3. Electronic Theses and Dissertations (ETDs) - All submissions

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    Variations in conformational stability and thermodynamics of the 28-kDa and a pseudo-26-kDa glutathione transferases from Schistosoma haematobium
    (2024) Seeletse, Malefo Tshepiso
    Schistosomiasis is a neglected tropical disease caused by blood flukes of the genus Schistosoma, affecting over 240 million individuals worldwide. The key detoxification enzymes of the parasite, glutathione-S-transferases (GSTs), play a crucial role in its survival by helping it evade the host immune system. Two isozymes, the Schistosoma haematobium 28- kDa glutathione transferase and the Schistosoma bovis/haematobium 26-kDa glutathione transferase are expressed during the parasite's life cycle, and the reason for encoding two similar enzymes within the genome is not fully understood. This study aims to comprehensively investigate and reveal the conformational stability and thermodynamics variations of the 28-kDa and a pseudo-26-kDa glutathione transferases from Schistosoma haematobium. Recombinant Sh28GST and Sbh26GST were overexpressed in Escherichia coli T7 cells and purified using immobilised metal affinity chromatography. The specific activity of the enzymes was determined using glutathione-1-chloro-2.4-dinitrobenzene (GSH-CDNB) assay, which was subsequently employed for Michaelis Menten kinetics of the two GSTs. The secondary structure of each enzyme was evaluated using far-ultraviolet (UV) Circular Dichroism (CD) and the tertiary structure using intrinsic tryptophan and extrinsic ANS fluorescence, in the presence and absence of urea. The conformational stability of the two GSTs was assessed under two different denaturing conditions: chemically induced denaturation using urea and far-UV CD, as well as heat-denaturing studies using Differential Scanning Calorimetry. Both proteins were successfully overexpressed, and high yields of purity were achieved. The secondary structural content of both proteins was predominantly alpha-helical. The tertiary structure showed that both proteins had most of their tryptophan residues exposed to the polar environment, and fluorescence spectra resulted in minor intensity changes from native to denatured protein. The specific activity of each enzyme was determined to be 40 μmol/min/mg for Sbh26GST and 67 μmol/min/mg for Sh28GST, indicating that Sh28GST had the higher specific activity among the two enzymes. Furthermore, when comparing their overall catalytic efficiency, Sbh26GST exhibited a higher value of 200 mM-1min-1 , whereas Sh28GST showed a lower catalytic efficiency of mM-1min-1 . Conformational stability and thermodynamics studies revealed interesting findings: Sbh26GST was more stable in the presence of urea, while Sh28GST was more stable in the presence of heat. Our collective findings led us to conclude that greater chemical denaturant stability observed in a protein does not always correlate with its stability in the presence of heat. These findings were attributed to the number and location of tryptophan residues found in each GST
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    Biophysicochemical properties of the 28-kDa Schistosoma haematobium and pseudo-26-kDa Schistosoma haematobium/bovis glutathione transferase
    (2022) Padi, Neo
    Schistosomiasis is a debilitating parasitic worm-induced, neglected tropical disease with veterinary and medical concerns in areas with poor socio-economic establishments. S. haematobium is one of the species that mainly affect humans and it has a zoonotic character enabling it to form hybrids with S. bovis thus justifying their common ancestor. In schistosomes, GSTs are primary detoxification enzymes. Moreover, they are involved in host immune response which makes them attractive drug candidates. However, studies are limited by an incomplete sequence of S. haematobium and there is a drug-resistant threat that calls for a new generation of anthelmintics. In this study, we designed a pseudo-Sbh26GST to elucidate its structural and functional properties in response to potential inhibitors, praziquantel (PZQ); artemisinin (ART) and bromosulfophthalein (BSP) in comparison to the well-studied Sh28GST. Several sequence analysis tools were used to complete the sequence of S. haematobium/bovis and generate the pseudo-Sbh26GST which was overexpressed successfully in E. coli with vector, pMAL-c5x while Sh28GST was expressed in pET-11a. All proteins were in the soluble fraction and then purified to ˃95% homogeneity. Functional characterisations were based on the classical GSTs glutathione (GSH) and 1-chloro-2,4 (CDNB) conjugation assay. Sh28GST had a higher 44 µmol/min/mg activity towards CDNB relative to 13 µmol/min/mg for Sbh26GST. PZQ and ART slightly increased the activity insignificantly while BSP inhibited Sbh26GST with an IC50 of 27 µM and 0. 88 µM for Sh28GST. The mode of inhibition, determined by kinetics was found to be random and non-competitive respectively. Structural characterisations were studied through Far-UV circular dichroism which showed that both proteins have a predominantly α-helical secondary structure content. Fluorescence spectroscopy studies showed that BSP and ART disturbed the local Trp environment whereas PZQ had an insignificant effect, all in the presence and absence of co-substrate, GSH and its similar structure, S-hexylglutathione (GTX). Additionally, extrinsic 8-anilino-1- naphthalenesulfonate fluorescence proved that BSP outcompetes it, while PZQ enhances it thus showing competition for the dimer interface and hydrophobic binding site. Thermodynamics parameters obtained by isothermal titration calorimetry indicate that the interaction between dimeric Sbh26GST and Sh28GST with one molecule of BSP is spontaneous and enthalpically driven, with additional entropy support in Sbh26GST but entropy cost in Sh28GST. Stability inferences from SYPRO Orange-based thermal shift assay demonstrated Sbh26GST had increased stability in the presence of BSP and GSH. BSP proved to bind and inhibit both proteins which translate to possibly rationalized new generation anthelmintics for the treatment of schistosomiasis.
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    Western blot analysis of bilharzial infections
    (1997) Brant, Cheryl Dawn
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