Molecular and Biochemical Parasitology, 49 (1991) 99-110 © 1991 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/91/$03.50 ADONIS 016668519100380E

99

MOLBIO 01606

Primary structure of a Plasmodium falciparum rhoptry antigen Heidi J. Brown and Ross L. Coppel The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia (Received 3 April 1991; accepted 4 June 1991)

The high-molecular-weight rhoptry complex of Plasmodium falciparum consists of 3 non-covalently associated polypeptides of 150, 135 and 105 kDa. We present the complete nucleotide sequence of the 105-kDa (RhopH3) component of this complex derived from analysis of genomic and cDNA clones. The genomic structure is unusually complex for P. falciparum, consisting of 7 exons including 2 mini-exons of 19 and 21 amino acids. The sequence lacks tandem repeats and is conserved among several parasite isolates. B cell epitopes that induce antibody responses during natural infection were mapped to five different regions of the polypeptide. Key words: Plasmodium falciparum; Rhoptry antigen; Intron; RhopH3

Introduction

Rhoptries are paired, ducted organelles located at the apical end of Plasmodium merozoites. During invasion of red blood cells by merozoites, rhoptry contents, including protein and possibly lipids, are discharged [13]. Evidence from indirect immunofluorescent assays (IFA) and immunoelectron microscopy suggests that rhoptry molecules may be involved in the invasion process and formation of the parasitophorous vacuole (reviewed in ref. 2), but no specific function for any rhoptry molecule has been determined. However, antibodies directed to certain rhoptry molecules are protective in active immunization experiments and passive transfer assays in Correspondence address: Ross L. Coppel, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M65059. Abbreviations: GBP, glycophorin binding protein; FIRA, falciparum interspersed repeat antigen; RESA, ring-infected erythrocyte surface antigen; KAHRP, knob-associated histidine-rich protein.

vivo and inhibit parasite growth in vitro. [4,5]. A number of rhoptry proteins have been characterized using both monoclonal antibodies and immune sera [4-14]. Several of these proteins are components of two distinct complexes; a low-molecular-weight complex comprised of polypeptides of 80 kDa, 60, kDa and a 42-kDa and 40-kDa doublet [5,6,9,10] and high-molecular-weight complex comprised of polypeptides of 150, 135 and 105 kDa [7,11,12,15]. Additionally, polypeptides of 225 and 55 kDa have been described and located in rhoptries but not apparently associated with either of the 2 antigen complexes [13,14]. The 150-, 135- and 105-kDa polypeptides (referred to as RhopH1, RhopH2 and RhopH3, respectively) are unrelated gene products which form a stable, noncovalent complex that is immunoprecipitated by antibodies directed to any of the individual proteins [7,12]. Pulse chase studies revealed that the three proteins are synthesized and associate during the mid-trophozoite stage prior to formation of the rhoptry organelle. At the time of merozoite invasion the complex is transferred to the ring where the RhopH3 polypeptide is modified in some way resulting in a shift in apparent molecular weight from

3

Sheared G e n o m i c

,I

~coR1

~xpresssionClone A

Amino Terminus

~enomic Clones

#15

:DNA C L O N E S

3

~TG AminoTermlnus

Tm'l

SQpl

~pression Clone B

|n~l m

Mini-2 m

~pression Crone C

~ -x/'-X/-~

,,,/-

273

I

=pl

3270

-coR1 G e n o m i c

ind3

lind3

~g44

~14

I

ind3

470

"mql klin~t'.t

xpression C~ne O

V

aql

Expression Clone E

Stop

O68

TAA

J

~coR1

101

105 kDa to 110 kDa [7]. Antibody screening of a cDNA expression library identified a 494-bp cDNA coding for the carboxy terminus of RhopH3 [16]. We have used this cDNA as a probe to detect genomic and other cDNA clones, and present here the complete gene structure deduced for RhopH3. The structure is an unusual one for a P. falciparum antigen gene consisting of 7 exons including 2 mini-exons encoding 19 and 21 amino acids. There are no regions of repetitive sequence and protein size, antigenicity and sequence are conserved among isolates. During infection of the human host, antibodies are induced to many different regions of the molecule.

Materials and Methods

Parasites and protein methods. P. falciparum isolates were maintained in culture in vitro as described [16]. Isolate FCQ27/PNG (FC27) was obtained through collaboration with the Papua New Guinea Institute of Medical Research. Cell extracts were prepared and electrophoresed on SDS/PAGE and transferred to nitrocellulose as previously described [16]. Patient sera were collected with informed consent from individuals living in the Madang area of Papua New Guinea. DNA methods. Parasite DNA was isolated from in vitro cultured trophozoite and schizont stage parasites and purified as previously described [16]. Southern blotting, sequencing protocols, labelling of DNA fragments and Fig. 1. The gene structure of RhopH3. (A) Scheme of the gene structure of RhopH3. Numbering corresponds to the nucleic acid sequence presented in Fig. 2. White boxes depict noncoding regions, black boxes denote coding sequence derived from both genomic and cDNA clones, whereas the shaded box denotes coding sequence derived from cDNA clones only. Restriction enzyme sites, the initiator methionine, amino terminus of the mature protein and the terminator codon are indicated. (B) Clones used in determination of the sequence. The intron sequences missing from cDNA clones are indicated by diagonal lines. (C) Position of expression clones relative to the gene structure is shown. Expression clones were derived from cDNA sequences and do not include introns.

hybridization conditions were as previously described [16]. Homology searches were performed using the FASTA programme [17] against the GenBank version 65, EMBL version 24, and NBRF version 35 nucleic acid databases and the NBRF PIR version 26, PSD-Kyoto (Ooi) version March 1986, GBtrans version 63 and Swissprot version 15 protein databases.

Construction of expression clones and specific antisera. The cDNA clones were cut with restriction enzymes, fragments were gel-purified and cloned into the pGEX vectors [18]. Expression clones were identified using pooled hyperimmune Papua New Guinea (PNG) sera in a colony immunoassay [16]. All clones except that expressing the Ag44 cDNA produced insoluble fusion proteins. The soluble fusion protein was purified according to Smith et al. [18]. Fusion proteins were analyzed by fractionating the bacterial lysate on 10% SDS-PAGE gels and immunoblotting with pooled PNG sera. The bands were cut out and electroeluted in a 0.5 M NH3CO3/0.01% SDS buffer at 50 mA for 48 h and ethanol precipitated at -70°C. The fusion protein was then resuspended in phosphate-buffered saline analyzed on SDS-PAGE and emulsified with adjuvant, Freund's complete for the first injection and incomplete for booster injections. Rabbits were injected 4 times at 2-week intervals and bled 10 days after the last injection. Rabbit sera against Ag44 were prepared as described [16]. Construction of mini-exons as expression clones. Complementary oligonucleotides corresponding to the mini-exons of RhopH3 were synthesized with the codon usage designed to incorporate the most frequently used codons of Escherichia coli instead of that of P. falciparurn. BamHI and BglII sites were added to either end to facilitate cloning. Oligonucleotides in solution were heated to 100°C and allowed to cool to room temperature overnight. The 5' ends were then phosphorylated and the oligonucleotides were purified by gel electrophoresis to remove small fragments and

102

tion, larger inserts containing 4-5 copies of the sequence were purified and ligated into the pGEX2T vector. Expression clones were

single-stranded oligonucleotides. The doublestranded fragments were ligated to obtain a series of multimers. By agarose gel purificaAAAAAAGGAAATTACACAATAAGATATGTACGAGTATATTAATATATATATATATATATAT M

R

S

K

H

L

V

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A~TATGCGTAGTAAGCATTTAGTAACATTATTTATTATAACT

L

S

CAACTTATATTAAATTATATTCATTTGTCT

F

S

T

V

T T T CCT T T T T T T A G G A T G A T A T A G T T A A A

21

TTTTTATCATTTTCAACCGTCAAGGGTAATATATT

TTTTT TTTTAAT~GTACAC

TGTTT CA~TGATT V

ACGAGTGCACTGATAGTTGAGTATATAATATATATATATATATATATT G

F

V

T

K

K

L

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T

C GGTTTTGTAACAAAGAAATTAAIt%AC

L

L

D

C

N

TATATATATTCATGTGTTATTTTATTTTATTTTATTTTATTTT F

A

CCTTTTAGACTGTAATTTTGC

L

Y

Y

N

F

K

G

N

G

P

D

A

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K

H

K

F

L

D

K

E

F

TTTTTTTTTTTTATTATTTTATTGTAGTCCT

L

K

D

K

N

I

H

N

F

Q

R

L

F

F

T

V

N

T

F

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L

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840

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81 960 121

TGTTATGA

TAGAACCACAAGGTAAATATACATATTTCCAGTTAAGGAATTTCACCAAATATTGTATACTACCCCCCT

K

K

TTTGTCTGTGAAAT F

K

600 720

TTTTAGATTTTGTGGATGAACCTGAACAATTTTACTGGTTCGTGGAACATTT

C

480

TTTATTTATTTATTT T

TATTTTTTACATATAACTTTGGAGATG

I

29 360

GACACA

AATTTCTAGATAAAGAGCAAAGAT TAATAAAAACTAATATAA~GATGGT

A GC

TATTTTTATATATGTGTACAAA

T T T A C A C CTT G C T T A A A T A G A T C A T G G G T A T C T G A A T T T T T A A A A G A A T A T G A A G A G C C A T T T G T A A A T C C

Y

F

CTTTGTGTAACTCCTTTTATTTTATGTATATATTTAATATGAATTCATATTATATT

TTATT TATTTATTTTATTTATTTATTTTATTTATTTATTTTATTTTATTTATTTATTTAT S

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N

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S

T

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D

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C

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T

Y

N

I

K

L

1080 161 1200

K

N

201

GAAAGTGGAGAATTTTTAAAATATCAATTAAATAAAGAAGAATATAAAGTTTTTCTTTCTTCGGTTGGTTCCCAAATGACAGCTATAAAAAATT

E

240

58

GTAACATAA~TTTATTCT

TTC GAGTTCCAAAGCATCTTAAAGATAAAAACATTCATAAT

K

TCTTTATTATAATT TTAAAGGAAATGGC CCAGACGCTGGAGTAAGAGAAAATTGAATCTTGAATTTAAAGAT

TTTTT TTTTTAAAAATAAT TATATGAACTGTACCTCAA~GGTGTACTTTTTTTTTTTT

ATTTATTTATTTTATTTATTTAT

G

TTTTTAGTTTGGGGAAAAGATGTATTC

AAA~CGTGACCATAAATATTTTTATATATAT

TGTATGTAAATTTTATATGTCT

W

GT

G

GAAATGAGCATATTATGGACATACATATATATGTATTTT TCATAATATTA~TGTATATT

120

K

1320

H

Y

T

241

TATATTCAACAGTTGAAGATGAACAAAGAAAACAATTATTAAAAGTTATCATAGAAAATGAAAGTACAAATGATATATCTGTTCAATGCCCAACTTATAACATAAAATTACATTATA•TA1440 K

E

C

A

N

S

N

N

I

L

K

C

I

D

E

F

L

R

K

T

e

E

AAGAATGTGCTA_%TAGTAATAATATATTA~TGTATTGATGAATTTCTTAGA~CATGT L

F

E

S

L

K

N

P

TTTTTGAATCATTA~GAATCC P

K

N

Y

L

D

Y

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V

Q

CAAAAAATTATTTAGATAGTGTC V

E

L

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I

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F

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C TT T T T G T A G A T A ~ T T G

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F

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TATGACTAACAGTGATTTTAC

E

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K

H

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L

N

S

K

L

I

K

P

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281

TGCAGACTTAT GTGAACACTTACAATTTC

A

1560

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N

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361

GATATACCTAACAATCCATATTACAATG

1800

S

G

A

P

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S

P

V

CCA~TAT

GTTTTAT TA~

1920

T T T A T T T T T T T T A T T T T A T T A T A T T T T T T T T T TAT T T T T T G T T T C T T C

2040

$

399

CATTAGT CCAGTGTCAGGTAT GCTA~GGA~GAATAATTAAATAATACC

TGATGAAATA~T V

L

S

H

N

TGT G A ~ C

I

T

D

F

S

CTAGAGATGCTTTAATA~GGATGCTATGATATATATATATATAT

F

ATTGAGCCACAATATCACCGATTTTAGCT

K

E

D

N

1680

378

T A T A A T T T C T TCT T C G TTT T T A ~ G T G G A G C T C C

AAGA~TACA~TA~TATT

321

CGATATATATAAAC

F

GTAAA~TATTTATCTGTTTATTTAATTATTAT F

E

CTTAATCAAACCTCAATCAGTATGGAATGTACCTATATT

TT T A A G A A A T T A A A T A G C A A A A A T T T G A T C T T C T T A T C A T T C C A T G A T

S

D

CTACACATATAGCATATTTGGTAATATATCATATATATA~TGTATATATATATATATATAATACATAC

ATATATAAATATATATTTGTGTATCTCTGTATCATCATT K

F

CAAAATTTAGATACAGAATGT

T GGAACT TCAAGAAATTGTT~ATTGAGTAC D

K

TTACTTGGATAATTTTA~TT

K

GA~GA.~CCC4%AAGTA~CACCCTTC

S

TTATTTATTTATTTATTTTAATTTTTTTTC

2160

K

E

CTTCAGTTA~GA

418

TTAAAGAGGACAACAGTGAAAGTAAGA~CC~TTATTAGTAAATATATTTGTTTATTTGTATAATATAATATATGGAAGATGTAcTAAT

2400

TTATTTCGTGAATATATTTTATATAAA~TAAATGATAAATA~ATGATAAATAAAAATATATATATATATATATTTGTTTATTTATATATATACTTATACATATTTTTTTTTATGTTAT I

Q

C

Q

N

V

R

K

S

L

D

402 2280

E

L

E

V

D

V

E

T

M

K

G

I

A

A

E

K

L

e

K

I

I

T E

K

F

I

2520 453

TTTTTACAATTTTTAGTTCAATGCCAAAATGTAAGAAAGAGTTTAGATTTAGAAGTAGATGTAGAAACAATGAAAGGTATT GCGGCAGAAAAGTTATGTAAGATCATTGAAAAATTTATT

2640

L T K D D A D K P E K S D I H R CTTACA~GATGATGCAGATAAACCAG;t~GAGTGATATACACAGAGGTT

493 2760

M

E

S

M

I

S

L

ATGGAAAGTATGATATCAT V

L

V

V

K

P

T

R

Y

T

S

P

F

F

D

H

T

Y

T

F

I

I

E

H

F

AGTCCAT TTTTT GATACAATTATAGAAT E

Q

L

S

C

F

Y

I

H

F R I L C I L I S T H V E A Y N I V R Q L L N TCCGTATCTTAT GTATATTAATATCTACTCATGT GGAAGCTTAT~.%CATAGTTAGACAATTATTA~T

K

F

K

M

R

T

P

Y

M

K

S

P

K

F

Y

T

E

E

I

L

V

T

Y

Y

N

K

G

TTTTAGTAACTTATTACAATAAGGG E

Y

L

D

F

F

Y

I

V

F

CCGTTTTATACAGAAGAAATATATATATT

T

L

L

K

G

N

F

E

L

I

K

S

R

E

S

S

D

GATA~TGCGTACAATGTATTTGGATTTCGA~GTTCC

I

S

E

L

K

H

L

I

L

Y

K

N

N

K

M

I

I

I

L

G

L

S

N

L

V

F

V

L

Y

F

I

S

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N

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D

R

I

L

K

G

I

D

N

L

R

Y

H

533

S

N

N

S

573

CAAATCCTAGT

3000

E

P

2880

Y

613

CAACGAATATTATTAT

3120

M

GATACATCCAAATATAGTAGATCGTATATTAAAGGGTATAGATAACTTAAT F

I

CCTGA~CATCTTATCATTATACTAGGATTATCAAATTTAGTATCTT K

H

A

TTTTTATATAAAAACAATAAGGCTATAAGATATCAC

TTCTGAAAAATTCGTTCTTTATTTTATATCTATTATATCAGTATTATATAT

GAACAAC TTTCATGTTTC TATCCA~GAATTTGAATTAATA~TCCAC~T D

F

TAACAAGATATACTTCATTATATATCCATAAATTTTTTAAGAGTGTAACATTATTAAAAGGAAAC

GTGCTTGTAGTIL~AGCCTCATTACACGTTCCAT S

L

G

K

Y

S

Y

T

R

Y

653

GA~GTACAAGATAT

3240

K

673

GATAT CTTCTCCAGAGA~GGTA~GAATTGATI~TATGAGCATAATAATTTGAT V

F

CA~TGAAT T

A

L

Y

N

F

G~d~TATATA D

S

F

I

K

TATATATATATATATATATATTTTATTTACTTATATTTATTTATTTTTATATTTATTTATTTTTTTTTTTTTTTTTGTTTCAGTTTTCACCGCCTTATACAACTTCGATAGCTTCATTAA T

N

E

Q

L

K

K

K

N

L

E

E

I

S

E

I

P

V

Q

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S

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A G

H H V N E K Q H S A H TCACCATGTTAATGA~CAACACAGTGCCCACTTCT P

AGA~CCAAGGATCAAGATTTAGA~TAGAATTATAC~ATATATGGGACCAT

L

K

E

Q

S

K

S

T

S

A

E

T

D

K

F

L D A I A E K D I TAGATGCTATTGCGGA~GACATATTAGA

L

E

S

E

G

S

D

F

Y

K

S

S

L

K

L

D

Q

L

D

K

E

L

S

G

TA~GAACAATCT~a%AAGTACAAGTGCTGCATCTACTAGTGATGAATTATCAGGT

P S T E S T S T G N Q G E D K T T D N T Y K E M E E L E E A E G T TCCATCTACTC-AATC T A C A A G T A C A G G A A A T C A A G G T G A A G A T A A A A C A A C A G A T A A T A C A T A C A ~ G A A A T G G A A G A A T T A G A A G A A G C T G A A G G A A C T T C A ~ T C E

E

K

P

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K

R

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K

K

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D

S

S

S

D

$

N

R

I

726 3600

A

T

686 3480

GACCAATGAACAATTAAAGAAGAAGAACTTAGAAGA~TATCAGAAATACCTGTACAATTAGAAACATCTAATGATGGTATTGGATACAGA~CAAGACGTTCTTTATC~CTGATAA P Q T M D E A S Y E E T V D E D ACCACAAACTATGGATGAAGCTTCATATGAAGA.%ACTGTAGATGAAGATGC

3360

S

TCTGAAGG

L K K TTA~GGTTT L

L

E

766 3720 806 3840

G

L

846 3960

E

S

886

AGAATTTTATAAATCTTCTCTAAAACTTGATCAATTAGATAAAGAAAAACCTAAAAAGAAAAAATCTAAAAGAAAAAAAAAGAGAGACAGTTCTAGTGACAGAATATTATTAGAAGAATC 4080 K

T

F

T

S

E

N

E

T;t~AACCTT T A C T T C T G A A A A T G A A T T A~qATACATTTATAA~T

L

* GTAAATTA~TTT~TCC

895 TACAT GTAGATTTTATTATATTACATCATGTAATCATATTATAGAATTTATTTTTAAGA~

TGA~TTTTTTTTTTTTTTTTTGTATGCATTTTAATTTTTATGTG~AACATGATAAATATTATTTTTTTTTTTTAATATATATATTAATGTATGAATA

TTTTTTTCTTTCA~TATT~CTTGAACTCTTA~CTTATATGTGAGCATGATTTGAAGCA~CGGAATTC

4200 4320 4395

Fig. 2. The complete nucleotide and predicted amino acid sequence of RhopH3. The signal sequence is underlined and the cysteines are in bold type.

103

detected with pooled PNG sera.

Results

Gene structure. The cDNA clone designated Ag44 [16] corresponding to the putative carboxy terminus of RhopH3 was used as a probe to screen an EcoRI genomic library and clones containing a 5-kb insert, the expected size, were isolated. Analysis of the sequence of a 2.2-kb EcoRI-HindIII fragment failed to reveal any extensive open reading frames and so regions of the 5-kb genomic clone were used to detect hybridizing cDNA clones. One of the cDNA clones isolated extended beyond the 5' end of the EcoRI fragment and was used to identify an overlapping genomic clone from a sheared genomic DNA library. Genomic and cDNA clones used to obtain the complete structure are shown in Fig. lB. The complete nucleotide sequence of 4390 bp and predicted amino acid sequence of RhopH3 are presented in Fig. 2. Comparison of the cDNA and genomic sequences reveals the presence, in the genomic sequence, of 6 regions of high (98%) A + T content not found in the cDNA clone, consistent with the occurrence of introns. The location of the 6 introns, which vary in length from 143 to 445 bp, is shown schematically in Fig. 1A. The splice acceptor and donor sequences proposed for the RhopH3 gene are typical of those found in other P. falciparum proteins; these are listed in Table I. The 3' region of the gene from nucleotides 3617-4395 were derived from cDNA sequences only, as genomic clones of this region were consistently unstable in M13 vectors. To search for the presence of introns in this region, multiple restriction digests were performed on the cDNA clones and the fragments obtained was compared to those derived from similar digests of D10 genomic DNA by Southern hybridization. No size differences were detected and we conclude that no introns are present in this region of the RhopH3 gene (data not shown). The presumptive initiating methionine is at

position 125 and is followed by a stretch of 16 uncharged and hydrophobic residues, consistent with a signal sequence. The N-terminus of RhopH3 has been determined by amino acid sequencing of the purified protein [19] and this sequence KDVFAGFV commences 24 aa after the presumed initiator methionine. This site of signal sequence cleavage occurs at the second charged residue after the hydrophobic region. The nucleotide sequence encoding the signal sequence is interrupted by an intron, a feature of several exported P. falciparum sequences including antigens located in the erythrocyte cytoplasm such as GBP and FIRA or associated with the erythrocyte membrane skeleton RESA, KAHRP and 11-1 (although, it should be emphasized that in the case of these other antigens the position of the signal sequence is predicted from hydrophobicity profiles and has not been experimentally verified) [20-24]. The sequence predicts a mature polypeptide of 871 amino acids with a molecular weight of 101975 and a nett negative charge of 14. The amino acid composition of the mature polypeptide is not as rich in asparagine (6.1%) as are many other P. falciparum proteins [25]. Negatively charged amino acids account for 15.2% of residues and positively charged residues account for 13.6% of residues. The percentage of hydrophobic residues is higher than average for P. falciparum antigens and is closer to that of a typical eukaryotic protein. Several stretches of hydrophobic residues may be found in the sequence of the mature protein (e.g., 557-577 and 595-609), but none have the properties of conventional membrane-spanning regions. Thus, it is not possible to designate an obvious structural feature that may be responsible for the reported binding of RhopH3 to the erythrocyte surface [15]. The 7 exons that encode the protein vary in length from 57 to 960 bp. An unusual feature is the presence of two-mini exons of 57 and 63 bp, a structural feature not previously described for P. falciparum proteins. Organization of the RhopH3 locus was examined in the following isolates and cloned lines: FC27, IMR 143, IMR 147 and MAD 71

104 TABLE I The splice acceptor and donor sequences for RhopH3 and other known P. falciparum genes Gene

No.

exonl

intron

[exon

Size

(bp)

Ref.

AGGIGTAATAT...TTTATTTTATTTTTTTTTAGITTT

150

GGAIGTAAGAG...TTTTTATTATTTTATTGTAGITCC

442

TTGIGTAATAT...TTGTTTCTTCCTTTTTGTAGIATA

199

CAGIGTATGCT...TTTAATTTTTTTTCCTTCAGITTA

160

AAAIGTAAGAA...TATTTTTTTACAATTTTTAGITTC

206

AAGIGTAAAAG...TTTTTTTTTTTTTGTTTCAGITTT

142

TGTIGTAAGAA...TTGTCATTATTTTTTTTTAGIGTG

158

AAAIGTATAAA...TTTATTTATTTTTTTTTTAGiATA

175

CAGIGTAAATA...TTT']?AAT'F'I!TTTTGTTTTAGtAAA

129

RESA

AATIGTAAGTT...TTTTTTTTTTTTTTTCATAGIGGT

203

[22]

FIRA

TGTIGTAAGGA...TTTTTATATTTTTTCTTTAGICGA

175

[21]

HRP II

A A C I .......... C T T T T T T A T T T T T A T T A T A G IAAT

KAHRP

AAC iG T A A G T T . . .T T A T T T T T T T T T T C A T A T A G

TGC

430

[23]

GBP

T T G I G T A T G ..... T G T G T A T T G T T T A T T T T T A G

AAT

179

[20]

ii-i

AATIGTAGGAT...AAATTTTCCTTTTTTTATAG

TGT

107

[24]

Exp-I

AAAIGTAAGTC...TATTTTCTTTTTGTTAATAG

AAG

313

[46]

RhopH3

SERA

[26]

[45]

GAGIGTATGAT...ATTTTTCTTTATTTTTATAGICAA

134

GARP

AAGIGTAACAA...TATATGTATTTTTTTTTTAGITGC

214

[47]

Pf-~tub

AAGi .......... T T T T T T T A T T T A T T T T T T A G [ T G G

-340

[48]

AAAiGTAAGAG...TTGTTTTTTTTTTCTTTTAGiATC

174

105 Tubulin ~ - I I

Tubulin ~

CTGIGTAAAAA...TATATATATTTTTTTTTTAGIGGA

211

2

CATIGTAATAA,..TTTGTCGTTACTCATTTTAGIGTA

119

1

CCAIGTAAGTT...TTTTATTTTATTTTTTTTAGIAGT

344

2

AAAIGTAAGAA...TTTTTTTGTTTTTTTCTTAGIATC

164

ATGIGTAAGAA,..TATTTTTATATTTTTTTTAGIGCT

452

[52]

CAGIGTATAGA...TCCTAAATGGTTCCCTGTAGIGTA

365

[53]

Aldolase

Actin II

[50]

1

1

[51]

M a l a r i a consensus A A G I G T A A A G A ........... T T T T T T T T T T A G I N N N

(Papua New Guinea), NF7 (Ghana), K1 (Thailand), ItG2 (Brazil), V1 (Vietnam), CSL-2 (Thailand), 3D7 cloned derivative of NF-54 (Schiphol Airport strain), Indochina 1 (Indochina) and HB3 (Honduras). Genomic DNA was restricted with EcoRI, HindIII or both enzymes, and in all cases the number and size of RhopH3 hybridizing fragments revealed by Southern hybridization with the 5-kb EcoRI RhopH3 probe were identical (data not shown). PCR analysis using oligonucleotide primers flanking the mini-exon region (primer 1, nucleotides 1939-1956; primer 2, nucleotides 2544-2562) gave a single band of about 620 bp in all isolates examined, suggesting that this region is also conserved in genomic DNA (data not shown). Seven of the isolates were examined by Northern blot analysis using the 5-kb RI fragment and all revealed a single m R N A species of 3.4 kb in all lanes. Finally, 10 isolates and cloned lines were examined by immunoblotting using a rabbit anti-Ag 44 antiserum and an identically sized band of 105 kDa was detected in all isolates (data not shown). The relative amount of RhopH3 protein was comparable in all isolates examined. Thus the RhopH3 gene and protein product appear conserved in all isolates examined, although this analysis does not

exclude the presence of minor changes such as single point mutations occurring in the RhopH3 gene of different isolates.

Expression clones. The clone Ag44 encoding the carboxy terminus of RhopH3 was initially detected in an immunoassay using antibodies purified by affinity chromatography from hyperimmune sera, suggesting that this region of RhopH3 is immunogenic during natural infection. In order to examine whether other regions of the protein are immunogenic during infection, clones expressing various regions of RhopH3 were constructed by two methods (see Fig. IC). cDNA clones 15 and 28 (Fig. 1B) were digested with the restriction enzyme SspI, to generate fragments ranging in size from 400-1000 bp and the fragments ligated in frame into the appropriate pGEX vector [18]. Clones expressing one or other of the miniexon regions were constructed from polymerized double-stranded oligonucleotides ligated into pGEX in order to create a better immunogen from the relatively short 19- and 21-residue sequences. Recombinant clones were grown in liquid culture, induced with IPTG, lysed, fractionated by SDS-PAGE and transferred to nitrocellulose for immunoblotting (Fig. 3). The pooled hyperimmune sera

106

1 93

--

67

--

43

--

30

20

I

2

3

4

5

6

7

8

!~il

--

Fig. 3. Multiple regions of RhopH3 induce antibodies during natural infection. Lysates of induced E. coli expression clones were immunoblotted and probed with pooled PNG sera that had been pre-incubated with the non-recombinant pGEX2T bacterial sonicate. Tracks were loaded as follows; 1. pGEX2T, 2. E, 3. A, 4. D, 5. C, 6. mini-l, 7. mini-2, 8. B.

reacted strongly with expression clones derived from the amino-terminal and carboxy-terminal regions. (clones A, E and D) and with miniexon 1. In addition there was weaker but definite reactivity with mini exon 2 and clones B and C. Several of the clones overlap (Fig. 1C), thus there is definite evidence for 4 epitopes from this experiment; clone A, mini1, mini-2 and D. In a second experiment, immunoblots of these various expressing clones were probed with individual sera collected from people living in an endemic region. The sera were scored for reactivity with each individual protein on a simple numerical scale. The results from this experiment indicated that there was an additional antigenic domain in RhopH3, as a number of individual sera reacted with clone B but had no detectable reactivity against mini-1 or clone C (data not

shown). Antibody responses to these antigenic domains are prevalent among individuals living in an endemic region, with all of 80 individual sera tested having reactivity to at least one of the RhopH3 expression clones.

Discussion

The most striking feature of the RhopH3 sequence is the complex intron-exon structure, a gene organisation more reminiscent of that found in higher eukaryotes, with 6 introns punctuating the open reading f r e ~ e of the R h o p H 3 protein. Many genes for P. falciparum antigens contain a single intron of 106-200 bp which is usually located within the signal sequence or within the amino terminal region of the protein, e.g., RESA [22], F I R A

107

[21] GBP [20] and K A H R P [23] whereas others such as the serine-rich antigen [26] have as many as 3. We have no indication as to the reason for such a large number of exons in the RhopH3 protein and have no evidence that the exons of RhopH3 represent functional domains as seen, for example, in the immunoglobulin genes [27]. The point of cleavage of the RhopH3 signal sequence is known precisely from direct protein sequencing of the amino terminus of the mature polypeptide [19]. The N-terminus of the mature RhopH3 begins with residues K D V F A G which commence 24 residues from the initiator methionine. Lysine residues flank a predominantly hydrophobic core of 19 residues and cleavage occurs between glycine and lysine. MSA-1 is the only other P. falciparum polypeptide for which N-terminal protein sequence is available and here a 19residue signal sequence comprising a hydrophobic region of 17 residues flanked by lysine on the N-terminal side is cleaved between cysteine and valine [28]. There appears to be little similarity between the cleavage sites of these two proteins and further data from other sequences is required to allow accurate prediction of signal cleavage sites. In contrast to most P. falciparum antigens, RhopH3 does not contain any repetitive elements or highly charged domains. There appear to be no regions of similarity between RhopH3 and the two other rhoptry molecules for which sequence data exists, AMA-1/Pf83 [29,30] and RAP-1 [31]. The arrangement of cysteines is such that the 13 cysteines of

RhopH3 Hog

Cholera

Bovine

Viral

Virus Diarrhea

RhopH3 Atpase

6

(P.

lividus)

Atpase

6

(P.

anserina)

370 L S T Y T 1600 B E R C T 1690 Virus H E K C H

RhopH3 are all located within the N-terminal half of the molecule. A similar arrangement is present in at least two classes of molecules; the preprohormones opioid peptide precursors and in some receptors [32]. A search of the available databases failed to detect any overall similarity between RhopH3 and any known proteins. However, 2 short regions of RhopH3 showed similarity to known proteins (Fig. 4). The first region (residues 370-404), encompassing the first mini-exon shows 34% identity over a span of 34 residues with a region of bovine viral diarrhoea virus and the closely related hog cholera virus [33,34]. This region of the virus genome encodes a portion of p125, a 125-kDa protein of unknown function that is processed into two smaller products of 54 and 80 kDa. The second sequence similarity of 33% identity over a 30-residue stretch is between residues 591-620 of RhopH3 and a region of the protein 6 subunit of the mitochondrial ATPase complex. Of interest is that within this region RhopH3 is as similar to this protein in the sea urchin Paracentrotus lividus [35] and to the protein in the filamentous fungus Podospora anserina [36] as these latter proteins are to each other. The protein 6 subunit appears to be important for H ÷ translocation [37] and for correct binding of other subunits of the ATPase complex [38]. Whether this sequence motif is important in stabilizing the other members of the highmolecular-weight rhoptry complex is not known. We used the Dayhoff Align programme to assess the significance of these similarities [39]. In this analysis, an alignment

F

D K L Y N F F F V F K K S G A P I S P V S

T S

I M

D K L T A F F G V M P R G T T P R A P V R

I N

I

D K L T A F F G I M P R G T T P R A P V R

Y S I

L

404 K E L S 1634 F P T S L 1724 F P T S L

V

591 620 G S E K F V L Y F . I S I . I S V L Y I N E Y Y Y E Q L S C F Y 198 227 I F V I F V L L r . I L E . I G V A C I E A Y V F T A L V B F Y 36 65 S T S H F V L T F A L S F T I . V L G . A T I L G F Q K H G L E

Fig. 4. Sequence similarities between RhopH3 and other proteins. Identical residues are in bold type and residue numbers are as indicated. Mini-exon 1 is boxed. Note also a number of conservative changes in amino acids.

108 between the sequences is performed and the matching score obtained. Two hundred random sequences with the same amino acid composition are then generated and compared to the target sequence and the mean matching score and the standard deviation are obtained. This is compared to the observed score for the original alignment and the number of standard deviations of the observed score above the random score is calculated. There is some debate over the cut-off levels for significance but a statistically significant level of sequence similarity must be at least three standard deviations higher than the random score. The bovine viral diarrhoea virus and the hog cholera virus comparisons lie 6.18 and 6.87 standard deviations from the mean of the random comparisons. The Paracentrotus lividus comparison lies 4.62 standard deviations from the mean of the random comparisons. The RhopH3-Podospora anserina comparison and comparison of the two ATPase 6 subunit sequences to each other give similar scores, but neither comparison is statistically significant. RhopH3 is apparently well conserved among isolates of P.falciparum. Western blots and D N A restriction mapping indicate that the epitopes and the overall structure is conserved, unlike the dramatic diversity seen in the S-antigens [40] or MSA-2 [41]. Rhoptry molecules as a group appear to have conserved molecular weight and this conservation has been demonstrated at the nucleotide level in the case of AMA-1 [29,30] and RAP-1 [31]. RhopH3 is a natural immunogen, with at least five antigenic domains that react with P N G immune sera. There is little detailed data on natural B cell epitopes of P. falciparum antigens during infection. Most P. falciparum antigens characterized to date have been identified by reactivity of expression clones with immune sera and such a procedure by its very nature identifies at least one B cell epitope, often a region of repetitive sequence [40]. However, few antigens have been epitope mapped in detail. Analysis of S-antigens and RESA suggested that the majority of the antibody response is directed against the

tandem repeats [42,43]. In the case of Santigen there is no detectable antibody response against non-repetitive regions and it has proven difficult experimentally to raise anti.. sera to the non-repetitive region in laboratory animals (our unpublished results). DetailecL epitope mapping has been performed on RESA using the epitope scanning procedure in which twenty-eight peptides were reacted with individual sera [43]. The predominant antibody response was to the 3' terminal repeat region but there was also strong reactivity with the 5' repeat region. In addition there appeared to be weaker but definite antibody binding to peptides derived from near the N-terminus and near the 3' repeats. RhopH3 does not contain any regions of repetitive sequence and this may explain the relatively large number of epitopes detected as there has been no focusing of the immune system on repeats. We have not quantitated the relative immunodominance of the different RhopH3 B-cell epitopes, but preliminary data suggests that of the human antibodies reacting with RhopH3, the most prevalent are those reacting with the carboxy terminal domain, encompassed by c D N A clone Ag44 [ 16]. Two reports suggesting a protective role for the high-molecular-weight rhoptry complex have been published. Cooper et al. [12] found that a monoclonal antibody to the 105-kDa molecule gave up to 35% inhibition during an in vitro inhibition assay and Siddiqui et al. [44] found that immunization with the 140-, 130-, and 105-kDa molecules conferred partial protection upon subsequent challenge in monkey trials. The cloning of the complete RhopH3 molecule will allow testing of this protein for protective efficacy against P. falciparum infection.

Acknowledgements We thank Robin Anders and Dave Kemp for useful discussions throughout the course of this work, and for critical review of the manuscript, Fiona Smith for expert technical assistance, Tony Kyne for assistance with

109

computing and Heather Saunders and Etty Bonnici for typing the manuscript. This work was supported by the Australian National Health and Medical Research Council, Australian National Biotechnology Program Research Grants Scheme and the John D. and Catherine T. MacArthur Foundation. H.J.B. was supported by an Australian Postgraduate Research Award. References 1 Bannister, L.H., Mitchell, G.H., Butcher, G.A. and Dennis, E.D. (1986) Lamellar membranes associated with rhoptries in erythrocytic merozoites of Plasmodium knowlesi: a clue to the mechanism of invasion. Parasitology 92, 291-303. 2 Mitchell, G.H. and Bannister, L.H. (1988) Malaria parasite invasion: interactions with the red cell membrane. CRC Crit. Rev. Oncol. Hematol. 8, 225310. 3 Stewart, M.J., Schulman, S. and Vanderberg, J.P. (1986) Rhoptry secretion of membranous whorls by Plasmodium falciparum merozoites. Am. J. Trop. Med. Hyg. 35, 37~,4. 4 Holder, A.A. and Freeman, R.R. (1981) Immunization against blood-stage rodent malaria using purified parasite antigens. Nature 294, 361 364. 5 Schofield, L., Bushell, G.R., Cooper, J.A., Saul, A.J., Upcroft, J.A. and Kidson, C. (1986) A rhoptry antigen of Plasmodium falciparum contains conserved and variable epitopes recognized by inhibitory monoclonal antibodies. Mol. Biochem. Parasitol. 18, 183 195. 6 Bushell, G.R., Ingram, L.T., Fardoulys, C.A. and Cooper, J.A. (1988) An antigenic complex in the rhoptries of Plasmodium falciparum. Mol. Biochem. Parasitol. 28, 105 112. 7 Lustigman, S., Anders, R.F., Brown, G.V. and Coppel, R.L. (1988) A component of an antigenic rhoptry complex of Plasmodium falciparum is modified after merozoite invasion. Mol. Biochem. Parasitol. 30, 217224. 8 Oka, M., Aikawa, M., Freeman, R.R., Holder, A.A. and Fine, E. (1984) Ultrastructural localization of protective antigens of Plasmodium yoelii merozoites by the use of monoclonal antibodies and ultrathin cryomicrotomy. Am. J. Trop. Med. Hyg. 33, 342 346. 9 Campbell, G.H., Miller, L.H., Hudson, D., Franco, E.L. and Andrysiak, P.M. (1984) Monoclonal antibody characterization of Plasmodium falciparum antigens. Am. J. Trop. Med. Hyg. 33, 1051-1054. 10 Howard, R.F., Stanley, H.A., Campbell, G.H. and Reese, R.T. (1984) Proteins responsible for a punctate fluorescence pattern in Plasmodium falciparum merozoites. Am. J. Trop. Med. Hyg. 33, 1055 1059. 11 Holder, A.A., Freeman, R.R., Uni, S. and Aikawa, M.

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