Plasmodium falciparum asparagine and aspartate rich protein 2 is an evolutionary conserved protein whose repeats identify a new family of parasite antigens1

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Abstract

We describe here a new Plasmodium falciparum antigen, asparagine and aspartate rich protein 2 (PfAARP2) of 150 kDa, which is encoded by a unique gene on chromosome 1. PfAARP2 is first expressed 12 h post-invasion and accumulates in trophozoites and schizonts. Immunofluorescence studies indicate that PfAARP2 is translocated into the red blood cell cytoplasm. The central region of Pfaarp2 contains blocks of repetitions encoding asparagine and aspartate residues, which define a new family of related genes dispersed on different chromosomes, and two members of this family have also been identified. Interestingly, the non-repeated N- and C-termini of PfAARP2 display significant similarity to two yeast and human predicted proteins, and its possible function is discussed.

Introduction

The role of antibodies in the protective immune response against Plasmodium falciparum has been clearly established by passive transfer experiments 1, 2, 3and protection may in part be mediated by cytophilic antibodies. In in vitro tests, cytophilic antibodies have been shown to mediate either phagocytosis 4, 5, or inhibition of parasite proliferation in cultures containing monocytes 6, 7. Recombinant proteins rich in asparagine residues which were originally isolated by screening an expression library with a serum raised against infected red blood cell membranes, have been shown to inhibit the phagocytosis of parasitised Saimiri erythrocytes [8]. This indicates that the asparagine motifs might be the target of the opsonising antibodies. A number of different asparagine rich parasite antigens have been identified. For example, three antigens particularly rich in asparagine residues (asparagine rich protein (ARP), clusters of asparagine rich protein (CARP) and 10b) have been described following the isolation of their corresponding genes using antibodies selected on the surface of either merozoites or infected red blood cells respectively 9, 10, 11.

Whatever the role of antibodies in protection, it is assumed that the target antigens are accessible to the immune system. Interestingly, parasite proteins associated with the erythrocyte membrane share a number of characteristics possibly related to immune evasion. First, they belong to families, either directly like PfEMP1 12, 13, 14, or indirectly via the presence of related repeated motifs which contain histidines (KAHRP, [15]), asparagines (PfEMP3, [16]), or glutamates (RESA, Pf332, Pf11-1, MESA/PfEMP2, 17, 18, 19). The variant proteins (PfEMP1) are encoded by a multigene family (50–100 var gene members) related by Duffy-binding-like (DBL) domains. Furthermore, exon 2 of var is highly homologous to an even larger (150 member) family (Pf60, [20]). The second group of erythrocyte membrane associated antigens (PfEMP3, RESA, KAHRP, MESA/PfEMP2) have blocks of repeated amino acids organised, either as clusters disseminated amongst unique sequences, or grouped into large blocks of repeats, e.g. Pf332 and Pf11-1. In the case of RESA, its capacity to bind to the cytoskeleton resides outside the repeats [21]. This interaction is followed by a phosphorylation of RESA, while MESA/PfEMP2 is phosphorylated prior to association with the cytoskeleton, differing it from RESA 22, 23.

In an attempt to describe novel parasite antigens associated with the erythrocyte membrane, we have identified three proteins rich in asparagine and aspartate, hereafter named PfAARP1, PfAARP2 and PfAARP3 for P. falciparum asparagine and aspartate rich proteins. We describe here, the detailed characterisation of PfAARP2 which is expressed during P. falciparum asexual blood stage development.

Section snippets

Parasites

Palo Alto and 3D7 strains of P. falciparum were cultivated according to Jensen and Trager [24]. Synchronous cultures were obtained by alanine treatment: parasitised red blood cells were centrifuged and then resuspended in two volumes of pre-warmed (37°C) 0.3 M alanine in 10 mM Hepes for 3 min [25]. The lysis was stopped by adding an excess of RPMI-1640 containing serum, and after centrifugation, the parasites were again placed in in vitro culture.

Genomic and cDNA library screenings

The original clone, Pfaarp3, was isolated from

Sequence analysis of Pfaarp2 cDNA

As part of our ongoing analysis of membrane associated antigens, we sequenced part of an antigen gene, Pfaarp3 (accession no. Y08925). This genomic clone was used to screen a cDNA library and a related gene Pfaarp2 was isolated. By the use of genomic fragments generated by asymmetric PCR on vectorette libraries and rescreening of cDNA libraries, the complete cDNA sequence of Pfaarp2 was obtained (accession no. Y08924). The deduced amino acid sequence is presented in Fig. 1.

If the N-terminus is

Discussion

PfAARP2 is an evolutionary conserved protein of 150 kDa which is first synthetised in 12 h ring stage parasites and subsequently accumulates in trophozoites and schizonts. Failure to detect PfAARP2 in parasites 6 h post invasion indicates that the protein is not carried into the erythrocyte at the time of invasion. Pfaarp2 is a unique gene carried on chromosome 1 and its deduced amino acid sequence reveals the presence of four blocks of repeated residues, three of them rich in asparagine and

Acknowledgements

We thank Denise Mattei and Serge Bonnefoy for their helpful discussion and Alain Cosson and Eduardo Santos Lima for their help in immunising mice. J.C.B. was supported by a Bourse Roux fellowship from the Pasteur Institute.

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    Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBankTM and DDJB data bases under the accession numbers Y08924 and Y08925.

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