Abstract

Malaria is one of the world’s major health problems, causing millions of deaths every year, primarily in Africa. The disease is caused by Plasmodium parasites, which invade and destroy human erythrocytes. Of the four species infecting humans, Plasmodium falciparum is responsible for the greatest morbidity and mortality burden. The erythrocyte membrane plays a vital role in all aspects of the pathogenic phase of the parasite’s life cycle and the protein-protein interactions between host and parasite are a key focus of research. Spectrin is the main structural protein in the erythrocyte membrane skeleton and phage-display technology was used to probe the interaction between P falciparum peptide fragments and human erythrocyte spectrin. A phage-display library was constructed by isolating mRNA from P falciparum strain FCR-3, which was reverse transcribed using two-base anchored oligodT primers. Linkers facilitating directional cloning were added to the cDNA, followed by insertion into a gene encoding the 10B capsid protein of the T7 bacteriophage vector. The vector was packaged into viral particles and the library amplified using Escherichia coli as a host. The presence and size of inserts were determined by PCR amplification with T7 bacteriophage vector arm specific primers. Human erythrocyte membranes were prepared from whole blood by hypotonic lysis and spectrin was extracted with a low ionic strength buffer and purified by size exclusion chromatography. The protein was biotinylated, immobilized on streptavidin-coated magnetic beads and biopanned against the phage library. Bound phage were eluted and amplified in E coli for three additional rounds of biopanning to eliminate non-specific protein-protein interactions. The P falciparum cDNA inserts of interacting phage were sequenced and compared to the PlasmoDB database. One of the sequences was identified as a putative aminopeptidase (PFI1570c), which has a 30.7% homology to a human aspartyl aminopeptidase, an enzyme catalysing the release of N-terminal amino acids from a peptide. The parasite protein contains a putative transmembrane domain at the C terminal end and is larger than the human form, with an estimated molecular weight of 65 kD. Several features that are critical for enzyme activity are conserved in the P falciparum aminopeptidase. These include twelve amino acids (four histidine, three glutamic acid and five aspartic acid residues), which are involved in the binding of catalytic zinc ions in the active site, as well as a putative N-myristoylation site and phosphorylation sites for casein kinase II and protein kinase C. Interestingly, the peptide fragment that bound to spectrin in the initial phage display screening, corresponds to a 33 amino acid fragment that is not found in the human aspartyl aminopeptidase. This suggests an evolutionary development of the parasite that allows the protease to bind to human spectrin. Mass spectrometry and microarray data from the PlasmoDB database indicate that the protein is present at the erythrocyte membrane and is expressed in all the developmental stages of the parasite’s erythrocytic life cycle. During the trophozoite stage the parasite modifies the erythrocyte membrane to allow transport of nutrients and waste products. The aminopeptidase could cleave spectrin and destabilise the membrane skeleton to facilitate the insertion of parasite protein channels during development. It may also play a role in proteolysis of the skeleton to enable the release of schizonts from infected erythrocytes.

Author notes

Corresponding author