Characterization and Mutational Analysis of Omega-Class GST (<i>GSTO1</i>) from <i>Apis cerana cerana</i>, a Gene Involved in Response to Oxidative Stress

<div><p>The Omega-class of GSTs (GSTOs) is a class of cytosolic GSTs that have specific structural and functional characteristics that differ from those of other GST groups. In this study, we demonstrated the involvement of the <i>GSTO1</i> gene from <i>A. cerana cerana</i> in the oxidative stress response and further investigated the effects of three cysteine residues of GSTO1 protein on this response. Real-time quantitative PCR (qPCR) showed that <i>AccGSTO1</i> was highly expressed in larvae and foragers, primarily in the midgut, epidermis, and flight muscles. The <i>AccGSTO1</i> mRNA was significantly induced by cold and heat at 1 h and 3 h. The TBA (2-Thiobarbituric acid) method indicated that cold or heat resulted in MDA accumulation, but silencing of <i>AccGSTO1</i> by RNAi in honeybees increased the concentration of MDA. RNAi also increased the temperature sensitivity of honeybees and markedly reduced their survival. Disc diffusion assay indicated that overexpression of AccGSTO1 in <i>E. coli</i> caused the resistance to long-term oxidative stress. Furthermore, AccGSTO1 was active in an <i>in vitro</i> DNA protection assay. Mutations in Cys-28, Cys-70, and Cys-124 affected the catalytic activity and antioxidant activity of AccGSTO1. The predicted three-dimensional structure of AccGSTO1 was also influenced by the replacement of these cysteine residues. These findings suggest that <i>AccGSTO1</i> plays a protective role in the response to oxidative stress.</p></div

Comparison of specific activities of AccGSTO1 and other GSTO.

<p>Specific activity shown in μmol/min/mg. ND, not detectable. “_”indicated not determined. C38A, P39A and Y40A are mutants for bmGSTO.</p

The expression profile of <i>AccGSTO1</i> determined using qPCR.

<p>(A) <i>AccGSTO1</i> mRNA expression at different developmental stages. Total RNA was isolated from larvae (L3-third instars, L4-fourth instars, and L5-fifth instars), pupae (Pw-white eyes pupae, Pp-pink eyes pupae, and Pb-brown eyes pupae), and adults (Em-newly emerged bees, Nu-nurse bees, Fo-foragers, Oa-old bees, and De-dead bees). (B) The tissue distribution of <i>AccGSTO1</i> expression. Total RNA was extracted from dissected brain (Br), epidermis (EP), flight muscle (Fm), midgut (Mi), and mandibular gland (Mg) tissues of foragers. (C) Expression of <i>AccGSTO1</i> after different temperature treatments (4°C, 15°C, 25°C, 34°C, and 43°C). Samples were collected at 0.5, 1, 3, 5, and 7 h. Bees at 34°C were used as a control. The vertical bars represent the mean ± S.E.M. (n = 3). The different letters above the columns indicate significant differences (P<0.05) according to Duncan's multiple range test performed using SAS version 9.1 software.</p

The preidicted tertiary structure of the wild-type AccGSTO1 and C28A, C70A, and C124A mutant proteins.

<p>The N-terminus, C-terminus, α-helices, and β sheets are marked. Helices α2 and α8 are 3₁₀ helices, and the other helices are α-helices. The Cys and mutagenic Ala residues are highlighted by a ball and stick representation. These images were generated using Swiss-PdbViewer.</p

The distribution of H-bonds and different amino acid residues on the surface of the protein.

<p>The H-bonds are highlighted in red. The different colors indicate the different locations of the amino acids within the protein. Blue and green represent interior amino acids. The amino acids on the surface of the protein are shown in ball representation. Orange represents the superficial amino acids that were highly contacted by solvent. These images were drawn using Swiss-PdbViewer.</p

The detection of the MDA content at different temperatures.

<p>In all experiments, foragers at 34°C were used as the controls. The effects of temperature-induced oxidative stress on the MDA concentration (nmol mg⁻¹ protein) in foragers. The vertical bars represent the mean ± S.E.M. (n = 3).</p

Protection of DNA from oxidative damage by purified proteins in the MFO system.

<p>Lane 1, pUC19 plasmid DNA only; lane 2, pUC19 plasmid DNA + FeCl₃; lane 3, pUC19 plasmid DNA + FeCl₃ + DTT; lane 4–10, pUC19 plasmid DNA + FeCl₃ + DTT + purified proteins (5, 10, 20, 40, 60, 80, and 100 μg/mL, respectively). SF, supercoiled form; NF, nicked form. SF% is SF/(SF + NF) for each lane.The expression level was determined by the signal intensities of the bands.</p

The effects of the RNAi-mediated silencing of AccGSTO1.

<p>(A) The survival rate of <i>AccGSTO1</i> RNAi-treated foragers compared with controls at 50°C. The foragers were collected and divided into nine groups (n = 80/group). Groups 1-3 and Groups 4-6 were used as the controls and were fed a normal diet or a normal diet supplemented with ds<i>GFP</i>, respectively. Groups 7–9 were fed ds<i>AccGSTO1</i>. All the foragers were placed in incubators at a constant temperature (50°C) and humidity (75%) for 1–5 h. (B) The MDA content of RNAi-treated foragers compared with controls at 50°C. All the foragers were placed in incubators at a constant temperature (50°C) and humidity (75%) for 0.5–2 h.</p

Disc diffusion assays using <i>E. coli</i>-overexpressed proteins.

<p>The halo diameters of the killing zones were detected after an overnight exposure. LB agar plates were flooded with bacteria overexpressing AccGSTO1, C28A, C70A, or C124A. Bacteria transfected with BL21 (pET-30a (+) only) was used as a control. The data presented are the mean ± S.E.M. of three independent experiments (n = 3). The different letters above the columns indicate significant differences (P<0.05) according to Duncan's multiple range test performed using SAS version 9.1 software.</p

Sequence analysis of the AccGSTO1.

<p>(A) Alignment of AccGSTO1 with the Omega-class GSTs from <i>Apis florea</i> (AfGSTO1), <i>Bombyx mori</i> (BmGSTO1), <i>Homo sapiens</i> (HsGSTO1), <i>Megachile rotundata</i> (MrGSTO1) and <i>Nasonia vitripennis</i> (NvGSTO1). Identical amino acids are shaded in black and similar amino acids are shown in gray. The putative secondary structures are displayed, and the H-site regions are boxed. The Cys residues in AccGSTO1 are marked by asterisks. The intron/exon junctions are indicated by triangles. (B) Phylogenetic relationships among GSTO1 and other GST families. Six main groups are shown. Acc: <i>Apis cerana cerana</i>; Af: <i>Apis florea</i>; Am: <i>Apis mellifera</i>; Bm: <i>Bombyx mori</i>; Bt: <i>Bombus terrestris</i>; Dm: <i>Drosophila melanogaster</i>; Hs: <i>Homo sapiens</i>; Mm: <i>Mus musculus</i>; Mr: <i>Megachile rotundata</i>; Nv: <i>Nasonia vitripennis</i>. (C) Comparison between the genomic structures of <i>AccGSTO1</i> and <i>AccGSTO2</i>. The translational initiation codons (ATG) and termination codons (TAA) are marked with dots and asterisks, respectively.</p