CLONING AND CHARACTERIZATION OF A PLASMODIUM-FALCIPARUM GENE ENCODING A NOVEL HIGH-MOLECULAR-WEIGHT HOST MEMBRANE-ASSOCIATED PROTEIN, PFEMP3

The rat monoclonal antibody, mAb 12C11, reacts with numerous proteins from mature asexual stages of Plasmodium falciparum. The largest is 315 kDa and is designated PfEMP3. A lambdagt11 expression library, generated from genomic DNA of Malayan Camp strain parasites, was screened with mAb 12C11. One positive clone, lambda12.1.3, contained a 1.4-kb fragment in frame with the beta-galactosidase gene of lambdagt11. The deduced 455-amino acid sequence is a novel, highly charged sequence encoding two 15-amino acid repeats at the N-terminus followed by 27 repeats of 13 amino acids. The last 59 C-terminal residues are non-repetitive. Two in-frame stop codons at the 3' end of the DNA suggests that this DNA fragment encodes the C-terminus of the protein. Southern blotting with the cloned fragment identified two copies of this fragment per haploid genome in knob-positive, parasitized erythrocytes (K+ PE). Both DNA fragments are absent from K- PE. Northern blotting of trophozoite-stage PE total RNA revealed mRNAs of 10, 4.4 and 2 kb in K+ PE, but no hybridization with K- PE. Immune sera were elicited against the lambda12.1.3 beta-galactosidase fusion protein and peptides generated from the predicted lambda12.1.3 amino acid sequence. These sera and mAb 12C11 reacted specifically with PfEMP3 in Western blots of mature K+ PE but not with K- PE. Rat and mouse sera against the recombinant protein produced an immunofluorescence pattern in fixed mature K+ PE almost identical to the pattern produced by a monoclonal antibody against the knob-associated protein, Histidine Rich Protein 1. The same antibodies were immunofluorescence negative with fixed K- PE. Mouse antibodies against the recombinant protein reacted on immunoelectron microscopy with the erythrocyte membrane of K+ PE, labeling knobs as well as the membrane between knobs. In contrast, a mAb against Histidine Rich Protein 1 reacted only under the electron dense material of knobs. We conclude that the lambda12.1.3 clone encodes the C-terminal portion of the 315 kD PfEMP3 antigen and that PfEMP3 may be involved in knob formation or other perturbations of the erythrocyte membrane.