Two Plasmodium falciparum genes express merozoite proteins that are related to Plasmodium vivax and Plasmodium yoelii adhesive proteins involved in host cell selection and invasion

Abstract

Two related Plasmodium falciparum genes and their encoded proteins have been identified by comparative analyses with Plasmodium vivax reticulocyte binding protein 2 (PvRBP-2). The P. falciparum genes have a structure which suggests that they may be the result of an evolutionary duplication event, as they share more than 8 kb of closely related nucleotide sequence but then have quite divergent unique 3' ends. Between these shared and unique regions is a complex set of repeats, the nature and number of which differs between the two genes, as well as between different P. falciparum strains. Both genes encode large hydrophilic proteins, which are concentrated at the invasive apical end of the merozoite and are predicted to be more than 350 kDa, with an N-terminal signal sequence and a single transmembrane domain near their C termini. Importantly, they also share gene structure and amino acid homology with the Plasmodium yoelii 235-kDa rhoptry protein family, which is also related to PvRBP-2. Together these Plasmodium proteins define an extended family of proteins that appear to function in erythrocyte selection and invasion. As such, they may prove to be essential components of malaria vaccine preparations.

Figure 1

PfRBP2-H genes are composed of shared, repetitive, and unique domains. (A) Schematic diagram of PfRBP2-H DNA clones and the PfUMP GenBank sequence. Sequences are grouped according to their relationship to PfRBP2-Ha (PFUMP, 6A1, 1.1) or -Hb (4D1, 18.2). Clone 20.2b could be a fragment of either. Regions shared by all sequences (gray), repeat regions (black), and regions unique to PfRBP2-Ha or -Hb (hatched) are noted. (B) clustal alignment of predicted amino acid sequence from clones 6A1 and 4D1. Areas of identity and similarity are boxed, with identical residues highlighted in gray. Not all of the 6A1 or 4D1 sequences are shown. Solid lines indicate the boundaries of repeated motifs.

Figure 2

PfRBP2-Ha and -Hb share >8 kb of common sequence. (A) PfRBP2-Ha (1.1 and 08813) and -Hb (18.2 and 04278) schematic. Gray, black, and hatched boxes define shared, repeat, and unique regions, respectively. Primers used in long-range PCR are marked (SF7, SF5, SF3, AR2, and BR2), as are EcoRV (RV) and HindIII (H) restriction sites and fragments used as probes in Southern blots (Sh1, Sh2, A, and B). (B) Long-range PCR performed on FVO DNA by using reverse primers from the unique regions (AR2 or BR2) paired with forward primers designed from contig 04278 (SF3, SF5, and SF7). (C) Southern blots of EcoRV (RV) and HindIII (H) -digested FVO DNA, probed with fragments Sh1, A, B, and Sh2. DNA 1-kb ladder markers from GIBCO/BRL are indicated.

Figure 3

Structure of the PfRBP2-H genes and their relationship with each other and with other Plasmodium proteins. (A) Schematic of the PfRBP2-H genes, highlighting structural features as marked. Shared regions (gray), repeats (black), and unique regions (hatched) are also noted. (B) Sequence of the 5′ end of the PfRBP2-H genes, showing the boundaries of the intron (arrows) and sequence of the short exon 1 that encodes a signal peptide. (C) clustal alignment of the C terminus of the PfRBP2-H proteins, the P. vivax reticulocyte-binding proteins, and the E-3 member of the P. yoelii p235 family. Identical residues are highlighted in gray and similar residues in light gray.

Figure 4

Alignment of PfRBP2-H repeated motifs. Clones 4D1 and 6A1 were derived from P. falciparum strain FVO, whereas 04278 and 08813 sequences originate from strain 3D7. One-letter code refers to sequences as follows: A, QE(K/Q)ERL (blue); B, EREKQEQL (red); C, QKEE(E/A)LKR (black); and D, EQQ (green). Divergences from these consensus sequences are noted with underlines.

Figure 5

PfRBP2-Ha and -Hb are expressed at the apical end of merozoites. (A) PfRBP2-Ha (1.1 and 08813) and -Hb (18.2 and 04278) schematic. Gray, black, and hatched boxes define shared, repeated, and unique regions, respectively. Primers used in reverse transcription–PCR are marked (SF1, SF2, AR1, AR2, BR1, and BR2), as are the fragments of the genes expressed to raise antisera (S1, S3, A, and B). (B) Nested PCR was performed by using primers specific to PfRBP2-Ha (SF1/AR2 followed by SF2/AR1, lanes 1, 3, and 5) or -Hb (SF1/BR2 followed by SF2/BR1, lanes 2, 4, and 6), with either FVO DNA (lanes 1 and 2) or cDNA as templates. The cDNA was generated from schizont stage total RNA and primers specific to PfRBP2-Ha (lanes 3 and 4) or PfRBP2-Hb (lanes 5 and 6) for reverse transcription reactions. (C) Western immunoblots prepared with anti-S3 (S3), anti-PfRBP2-Ha (A), anti-PfRBP2-Hb (B), or preimmune (Pre) antiserum as detection agent. Lanes contain either P. falciparum mature schizont SDS extracts (1 and 2) or uninfected erythrocyte ghost preparations (3). Protein size standards from GIBCO/BRL and Coomassie-stained apolipoprotein B (520 kDa) are noted. (D) Immunofluorescence assays performed with antisera against fragments of the shared domain (S1), fragments of the unique regions, anti-PfRBP2-Ha (A) and anti-PfRBP2-Hb (B), and the rhoptry antigen Pf-140, on air-dried, acetone-fixed, P. falciparum-infected erythrocytes. The lower center panel (B) is at a higher magnification (×3,750; others are ×1,250), with the outline of the merozoites marked. The lower right shows costaining of single merozoites with rabbit anti-A serum (A) and rat anti-B serum (B).