Identification of Plasmodium Falciparum proteins interacting with the erythrocyte membrane skeleton protein spectrin

Show simple item record Lauterbach, Sonja Brigitte 2009-04-08T12:04:43Z 2009-04-08T12:04:43Z 2009-04-08T12:04:43Z
dc.description.abstract Malaria, which is caused by Plasmodium parasites, is responsible for the death of millions of humans every year in the tropical and subtropical regions of the world. Specifically P. falciparum, one of four malaria species infecting humans, is responsible for the greatest morbidity and mortality burden in African populations. The Anopheles mosquito transmits the parasite to the human host, where it infects and destroys human erythrocytes. The erythrocyte membrane therefore plays a vital role in all aspects of the pathogenic phase of the P. falciparum life cycle and protein-protein interactions between host and parasite are thus a key focus of research. The human erythrocyte maintains its shape with a structural network of proteins underneath the plasma membrane and the main protein component of this erythrocyte membrane skeleton is spectrin. To investigate host-parasite protein interactions, a novel application of phage display technology was developed, whereby purified human erythrocyte spectrin was biopanned against a P. falciparum phage-display library. The P. falciparum DNA inserts of interacting phage were compared to the PlasmoDB database and five interacting proteins were identified: a putative aminopeptidase (PfM18AAP); a putative Ebl-1 like protein, which is proposed to participate in erythrocyte invasion; and three hypothetical proteins. The interaction of the hypothetical proteins with spectrin is the first information available on the function of these proteins. The five gene sequences were cloned into the pET-15b or pGEX-4T-2 expression vectors for purification of the recombinant proteins from Escherichia coli. Only the 6His-PfM18AAP fusion protein was expressed in soluble form and purified by affinity selection. PfM18AAP migrated as a 67 kDa peptide on SDS-PAGE and native gel analysis revealed multiple subunits of the enzyme, predominantly a tetramer and higher oligomers. Cleavage of the 6His-tag and subsequent IEF SDS-PAGE revealed three 65 kDa entities with pI ~6.6, ~6.7 and ~6.9. An in vitro coupled enzyme assay showed that PfM18AAP cleaved an N-terminal aspartate from a peptide substrate with a maximum activity at pH 7.5 and 37 ºC. Inhibitor studies confirmed that the enzyme is a metalloprotease. Blot overlay assays with PfM18AAP against spectrin and erythrocyte membrane proteins verified that PfM18AAP binds strongly to β-spectrin, as well as protein 4.1, protein 4.2, actin and glyceraldehyde-3-phosphate dehydrogenase. Comparison of the PfM18AAP protein sequence to ten other M18 aminopeptidase sequences, including human and three other Plasmodium species, revealed that all the critical amino acids responsible for the binding of two catalytic metal ions, enzymatic catalysis and quaternary structure stabilisation are conserved. The peptide fragment, which initially bound to spectrin during phage display, is not found in other M18 aminopeptidases, suggesting that the presence of this fragment is an evolutionary development of P. falciparum that allows the protease to bind to human spectrin. Analysis of four M18 aminopeptidase crystal structures revealed that the spectrin-binding region forms an external loop on the protein and would thus be accessible to spectrin. Results from this study suggest that, apart from haemoglobin digestion, PfM18AAP performs additional functions in the parasite and infected erythrocyte by cleaving spectrin and other erythrocyte membrane proteins. This would destabilise and disrupt the erythrocyte membrane skeleton to facilitate entry or exit from the host cell, or the insertion of parasite proteins into the host cell membrane. Further analysis and characterisation of PfM18AAP and its interactions with the erythrocyte membrane proteins will shed more light on the multifunctional role of this parasite enzyme. Studies of this enzyme and the hypothetical proteins may also aid in the quest to discover new therapeutics to combat this killer disease. en
dc.language.iso en en
dc.title Identification of Plasmodium Falciparum proteins interacting with the erythrocyte membrane skeleton protein spectrin en
dc.type Thesis en

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