Variations in the C-terminal repeats of the knob-associated histidine-rich protein of Plasmodium falciparum

BIOCHIMICA ET BIOPHYSICA ACTA

g

ELSEVIER

Biochimica et Biophysica Acta 1360 (1997) 105-108

Short sequence-paper

Variations in the C-terminal repeats of the knob-associated histidine-rich protein of Plasmodium falciparum l Hiroko Hirawake

a, K i y o s h i K i t a a, Y a g y a D. S h a r m a b,.

a Department of Parasitology, Institute of Medical Science, Unieersity of Tokyo, Tokyo 108, Japan b Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110029, India

Received 6 November 1996; revised 20 January 1997; accepted 20 January 1997

Abstract

The knob-associated histidine rich protein (KAHRP) of Plasmodium falciparum plays an important role in the pathophysiology of cerebral malaria. In the present study, the immunogenic C-terminal repeat domain of the KAHRP gene was amplified, cloned and sequenced from the Indian (RJ 181) and Honduran (HB3) isolates of P. falciparum. Based on the number and types of repeats in the domain, we report here the presence of three unique variant forms of KAHRP among these isolates. The Indian isolate (RJ181) contained four units of the decapeptide repeats whereas the Honduran isolate (HB3) contained two forms i.e. one form containing four decapeptide repeats plus a tetrapeptide subunit and the other form containing three decapeptide repeats plus a tetrapeptide subunit. Thus, all together, the number of KAHRP variants is increased to five which includes previously described two variants, each containing either 3 or 5 decapeptide repeats. This high rate of variability in the antigenic domain of the KAHRP gene via deletion or addition of whole or part of the decapeptide units could be involved in the evasion of host immune system possibly by providing the speculative complementarity to the vargene product. The results of the present study will be useful in designing the suitable molecular therapeutic reagents for cerebral malaria. © 1997 Elsevier Science B.V. All rights reserved. Keywords: Cerebral malaria; Knob protein gene; Falciparum isolate; Polymerase chain reaction; Nucleotide sequence

P l a s m o d i u m f a l c i p a r u m is the main killer among all human malaria parasites. The surface of this parasite infected RBC (IRBC) becomes rough due to the presence of knobs. These knobs are 60 to 100 nm in

Abbreviations: KAHRP, knob-associated histidine rich protein; PFEMP-1, P. falciparum infected erythrocyte membrane protein-l; PCR, polymerase chain reaction; IRBC, P. falciparum infected erythrocytes *Corresponding author. Fax: +91 11 6862663; E-mail: yds @aiims.ernet.in i EMBL Data Bank accession numbers are X94295 for RJ181, X94294 for HB5 and X94293 for HB6.

diameter, contain electron dense material and are cup shaped in structure. Their presence has been implicated in the pathogenesis of the disease. Because, these knobs can mediate the cytoadherence phenomenon between IRBC and host endothelial cells resulting in the sequestration of mature forms of the parasite in deep vasculature reviewed in [1]. The induction of these knobs is associated with the expression of K A H R P [2]. This protein, therefore, plays an important role in the pathophysiology of cerebral malaria. The primary structure of K A H R P contains 3 domains; the N-terminal histidine- rich domain, central lysine-rich domain and the C-terminal decapep-

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tide repeat domain [3]. The latter two domains are antigenic in human [4]. The sequence comparision from various isolates showed variation in the Cterminal domain, elsewhere the gene was conserved. So far only two variant forms of the KAHRP have been reported in the literature; each one containing either 3 or 5 decapeptide repeats in the C-terminal region [1,5]. In the present study we show three more variant forms of the KAHRP gene and thus the existance of multiple alleles of this gene. The PCR was carried out for the central constant and C-terminal variable regions of the KAHRP gene on 5 clinical and 5 laboratory adapted regional isolates of P. falciparum. The clinical isolates were from the Rajasthan epidemic [6]. The other regional isolates have been described earlier [3,5,7,8]. The primer sequence for the constant region is 5'-GTA CCA CAT GTG GAC CTG CC-3' for forward and 5'-TTC TCC TTC ACC GTC ATT TCC-3' for reverse primer. The primer sequences for variable Cterminal region are; forward primer 5'-GAA ACA AAA AAC ACC GCT G-3' and reverse primer 5'GTA CTG CAT TAG CTC CTG TAG TTG-3'. The PCR amplification was carried out under the same conditions as described before, except that the annealing temperatures for constant and variable region primers were 51°C and 45°C respectively [5]. The PCR products were analysed on the 1.5% agarose gel (Fig. 1). The PCR products of the variable region of KAHRP from Indian isolate R J181 and Honduran isolate HB3 were recovered from the agarose gel and cloned into the plasmid vector by using TA cloning kit (Invitrogen, USA). The nucleotide sequencing of 2 to 4 independent clones was carried out on a DNA sequencer (Applied Biosystems, USA, Model 373A) by using universal M13 forward and reverse primers and the dye terminators. We decided to sequence only the variable region and not the entire gene from these isolates. The sequencing results are shown in Fig. 2 where they are compared with the previously reported sequences of the KAHRP gene from Indian isolate PF3-92 [5]. The results show that RJ181 and HB3 contain variable number of imperfect decapeptide repeats (underlined in Fig. 2). These decapeptide units are composed of two smaller subunits, the perfect tetrapeptide (Thr-Lys-Glu-Ala) and an imperfect hexapeptide. As a result these subunits are also

1 2 3 4 5 6 7 B 910

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--360 bp

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5 6 7 8 910

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Fig. 1. The PCR amplification of (A) constant central region and (B) variable C-terminal region of the KAHRP gene from various isolates of P. falciparum. Lanes 1 to 5 represents the clinical isolates from Rajasthan and lanes 6-10 represents culture adapted regional isolates from India and abroad. The sizes of the amplified products are indicated on the right hand side of the gel. Lane 1, RJ32; lane 2, RJ58; Lane 3, RJI81; lane 4, RJ183; lane 5, RJ220; lane 6, PF392 from Delhi; lane 7, PF2992 from Delhi; lane 8, HB3 from Hondura; lane 9, FCR3 from Gambia; lane 10, FDLI from Delhi.

repeated several times in this domain. A tetrapeptide (Thr-Lys-Gly-Ala) flanks both ends of these repeats whose role is not known. So far, either three or five units of such decapeptide repeats have been reported [1,5]. For the first time we show here a different pattern in these repeats. The isolate RJ 181 showed an intermediate form, i.e. the presence of four decapeptide units which have not been reported earlier. We also report here, for the first time, that the addition (tetrapeptide) or deletion (hexapeptide) of a subunit of the decapeptide can also occur in the decapeptide repeat region. This was found in case of the Honduran isolate. In this isolate there were two variant forms (HB5 and HB6). One form (HB5) contained four decapeptide repeating units plus a tetrapeptide subunit, whereas, the other form (HB6) contained three decapeptide repeating units and a tetrapeptide subunit (Fig. 2). The other change in HB6 was the deletion of a tetrapeptide in the downstream region of the repeats. The results of the present study indicate

H. Hirawake et al. / Biochimica et Biophysica Acta 1360 (1997) 105-108

that deletion or duplication of whole or part of the decapeptide repeats generates a higher rate of variability in the C-terminal antigenic domain of KAHRP. Peptide repeats are very common among malarial antigens and they are the major source of antigenic variation in malaria so as to evade the host immune system. However, in case of KAHRP one would not expect such variations to occur because this protein has to interact with several host and parasite molecules in the knobs. These interactive sequences are not yet identified in the KAHRP except, the putative spectrin/actin binding region which is in the 448

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central lysine-rich domain and is conserved among the isolates studied so far (Fig. 1A and [1,5,9]). Although this region is also antigenic, like the Cterminal domain, but the parasite has allowed variations to occur only in the latter domain (Fig. 1B and [1,5]). The reason for this selective variation remains unknown. However, it can be speculated that such variation in this part of the KAHRP is providing a complementarity to the another highly variable knob antigen, the P. falciparum-infected erythrocyte membrane protein-1 (PFEMP-1). The PFEMP-1, a var gene product, is involved in the evasion of the host

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Fig. 2. Comparative sequence analysis of the C-terminal domain of the KAHRP. The D N A encoding this domain of the KAHRP gene was amplified, cloned and sequenced from Indian isolate RJ181 and Honduran isolate HB3. The sequences are compared with the previously published sequences from another Indian isolate 'PF392' [5]. The imperfect decapeptide repeats are underlined. Stars(*) indicate the same amino acid, dashes ( - ) indicate the deletions and the changed amino acids are identified. The P. falciparum isolates used in the present study are indicated as RJ181 from Rajasthan, India; HB5 and HB6 from the Honduran isolate (HB3). The numbers on the left hand side indicate the amino acid residue number of KAHRP.

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immune system [10]. S u e t al. [11] have stated that the PFEMP-1 could interact with the KAHRP, through the salt bridges. This is possible because the C-terminal end of PFEMP-1 is acidic and KAHRP, including the C-terminal domain, is positively charged [3]. However, further work is required to identify the interactive regions of KAHRP to the other knob molecules so that a therapeutic reagent, such as ribozyme, can be developed to treat falciparum malaria. Financial assistance (to YDS) came from the Department of Biotechnology (DBT) and the Council of Scientific and Industrial Research (CSIR), Government of India. This work was also supported by Grant-in-Aid for Scientific Research on priority Areas from the Ministry of Education, Science and Culture of Japan. One of us (YDS) was supported by the DBT overseas short term Associateship to Japan. We thank Prof. S. Kojima, Dr. T. Kuramouchi, Prof. Indira Nath and Dr. V.P. Sharma for their help and fruitful discussions. We also thank Dr. Araxie Kilejian for providing P. falciparum DNA of HB3 and FCR3 isolates.

References [1] Sharma, Y.D. (1991). int. J. Biochem. 23, 775-789. [2] Kilejian, A. (1979). Proc. Natl. Acad. Sci. USA. 76, 46504653. [3] Sharma Y.D. and Kilejian A. (1987). Mol. Biochem. Parasitol. 26, 11-16. [4] Rashid, M.A. Yang, Y.F. and Kilejian, A. (1990). Mol. Biochem. Parasitol. 38, 48-56. [5] Kant, R. and Sharma, Y.D. (1996). FEBS Lett. 380, 147151. [6] Sharma Y.D., Kant, R., Pillai, C.R., Ansari, M.A. and Pillai, U. (1995). Nature 376, 380. [7] Kilejian, A., Sharma, Y.D., Karoui, H. and Naslund, L. (1986). Proc. Natl. Acad. Sci. USA. 83, 7938-7941. [8] Kilejian, A., Sharma, Y.D. and Yang, Y.F. (1987) In: host-parasite cellular and molecular interactions in protozoal infections (Chang, K.P. and Snary, D., eds.) NATO-ASI series, pp. 307-314, Springer, Heidelberg. [9] Kilejian, A., Rashid, M.A., Aikawa, M., Aji, T. and Yang, Y.F. (1991). Mol. Biochem. Parasitol. 44, 175-182. [10] Borst, P., Bitter, W., Mc Culloch, R., Leeuwen, F.V. and Rudenko, G. (1995) Cell 82, 1-4. [11] Su, X.H., Heatwole, V.M., Wertheimer, S.P. Guinet, F., Herrfeldt, J.A. Peterson, D.S., Ravetch, J.A. and Wellems, T.E. (1995). Cell 82, 89-100.