The diverse biological functions of glutathione S-transferase omega in Drosophila

Introduction

Glutathione S-transferases (GSTs) are phase II detoxification enzymes that catalyze the conjugation of glutathione (GSH) to an electrophilic substrate. These enzymes provide protection against carcinogens, therapeutic drugs, and several types of cellular oxidative damage [1]. Based on their amino acid sequences and substrate specificities, GSTs are grouped into at least ten classes: α, δ, ε, κ, μ, π, σ, θ, ζ, and ω [2].

The GST omega (GSTO) is the most recently defined GST class [3]. The active sites of GSTOs have a cysteine residue at the N-terminus that can bind to GSH, whereas other GST classes have tyrosine or serine residues in their active sites. GSTOs have thiol transferase and dehydroascorbate (DHA) reductase activities, the latter of which is similar to the reactions catalyzed by thioredoxin and glutaredoxin [3]. In addition, GSTOs catalyze the reduction of monomethylarsonic acid [4, 5]. Human GSTO1 modulates the ryanodine receptor, which is a Ca²⁺ release channel modulator and is involved in the activation of interleukin-1β, an important mediator of the inflammatory response [6, 7]. Variations in the human GSTO1 gene may be associated with the risk of breast cancer and hepatocellular carcinoma [8]. Furthermore, a polymorphism in the human GSTO2 gene is associated with the risk of ovarian cancer [8, 9]. The following is a mini-review of the recent studies investigating the novel biological functions of GSTOs in Drosophila.

GSTO genes in Drosophila

Currently, the Drosophila GST genes are divided into six classes: δ, ε, σ, ω, ζ, and θ. Drosophila harbor 36 GST genes that encode 41 proteins [2, 10]. The GST δ and ε classes contain 11 and 14 genes that encode 12 and 14 proteins, respectively. The GST ζ and θ classes contain two and four genes, respectively. Four different GSTO genes in Drosophila are located on chromosome 3L 66D5: CG6781, CG6662, CG6673, and CG6776. The CG6673 gene produces two alternatively spliced products, isoforms A and B (Figure 1). Recently, these four GSTO genes were named sepia, GstO1, GstO2, and GstO3, respectively [2]. The average sequence identities/similarities are high, at 43%–65%/66%–82%, based on the amino acid sequence alignment of the different isoforms of GSTO [11]. All isoforms of Drosophila GSTO have N-terminal extensions and cysteine residues in the GSH-binding site rather than tyrosine or serine residues, which are found in the active sites of other classes of GSTs. In addition, Drosophila GSTOs all have high thiol transferase and DHA reductase activities, characteristic of GSTOs, and low activity towards 1-chloro-2,4-dinitrobenzene (CDNB), a general GST substrate [11].

Figure 1

Genomic organization of the DrosophilaGSTO genes.

Four DrosophilaGSTO genes are located on chromosome 3L. The CG6673 (GstO2) gene has two alternative splice variants, isoforms A and B. The arrow indicates the direction of transcription.

CG6781 is the structural gene for sepia and encodes pyrimidodiazepine (PDA) synthase, a key enzyme in drosopterin biosynthesis

Eye color in Drosophila is due to the presence of two types of pigments, the brown ‘ommochromes’ and the red ‘drosopterins’. Drosopterins consist of five compounds, which have been referred to as drosopterin, isodrosopterin, neodrosopterin, aurodrosopterin, and fraction e [12]. The eye color mutant se¹ is defective in the PDA synthase that catalyzes the conversion of 6-pyruvoyltetrahydropterin (6-PTP) into PDA, a key intermediate in the drosopterin biosynthetic pathway [11]. Therefore, se¹ mutant flies have dark brown eyes. In a recent study, our group determined that CG6781 is the structural gene of the Drosophila eye color mutant sepia and encodes a PDA synthase. se¹ mutant flies have dramatically decreased levels of all red eye pigments in the head. This eye pigment-defective phenotype of the se¹ mutant was rescued by the transgenic expression of CG6781 in the se¹ mutant background (Figure 2).

Figure 2

Transgenic GSTO expression Drosophila rescues eye color defect in sepia mutants.

Analysis of Drosophila eye pigments by two-dimensional thin layer chromatography (2D-TLC). The identity of each spot is as follows: 1, neodrosopterin; 2, drosopterin; 3, isodrosopterin; and 4, aurodrosopterin. Eye pigments from wild type, Oregon-R, heads (left panel). Eye pigments are not present in the extract of the se¹ mutant heads (middle panel). The overexpression of CG6781, sepia, in the se¹ mutant background rescues the phenotype of se¹ mutants (right panel).

CG6673 regulates mitochondrial F₁F₀-ATP synthase activity in a Drosophila model of Parkinson’s disease (PD)

It has been reported that single nucleotide polymorphisms in human GSTO genes are associated with the age at onset for Alzheimer’s disease, PD, vascular dementia, and stroke [13, 14]. However, many studies have failed to demonstrate the molecular function of GSTOs in vivo. Recently, we found that GstO2A is a novel genetic suppressor of the Drosophila parkin mutant [15]. In this study, we showed compelling evidence that GstO2A catalyzes the glutathionylation of the ATP synthase β subunit, which is a catalytic component of the mitochondrial F₁F₀-ATP synthase complex (Figure 3). The glutathionylation of the ATP synthase β subunit induced by GstO2A expression in parkin mutants is important for the rescue of F₁F₀-ATP synthase activity in these mutants. These findings strongly suggest that enhancing the activity of GstO2A could alleviate neurodegeneration in parkin mutants. However, GstO2B expression was not able to rescue the defective phenotype in parkin mutants [15], suggesting that the two isoforms of GstO2, A and B, have different functions. Because these two isoforms differ only in the proportion of the C-terminal domain that binds the hydrophobic substrate, this region may influence substrate preference.

Regulation of ascorbic acid recycling by GstO2

Ascorbic acid (AsA) is a critical cofactor in various enzymatic reactions and plays an important role in protecting cells against oxidative stress. In most cells, ascorbic acid is regenerated from the oxidized form of ascorbic acid, DHA [16, 17]. This recycling pathway of DHA to AsA is known to be mediated by GSH- or NADPH-dependent DHA reductases [18–20]. Recently, using an in vitro enzyme assay, the Board group [4] showed that the DHA reductase activity of human GSTO2 is approximately 70 to 100-fold higher than that of human GSTO1. GstO2 has the highest GSH-dependent DHA reductase activity among the GSTO genes [11]. GSH-dependent DHA reductase activity is decreased in GstO2 mutant flies. Furthermore, the AsA redox state, determined by the AsA/DHA ratio, is also dramatically decreased in GstO2 mutants. These defective phenotypes in GstO2 mutants can only be suppressed by the overexpression of GstO2B [15]. These data suggest that GstO2B plays a protective role against oxidative stress by regulating the AsA recycling pathway in Drosophila in vivo (Figure 3).

Figure 3

The proposed model for the different functions of Drosophila GSTOs.

CG6673 isoform A (GstO2A) regulates mitochondrial F₁F₀-ATP synthase activity through the glutathionylation of the ATP synthase β subunit. CG6673 isoform B (GstO2B) catalyzes the reduction of DHA to AsA. Reduced AsA decreases the cellular oxidative stress level.

Functions of other GSTOs in Drosophila

The in vivo functions of GstO1 and GstO3 remain unknown. Interestingly, GstO1 transcripts are highly expressed in the ovaries and testes [21] (Figure 4). The spatial expression pattern of GstO1 in adult tissues indicates that GstO1 most likely has different substrates and different functions in vivo. Studies on the molecular function of GstO1 in reproductive organs are currently underway in our laboratory.

Figure 4

Tissue distribution of DrosophilaGSTO mRNAs.

The relative mRNA expression levels of GSTO genes in 12 different adult tissues are shown. CG6781 (sepia) mRNA is detected only in head and eye tissue. CG6662 (GstO1) mRNA is primarily expressed in reproductive organs. The data on GSTO mRNA expression in adult tissues were compiled from Fly9Atlas.

The level of GstO3 transcript of Drosophila is increased in response to heat shock, heavy metal stress, and exposure to rotenone [22–24]. These results suggest that GstO3 has a wide range of antioxidant activities. Further studies are required to understand the molecular mechanism by which GstO3 protects cells from these oxidative stresses.

Conclusion

Sequence alignment analyses revealed that GSTs, including GSTOs, exist in a wide range of organisms. This wide distribution may reflect diverse biological functions in various organisms. Previously, we provided evidence for novel diverse roles of GSTOs based on genetic and molecular studies in Drosophila. One of the Drosophila GSTOs, CG6781 (sepia), has been identified as the sepia gene. We also found that CG6673 (GstO2) is required for the activation of mitochondrial F₁F₀-ATP synthase activity and for the regeneration of AsA. The C-terminal domain of Drosophila GSTOs is less similar than the N-terminal domain, which contains a cysteine residue in the GSH-binding site. Therefore, the differences in the C-terminal domain, which binds to the second substrate, may be responsible for the differences in the functions of various Drosophila GSTOs. Further studies identifying the in vivo substrates of the Drosophila GSTOs will provide a better understanding of the functional diversity of GSTOs. Because many biological functions are conserved in Drosophila and mammals, we expect that the elucidation of diverse physiological functions of Drosophila GSTOs will have broad biological implications.