Bl. Pasloske et al., CLONING AND CHARACTERIZATION OF A PLASMODIUM-FALCIPARUM GENE ENCODINGA NOVEL HIGH-MOLECULAR-WEIGHT HOST MEMBRANE-ASSOCIATED PROTEIN, PFEMP3, Molecular and biochemical parasitology, 59(1), 1993, pp. 59-72
The rat monoclonal antibody, mAb 12C11, reacts with numerous proteins
from mature asexual stages of Plasmodium falciparum. The largest is 31
5 kDa and is designated PfEMP3. A lambdagt11 expression library, gener
ated from genomic DNA of Malayan Camp strain parasites, was screened w
ith mAb 12C11. One positive clone, lambda12.1.3, contained a 1.4-kb fr
agment in frame with the beta-galactosidase gene of lambdagt11. The de
duced 455-amino acid sequence is a novel, highly charged sequence enco
ding two 15-amino acid repeats at the N-terminus followed by 27 repeat
s of 13 amino acids. The last 59 C-terminal residues are non-repetitiv
e. Two in-frame stop codons at the 3' end of the DNA suggests that thi
s DNA fragment encodes the C-terminus of the protein. Southern blottin
g with the cloned fragment identified two copies of this fragment per
haploid genome in knob-positive, parasitized erythrocytes (K+ PE). Bot
h DNA fragments are absent from K- PE. Northern blotting of trophozoit
e-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 lam
bda12.1.3 beta-galactosidase fusion protein and peptides generated fro
m the predicted lambda12.1.3 amino acid sequence. These sera and mAb 1
2C11 reacted specifically with PfEMP3 in Western blots of mature K+ PE
but not with K- PE. Rat and mouse sera against the recombinant protei
n 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 antibodie
s were immunofluorescence negative with fixed K- PE. Mouse antibodies
against the recombinant protein reacted on immunoelectron microscopy w
ith the erythrocyte membrane of K+ PE, labeling knobs as well as the m
embrane between knobs. In contrast, a mAb against Histidine Rich Prote
in 1 reacted only under the electron dense material of knobs. We concl
ude 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 formatio
n or other perturbations of the erythrocyte membrane.