Biochemical and biophysical investigations into key malaria parasite proteins
Document TypePhD thesis
Access StatusOpen Access
© 2017 Dr. Christopher Neil Haggarty-Weir
Completed under a Cotutelle arrangement between the University of Melbourne and the University of Edinburgh (School of Chemistry)
Plasmodium falciparum, the most pestilential of the malaria parasite species, is responsible for ~450,000 direct deaths annually. Clinical disease is a consequence of the blood stage of the parasite’s lifecycle involving a plethora of host-parasite interactions. Key to these interactions are the P. falciparum reticulocyte binding-like homologue (PfRh) proteins responsible for binding erythrocyte receptors and gaining entry to host cells. For example, PfRh4 binds to human complement receptor-1 (CR1) on erythrocytes for sialic-acid-independent invasion. Another protein important for invasion is the PfRh5-interacting protein (PfRipr), an essential member of the PfRh5-associated invasion complex (PAIN-complex) along with CyRPA, the cysteine-rich protective antigen. Loss of function of PfRipr in P. falciparum parasites prevents erythrocyte entry and ablates Ca2+-influx into the erythrocyte; essential events during invasion. This study aimed to biochemically and structurally investigate truncated recombinant versions of PfRh4 and PfRipr. Homology modelling suggested that PfRh4 is rich in alpha-helical secondary structure. The sequence of PfRipr suggested the presence of ten epidermal growth factor-like (EGF) modules, two towards the N-terminus and eight in the C-terminal domain. In this project, monoclonal antibodies made against recombinant PfRh4 were shown, via indirect immunofluorescent assays, to localize to the apical tip of merozoites. Monoclonal antibody 5H12, raised against PfRh4, reduces parasite invasion of erythrocytes by ~75% in growth-inhibition assays with neuraminidase pre-treated erythrocytes. Attempts to produce a stable truncated recombinant PfRh4 protein for structural studies were unsuccessful. An ELISA-based assay using ten alanine-scan mutants suggested the CR1-binding site lies outside of amino acids 283 – 341 of PfRh4. PfRipr truncations, defined by the boundaries of EGF-like repeats predicted based on sequence homology, were produced recombinantly in Escherichia coli and Pichia pastoris. These proteins had a circular dichroism signature suggestive of β-strand-containing proteins with disordered regions. EGF-containing PfRipr truncations did not bind recombinant PfRh5 according to ELISA and size-exclusion chromatography assays. EGFs 1-2, 5-7 and 7-10 of PfRipr did not bind CyRPA via size-exclusion chromatography or NMR. Crystallisation trials performed on EGF modules failed to yield crystals suitable for data collection. A 15N isotopically-labelled sample of EGF5-7 gave good quality HSQC NMR spectra. A 15N isotopically-labelled sample of EGF5-7 gave good quality HSQC NMR spectra. A suite of three-dimensional NMR spectra collected on a 13C,15N-EGF5-7 sample, at three different temperatures, allowed for >86% of backbone assignments. T1/T2 relaxation analysis and heteronuclear NOE data were suggestive of an elongated, rigid protein undergoing intermolecular self-association. Further evidence for EGF5-7 being an elongated protein was provided via SAXS analysis. Chemical shifts facilitated prediction of secondary structure in EGF 5-7 consistent with an EGF-like fold. Melting studies performed on EGF5-7 showed no evidence of denaturation over the temperature range 20˚C - 95˚C indicating a thermally-stable protein. The addition of Ca2+ to the 15N-EGF5-7 sample caused chemical shift perturbations consistent with high-affinity binding. The discovery of inhibitory monoclonal antibodies recognising a conformational epitope on EGF7 provided evidence of the functional importance of this region within PfRipr. The work described in this thesis provides methods for the industrially-scalable production and biophysical investigations of P. pastoris or E. coli-produced disulfide-rich P. falciparum antigens of interest to vaccinologists.
Keywordsmalaria; vaccines; parasitology; biochemistry; biophysics
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