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Cloning and characterization of a Plasmodium falciparum gene encoding a novel high-molecular weight host
B L Pasloske1, D I Baruch, M R van Schravendijk
1DNAX Research Institute, Palo Alto, CA 94304.
Abstract:
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.
Insights
Researchers identified PfEMP3, a 315 kDa protein in Plasmodium falciparum, using monoclonal antibody 12C11. The study characterized its gene and protein, suggesting PfEMP3
Area of Science:
- Malariology
- Molecular Biology
- Immunology
Background:
- Plasmodium falciparum causes severe malaria.
- Erythrocyte membrane perturbations are crucial for parasite virulence.
- PfEMP3 is a large protein found in mature asexual stages of P. falciparum.
Purpose of the Study:
- To clone and characterize the gene encoding PfEMP3.
- To investigate the role of PfEMP3 in infected erythrocyte membranes.
Main Methods:
- Screening of a lambda gt11 expression library with a monoclonal antibody (mAb 12C11).
- DNA sequencing and analysis of the cloned fragment.
- Southern and Northern blotting to assess gene copy number and expression.
- Western blotting and immunofluorescence assays using antibodies against recombinant PfEMP3.
Main Results:
- A 1.4-kb fragment encoding the C-terminus of PfEMP3 was identified.
- The gene for PfEMP3 exists in two copies per haploid genome in knob-positive erythrocytes.
- PfEMP3 is expressed in trophozoite stages and localized to the erythrocyte membrane, including knobs.
Conclusions:
- The lambda 12.1.3 clone encodes the C-terminal portion of the 315 kDa PfEMP3 antigen.
- PfEMP3 is associated with erythrocyte membrane knobs.
- PfEMP3 may play a role in knob formation or other erythrocyte membrane alterations during P. falciparum infection.