Structure of the knob protein (KP) gene of Plasmodium falciparum

Molecular and Biochemical Parasitology, 26 (1987) 11-16 Elsevier

11

MBP 00863

Structure of the knob protein (KP) gene of Plasmodium falciparum Yagya D. Sharma and Araxie Kilejian The Public Health Research Institute, New York, NY, U.S.A. (Received 9 June 1987; accepted 10 June 1987)

We have determined the nucleotide sequence of the gene encoding the knob protein (KP) of Plasmodium falciparum (FCR3/Gambia). The gene is interrupted by an intron which contains 34 imperfect tandemly repeated ATTTT sequences. The first exon encodes 33 amino acids with a hydrophobic core typical of signal peptides. The second exon has an open translational reading frame for 597 amino acids. The deduced protein sequence indicates that KP has multiple structural domains; unlike the N-terminal histidine-rich domain which we described previously, the C-terminal half is rich in lysine residues. Consistent with the apparent association of KP with the cytoplasmic surface of the host erythrocyte membrane, the protein is highly charged and hydrophilic. Key words: Plasmodiumfalciparum; Knob protein; Malaria; Histidine-rich protein

Introduction T h e k n o b protein (KP) of Plasmodium falciparum is essential for the f o r m a t i o n of the characteristic knob-like protrusions on the host erythrocyte membrane. It has been identified in several geographical isolates and has b e e n characterized as a red cell m e m b r a n e - a s s o c i a t e d polypeptide which incorporates relatively large a m o u n t s of e x o g e n o u s histidine [1-7]. In a previous study we described a c D N A clone e n c o d i n g the N-terminal histidine-rich d o m a i n of the K P of isolate F C R - 3 [8]. W e now report the c o m p l e t e nucleotide sequence of the gene and the d e d u c e d primary structure of KP.

cloned k n o b - p r o d u c i n g G a m b i a n isolate F C R - 3 [1]. The D N A was digested with E c o R I and fractionated on a 10-40% sucrose gradient. Aliquots of the fractions were analyzed on a 0.8% agarose gel and transferred to nitrocellulose paper [9]. The filter was p r o b e d with nick-translated pfc43 [10]. A fraction which gave a strong hybridization signal was ligated to C h a r o n 4 A E c o R I arms, packaged in vitro and used to infect Escherichia coli LE392 [10]. R e c o m b i n a n t phage were screened with pfc43 and positives were plaque-purified. R e c o m b i n a n t phage D N A was isolated [10] and PstI and B a m H I fragments were subcloned into p U C 8 [11]. T h e 3' end fragment of a PstI subclone was nick-translated and used to rescreen the c D N A library [8] for the isolation of clone pfc24.

Materials and Methods Isolation of c D N A and genomic D N A clones. The isolation and characterization of a c D N A clone, pfc43, from a c D N A library was described previously [8]. D N A was p r e p a r e d [8] f r o m an unCorrespondence address: Araxie Kilejian, The Public Health Research Institute, 455 First Avenue, New York, NY 10016, U.S,A. Abbreviations: bp, base pair; KP, knob protein; HRP, histidine-rich protein.

Sequence analysis. The chemical degradation [12] and the dideoxy chain termination m e t h o d s [13] were used for sequence analysis. Results A c D N A clone, pfc43, encoding the N-terminal half of KP was used to isolate a cross-hybridizing E c o R I genomic D N A fragment f r o m a partial library constructed in Charon 4A. To facilitate further analysis, PstI and B a m H I subfragments of

0166-6851/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. Partial restriction map of the KP genc and sequencing strategy. The boxed areas indicate the coding regions: the first exon coding the signal peptide, S, separated by intron, I, from second exon, C. Closed circles indicate the origin of end-labeled fragments sequenced by the method of Maxam and Gilbert [12] in the direction and to the extent indicated by the arrows. Arrows with no circles indicate sequencing by the dideoxy chain termination method. The origin of the sequenced fragments is indicated with the underlined respective cDNA (pfc24) and genomic DNA clones (Pg43 and Bg43) described in the text.

the insert D N A were cloned in plasmid pUC8. A PstI subclone, denoted Pg43, and a B a m H I subclone, denoted Bg43, were used for further analysis. All of the c D N A clones which we had identified previously [8] proved to be lacking the 3' end of the KP gene. Therefore, a 3' end fragment of Pg43 was used to isolate a c D N A clone, pfc24, encoding the C-terminal half of the KP gene. The structure of the KP gene was determined by comparative restriction enzyme analysis and sequencing of selected fragments of the genomic and c D N A clones as summarized in Fig. 1. The complete nucleotide and deduced amino acid sequence shown in Fig. 2 was derived by combining our previous data on c D N A pfc43 [8] and the sequence of fragments indicated in Fig. 1. A comparison of the 5' end of pfc43 [8] with that of genomic clone Pg43 clearly shows that the gene is interrupted with a 438 bp intron. The first exon encodes 33 amino acids with a hydrophobic core typical of signal peptides. The sequences of the splice sites (Fig. 2, boxed) are similar but not identical to published consensus sequences for 5' (AG { G T A ) and 3' ( T X C A G { ) junctions [14]. The intron shows a unique feature: it contains 34 imperfect repeats of A T T T T (Fig. 2, overlined). The second e x o n of the KP gene has an open translational reading frame of 1791 bp encoding 597 amino acids, with a calculated molecular weight of 64 843. In the absence of chemical data

on the N-terminal amino acid of mature KP, we have n u m b e r e d the first amino acid following the intron as 1 for the convenience of discussion (Fig. 2). On polyacrylamide gels the precursor of KP has a molecular weight of 75000 [15]. The apparent difference in the estimated and calculated molecular mass may be due to anomalous electrophoretic mobility as has been noted for several other malarial polypeptides. The deduced structure of KP shows some interesting features; it is distinctly a multidomain polypeptide with limited regions of repeated sequences (Fig. 2, underlined). The outstanding repeats are the three polyhistidine stretches characterizing the N-terminal histidine-rich domain (residues 24-80), three lysine-rich fragments (residues 336-398), and three imperfect repeats of the sequence Ala Thr Lys Glu Ala Ser Thr Ser Lys (residues 507-532). The latter two repeated sequences form the rough boundaries of a lysinerich domain. The multidomain nature of KP is more clearly apparent in Fig. 3 which shows the hydropathy index and charge distribution of the polypeptide; KP is clearly a highly charged and hydrophilic protein. The amino acid composition of KP deduced from the nucleotide sequence is as follows: Lys 84, Gly 65, Ala 55+ His 49, Glu 48, Asn 43, Set 43, Thr 39+ Gin 30, Asp 30, Pro 22, Tyr 20, Val 19, Leu 13, 1/2 Cys 11, Arg 10, Phe 9+ lle 4, Met 3. Although the 49 histidine residues account for

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Fig. 2. Nucleotide sequence of the KP gene and the deduced amino acid sequence of the coding region. The gaps in the sequence analysis shown in Fig. 1 were completed with our previously published data [8]. The splice junction sequences are boxed. The tandemly repeated elements of the intron are overlined. The major repeated coding sequences are underlined. Asterisks mark the termination codon.

14 A

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the knob protein. Basic residues of arginine and lysine are represented by long bars and histidine by half bars. The positions of acidic aspartic and glutamic acid residues are represented by long bars. The hydropathy index was calculated by the method of Kyte and Doolittle [28]. only 8% of the total amino acids, considering the fact that the average histidine content of analyzed polypeptides is around 2% [14], KP is a relatively histidine-rich protein. Discussion

We have determined the nucleotide sequence of the KP gene of P. falciparum and thereby deduced the amino acid sequence of the polypeptide. KP is one of 4 known malarial histidine-rich proteins (HRPs). The structure of each of the three other proteins, the H R P of Plasmodium lophurae [17,18] and two closely related alaninehistidine rich proteins ( S H A R P or P f H R P I I and PfHRPIII) of Plasmodium falciparum [19,20], llas been described previously. Although a c o m m o n physiological function cannot be assigned to these proteins, the structural organization of the 5' end of their genes shows a striking similarity; they are

all interrupted and the first exon contains a short open translational reading frame with hydrophobic amino acids typical of signal peptides. A similar organization has been reported for the gene of the R E S A antigen [21]. The only apparent c o m m o n attribute of KP, P f H R P l l and R E S A is their eventual association with the host erythrocyte m e m b r a n e [5,7,22]. It seems unlikely that a spliced leader peptide is correlated with the processing of malarial polypeptides through particular c o m p a r t m e n t s of infected erythrocytes; the sequences of the leader peptides of these proteins do not show any obvious similarity. Furthermore, the H R P of P. lophurae is localized in m e m b r a n e bound granules and there is no experimental evidence for the export of this polypeptide to the host cell m e m b r a n e [23]. The only m e m b r a n e barrier which these granules may possibly cross is the m e m b r a n e of the food vacuole; they apparently accumulate within residual bodies of mature schizonts. The repeated A T T T T elements of the Jntron of the KP gene appear to be a unique feature; an identical sequence is also present in the KP gene of a Honduran isolate HB3 [24]. However, the splice junction sequences of all the H R P s are identical, while that of the R E S A shows a single C for T substitution (Table I). The structure of KP is very different from the other HRPs. The H R P of P. lophurae as well as the two small histidine-alanine-rich polypeptides of P. falciparurn are dominated by tandem repeats of a few amino acids, while KP shows distinctly different structural domains with limited areas of repeated sequences. This may have functional relevance; although the details of the interactions of KP with the erythrocyte m e m b r a n e are not known, it has been suggested that it is associated with the cytoskeleton of erythrocytes [4].

TABLE I Splice junction sequences 5' junction Consensus HRP, P. lophurae PfHRP, P. ,[~llciparum RESA, P. falciparum KP. P. falciparurn

AG { GTA GC ~,GTA AT { GTA AC ~,GTA

3' junction TXCAG ,L TATAG $ TATAG ~ CATAG $ TATAG ,L

References [14] [ 17] [20] [21] This study

15 We speculate that the different structural domains of KP may be essential for specific interactions with cytoskeletal polypeptides which lead to formation of knobs. A computer comparison of the amino acid sequence of KP with sequences of other proteins revealed limited areas of homology with several proteins. The most extensive homology was with the histidine-rich glycoprotein of human serum [25]. This is expected in view of the presence of histidine-rich domains in these polypeptides.. The HRPs of P. falciparum, KP and the two related small histidine-alanine rich polypeptides, are very different from each other; however, they each share some common sequences with the H R P of P. lophurae. The small polypeptides and H R P contain the repeated AlaHisHis sequence while the relationship of KP and H R P resides in repeated polyhistidine sequences. Since incorporation of relatively large amounts of histidine is one of the attributes of KP from several geographical isolates, it could be inferred that this domain is conserved. The evolutionary relationship of the polyhistidine sequences of KP and H R P can be only speculative. Although the function of H R P remains unknown, the gene encoding this peculiar polypeptide (over 70% histidine) has been conserved after more than 4 decades of maintenance of P. lophurae in an experimental host, the duck. The serological cross-reactivity of immune human sera with H R P [26] raises the possibility that the histidine-rich domain of KP may be immunogenic in man. H R P was the first purified malarial polypeptide used to test the feasibility of using a single defined antigen as a malaria vaccine [27]. The role of KP, a functionally important, immunogenic [7,26] and apparently conserved malarial polypeptide, in the immunity to P. falciparum remains to be tested. Since the completion of this study, there have been two reports of the sequence of the KP from two different isolates. The partial sequence of the KP of Honduras I [29] is almost identical to that of FCR-3, with the exception of 3 bp substitutions which change Gly 254 to Asp, Asn 421 and 423 to Lys (Fig. 2). The most notable difference between the complete sequence of the KP of isolate NF7 [30] and FCR-3 is at residues 399-430 (Fig. 2). The apparent difference in the transla-

tional reading frame of this region is due to an additional A in NF7 preceding Ser 399 and the deletion of an A following Lys 426 (Fig. 2); the authors have already discussed the rationale for adding an A at position 399. It is of interest that NF7 lacks the DraI restriction site at position 429-431; our studies indicate that this sequence is conserved in a Thai as well as a Honduran isolate (unpublished). A second difference between the two isolates is the substitution of the sequence Pro Pro His Gly Ala (residues 284-288, Fig. 2) by the sequence His Pro Trp Ser in NF7. The apparent minor differences in the histidine-rich domains of the two isolates are of interest from the point of view of the evolution of differences in repeated sequences of malarial polypeptides. Single bp substitutions have changed Gln 33, 35, 80 and 83 to His, resulting in longer stretches of polyhistidine; furthermore, the sequence Pro His Glu Ala (residues 41-44, Fig. 2) has been tandemly repeated in NF7. Despite minor apparent differences, the basic primary structure of KP appears to be highly conserved in the isolates studied.

Acknowledgments This study was supported by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and NIH Grant AI19845. We thank Dr. Habib Karoui for screening the c D N A library and Lauren Naslund for technical assistance.

References 1 Kilejian, A. (1979) Characterization of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodiumfalciparum. Proc. Natl. Acad. Sci. USA 76, 4650-4653. 2 Kilejian, A. (1980) Homology between a histidine-rich protein from Plasmodium lophurae and a protein associated with the knob-like protrusions on membranes of erythrocytesinfected with Plasmodiumfalciparum. J. Exp. Med. 151, 1534-1538. 3 Hadley, T.J.. Leech, J.H., Green, T.J., Daniel, W.A., Wahlgren, M., Miller, L.H. and Howard, R.J. (1983) A comparison of knobby (K+) and knobless (K-) parasites from two strains of Plasmodiumfalciparum. Mol. Biochem. Parasitol. 9,271-278.

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