Biophysicochemical properties of the 28-kDa Schistosoma haematobium and pseudo-26-kDa Schistosoma haematobium/bovis glutathione transferase
Date
2022
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Abstract
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.
Description
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Molecular and Cell Biology to the Faculty of Science, University of the Witwatersrand, Johannesburg, 2022
Keywords
Schistosomiasis, Biophysicochemical properties, Schistosoma haematobium