Structural and Functional Investigation of Two Parasite Protein Systems, Alpha Giardins from Giardia lamblia and Pathogenesis-Related Proteins from Ancylostoma caninum
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Investigation of structure and function of parasite proteins is the major goal of this thesis. The two protein systems studied in this work, parasite annexins and pathogenesis-related proteins (PRPs), have both been speculated to be involved in host-parasite interactions. Parasite annexins are members of the annexin superfamily; the hallmark feature of annexins is their calcium-dependent binding to phospholipid membranes. PRPs are among the excretory/secretory (ES) products that are highly up-regulated in the transition from a developmentally arrested free-living form to a tissue-penetrating larva (L3 stage) of the parasite. Some parasite annexins and PRPs have been reported to have immunogenic properties, thus making them potential vaccine candidates. The first part of the thesis covers the structural and functional investigation of parasite annexins. The expression, purification and biophysical properties of alpha-1 giardin and alpha-3 giardin (from G. lamblia) and Anx(Sm)1 from S. mansoni are discussed in chapter 3. This chapter describes an efficient expression and purification protocol that was used to successfully produce large amounts of highly pure untagged alpha-1 and alpha-3 giardins. A liposome affinity and anion exchange chromatography were used for purification of alpha-1 giardin, whereas ion exchange chromatography was used to purify alpha-3 giardin. The identity and molecular mass of all parasite annexins were validated using mass spectrometry and SEC-MALS, and were found to be in excellent agreement with the theoretical value of each protein. SEC-MALS results also showed that alpha-1 giardin is monomeric, whereas alpha-3 giardin and Anx(Sm)1 exist as a monomer-dimer mix in solution. CD spectroscopy was used to investigate the effect of phospholipid vesicles and calcium on the conformational and thermal stability of parasite annexins. Differential effects on the alpha-helical content of alpha-1 and alpha-3 giardin are observed with varying lipid compositions. While calcium induced conformational change of alpha-1 giardin, it has no effect on the CD spectra of alpha-3 giardin. Calcium was also found to destabilise the folding stability of alpha-1 giardin and alpha-3 giardin, but had no significant effect on the folding stability and conformation of Anx(Sm)1. The destabilisation effect of calcium on protein folding stability was also observed in annexins that lack type II calcium in some of their repeats.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
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Parasite protein systems