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 lambda gt11 expression library, generated from genomic DNA of Malayan Camp strain parasites, was screened with mAb 12C11. One positive clone, lambda 12.1.3, contained a 1.4-kb fragment in frame with the beta-galactosidase gene of lambda gt11. 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 lambda 12.1.3 beta-galactosidase fusion protein and peptides generated from the predicted lambda 12.1.3 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 lambda 12.1.3 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.
DNAX Research Institute, Palo Alto, CA 94304.
B L
BL
Pasloske
D I
DI
Baruch
M R
MR
van Schravendijk
S M
SM
Handunnetti
M
M
Aikawa
H
H
Fujioka
T F
TF
Taraschi
J A
JA
Gormley
R J
RJ
Howard
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004910:Erythrocyte Membrane / Q000469:parasitology; D017125:Genes, Protozoan; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D010963:Plasmodium falciparum / Q000235:genetics*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D016054:DNA, Protozoan; D008565:Membrane Proteins; D010446:Peptide Fragments; D015800:Protozoan Proteins