Structural and functional analysis of NPA and far nematode proteins

Martin, Claire Patricia (2003) Structural and functional analysis of NPA and far nematode proteins. PhD thesis, University of Glasgow.

Abstract

Parasitic nematodes cause a wide range of diseases in humans, including river blindness caused by Onchocerca volvulus and elephantiasis caused by Ascaris lumbricoides (www.medic-planet.com). The greatest proportion of agricultural loss is in potato crops and is caused by potato cyst parasitic nematodes including Globodera pallida. For survival, nematodes need to sequester fatty acids (usually from the host) and therefore must produce proteins capable of binding essential fatty acids (Kennedy et al, 1995). The nematode proteins studied in this project have been classified into two new types of fatty acid and retinoid binding proteins. Firstly, nematode polyprotein allergens/antigens (NPA), an example of this class of protein is the Ascaris body-fluid allergen (ABA-1) of the parasitic nematode Ascaris. The second class are known as fatty acid and retinol binding proteins (FAR), an example of this class is OvFAR1 from the parasitic nematode Onchocerca volvulus. The nematode proteins are believed to be a cause of severe allergic-type reactions in nematode infections (Christie et al, 1990). Since the pathology of these allergic reactions dictate the involvement of the immune response system, it is thought that these classes of protein are potential candidates for vaccines containing recombinant protein antigens (McDermott, 1999). ABA-1 was purified from Ascaris suum (pABA-1) and produced recombinantly (rABA-1). Fluorescence studies using DAUDA showed that rABA-1 is similar in binding to the pABA-1. Secondary structure studied using circular dichroism indicated rABA-1 and pABA-1 have similar secondary structures. Crystallisation of pABA-1 and rABA-1 using a large range of conditions failed to produce crystals suitable for diffraction and was investigated using dynamic light scattering (DLS), which suggested a dimer structure. GpFAR1, OvFAR1, CeFAR2 and CeFAR3 proteins from the FAR family of proteins were overexpressed and purified. Ligand binding properties of rOvFAR1, rCeFAR2 and rCeFAR3 established that the CeFARs from Caenorhabditis elegans were good functional homologues of rOvFAR1, a major antigen of Onchocerca volvulus. Binding DAUDA suggested that the binding site is in an apolar environment and isolated from solvent. Competition between retinol and oleic acid indicated that the binding site was interactive or congruent. The FAR proteins also showed a preference for fatty acids with chain length C14-C18, investigated by competition of the DAUDA-FAR protein complex. This would indicate that the recombinant proteins are behaving as they do in vivo. The biophysical properties of rOvFAR1, rCeFAR2, rCeFAR3 and rGpFAR1 were investigated. Circular dichroism suggested that these FAR proteins have essentially the same secondary structure. MALDI analyses suggested that fatty acids are bound to the proteins and were persistently bound after a multistep purification, possibly sequestered from E. coli during overexpression. DLS measurements indicated that the proteins were heterogeneous. The objective of the project was to characterize a number of these NPA and FAR proteins with the ultimate goal of determining their 3-D structure. Initially, seven proteins were investigated (ABA-1, GpFAR1, OvFAR1, CeFAR1, CeFAR2, CeFAR3, CeFAR5) and only the more viable and attainable were carried forward for further characterization and attempts at crystallisation. However, rGpFAR1 was the only FAR protein that grew protein crystals, diffraction of these needle crystals showed that they were twinned or hollow. For future crystallisations to be successful, new purification methods are required for the FAR proteins.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Andy Freer
• Keywords: Parasitology
• Date of Award: 2003
• Depositing User: Enlighten Team
• Unique ID: glathesis:2003-74062
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 23 Sep 2019 15:33
• Last Modified: 23 Sep 2019 15:33
• URI: https://theses.gla.ac.uk/id/eprint/74062