Plasmodium falciparum: Characterization of gene R45 encoding a trophozoite antigen containing a central block of six amino acid repeats

EXPERIMENTALPARASITOLOGY

74,441-451

(1992)

Plasmodium falciparum: Characterization of Gene R45 Encoding a Trophozoite Antigen Containing a Central Block of Six Amino Acid Repeats’ SERGEBONNEFOY,2 MICHELINE GUILLOTTE, GORDON LANGSLEY, AND ODILE MERCEREAU-PUIJALON Unit6

de Parasitologic

BONNEFOY,S.,GUILLOTTE, Plasmodium falciparum:

ExpCrimentale,

Institui

Pasteur,

M.,LANGsLEY,G.,

75724 Paris

Cedex

15, France

AND MERCEREAU-PUUALON,~.

1992.

Characterization of gene R45 encoding a trophozoite antigen containing a central block of six amino acid repeats. Experimental Parasitology 74,441-451. We describe here an antigen, called R45, expressed by the young trophozoites of Plasmodium falciparum. This antigen contains a block of tandem repeats of six amino acids which are recognized by sera from humans living in endemic areas. The R45 gene is located on chromosome 3. It is present in all strains examined and shows limited size polymorphism. The C-terminal unique region of the protein shows a strong homology with the catalytic domain of the serine protein kinases. Interestingly, the central repeats contain a large number of putative phosphorylation sites. The implications of these features are discussed. 0 1992 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Malaria; Protozoa; Plasmodium falciparum; Trophozoite; Hexapeptide repeats; Protein kinase; Phosphorylation; Messenger ribonucleic acid (mRNA); Deoxyribonucleic acid (DNA). INTRODUCTION

A large number

of genes from Plasmoto date contain regions of tandem repeats (Kemp et al. 1990). Repeated peptide motifs have been described in antigens expressed at a variety of different developmental stages, in the mosquito and in the human host. The reasons for the presence of repeats are still obscure. They clearly are highly immunogenic and often constitute the major target of antibodies on an antigen (Anders et al. 1988). They may be an evasion mechanism by diverting the immune response away from important sites (Anders 1986). Furthermore the presence of repeats of similar

dium falciparum described

1 Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. M83790, M83791, M83792, M83793, M83794, and M83795. * To whom correspondence should be addressed at Institut Pasteur, Unite de Parasitologie Experimentale, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.

sequence, or with significant homology on several distinct antigens, creates a complex network of immunological crossreactivities. This may participate in immune evasion, as proper maturation of antibodies with high affinity may be prevented (Anders 1986; Mattei et al. 1989) and the various antigens may compete for antibody binding (Mercereau-Puijalon et al. 1991). Interestingly, repeats are present not only on antigens but also on enzymes such as RNA polymerase III (Li et al. 1991) and RNA polymerase II (Li et al. 1989; Giesecke et al. 1991). We report here the analysis of a gene of P. falciparum encoding a protein containing tandem repeats of six amino acids. The gene is expressed at the early stage of the erythrocytic cycle. It is poorly polymorphic and presents at the C terminus, a region of strong homology with the catalytic domain of serine protein kinases (Hanks et al. 1988). Furthermore, the repeats contain numerous putative phosphorylation sites (Pearson et al. 1985).

441 0014-4894192 $5.00 Copyright Q 1992 by Academic Press, Inc. Au rights of reproduction in any form reserved.

442

BONNEFOYETAL. MATERIALSAND

METHODS

Parasites and culture conditions. The Uganda Palo Alto FUPKB strain selected on plasmagel was used (Fandeur et al. 1991). In vitro cultures were performed in RPM1 1640 medium supplemented with 10% normal human sera and 5% human A+ red blood cells under the conditions described by Trager and Jensen (1976). Plasmagel flotation was done once a week (Jensen and Trager 1978). The other strains used were Tak 9-96 (provided by Dr. D. Walliker), FCR3 (provided by Professor Trager), FCC1 (provided by Dr. Li Wen Lu), Sb27 Bandia (provided by Dr. J. Roffi), 7G8, and ItG2Gl (provided by Dr. R. Howard). Synchronization was done by concentrating late stages by plasmagel flotation. At 6 hr after the beginning of reinvasion, the culture was harvested and treated with sorbitol (Lambros and Vanderberg 1979). Construction

and

screening

of genomic

libraries.

The construction of the Tak 9-96 genomic library has been described elsewhere (Guerin-Marchand et al. 1987). Immunoscreening of the Tak 9-96 genomic expression library was performed using R96 rabbit antiserum (see below). The rabbit antiserum was identitied in the library 5 clones R45, R23, R5 1, R25, and R33. These clones (except R33) were also recognized by human sera from adults living in hyperendemic areas and by Saimiri monkey immune sera. The EcoRI insert of clone R45 was nick translated and used to screen DdeI and AccI minilibraries prepared as follows: The DdeI Palo Alto genomic library was obtained after excision of genomic fragments of 4 to 8 kb from a preparative agarose gel. Fragments were electroeluted, phenol was extracted, and then repair was by T4 DNA polymerase and Escherichia coli DNA ligase. After ligation with phosphorylated EcoRI linkers and digestion with EcoRI, the DdeI fragments were ligated with EcoRI-cut phosphatasetreated A gtWES vector and packaged under conditions recommended by the manufacturer (Promega Biotec). The library was plated on 1046 cells (Murray et al. 1976) and screened with R45 radiolabeled insert. Hybridizing clones were highly unstable. During amplification, clones forming large plaques were removed with a Pasteur pipette. DNA was prepared and digested with EcoRI, and the 5.6-kbp insert was subcloned in pUC 13 plasmid, in which the stability was improved. An AccI Palo Alto genomic library was obtained essentially as the DdeI library, except that fragments of I.54 kbp were ligated into A gtll vector and the library was plated on Y 1090 cells. DNA sequencing. Genomic Tak 9-96 clones were subcloned in the single-stranded phage Ml3 vectors mp9 and mp8 (Messing and Viera 1982) and both strands sequenced by the dideoxy chain termination

methods (Sanger et al. 1977). Restriction fragments of the DdeI and AccI inserts were subcloned in Ml3 vectors mp18 and mp19 or pUC13. DNA sequencing was performed by dideoxy chain termination methods in both Ml3 and pUC. When necessary, specific oligonucleotide primers were synthesized. Southern blot analysis. Genomic P. falciparum DNA was prepared from late-stage parasites according to standard methods (Maniatis et al. 1982). Three micrograms of DNA was digested for 3 hr with various restriction endonucleases under manufacturer’s conditions, fractionated on 0.8% agarose gel, and transferred to nylon membrane (Amersham). The R45 insert was labeled with [32P]dATP and used as a probe. Hybridizations were done at 65°C overnight in 6~ SSC (20x SSC = 3M NaCl, 0.3 Na citrate), 2.5% nonfat powder milk, 100 kg/ml herring sperm DNA and washed at the same temperature in 0.5X SSC, final concentration. Pulse

field

gradient

electrophoresis.

P. falciparum

parasites (Patarapotikul and Langsley 1988) were prepared in agarose blocks for electrophoresis as described by Van der Ploeg et al. (1985). Blocks were sealed in 0.9% agarose gel in Tris/borate/EDTA (TBE) buffer and electrophoresed for 62 hr at 50 V, 50 mA, with a IO-min pulse. After electrophoresis the gels were stained in 0.5~ TBE with ethidium bromide for 1 hr and then destained and photographed. Transfer of DNA to Hybond N membrane was performed as described above except that the gel was treated with 0.25 M HCI for 25 min prior to denaturation. Hybridization was performed overnight at 65°C with R45 radiolabeled probe or a CSP (kindly provided by Dr. T. McCutchan) radiolabeled probe in 6x SSC, 2.5% nonfat milk, 100 kg/ml herring sperm DNA and washed at 65°C in 2x SSC, final concentration. RNA preparation. Highly synchronous cultures were harvested and washed twice in RPMI, and parasites were released from infected erythrocytes by saponin lysis (0.025% for 10 min at 37°C). Free parasites were washed in RPMI, nucleic acids were extracted by the addition of sodium dodecyl sulfate (final concentration, 3%) in buffer A (50 mM sodium acetate, 100 mM NaCl, 1 mA4 EDTA), and an equal volume of phenol was equilibrated with buffer A. After successive extractions in phenol/CHCl, and CHC13 alone, salt concentration was adjusted to 300 mM sodium acetate and the nucleic acids were precipitated with 2 vol of cold ethanol. The DNA which forms a gelatinous precipitate was immediately collected with a Pasteur pipette. Following centrifugation, RNA and some contaminating DNA were resuspended in 100 pl sterile DEPC-treated water. The integrity of the RNA preparation was determined by electrophoresis on 1% agarose gel stained with ethidium bromide. Little or no DNA contamination, depending on the fraction, was seen in the RNA preparations.

P.falciparum

R45 BLOCK OF SIX AMINO

Northern blot analysis. Ten micrograms of each stage-specific RNA was loaded on a 1.5% agarose gel containing formaldehyde as described in Maniatis (1982). After size fractionation, RNA was transferred from the gel to a nylon Hybond N membrane using the protocol recommended by Amersham. Hybridization cocktail was 1% bovine serum albumin, 1 mM EDTA, 0.5 M sodium phosphate, pH 7.2, 7% SDS, 100 pg/ml herring sperm DNA. After overnight incubation at 65”C, washing was carried out at 65°C in 1 mM EDTA, 40 mM sodium phosphate, pH 7.2, 1% SDS. Expression teins. The

cloning

and purification

of fusion

pro-

Palo Alto A2 fragment was prepared by digestion of the purified DdeI insert with AluI and the 2-kbp fragment hybridizing with R45 insert probe was electroeluted from a gel. It was ligated into EcoRIdigested pMS plasmid vector (Scherf et al. 1990), yielding a functional B-galactosidase. Immunoblots and antibody selection. For immunoblots, synchronous parasites were resuspended in boiling SDS sample buffer, and the equivalent of 10’ parasitized cells were loaded on a 7.5% SDS polyacrylamide gel. After electrophoretic transfer on nitrocellulose filters, immunoblots were incubated with the appropriate sera or ascidic fluid in 5% nonfat milk (Regilait), 50 mMTris, pH 8,0.15 M NaCl, 0.05% Tween 20 and then with anti-species IgG conjugated with alkaline phosphatase (Promega Biotec). Immune human antibodies were affinity purified using B-galactosidase R23 fusion protein or synthetic peptide conjugated to CNBr-activated Sepharose 4B as recommended by Pharmacia. Specifically bound antibodies were eluted from the affinity column by 0.2 M glycine, pH 2.5, and immediately neutralized with 2 M Tris. Immunizations. Rabbit R96 was immunized with a purified lOO-kDa thermostable protein. This protein was purified as follows: boiled culture supematants were ultracentrifuged and the supematant was concentrated on Amicon XMSO and then resuspended in Laemmli sample buffer under reducing conditions (Laemmli 1970). One milliliter of concentrated supernatant was loaded on preparative 7.5% acrylamide gels and the lOO-kDa protein cut off from the gel and electroeluted according to (Hunkapiller et al. 1983). The rabbit was inoculated with 3 p.g of the electroeluted protein in Freund’s adjuvant followed by five injections at 2-week intervals with 3 pg of lOO-kDa protein together with 100 ug of rabbit’s own serum albumin and 2 mg of poly(A)-poly(U) (Hovanessian et al. 1988). Eight-week-old female BALB/c mice or outbred mice were injected with about 10 pg of A2 B-galactosidase prepared from IPTG-induced bacteria. Ninety percent of the protein was found insoluble in E. coli extracts. It was prepared by resuspending the bacterial pellet stepwise in 2, 4, and finally 6 M urea. The protein was highly enriched in 6 M urea. Injections were done SCusing the antigen emulsified in Freund’s com-

ACID REPEATS

443

plete adjuvant for the first immunization and in Freund’s incomplete adjuvant for the subsequent immunizations. They were performed at 4-week intervals, bleedings being done 8 days after the injection. RESULTS

Isolation of recombinant clones and immunological properties of recombinant proteins. DNAse-treated DNA from the P. falciparum clone Tak 9-96 was used to con-

struct an expression library in A gtll (Guerin-Marchand et al. 1987). This library was screened with a rabbit antiserum raised against a lOO-kDa thermostable antigen from culture supernatants, reacting with the 96tIUGBP130 as well as a 160-kDa antigen. Several clones were isolated. Antibodies affinity purified on each of the clones R45, R23, or R51, reacted with the two other ones, showing that the recombinants shared antigenic determinants. Furthermore, these clones reacted strongly with human immune sera. Southern analysis indicated that the three clones crosshybridized and were fragments of the same gene. This gene is called R45. The clones R23 and RS 1 produced a B-galactosidase fusion protein, whereas clone R45 produced a small 15-kDa protein upon IPTG induction, resulting from an internal restart of translation. The fusion proteins and the R45 polypeptide were shown to react with rabbit and human sera (data not shown). Nucleotide sequence of the R45 gene.

DNA sequence of the inserts of clones R45, R23, and R51 showed that they encode tandemly repeated hexapeptides (Fig. 1). Clones R23 and R45 overlap. The R45 radiolabeled insert was used to probe Southern blots of Palo Alto FUPlCB DNA digested with various restriction enzymes, and a restriction map of the R45 gene was constructed (Fig. 2). A DdeI fragment of about 6 kbp was chosen for further investigation and cloned into A gtWES. As recombinant phages were unstable, the insert was subcloned in pUC 13 plasmid vector. Fragments obtained after digestion with various

BONNEFOY

ET

AL.

P. fakiparum AC

R45 BLOCK OF SIX AMINO AC

AC

ACID REPEATS AC

445

AC

TAA

FIG. 2. Restriction map of the R45 gene and relative position of recombinant clones. Selected restriction sites are indicated forAcc1 (AC), DdeI (D), RsaI (R), and ALUM (A). Stop codon is indicated by TAA. The central repeat domain is visualized by hatching; 3’ of the R45 gene, the DdeI fragment ends with an open reading frame indicated with dotted shading. The location of the AZuI clone A2 is shown. Although the three clones R23, R45, and R51 were derived from a different strain, their relative positions were indicated.

restriction enzymes were subcloned and sequenced. This showed that the D&I fragment did not contain the entire gene. Several strategies were used to clone the 5’ end of the gene, However sequences upstream from the DdeI site proved to be highly unstable in E. c&i. An AccI restriction fragment provided an additional 42%bp sequence in 5’. To try to move further upstream, we have used inverse PCR and PCR using Uni-Amp adaptors (Clontech), but without success. Thus the 5’ end of the gene is still to be cloned. The coding sequence of the R45 gene spans about 3 kbp. A large open reading frame is observed from the AccI site, encoding a block of about 80 repeats of six amino acids embedded in unique sequences. The nucleotide and deduced amino acid sequence are shown in Fig. 1. A sequence of 50 repeats has been obtained so far. The position of the R45, R23, and

R51 fragments within the block of repeats is still unknown. The consensus sequence of the repeat is His Lys Ser Asp Ser Asn. At the C terminus, the protein ends with a short stretch of hydrophobic residues preceded by a series of seven lysine residues. The R45 coding sequence is followed by a large region of highly A + T-rich DNA. About 2 kbp 3’ to the putative stop codon, the DdeI fragment ends by a large open reading frame (Accession No. M83794). An RsaI fragment containing part of this coding sequence was used to probe a Northern blot. No hybridization was found (Fig. 3), showing that this putative coding sequence is not an exon of the R45 gene. Furthermore, this indicated that this region is not transcribed by blood-stage parasites. Chromosomal location of the R45 gene. To determine the chromosomal localization of the R45 gene, pulse field gradient electrophoresis was performed on various P.

FIG. 1. Nucleotide and translated amino acid sequence of the R45 gene. The Palo Alto FUP/CB gene sequence derived from DdeI and AccI fragment is shown on the left. Tandem repeats are boxed. The central core of the repeat domain has not been determined and is indicated with dotted lines. The putative N-glycosylation site is shaded and the stop codon is indicated by an asterisk. The three Tak 9-96 clones are shown on the right. Tandem repeats are boxed and the overlapping region between R23 and R45 clone is shaded.

446

BONNEFOY

A

3

8

16

B

24

32

36

R45

ORF

Ag44

FIG. 3. Northern blot analysis of the R45 gene. (A) Time course synthesis of the R45 mRNA production. Time 0 of the kinetic represent the last sorbitol synchronization and corresponds to parasites about 5 hr old. mRNA was prepared at times 3, 8, 16, 24, 32, and 38 hr after synchronization. Forty hours postsynchronization, the first new ring-stage infected erythrocytes were visualized. (B) Northern blot of asynchronous mRNA. mRNA was hybridized with the R45 insert probe, or Ag44 PCR radiolabeled probe. In order to verify that the ORF 3’ of the DdeI fragment was not an exon of R45 gene, an RsaI-D&I fragment containing a large part of this ORF was used as a probe.

fulciparum strains. The R45 labeled probe hybridized to chromosome 3 (Fig. 4). The chromosomal assignment was confirmed by hybridization with the CSP gene, which has been previously mapped to this chromosome (Walliker et al. 1987) (data not shown). R45 gene polymorphism. The R45 gene was detected in all strains tested so far. At the genomic level, limited size polymorphism was observed. This is illustrated in Fig. 4A, for RsaI and TuqI digestions. As the RsaI fragment contains the entire block of 18-bp repeats, this size polymorphism probably reflects different numbers of repeats. Time course of R45 expression. We next analyzed the kinetics of synthesis of the R45 mRNA. As shown in Fig. 3, the R45 probe hybridized to a mRNA of about 5 kb, whose synthesis begins rapidly after reinvasion (about 3 hr postsynchronization, corresponding to about 8 hr postinvasion) and peaks at about 13 hr after reinvasion. Twenty-one hours after reinvasion, the R45 mRNA is barely detectable. This indicates

ET

AL.

that the R45 mRNA is produced mainly at the early stage of the parasite (late ring and young trophozoite). Identification of the R4.5gene product. In order to identify the product of the R45 gene, we prepared immunoblots of parasites collected at various time points, together with those used to extract mRNA and analyzed above. The immunoblots were incubated with a mouse antiserum raised by immunization with the large recombinant A2 P-galactosidase, expressing unique regions in 5’ and 3’ as well as all the repeats (see Fig. 2). As shown in Fig. 6, a 160-kDa protein was detected at the early trophozoite stage. The amount of protein abcdefghii

klmn

W

2.3 2-

4B

a,

I

** 1

c

EsBl

B

FIG. 4. Southern blot analysis of R45 gene. (A) Size polymorphism. DNA from seven strains of P. fulciparum was digested with RsaI or TaqI. The P. fulcipurum strains were as follows: lanes a and b, Palo Alto FUP/CP from Uganda; lanes c and d, Tak 9-96 from Thailand; lanes e and f, FCR3 from Gambia; lanes g and h, FCC1 from China; lanes i and j, Sbd27 from Senegal; lanes k and 1,7G8 from Brazil; lanes m and n, Itg2Gl from Brazil. (B) Chromosome blot analysis showing lane 1, EB; lane 2, TB2H5; lane 3, Cl 1; lane 4, C3; lane 5, Palo Alto FUPKB; lane 6, 3D7; lane 7, A3; lane 8, G9; lane 9, B9. The R45 gene is located on chromosome 3.

P.falciparum 3 200

kDa

--)

96

kDa

--)

66

kDa

+

8

16

24

32

~45 BLOCK OF SIX AMINO 36

40

C

FIG. 5. Identification of the R45 antigen. After sorbitol synchronization (TO) corresponding to parasites of about 5 hr old, infected erythrocytes were collected at times 3, 8, 16, 24, 32, 36, and 40 hr after synchronization. After electrophoresis and transfer to nitrocellulose, the different parasite extracts were incubated with A2 mouse antisera (raised against A2 g-galactosidase expressing the entire repeat domain of R45 flanked by unique sequences).

ACID REPEATS

447

rabbit was immunized with the thermostable 96tR protein extracted from culture supernatant. We have shown that the reactivity on the 160-kDa protein and R45 repeats is due to immunological crossreactivity between the two antigens 96tR/ GBP130 and R45 (Bonnefoy et al. submitted for publication). The intracellular localization of the 160kDa antigen remains to be determined, as the antisera raised to the recombinant protein did not react on immunofluorescence slides. DISCUSSION

In this report, we describe a new blooddecreased during schizogony, to become barely detectable in segmented schizonts. stage antigen, R45, strongly recognized by In young parasites, the presence of the human immune sera. As for numerous P. 160-kDa protein was parallel to that of the falciparum antigens (Kemp et al. 1990), R45 mRNA. The protein was visualized on R45 contains tandemly repeated stretches immunoblots a few hours after the mRNA of amino acids inserted between unique sequences. These repeats are composed of could be detected. The mRNA disappeared six amino acids with the following consenafter 21 hr postinvasion, whereas the prosus sequence: HKSDSN. Some positions tein was detected for several additional are remarkably conserved, especially the hours. We interpret these data as indicating histidine residue, which was found in all rethat the product of the R45 gene is the 160peats. For the other amino acids, although kDa antigen. The short delay observed between the mRNA and the protein at the be- some changes occurred, few permutations were observed. ginning of expression may be due to differThe R45 messenger RNA and protein are ences in the sensitivity of the detection synthesized at the early trophozoite stage. techniques used. The immunoblot is less sensitive than the Northern blot and hence An immune serum raised against a recomthe small amounts of proteins present at the binant p-galactosidase expressing all the six onset of synthesis are probably not de- amino acids repeats as well as unique sequences located upstream and downstream tected. Alternatively, the delay may indiidentified a parasite protein of 160 kDa, cate that the mRNA is not translated immeat the trophozoite diately after its synthesis. The protein ac- present predominantly at reduced cumulates and remains stable several hours stage, but still persisting after the mRNA has totally decayed. In late amounts at the schizont stage. As the synschizonts, however, the protein was no thesis of the 160-kDa protein occurred at the same time as the R45 mRNA, we conlonger detected. The pattern observed with the R96 rabbit antiserum used to isolate the cluded that it is the product of the R45 gene. Recently, a large family of cross-reacting clones was consistent with this. The serum antigens was defined by a monoclonal antireacted with a 160-kDa antigen showing the body, M26-32, obtained from mice immusame profile during the kinetic, as well as a nized by P. yoelli, but reacting also with P. 105kDa protein identified as 96tR/GBP130 falciparum (Cheng et al. 1991). This mAb, (Bonnefoy et al. 1988) (not shown). R96

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AL.

FIG. 6. (A) Alignment of the translated 3’ unique sequence of the R45 gene with the catalytic domain of calcium calmodulin-dependent kinases (Hanks et al. 1988). Each sequence was aligned to GenBank. Gaps represented by dashes were introduced to optimize homologies. Similar amino acid residues are shown in boxes. Amino acids were grouped as (K,R,H), (D,E,N,Q), (W,F,Y), (V,I,L,A,M), (P), (C), (ST), (G) and shaded. Amino acid residues involved in ATP binding (Hanks et al. 1988) are indicated with an asterisk. Roman numerals above the lines indicate conserved subdomains of the set-me kinase family. (B) Homologies between R45 protein, phosphotransferases A,, and V,, (Brenner 1987) and the CaMII serine kinase. Amino acid residues described as involved in ATP binding are indicated with an asterisk. (C) Homologies between R45 repeats and the chondroitin sulfate core protein (Bourdon et al. 1985). Putative CaMII kinase phosphorylation sites are indicated with an overbar.

which inhibits the growth of P. falciparum in culture, reacted with several unrelated clones of a genomic expression library, among which was clone 3, that contained the sequence NNNKDNKNDDNDDS, a sequence identical to the short stretch of asparagine and aspartate residues contiguous to the tandem repeats of the R45 antigen. As the clone 3 recognized by monoclonal antibody M26-32 was described to encode 10 repeats of six amino acids, it is likely that it is part of the R45 gene. The clones R45, R23, and R51 reacted with rabbit antiserum raised to purified 96tR/GBP130. No strict homology was

found between the protein sequences. However, the N-terminal unique region of 96WGBP130 is particularly rich in S, D, and N, residues highly represented in the repeats of the R45 antigen. This may be sufficient to allow immunological crossreactions. Remarkable homologies were found between the C-terminus-unique domain of R45 protein and the catalytic domain of the serine protein kinases. A number of amino acids identified in these proteins as being involved in ATP binding (Hanks ef al. 1988) are conserved at the same position in R45 protein, as shown in Fig. 6A. However,

P.falciparum

R45 BLOCK OF SIX AMINO

some of the consensus sequences are missing, which reduces the probability that R45 might be an active serine protein kinase. However, the role of the R45 protein in ATP binding and phosphorylation of substrates remains a serious interesting possibility. Indeed, it has been claimed that many enzymes which confer antibiotic resistance by acting as phosphotransferases share a common amino acid sequence with the protein kinases, in particular the two aspartate residues and the asparagine involved in the Mg*+-dependent binding of the phosphate group of ATP (Brenner 1987). This is the unique homologous region within this phosphotranferase family. This consensus site is present in R45 (Fig. 6B). The tandem repeats also have interesting structural characteristics. The central block of repeats has some homology with the glycosaminoacid glycan core protein (Fig. 6C), in which most serine residues are substituted by glycosaminoglycan chains via 0-glycosyl linkage to serine (Bourdon et al. 1985). Furthermore, the R45 repeats contain numbers of putative phosphorylation sites for various kinases such as CaMII phosphorylation sites (H/R)XX(S/T) (Pearson et al. 1985; Kennelly and Krebs 1991). It is especially relevant here since a phosphorylated protein of 160.5 kDa has been recently described by Suetterlin et al. (1991). This protein may correspond to the R45 product. Phosphorylation of repeated motifs has already been described in the case of RNA polymerase II. It is one of the few examples where a functional role for repeats has been discovered. In this protein, the C-terminal domain contains heptapeptide repeats providing multiple phosphorylation sites that regulate the activity of the enzyme (Corden 1991). The possibility that phosphorylation of the R45 repeats might regulate an enzymatic activity is highly interesting. This point merits further investigation, as pro-

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tein phosphorylation by protein kinases is of prime importance in regulating proliferation and differentiation processes, in particular the GUS and the G2/M transitions in eukaryotic cells (Draetta 1990). Furthermore, the peak of phosphorylation in P. falciparum was observed at the midtrophozoite stage (Jones and McLemore Edmundson 1990). In this context, it is interesting to note that the R45 gene is expressed at the time of or just before the onset of DNA synthesis (Inselburg and Banyal 1984). ACKNOWLEDGMENTS This work was funded by grants from the Ministere de la Recherche et de .l’Enseignement superieur (87WOO43-EUREKA). We thank L. Pereira da Silva for support. We thank T. McCutchan for the generous gift of CSP DNA probe. REFERENCES ANDERS, R. F. 1986. Multiple cross-reactivities amongst antigens of Plasmodium falciparum impair the development of protective immunity against malaria. Parasite Immunology 8, 529-539. ANDERS, R. F., COPPEL, R. L., BROWN, G. V., AND KEMP, D. J. 1988. Antigens with repeated amino acid sequences from the asexual blood stages of Plasmodium faiciparum. Progress in Allergy 41, 148-172.

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Received 20 December 1991; accepted with revision 17 March, 1992