title: Functional analysis of genetic polymorphism in Wuchereria bancrofti glutathione S-transferase antioxidant gene: impact on protein structure and enzyme catalysis.
abstract: Wuchereria bancrofti glutathione S-transferase (Wb-GST) is referred as a promising chemotherapeutic target for lymphatic filariasis. GST represents the major class of detoxifying enzymes of the tissue dwelling parasitic helminths. Though many inhibition studies were carried out for Wb-GST, understanding its genetic distribution in parasite population is necessary to develop ideal inhibitor. Our genetic polymorphic studies exposed the existence of three variant Wb-GST alleles in the four endemic regions of India. Moreover, it also revealed the variability in the distribution of Wb-GST alleles in the studied population. Therefore we cloned, expressed and purified the recombinant variant Wb-GST proteins to study the mutation impact on its structure and hence on its catalysis. Among the studied mutations, the I60F/G78S substitutions in the N-terminal domain and loop region connecting the two domains of Wb-GST lowered the affinity for glutathione and its analog, S-hexyl glutathione. Moreover, molecular modeling and docking studies revealed that the I60F/G78S mutations affected the proximity of Trp38 and Arg95 in glutathione binding site resulting in weaker interaction with S-hexyl glutathione. Besides, the variants also had lower affinity (Ki) and higher IC50 values for well-known GST inhibitors. Interestingly, the Wb-GST variant proteins showed enhanced catalytic efficiency for lipid peroxidation products which are produced due to oxidative stress. Thus, our study provides evidence for the functional impact of mutations on Wb-GST protein and also spotlights the mechanisms of parasite survival against the host oxidative stress environment.
authors.affiliation: Centre for Biotechnology, Anna University, Chennai, Tamil Nadu 600025, India.
authors.forename: Moorthy
authors.initials: M
authors.lastname: Sakthidevi
authors.affiliation: Centre for Biotechnology, Anna University, Chennai, Tamil Nadu 600025, India.
authors.forename: Prince Rajaiah
authors.initials: PR
authors.lastname: Prabhu
authors.affiliation: Centre for Biotechnology, Anna University, Chennai, Tamil Nadu 600025, India.
authors.forename: Swati
authors.initials: S
authors.lastname: Chowdhary
authors.affiliation: Vector Control Research Centre, Indira Nagar, Puducherry 605006, India.
authors.forename: Sugeerappa Laxmanappa
authors.initials: SL
authors.lastname: Hoti
authors.affiliation: Centre for Biotechnology, Anna University, Chennai, Tamil Nadu 600025, India. Electronic address: pk.kaliraj55@gmail.com.
authors.forename: Perumal
authors.initials: P
authors.lastname: Kaliraj
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007194:India; D007700:Kinetics; D015227:Lipid Peroxidation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D016415:Sequence Alignment; D013816:Thermodynamics; D014958:Wuchereria bancrofti / Q000201:enzymology* / Q000235:genetics*
chemical_list: D005982:Glutathione Transferase
keywords: Docking; Enzyme kinetics; Nematode glutathione-S-transferase; Polymorphism; Site-directed mutagenesis; Thermostability