A novel Omega-class glutathione S-transferase gene in Apis cerana cerana: molecular characterisation of GSTO2 and its protective effects in oxidative stress

Abstract

Oxidative stress may be the most significant threat to the survival of living organisms. Glutathione S-transferases (GSTs) serve as the primary defences against xenobiotic and peroxidative-induced oxidative damage. In contrast to other well-defined GST classes, the Omega-class members are poorly understood, particularly in insects. Here, we isolated and characterised the GSTO2 gene from Apis cerana cerana (AccGSTO2). The predicted transcription factor binding sites in the AccGSTO2 promoter suggested possible functions in early development and antioxidant defence. Real-time quantitative PCR (qPCR) and western blot analyses indicated that AccGSTO2 was highly expressed in larvae and was predominantly localised to the brain tissue in adults. Moreover, AccGSTO2 transcription was induced by various abiotic stresses. The purified recombinant AccGSTO2 exhibited glutathione-dependent dehydroascorbate reductase and peroxidase activities. Furthermore, it could prevent DNA damage. In addition, Escherichia coli overexpressing AccGSTO2 displayed resistance to long-term oxidative stress exposure in disc diffusion assays. Taken together, these results suggest that AccGSTO2 plays a protective role in counteracting oxidative stress.

Fig. 1

Molecular properties of AccGSTO2. a The amino acid sequence alignment of AccGSTO2 and other GSTOs. The putative secondary structure of AccGSTO2 is shown. Identical amino acids are shaded in black. The conserved functional domains are boxed. The putative G-site, H-site and dimer interface of AccGSTO2 are denoted by (), (inverted filled triangle) and (), respectively. b Phylogenetic relationships between glutathione transferases (GSTs) from different insect species. The six primary classes are shown, and AccGSTO2 is boxed. c The tertiary structure of AccGSTO2. The conserved G-site residues (Y27, C28, P29, and F30), H-site residues (S115, I118, S119, P173, and E176), N-terminal, and C-terminal are shown

Fig. 2

Genomic structure of the Omega-class glutathione transferase genes (GSTOs). The lengths of the exons and introns of genomic DNA from A. cerana cerana, A. florea, B. impatiens, B. terrestris, and N. vitripennis are shown according to the scale below. Light grey and grey are used to highlighted the exons and introns separately. The translational initiation codons (ATG) and termination codons (TAA) are marked by (inverted filled triangle) and (asterisk), respectively

Fig. 3

The nucleotide sequence and putative cis-acting elements of the AccGSTO2 regulatory region. The translation and transcription start sites are marked with arrows. The cis-acting elements are boxed, with the exception of Hb, which is shaded in black

Fig. 4

Expression profile of AccGSTO2 as determined by qPCR. The relative expression of AccGSTO2 at different developmental stages (a) and in different tissues (b) is shown. The data are the means ± SE of three independent experiments. The different letters above the columns indicate significant differences (P < 0.01) according to Duncan’s multiple range tests

Fig. 5

Expression profile of AccGSTO2 under different stress conditions. These conditions included 4 °C (a), 16 °C (b), 25 °C (c), 42 °C (d), UV (30mj/cm²) (e), H₂O₂ (2 mM) (f), Cyhalothrin (20 μg/l) (g), Phoxim (1 μg/ml) (h), Pyridaben (10 μM) (i), and Paraquat (10 μM) (j). Untreated adult worker bees (Lane 0) were used as controls, and adult worker bees injected with PBS for 5 h were used as injection controls. The data are the means ± SE of three independent experiments. The different letters above the columns indicate significant differences (P < 0.01) according to Duncan’s multiple range tests

Fig. 6

Western blot analysis of AccGSTO2 in different tissues and developmental stages. aLanes 1–4 were loaded with an equivalent amount of protein: Lane 1 epidermis, Lane 2 muscle, Lane 3 brain, Lane 4 midgut. bLanes 1–3 were loaded with an equivalent amount of protein: Lane 1 day-3 larvae, Lane 2 day-6 pupae, and Lane 3 day-10 adults

Fig. 7

Expression and purification of AccGSTO2. An SDS-PAGE analysis was used to separate recombinant AccGSTO2 expressed in E. coli BL21 cells. Lane 1 low molecular weight protein marker; Lane 2 induced overexpression of pET-30a (+) in BL21. Lanes 3 and 4 non-induced and induced overexpression of pET-30a (+) -AccGSTO2 in BL21, respectively; Lanes 5 and 6 suspension and pellet of sonicated recombinant AccGSTO2, respectively; Lane 7 purified recombinant AccGSTO2

Fig. 8

Temperature (a) and pH (b) effects on the catalytic activity of AccGSTO2. The DHAR activities of recombinant AccGSTO2 were tested at different temperatures (5–55 °C) and pH values (4.0–9.0). The values are the means of three replicates

Fig. 9

AccGSTO2 protected DNA from oxidative damage in the mixed-function oxidation system. Lanes 1–4 pUC19 plasmid DNA + FeCl₃ + DTT + purified AccGSTO2 (150, 100, 50 and 10 μg/ml, respectively); Lane 5 pUC19 plasmid DNA + FeCl₃ + DTT; Lane 6 pUC19 plasmid DNA + FeCl₃ + DTT + purified AccGSTO2 (50 μg/ml) + NEM (5 mM ); Lane 7 pUC19 plasmid DNA + FeCl₃ + DTT + BSA (150 μg/ml); Lane 8 pUC19 plasmid DNA + FeCl₃; Lane 9 pUC19 plasmid DNA only. SF supercoiled form; NF nicked form

Fig. 10

Disc diffusion assays using E. coli overexpressing AccGSTO2. LB agar plates were inoculated with 5 × 10⁸ cells. AccGSTO2 was overexpressed in E. coli and bacteria transfected with pET-30a (+) were used as negative controls. Filter discs soaked with different concentrations of cumene hydroperoxide (a, d), t-butylhydroperoxide (b, e) or paraquat (c, f) were placed on the agar plates. After an overnight exposure, the killing zones around the drug-soaked filters were measured