structure and expression of the knob-associated histidine-rich protein of plasmodium falciparum
TRANSCRIPT
Molecular and Biochemical Parasitology, 26 (1987) 203-214 203 Elsevier
MBP 00902
Structure and e x p r e s s i o n of the knob- as s oc ia t e d hist idine-r ich prote in of Plasmodium falciparum
Joan Ellis 1,*, David O. Irving 1,*, Thomas E. Wellems 2, Russell J. Howard 2 and George A.M. Cross 1
ILaboratory of Molecular Parasitology, The Rockefeller University, New York, NY, U.S.A. and 2Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, U.S.A.
(Received 26 May 1987; accepted 10 August 1987)
cDNA clones encoding 473 amino acids of the knob-associated histidine-rich protein (PfHRPI) of Plasmodium falciparum clone FCR-3/A2 (Gambia) have been isolated and sequenced. Although a short region close to the amino terminus of the predicted sequence contains three blocks of six, seven or nine consecutive histidine residues, the most abundant amino acid is lysine. The predicted sequence contains a putative amino-terminal signal sequence and two potential asparagine glycosylation sites. A 1284 bp Sau3A cDNA fragment was expressed in Escherichia coli as a fusion protein that was recognized by an anti-PfHRPI mono- clonal antibody. Pulsed field gradient electrophoresis indicated that the PfHRPI gene is located on chromosome 2. The PfHRPI gene was present, apparently intact, in knobless parasites derived from one uncloned P. falciparum isolate (St. Lucia). In a knob- less derivative of another uncloned isolate (Malayan Camp) and in a cloned knobless line (FCR-3/D4) of a third isolate (Gam- bian), that part of the gene covered by the cDNA clone has been deleted. Loss of PfHRPI expression may therefore arise via several different mechanisms of gene alteration.
Key words: Plasmodium falciparum; Knob protein; Histidine-rich protein; DNA sequence; Expression
Introduction
The virulence of Plasmodium falciparum is, in part, due to the adherence of erythrocytes in- fected with mature parasites to endothelial cells of the postcapillary venules in several tissues [1-5]. This cytoadherence prevents the circula- tion of parasitized erythrocytes through the spleen and lymph nodes, thereby protecting the parasite from destruction [6]. Ultrastructural studies have implied that knob-like protrusions on the mem-
*Present addresses: J. Ellis, 7600 Glennon Drive, Bethesda, MD 20817, U . S . A . D . O . Irving, Biotechnology Australia, P.O. Box 20, Roseville, New South Wales 2069, Australia.
Correspondence address: Dr. G.A.M. Cross, The Rockefel- ler University, 1230 York Avenue, New York, NY 10021~399, U.S.A.
Abbreviations: HRP, histidine rich protein; PfHRPI, the knobby phenotype-associated HRP of P. falciparum; PIHRP, the HRP of P. lophurae; K ~, knobby; K-, knobless; SDS, so- dium dodecyl sulfate; bp, base pairs.
brane of infected erythrocytes may be involved in cytoadherence [7,8]. Only parasites that produce knobs (K +) can cytoadhere. Knobless (K-) var- iants do not [9], and are less virulent in non-sple- nectomized experimental hosts [2]. Knobs are probably composed of several host and parasite- derived proteins, including a histidine-rich pro- tein, PfHRPI, that is absent from K- variants [10,11]. However, the presence of knobs and expression of PfHRPI is a necessary but not suf- ficient condition for cytoadherence [12,13].
Several studies have indicated that PfHRPI shares some homology with the extremely histi- dine-rich protein of Plasmodium lophurae (P1HRP) at the biochemical [14], genetic [15] and immunological [16] levels. RNA from a K + strain of P. falciparum hybridized to a P1HRP cDNA probe while RNA derived from a K strain did not [15], indicating the loss of the PfHRPI gene or its expression. We have used a fragment de- rived from a P1HRP cDNA clone [15,17], con-
0166-6851/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
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taining the histidine-rich coding region, to screen a P. falciparurn cDNA library. Several positive clones were isolated, none of which contained the 3' end of the coding region. When a 1284 bp cDNA fragment isolated from one of these clones was expressed in Escherichia coli, a protein that cross-reacted with an anti-PfHRPI monoclonal antibody [18] was detected. The presence and or- ganization of the PfHRPI gene was studied in several P. falciparum isolates.
Materials and Methods
Parasites. FCR-3/A2 (K +) and FCR-3/D4 ( K ) parasites are cloned isolates derived from a Gam- bian strain of P. falciparum [19]. The 7G8 clone was derived from the Brazilian P. falciparum iso- late IMTM22 [20]. These parasites were main- tained in culture as described by Trager and Jen- sen [21]. Uncloned K + and K parasitized erythrocytes of the Malayan Camp [22] and St. Lucia [23] strains of P. falciparum were obtained from Aotus monkeys as ring-stages, cryopres- erved [24], thawed and cultured for up to 24 h to obtain late trophozoite-infected cells at 5-20% parasitemia [25]. The K + Malayan Camp strain had been passaged exclusively in Aotus monkeys after infection with the original human isolate. Malayan Camp K parasites, provided originally by Dr. David Haynes (Walter Reed Army Insti- tute of Research), had been passaged originally in Aotus monkeys, subsequently cultured in vitro in human erythrocytes for more than 1 year and then passaged into splenectomized Aotus mon- keys, where they remained K . St. Lucia para- sites were provided by Dr. William Collins (Cen- ters for Disease Control) and were passaged into splenectomized and intact Aotus monkeys. The parasites in intact animals remained K +. The knob phenotype of all samples obtained from splenec- tomized animals, and the cloned lines maintained in culture, was confirmed by transmission elec- tron microscopy.
Isolation of RNA and DNA. Sorbitol-synchro- nized cultures of FCR-3/A2 were harvested at various times after merozoite invasion to obtain K + RNA. Total cellular RNA was extracted by a modification of the method described by Chirg-
win et al. [26]. Parasitized cells were lysed with saponin [15] and the sedimented parasites were resuspended by vortexing in 10-20 volumes 4 M guanidine thiocyanate, 0.2 M 2-mercaptoethanol (pH 5.0). The homogenate was layered over a 5.7 M CsC1, 0.1 M E D T A (pH 6.5) cushion and cen- trifuged in a Beckman SW50 rotor at 30000 rev m i n t for 20-24 h at 18°C. The resulting RNA pellet was dissolved in 0.3 M sodium acetate (pH 5.5) and then precipitated by adding two volumes of ethanol. Polyadenylated RNA was isolated ac- cording to the method of Aviv and Leder [27], and stored in aliquots at -70°C. DNA was ex- tracted from cultures containing multinucleated parasites. The parasite pellet obtained after sa- ponin lysis of infected cells was resuspended in 5-10 volumes 0.1 M E D T A (pH 8.0), 0.5% sar- cosyl containing proteinase K (10 txg ml J) and incubated at 50°C for 2 h. After cooling to room temperature, DNA was extracted 2-3 times with an equal volume of phenol and chloroform (1:1), followed by precipitation with ethanol. Finally, the DNA was redissolved in 10 mM Tris-HC1 (pH 8.0), 10 mM EDTA, treated with pancreatic ri- bonuclease A (50 #,g ml l) for 60 min at 37°C, and ethanol precipitated.
Construction and screening of a cDNA library. Polyadenylated RNA derived from synchronous cultures of K + FCR-3/A2 parasites 16-20 h after invasion was used to construct a cDNA library, following the procedure described by Guebler and Hoffman [28] except that synthetic EcoRI adapt- ers (constructed by Dr. W. Fulford, Rockefeller University) were ligated to the double-stranded cDNA. The cDNA was then size-fractionated on a Sepharose CL-4B (Pharmacia) column in the presence of 0.3 M NaC1, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and the fractions were ethanol precipitated. The fraction containing cDNA ranging predominantly from 1-2 kb was ligated to EcoRI-digested ~.gtl0, the DNA was packaged and used to transfect E. coli Hfl C600 cells, all according to standard procedures [29]. A PstI-SfaNI fragment containing the histidine-rich coding region was isolated from a P1HRP cDNA clone [15,17], nick translated and used to screen the cDNA library according to the method de- scribed by Benton and Davis [30].
Hybridization analysis. Parasite DNA was di- gested with various restriction endonucleases ac- cording to the manufacturer 's specifications, elec- trophoresed in 0.8% agarose gels, and transferred to nitrocellulose filters by the method of South- ern [31]. The filters were hybridized in 1.0 M NaC1, 100 mM sodium citrate, 35 mM sodium phosphate pH 6.0 and 1 x Denhardt 's solution [29], with the nick-translated PfHRPI cDNA clone, p47, at 65°C overnight. The filters were washed thoroughly with 30 mM NaC1, 3 mM so- dium citrate and 0.1% sodium dodecyl sulfate (SDS) at 50°C. For RNA analysis, 4 Ixg of total RNA were denatured and electrophoresed in 1.2% forma|dehyde/agarose gels [29]. RNA was transferred to nitrocellulose filters and hybridized with a nick-translated EcoRI insert derived from the PfHRPI cDNA clone, p47, at 42°C overnight in the presence of 50% formamide and 10% dex- tran sulfate. The filter was washed extensively with 0.3 M NaC1, 30 mM sodium citrate, 0.1% SDS at 55°C.
Nucleotide sequencing. The cDNA clones p47 and p23-3B were subcloned into the pGem4 vector (Promega) [32] and a series of deletion deriva- tives were generated by unidirectional exonu- clease III digestion, as described by Henikoff [33]. The deletion clones were digested with HindIII and end-labelled with adenosine 5'-[y-3ZP] triphosphate (Amersham) to sequence the coding strand [29]. To sequence the noncoding strand, the deletion clones were digested with SphI and end-labelled with dideoxyadenosine 5'-[~- 32p]triphosphate (Amersham) according to the manufacturer 's specifications. All sequencing was performed according to Maxam and Gilbert [34].
Pulsed-field gradient electrophoresis. Purified P. falciparum-infected erythrocytes were prepared in agarose blocks for pulsed field gradient elec- trophoresis [35]. 2 mm slices from the blocks were sealed into a 1.5% agarose (Seakem ME) gel in 75 mM Tris-borate, 75 mM boric acid, 2 mM E D T A and electrophoresed for 24 h at 300 V, 18°C with a pulse field time of 90 s to separate chromosome-sized DNA [36]. The gels were stained with ethidium bromide and exposed to short wave ultraviolet light for 4 min before the DNA was transferred to nitrocellulose.
205
Construction and analysis of a PfHRP1 expres- sion clone. The expression vector pATH11, a de- rivative of one of the pKRS vectors [37], was kindly provided by T.J. Koerner and A. Tzago- loft, Columbia University, New York. A 1284 bp Sau3A fragment was isolated from the PfHRPI cDNA clone, p47, ligated to BamHI-digested p A T H l l and used to transform E. coli DH1 cells. The resulting recombinants, containing part of the E. coli trp E gene, which encodes anthranilate synthetase, fused in frame with the PfHRPI gene, were grown to stationary phase in M9 medium [29] containing 20 txg ml -l L-tryptophan, 5 g 1 l casamino acids and 50 Ixg m1-1 ampicillin. The cells were diluted into fresh medium without tryptophan for 1 h and then induced by the ad- dition of 3-13-indoleacrylic acid (20 ixg m1-1) and vigorously shaken at 30°C for 4 h. The cells were harvested and incubated in 50 mM Tris-HC1 (pH 7.5), 5 mM E D T A and lysozyme (3 mg ml-1) for 60 min at 0°C and lysed with 0.3 M NaC1, 0.75% Nonidet P-40 (NP-40) at 4°C for 10 min. The lysed cells were sonicated with a titanium microtip probe for 100 s at 170 W and centrifuged at 10000 x g for 10 rain. The pellet was washed once with 1 M NaC1, 10 mM Tris-HCl (pH 7.5), once with 10 mM Tris-HC1 (pH 7.5) and then resuspended in 2% SDS, 10 mM Tris-HCl (pH 7.5). Total protein extracts were prepared by solubilizing cell pellets in Laemmli sample buffer [38] at 100°C for 3-5 min. The protein samples were electro- phoresed on 7.5% SDS polyacrylamide gels. Electrophoretic transfer [39,40] was performed in 20 mM Tris base, 150 mM glycine and 20% methanol, for 2 h at 100 V and 4°C in a BioRad Transblot apparatus. The nitrocellulose filters were treated with Protoblot Immunoscreening System (Promega Biotec) according to the man- ufacturer's specifications. The IgG2a monoclonal antibody MeAb89 [18] was used at a dilution of 1:1000.
Results
Construction and screening of the cDNA library. Previous studies have shown that PfHRPI is syn- thesized by ring-stage parasites, 9-21 h after mer- ozoite invasion [41,42]. Thus a cDNA library was constructed from RNA extracted from synchro- nized cultures of FCR-3/A2 K + parasites 16-20 h
206
A B
28S
IBS
I 2 I 2 3 4 5
4 . 4 - -
2 . 4 - -
1 . 4 - -
m
Fig. 1. Northern blot analysis of stage-specific R N A derived from K- and K variants of FCR-3. (A) 4 FLg R N A derived from K + (lane 1) or K (lane 2) cultures 16-20 h after invasion; (B) 4 ~g K + RNA from cultures 12, 18, 26, 36 and 44 h after invasion (lanes 1-5, respectively). Both blots were hybridized with the nick translated 1820 bp EcoRI insert derived from the p47 clone. The size markers are the large and small ribosomal R N A s of P. J?dciparum (3720 and 1970 nucleotides, respectively [44]) (panel
A) or Bethesda Research Laboratories R N A standards (panel B).
after invasion. Thirty-three positive plaques were isolated from about 175000 recombinants screened with the P1HRP cDNA probe, purified and further characterized by hybridization of nick- translated purified DNA to Northern blots con- taining K + and K RNA. All of the recombinant clones hybridized to a 3500 nucleotide band pres- ent only in K + RNA (data not shown). The clone
containing the largest insert (1820 bp) was sub- cloned into pGem4 [32] for further analysis. This clone was designated p47. When it became evi- dent (see below) that p47 represented an incom- plete sequence, restriction analysis was used to identify an additional clone (p23-3B), among those originally screened, that extended beyond the 3' end of p47.
E R X p 4 7 I I I I
D
p23-3B IOObp ! i
R R N N II L ' r / ' / ' / ' /~ I I il i i ST T A A O S
E N / . . . . . . . . . . . . . . . . . . . . . . . I
Fig. 2. Diagram of the sequencing strategy and a schematic representat ion of the PfHRPI cDNA clones p47 and p23-3B. Arrows above the upper line represent the extent of the sequencing runs of the coding strand and the arrows below represent the same for the non-coding strand of p47. The coding region is indicated by the heavy line while the histidine-rich region and the 3' repeats are indicated by the hatched and cross-hatched boxes, respectively. Restriction enzyme sites were determined from the sequence and those marked below the line were confirmed to be present in parasite DNA: A, AvaII; D, DraI; R, RsaI; E, EcoRI; N, NcoI; S, Sau3A; T, TaqI; X, XmnI. The arrowhead denotes the region of the frameshift in p47. The EcoRI sites represent the ends of
the inserts. The dotted line in the p23-3B scheme represents the approximate length of the unsequenced region of this clone.
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Fig.
3.
Nuc
leot
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ence
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dict
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208
RNA analysis using p47. The 1820 bp p47 insert hybridized to a 3500 nucleotide transcript present in the K + RNA prepared from synchronous cul- tures 16-20 h after invasion, and not to K RNA prepared under similar conditions (Fig. 1A). The amount of PfHRPI RNA was highest in parasites harvested 18 h after invasion (Fig. 1B). The probe hybridized to a transcript of approximately 4000 nucleotides in the early (12 h) ring stages. The size of the RNA transcripts suggested that p47 did not represent the complete PfHRPI gene.
Nucleotide sequence analysis. Fig. 2 presents the sequencing strategy. The restriction enzyme sites shown in Fig. 2 were all determined from the nu- cleotide sequence and confirmed by digestion of the cDNA clone p47. In addition, the restriction sites indicated below the line were verified by comparison mapping with genomic digests of DNA derived from FCR-3 K + parasites (data not shown). The sequence of p47 contained a termi- nation codon at nucleotide 1578, indicating that this clone encoded a protein of 398 amino acids. However, inspection of the DNA sequence,
A
translated in all three frames, indicated that a single frameshift at any point up to about 143 nu- cleotides upstream of position 1578 would open up a new reading frame extending to the end of clone p47 (1820 bp). The possibility that a frame- shift had been introduced during the cloning processes was reinforced by examination of the A + T nucleotide bias throughout the clone, and by the observation that termination codons were present in two of the three alternative reading frames at an average frequency of one every 15 nucleotides between the initiation codon at posi- tion 385 and the end of the clone. Additional support for the possibility of a frameshift came from preliminary data suggesting that the corre- sponding gene from the P. falciparum NF7 strain from Ghana [35] encoded a protein of 657 amino acids (Walter and Eliza Hall Institute, Mel- bourne, Australia; presented at the 9th Rocke- feller Foundation Great Neglected Diseases of Mankind Biomedical Research Network, Sep- tember 14-19, 1986) [43]. Therefore, another cDNA clone, p23-3B, containing a 1400 bp in- sert, was sequenced through this region. Restric-
1 2 3 4 5 6 7 8 9 1 0 1 1
9.4 - "
6.7 . . . . .
B I 2 3 4 5 6 7 8 9 I0 II
9 . 4 ~
6 .7 - -
m
Fig. 4. Southern blot analysis of genomic DNA digested with EcoRI (A) or HindlII (B) and hybridized with nick-translated p47. DNA isolated from: P. lophurae (lane 1), P. falciparum FCR-3 K (lane 2), FCR-3 K + (lane 3), St. Lucia K + (lane 4), St. Lucia K- (lane 5), Malayan Camp K (lane 6), Malayan Camp K- (lane 7), clone 7G8 (lane 8), Rhesus monkey (lane 9), human (lane 10) and Aotus monkey (lane 11). No hybridization was detected to any bands less than 9.4 kb. Sizes of the markers are noted in
kb.
tion mapping indicated that p23-3B extended about 100 bp beyond the 3' end of p47 (Fig. 2), and was the only clone to do so. The sequence of p23-3B suggested that an additional A had been inserted in p47 between nucleotides 1504 and 1511. The frameshift that resulted from the re- moval of this single base opened the continuous reading frame shown in Fig. 3. With the excep- tion of that single nucleotide, the sequences of p47 and p23-3B were identical (in the sequenced re- gion) up to nucleotide 1804. Divergence of the sequences after this point (p47 extended only an- other 16 bp) indicated that either one or both of the cDNA clones had rearranged. Therefore, the presented nucleotide and amino acid sequences have been restricted to 1803 bp.
The open reading frame encodes a protein of 473 amino acids, with a predicted molecular weight of 53 193. The overall content of histidine in the partial sequence is only 10% and the most abundant amino acid is lysine (14%). A histidine- rich region containing three tracts of 6, 7 or 9 contiguous histidines is located close to the amino terminus of the putative mature protein while the carboxy-terminal sequence is repetitive and rich
A I 2 :5 4 5 6 7
209
in lysine. Another notable feature of the 5' end of the coding region is the predicted block of hy- drophobic amino acids that resembles a signal se- quence. A potential signal peptidase cleavage site [44,45] occurs prior to serine 35. Two potential glycosylation sites occur at asparagine residues 42 and 250.
The PfHRPI cDNA has a notable codon bias in the third position: A and T are used 77% of the time, in preference to G and C. The base composition is 63% A + T in the coding region and 91% A + T in the 5' untranslated region.
Southern blot analysis of PfHRPI. To determine whether the PfHRPI gene is conserved among different strains of P. falciparum, p47 was nick- translated and used to probe restriction digests of four different strains (Fig. 4). Restriction length polymorphisms were discernable in the HindlII digests (Fig. 4B).
The 7G8 clone and the St. Lucia K- strain also hybridized with the probe. The 7G8 clone has been shown by electron microscopy to have knobs scattered on the surface of infected erythrocytes, but PfHRPI has not been detected with McAb89
B I 2
S - -
_ _
5 4 .5 6 7
Fig. 5, Pulsed field gradient electrophoresis of K ÷ and K- chromosomal DNA. (A) The ethidium bromide stained gel and (B) the autoradiogram after hybridization with the nick-translated p47 cDNA clone. Parasites from: a culture adapted Malayan Camp K ÷ strain (lane 1); Malayan Camp K-- (lane 2) and Malayan Camp K (lane 3) from Aotus monkeys; FCR-3/A2 K- (lane 4) and FCR-3/D4 K (lane 5) from culture; St. Lucia K ÷ (lane 6) and St. Lucia K (lane 7) from Aotus. The three smallest chromosomes were resolved. In the compression region of this separation (C), the larger chromosomes were not distinguishable, but different
electrophoretic conditions resolved eleven additional chromosomes [61]. S denotes the sample slot.
210
or by biosynthetic labelling (R.J. Howard, W.A. Daniel and T. Wellems, unpublished data), or by immunoblotting with affinity-purified antibodies [46]. However, a faint RNA band of about 4000 nucleotides can be seen (data not shown). The St. Lucia K strain does not appear to have knobs in electron microscopic studies, nor can PfHRPI be detected by biosynthetic labelling [25].
Pulsed field gradient electrophoresis. To deter- mine the chromosomal location of the PfHRPI gene, pulsed field gradient electrophoresis was performed on several P. falciparum strains (Fig. 5). The nick translated p47 probe hybridized to DNA in the slot and to chromosomal DNA band 2 of the K + strains, but did not hybridize to the Malayan Camp K strain or the FCR-3/D4 K clone. These data indicated that the PfHRPI gene is located on chromosome 2 and is absent from some of the K- strains. The ethidium-bromide stained gels show a difference in the mobility of chromosome 2 between the K + and K variants of the Malayan Camp and FCR-3 isolates, con- sistent with the deletion of 5-150 kbp. Although the St. Lucia strain is phenotypically K- , its
chromosome 2 had the same relative mobility as the K ~ isolates and it hybridized to the p47 probe.
Expression of the PfHRP1 gene in E. coli cells. To obtain further evidence that p47 represents the PfHRPI gene, we inserted a 1284 bp Sau3A frag- ment (see Fig. 2) of the cDNA clone into the vec- tor p A T H l l . This clone (designated pA5) con- tained 90% of the coding sequence of p47, including the entire histidine-rich region and the 3' lysine-rich repeats. The predicted size of the fusion protein was 85 kDa. The monoclonal an- tibody McAb89 [18] recognized a 100 kDa pro- tein in the parasite extract and an 82 kDa protein in the induced E. coli cell extract (Fig. 6). The antibody did not react with extracts of induced E. coli containing p A T H l l alone, or a p A T H l l re- combinant containing another malaria gene [47]. By comparing Fig. 6A and 6B, it can be seen that very little parasite extract was necessary on the Western blot to obtain color reactions of approx- imately equal intensity with the parasite and E. coli fusion proteins, indicating that, after the pro- cedures involved in electrophoresis, blotting and immunodetection, McAb89 had a greater affinity for the natural protein.
A B I 2 3 4 I 2 3 4
t l 6
9 7 - - 4
6 6
Fig. 6. Analysis of the PfHRPI expressed in E. coli. Protein extracts from induced E. coli cells containing pATH11 (lane 1), from K + FCR-3/A2 cultures (lane 2), from induced E. coli cells containing pA5 (lane 3), or a pATH11 recombinant with another HRP- related gene from P. falciparurn [39] (lane 4), were transferred to nitrocellulose and probed with McAb89 (A) or stained with
Coomassie blue (B). The arrowhead indicates the pA5 induced fusion protein. The size markers are given in kDa.
211
Discussion
The cDNA clones that we have isolated appear to encode part of a protein with the biochemical attributes of the P. falciparum histidine-rich knob- associated protein. Firstly, the predicted protein has a histidine-rich region, although (unless the C-terminal portion missing from our sequence is also histidine-rich) histidine will probably repre- sent less than 10% of the total amino acid con- tent, in contrast to the 73% histidine content of P1HRP [48,49]. The deduced sequence is more lysine-rich than histidine-rich. Secondly, the fu- sion protein expressed in E. coli contained an ep- itope recognized by an anti-PfHRPI monoclonal antibody. Finally, p47 hybridized only with RNA from a K + isolate and did not hybridize to DNA from two of the three K- samples tested.
The deduced amino acid sequence of PfHRPI contains three blocks of 6-9 contiguous histidine residues similar to the blocks of 6-8 histidines comprising most of PIHRP [17,49,51], but absent from PfHRPII and PfHRPIII [52,53]. PfHRPII and PfHRPIII have extensive tandem repeats, containing high levels of histidine and alanine, which are not present in PfHRPI. The level of homology between PfHRPII and PfHRPIII indi- cates that they have probably originated from a common ancestral gene [52]. However, there is no evidence that PfHRPI is also a member of that gene family. The functions of PfHRPII and PfHRPIII are unknown, while PfHRPI is rele- vant to knob formation on infected erythrocytes, probably as a cytoskeleton-associated structural protein [25].
Outside the histidine-rich region there is little homology between PfHRPI and P1HRP. How- ever, there are structural 'similarities in the amino- terminal sequences encoded by the two genes. The P1HRP is synthesized as a prepro-protein, which undergoes two cleavages to produce the mature HRP [51,54,55]. The probable cleavage site of the secretory signal sequence can be in- ferred by applying the rules developed by von Heinje [44,45], which give the highest probability for cysteine 26 and serine 35 becoming the amino- termini of the signal peptide cleavage products of P1HRP and PfHRPI respectively. Asparagine 40 is probably glycosylated in the P1HRP precursor
[54]: the mature protein starts at valine 48 [55]. By analogy, asparagine 42 of PfHRPI may also be glycosylated in a precursor, but absent from the mature protein. However, comparison of the amino acid sequences of P1HRP and PfHRPI around this region does not indicate a homolo- gous processing site. It is also interesting to note that the initial 23 amino acids of P1HRP [51] and 37 amino acids of PfHRPI [43] are encoded by a short exon lying close upstream of the main exon.
Kilejian et al. have reported 901 bp of se- quence from a cDNA clone prepared from un- cloned FCR-3 K + parasites [50]. This published sequence can be aligned with nucleotides 294-1194 of our sequence. The only difference is at position 1161 in our sequence, where G re- places A, changing threonine 260 of Kilejian et al. to alanine. Also, the restriction map deduced from our sequence is consistent with another published map for cDNAs that were also derived from FCR-3 parasites [56]. Two recent publica- tions have presented the complete sequence of the PfHRPI gene of the NF7 isolate originating from Ghana [43] and 825 bp of coding sequence of a PfHRPI cDNA from a Honduras isolate [57]. The Honduras sequence is identical to nucleotides 940-1764 of our FCR-3 sequence except at posi- tions 1252, 1758 and 1764, where G, T and T in FCR-3 are all replaced by A in the Honduras se- quence. The differences between the NF7 and Honduras or Gambian sequences are much greater. Nucleotides 69-1803 of our FCR-3 se- quence can be aligned with nucleotides 1-2171 of the NF7 genomic sequence if the intron (nucleo- tides 425-854) is discounted. The optimal align- ment between the FCR-3 and NF7 sequences in- volves 23 nucleotide differences and the insertion of 13 gaps in the FCR-3 sequence and 7 gaps in the NF7 sequence. Two of these gaps produce a self-correcting frameshift, which completely changes the sequence of a 27 amino acid stretch of the protein. Also, the histidine-rich region of NF7 contains three blocks of 11, 6 and 10 con- secutive histidines, where the corresponding po- sitions of the FCR-3 sequence encode blocks of 6, 7 and 9 histidines.
The full extent of inter-isolate variation in the PfHRPI gene will only be revealed when addi- tional sequences are available. Such information
212
will help establish the rates of divergence of genes, isolates and species of malaria parasites.
Comparison of PfHRPI in parasite extracts and when expressed in E. coli suggested that the anti- PfHRPI monoclonal antibody was much less re- active with the E. coil product, possibly due to a problem in protein folding, although both pro- teins were denatured prior to SDS-polyacrylam- ide gel electrophoresis. McAb89 reacts with a conformation-dependent epitope on PfHRPI (A. Saul and R.J. Howard, unpublished data). Two short DNA clones were previously selected from a P. falciparum genomic DNA ag t l l expression library by screening with McAb89. From a com- parison of the short predicted amino acid se- quences, and a bias, due to reduced McAb89 reactivity after protein reduction, that cysteine residues might be involved, it was suggested that the tripeptide sequence Thr-Phe-Cys, encoded by both clones, might represent an epitope recog- nized by McAb89 (A. Saul and R.J. Howard, un- published observations). However , this tripep- tide is absent from that portion of the PfHRPI sequence expressed in E. coll. Thus, the concept that McAb89 may recognize a single epitope identified by consecutive amino acids may be na- ive.
The consistent presence of a slightly larger RNA in early ring-stage parasites could be due to a different gene product or to differential RNA splicing, which may lead to the generation of dif- ferent proteins from the PfHRPI gene. In this context it is interesting that the PIHRP [51], PfHRPI [43], PfHRPII and PfHRPIII [52] genes all contain introns.
Two published reports have also assigned the PfHRPI gene to chromosome 2 and shown that loss of the knobby phenotype can be attributed to a deletion in this chromosome, which can leave a remnant of the PfHRPI gene close to the te- lomere [46,56]. Deletion of all or part of the PfHRPI gene has been previously demonstrated in K parasites of the FCR-3/D3 and D4 clones [50,56] but not in an uncloned K- FCR-3 popu- lation, although the gene was apparently not transcribed in this population [50]. Deletions have been demonstrated in an FVO (Vietnam) K clone [56] and in a cloned K line (El2) of the FC27 Papua New Guinea isolate [46].
Our data confirm the published results with the FCR-3/D4 K clone. Since Culvenor et al. [46] noted fluctuations in the proportions of K + and K parasites in uncloned populations, we were cautious in interpreting the presence of a PfHRPI gene in our uncloned K St. Lucia population. However, electron microscopy and histidine la- beling of cells obtained from the same cryopres- erved sample used for DNA analysis showed no knobs or PfHRPI [58]. Clearly, it would be quite possible for transcription or translation to be al- tered by other means than gene deletion. Unfor- tunately, mRNA was not available to check whether PfHRPI gene transcription was occur- ring in the same parasite population from which DNA was obtained. Reversion from K- to K + phenotype has also been reported with a clone of the FVO isolate [59]. These observations suggest that the PfHRPI gene is in a quite unstable chro- mosomal location, which may be subject to a range of minor or major rearrangements during maintenance of the parasite in culture or in sple- nectomized hosts, under conditions where there is no selection for the K + phenotype. Such rear- rangements may only become evident following recloning of the populations. The probable te- lomeric location of the PfHRPI gene [56] may contribute to its instability, as is the case for te- lomeric genes in trypanosomes [60].
Acknowledgements
We thank Dr. Christine Clayton for assistance with the kgtl0 and her helpful suggestions throughout this work, Dr. Mary Morry for help with the expression vectors, R. Klein for techni- cal assistance and Dr. Diane W. Taylor for the monoclonal antibody. This work was supported by the Rockefeller 'Foundation and NIH grant AI 23848.
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