GGA GAA GAA
ACA TCC GCA
E E E G S T T N E A G E G T S N E A G E G "fAN
GAA GCT GCT
GAG GGT GGC
AGC GGT GGT
ACA ACA ACA
AAT AAT AAT
C
l
Fig. 3. Structure of the major tandem repeat array of PcEMA1 at the nucleotide (left) and amino acid (right) level.
64
in its central part an 8 amino acid sequence repeated in tandem 16 times, that constitutes almost 30% of the total protein. These tandem repeats are incompletely conserved (Fig. 3). An unrelated tandem repeat structure of 2 completely conserved nonapeptides occurs a little downstream of the major repeat area. (3) Immediately following the nonapeptide repeat structure and almost continuing to the Cterminal end is an area that is extremely rich in Lysine residues (31 out of 47 residues between amino acid positions 371-417). (4) Overall PcEMA1 is rather hydrophilic. One third of all residues are either Asp (7.0%), Glu (15.9%) or Lys (12.7%). The predicted pI of PcEMA1 is 4.4 (PC/GENE C H A R G P R O software). (5) Motif searching using the P C / G E N E PROSITE version 3.0 software revealed the presence of 21 potential casein kinase II phosphorylation sites (Fig. 2). 15 of these occur in the tandem repeat oligopeptides. There are also 4 potential protein kinase C phosphorylation sites. (6) Two potential Nglycosylation sites are present in the sequence (amino acid positions 21 and 112). A search of
the SWISS-PROT release 21.0 protein data bank using the FASTA programme [20] showed that PcEMA1 was not significantly similar to any protein in the data bank.
Immunofluorescence and immunoblotting. Sera raised against expressed parts of PcEMA1 (clones 81 and 44) reacted with the membrane of formaldehyde fixed infected red cells, showing a rim-like fluorescence on the surface of the cell (Fig. 4). No fluorescence was observed on unfixed infected red cells in suspension indicating that PcEMA1 is not 1 •
2
3
4
94--
I
I
67-
43--
30-
Fig. 4. Indirect immunofluorescence using antiserum (L81) to the expressed product of clone 81. Fluorescein staining of formaldehyde fixed erythrocytes infected with P. chabaudi. The noninfected erythrocytes are indicated by arrows.
Fig. 5. lmmunoblot analysis. Infected red cell ghosts were separated on a 12.5% SDS-PAGE gel and blotted onto a nitrocellulose filter. Filter strips were p r o b e d with antiserum (L81) to the fusion product of )~gtll clone 81 (lane 1), antiserum (L44) to the clone 44 (lane 2), antiserum (L43) raised against the fusion product of 2gtl 1 clone 43, which expresses part of an unrelated P. chabaudi antigen that is also associated with the membrane of the infected red cell (lane 3, our unpublished results) and L81 preimmunization serum (lane 4).
65
accessible from the outside. The rim-like fluorescent staining was observed on the surface of erythrocytes containing parasites at all stages of parasite development (rings, trophozoites and schizonts). Sera against the recombinant fusion protein of clones 81 and 44 recognized a 85-kDa band on Western blots of total membrane preparations from infected red cells (Fig. 5). Apart from the principal 85-kDa band both sera also immunostained a minor band of slightly higher electrophoretic mobility.
could be a substrate for kinases of parasitic or erythrocytic origin. Incubation of the crude parasitic fraction with [TYP]ATP resulted in the phosphorylation of many proteins of varying size (Fig. 6, lane 1). Serum L81 specifically immunoprecipitated a 85-kDa phosphoprotein (Fig. 6, lane 3) from the [7-32P]ATP-labeled parasitic fraction. Immunoprecipitation of [7-32p]ATP labeled membrane preparations from infected red cells with L81 yielded a very weak 85-kDa band that was only apparent after 14 days' autoradiography (data not shown).
Phosphorylation of PcEMA1.
Since analysis of the PcEMA 1 protein sequence indicated the existence of numerous potential phosphorylation sites, we investigated whether PcEMA1
Discussion We have identified by molecular cloning a P.
chabaudi protein that is associated with the 2
3
4
!i~~iiiiii
94" 67.
43
Fig. 6. Overnight autoradiography of the [y-32p]ATPlabeled parasite fraction. Lane 1, total [7-32P]ATP labeled parasite fraction. Lanes 2-4, immunoprecipitations of radiolabeled parasite fraction using preimmunization serum L81 (lane 2), L81 serum against the expressed fusion product of clone 81 (lane 3) and polyclonal rabbit antiserum L43, raised against part of a P. chabaudi antigen, unrelated to PcEMA1, that is also associated with the membrane of the infected red cell (lane 4; see also Fig. 5).
membrane of infected erythrocytes and have determined its primary structure. The discrepancy between the predicted Mr of PcEMA1 (50000) and its apparent Mr on SDS-PAGE (85000) can most likely be attributed to the pronounced hydrophilic character of this protein and the extensive array of tandem repeats that it contains. This has been commonly observed for malarial antigens of this type [21]. There is considerable diversity between the individual octapeptide units of the major repeat region. Except for the first AA of each unit, which is invariably Glu, at least 3 and sometimes up to 6 different AA can be found at any given position in the octapeptide unit (Fig. 3). Units 4 and 5 are the only ones that are repeated exactly and in tandem, both at the AA and at the nucleotide level. Units 6, 12 and 13 are completely conserved at the AA level but not at the nucleotide level (there is a silent thirdbase substitution in codon 5 of unit 13). Likewise, units 3 and 10 are identical at the AA level but not at the DNA level (two silent third base substitutions). The remaining 10 octapeptide units each have a unique structure. It is however possible to subdivide the major repeat region of PcEMA1 into a number of blocks on the basis of local homology between adjacent units.
66
Units 3-6 and 10-13 show close homology and have the general structure: E(Y/G)DA(E/ D / G ) T L N (denoted here as blocks B and B' respectively). Blocks B and B' are interrupted by 3 units (7, 8, 9; = block C) which are less homogeneous and appear to be repeated in units 14-15-16 (block C') although a number of point mutations have occurred between blocks C and C'. Units 1 and 2 are also clearly related and can be considered to constitute a separate block ( = block A). The overall structure of the repeat region can thus be represented as: ABCB'C'. It is clear from the sequence data of the major repeat area that a mechanism is operating to homogenize the repeat units at the D N A level. As well as from the global homogenization throughout the entire repeat array, this can also be seen from the apparent spreading of mutations to equivalent positions in neighboring units; see, e.g., codon No. 2 in units 4 and 5; codon 7 in units 7 8 and codons 3~4 in units 8-9). Unequal crossing-over, gene conversion or slippage replication can all be invoked to explain the concerted evolution of repeat units as well as the internal duplication that presumably took place in the repeat array (ABC ~ ABCB'C'). Antisera raised against a large portion of PcEMA1 stained the membrane of formaldehyde fixed infected red cells in an indirect immunofluorescence assay in suspension. No fluorescent staining of the membrane was observed when the infected erythrocytes were not fixed. This finding is in keeping with the determined structural feature of PcEMA1. Apart from the N-terminal secretory signal peptide no hydrophobic transmembrane anchor sequences can be found in PcEMA1. This suggests that PcEMA1 is not an integral but rather a peripheral membrane protein. The pronounced hydrophilicity of PcEMA1 is also consistent with this notion. We conclude therefore that PcEMA1 resides on the cytoplasmic face of the erythrocyte membrane. We have shown that PcEMA1 is a potential substrate for kinases. PcEMA1 is readily phosphorylated in the parasitic fraction but only very faintly in an equivalent (prepared
from an equal number of infected erythrocytes) ghost membrane preparation. From these data we cannot conclude whether the kinase that is responsible for this phosphorylation is of parasitic or of erythrocytic origin. The fractionation method used to separate membranes from parasites is only crude and it is likely that the parasite fraction is contaminated with 'membrane' material and vice versa. It remains to be determined whether PcEMA1 phosphorylation also takes place in live parasites. One could indeed argue that the disruption of the intracellular structure as in our parasitic fractions will artifactually expose substrates to 'opportunistic' kinases. However since PcEMA1 is associated with the red cell membrane and since many erythrocyte membrane associated parasite antigens that have been identified to date are phosphorylated it is not unlikely that PcEMA1 is also phosphorylated under in vivo conditions. The characteristic features of P c E M A I as outlined here, resemble very closely those of a family of acidic phosphoproteins identified by Wiser et al. on the membranes of P. chabaudi [Pc(em)93; Pc(em)90 and Pc(em)76] and P. berghei [Pb(em)46 and Pb(em)65] infected red cells [15,16]. These include the acidic nature of PcEMA1, its close association with the red cell membrane possibly as a phosphoprotein, its presence throughout the entire erythrocytic cycle and the approximate Mr. The difference in apparent Mr between PcEMA1 (85 000) and any of the P. chabaudi phosphoproteins described by Wiser et al. (93 000, 90000 and 76000) might be explained by the fact that both proteins have probably been characterized from different strains. It can be expected that the large tandem repeat array is liable to rapid diversification between different strains (as is the case for most malarial tandem repeat arrays) both in terms of its encoded sequence as in terms of its length. Pb(em)65 was actually reported to exhibit Mr heterogeneity between different strains [16]. A similar phenomenon is observed for other red cell membrane associated malarial proteins such as MESA and K A H R P . These antigens also contain extensive tandem repeat areas and do exhibit a
67
variant Mr between different isolates [8,11]. We can however definitely exclude that PcEMA1 is identical to Pc(em)93 on the following grounds. Monoclonal antibody 43 (mAb 43), that was made in this laboratory and recognizes an unrelated P. chabaudi antigen that is also associated with the red cell membrane, reacts with Pc(em)93 (M. Wiser, personal communication). In our hands mab 43 detects a band of 105 kDa on Western blots of P. chabaudi IP-PC1/C extracts and does not react with the PcEMA1 85-kDa band. This 105-kDa band is most probably the murine equivalent of RESA (our unpublished results). Also, the reported amino acid composition of Pc(em)93 [14] is different from the predicted amino acid composition of PcEMA1. The occurrence of an acidic phosphoprotein (pI = 5.0; Mr = 88 000) on the membranes of P.falciparum infected erythrocytes has recently been reported [22]. It is also possible that PcEMA1 is homologous to this antigen or/and a 85-kDa P. fi~lciparum phosphoprotein detected by Murray et al. [23] on the membrane of infected red cells. The identification and characterization of PcEMA1 adds another member to the expanding family of parasite antigens that interact with the membrane of the infected red cell. Although it is probably safe to say that the function of these proteins during infection, is to modulate the structure of the red cell membrane to the advantage of the parasite, the precise role of most of these proteins and especially PcEMA1 has still to be defined. The cloning, sequencing and partial expression of PcEMAI, as reported here, will provide the molecular tools to delineate its precise biological role during future work.
Acknowledgements This work was supported by EEC contracts TSD-M-152 and TS-2-0148 and Belgian government fund 'BIO 10'. The author wish to thank Jos6e Hanegreefs for skillful technical support and Andre van Dormael for photographic work.
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68 Cell Biol. 97, 196 201. 16 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. 17 Deleersnijder, W., Hendrix, D., Bendahman, N., Hanegreefs, J., Brijs, L., Hamers-Casterman, C. and Hamers, R. (1990) Molecular cloning and sequence analysis of the gene encoding the major merozoite surface antigen of Plasmodium chabaudi chahaudi IPPCI. Mol. Biochem. Parasitol. 43, 231 244. 18 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 5467. 19 Von Heijne, G. (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14,
4683 4690. 20 Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444~-2448. 21 Anders, R.F., Coppel, R.L., Brown, G.V. and Kemp, D.J. (1988) Antigens with repeated amino acid sequence from the asexual blood stages of Plasmodium Jalciparum. Prog. Allergy 41, 148 172. 22 Suetterlin, B.W., Kappes, B. and Franklin, R.M. (1991) Localization and stage specific phosphorylation of Plasmodium Jalciparum phosphoproteins during the intraerythrocytic cycle. Mol. Biochem. Parasitol. 46, 113 122. 23 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.
