Characterization of a glutamate-cysteine ligase in Bombyx mori

Glutamate-cysteine ligase (GCL) is a crucial enzyme involved in the synthesis of glutathione (GSH). Despite various studies on glutathione transferase, and its essential role in detoxification and resistance to oxidative stress, GSH synthesis has not been described in Bombyx mori (silkworms) to date. Silkworms form part of the lepidopterans that are considered as a model of agricultural pests. This study aimed to understand the GSH synthesis by GCL in silkworms, which may help in developing insecticides to tackle agricultural pests. Based on the amino acid sequence and phylogenetic tree, the B. mori GCL belongs to group 2, and is designated bmGCL. Recombinant bmGCL was overexpressed and purified to ensure homogeneity. Biochemical studies revealed that bmGCL uses ATP and Mg2+ to ligate glutamate and cysteine. High expression levels of bmgcl mRNA and GSH were observed in the silkworm fat body after exposure to insecticides and UV-B irradiation. Moreover, we found an increase in bmgcl mRNA and GSH content during pupation in the silkworm fat body. In this study, we characterized the B. mori GCL and analyzed its biochemical properties. These observations indicate that bmGCL might play an important role in the resistance to oxidative stress in the silkworms.


Introduction
Glutathione (GSH) is a non-protein thiol tripeptide, γ-Lglutamyl-L-cysteinylglycine [1], which is involved in several vital physiological functions such as detoxification of xenobiotics, antioxidant reactions, regulation of cell cycle progression, and control of the redox balance [2,3].
Despite evaluation of the significance of GSH synthesis in mammalian tissues, to date there are no investigations reported on the identification and function of the enzymes involved in GSH synthesis in silkworms (Bombyx mori). The silkworm is a model lepidopteran organism; therefore studies on GSH synthesis in B. mori could give us better insights about how to combat these species, considered agricultural pests. GSH transferases (GST, EC 2.5.1.18) participates in GSH conjugation to a wide range of hydrophobic and electrophilic molecules, and is vital for the protection against several xenobiotics and oxidative stress. We have shown earlier that unclassified GST2 of B. mori catalyzes diazinon metabolism, indicating its role in insecticide resistance of these insects [8]. The B. mori omega-class GST (bmGSTO) is expressed on exposure to various environmental stresses [9]. Furthermore, bmGSTO functions as an antioxidant by expressing thiol transferase, GSH peroxidase, dehydroascorbate reductase, and GST [9]. These findings indicate that bmGSTO is involved in scavenging reactive oxygen species 1 3 via its peroxidase activity, and its thiol transferase and dehydroascorbate reductase activities increases resistance to oxidative stress.
For better understanding of the molecular basis of synthesis of GSH, which is utilized by GST in silkworms, the identification and functional characterization of the involved enzymes are necessary to elucidate the mechanisms underlying its production. As a first step towards this, we have identified and characterized GCL from B. mori, in the present study. In addition, we have assessed the tissue distribution of this enzyme.

Insects and tissue
Silkworms (p50 strain) used in the current study was maintained at Kyushu University Graduate School (Fukuoka, Japan). Silkworm larvae and pupae were anatomized on ice to collect the tissues (hemocytes, testes, ovaries, silk glands, midgut, and fat bodies), which were stored at −80 °C until use. Total RNA was isolated from tissues by an RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) as per the manufacturer's instructions.

Cloning and sequencing of the bmGCL-encoding cDNA (bmgcl)
First-strand cDNA was synthesized with the total RNA extracted in the aforementioned step by performing reverse transcription-polymerase chain reaction (RT-PCR), using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), and an oligo-dT primer. The bmGCL-encoding cDNA was amplified by PCR using the first-strand cDNA as a template and the following oligonucleotide primers: 5′-ACG CCA TGG GTT TGC TAA CTG AAG GTA GCC − 3′ (sense) and 5′-ACG GGA TCC AGA GCA ATC CCT GCT CAT CAT − 3′ (antisense). Primers for PCR were prepared, based on the sequence obtained from the SilkBase EST database [10]. NcoI and BamHI restriction enzyme sites are indicated by the underlined and double-underlined nucleotide sequences in the primers, which were used to clone the PCR product into an expression plasmid. The PCR program in the presence of KOD FX Neo (TOYOBO, Osaka, Japan) was carried out with the following steps; pre-incubation at 94 °C for 2 min, followed by 35 cycles, each; denaturation at 94 °C for 1 min, annealing at 64 °C for 1 min, and extension at 72 °C for 2 min, with a final extension step at 72 °C for 10 min. The bmGCL-encoding cDNA (bmgcl) was inserted into the pGEM-T Easy Vector plasmid (Promega, Madison, WI, USA). The recombinant plasmid was transformed to Escherichia coli DH5α cells. The sequence of the insert in the recombinant plasmid was confirmed by a DNA sequencer (Applied Biosystems, Foster City, CA, USA). The ClustalW2 software (http:// www. ebi. ac. uk/ Tools/ msa/ clust alw2/) was used to deduce the amino acid sequence, align it, and create a phylogenetic tree. The boot strap values were calculated using the neighbor-joining plot.

Preparation of recombinant bmGCL
The pGEM-T Easy Vector harboring the bmgcl insert was digested with the restriction enzymes NcoI and BamHI. The fragment obtained was subcloned into pTYB21 expression vector (New England Biolabs, Ipswich, MA, USA) for the expression of recombinant bmGCL. Unless otherwise specified, all the subsequent experiments were done at 4 °C. The disrupted cells were centrifuged at 10,000 × g for 20 min, and the supernatant obtained, containing the chitin-binding-domain intein-bmGCL fusion protein, was poured into a chitin-affinity chromatography column (New England Biolabs, Ipswich, MA, USA) equilibrated with buffer A. After impurities were washed out with the same buffer, the column was filled with 50 mM 2-mercaptoethanol in buffer A at 4 °C for 64 h to cleave the inteintag. The mature bmGCL was eluted with 50 mL of buffer A, and applied to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) on 12.5% polyacrylamide slab gels containing 0.1% SDS, as previously described [11]. Protein bands were visualized by staining with Coomassie Brilliant Blue R250. Protein amounts were determined by the Bradford's method, using bovine serum albumin as the standard [12].
The bmGCL activity was monitored spectrophotometrically at 340 nm. Kinetic constants were measured to fit the data into the Michaelis-Menten equation by KaleidaGraph (Synergy Software, Reading, PA, USA).

Analysis of gene expression
The expression of bmgcl messenger RNA (mRNA) was examined by real-time quantitative PCR (qPCR). Total RNA was prepared using SuperScript III reverse transcriptase (Invitrogen) and random hexamers, according to the manufacturer's instructions. The primer sequences for bmgcl and the reference gene rp49 (ribosomal protein 49) were as follows: bmgcl forward primer 5′-AGA GTT CCG CCC CTG TGA AG-3′ and reverse primer 5′-CAC GCT GCA TGT TCT CGT CC-3′; rp49 forward primer 5′-CAG GCG GTT CAA GGG TCA ATAC-3′ and reverse primer 5′-TGC TGG GCT CTT TCC ACG A-3′; and bmgsto forward primer 5′-TGC GTA CCA CAA GGC TTT AGATT-3′ and reverse primer 5′-CCA GGT TCA CTT CCG TCC A-3′. The amounts of bmgcl mRNA expressed were measured by qPCR, performed on a StepOne Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) following the manufacturer's instructions. The PCR program included preincubation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Three biological replicates with technical replicates were analyzed using the comparative C T method [14].

GSH content
GSH content in the silkworm fat body was measured using an oxidized GSH (GSSG)/GSH quantification kit (DOJINDO, Kumamoto, Japan). The fat bodies of the silkworms were homogenized in phosphate-buffered saline, and centrifuged at 6000 × g for 20 min. From the resultant supernatant, 20 µL was mixed with 7.2 mL of reaction mixture containing a substrate (5,5'-dithiobis [2-nitrobenzoic acid]). The production of 5-mercapto-2-nitrobenzoic acid ( λ max = 412 nm) derived from the substrate was measured by the absorbance at 412 nm. GSH content was evaluated as arbitrary units normalized to mg of protein used in the assay.

Statistical analysis
Student's t-test and one-way analysis of variance (ANOVA) were used to verify the differences between each group. A P-value of < 0.05 was considered to be statistically significant.

Identification of bmGCL
bmgcl was amplified by RT-PCR using total RNA isolated from the fat bodies of B. mori. The nucleotide sequence of bmgcl was registered in GenBank (accession number: LC719986). The gene contains a 1902 bp coding region that encodes a deduced protein containing 633 amino acid residues (Fig. 1). The calculated molecular mass and isoelectric point of bmGCL are 71,875 and 5.95, respectively. A BLAST search revealed that the deduced bmGCL amino acid sequence shares 61, 60, and 41% identity respectively, with the GCL homologs of Homo sapiens (P48506), Rattus norvegicus (P19468), and Saccharomyces cerevisiae (P32477), respectively (Fig. 1).
Analysis of the phylogenetic analysis (Fig. 2) suggest that bmGCL is closely related to non-plant eukaryotic organisms and is a member of Group 2 GCL.

Overexpression and enzymatic properties
The recombinant bmGCL overexpressed in soluble form in E. coli and it was purified to homogeneity (Supplementary Fig. S1). Approximately 4.0 mg of purified bmGCL was obtained from 1 L of bacterial culture. The enzymatic characterization of bmGCL was performed using cysteine and glutamate as the substrates and the kinetic constants were calculated. bmGCL had K m values of 2.4 and 6.4 mM with glutamate and cysteine, respectively.

bmgcl mRNA distribution
The distribution of bmgcl mRNA was estimated in different organs. A ribosomal protein 49 (rp49) is one of housekeeping genes and found in tissues of B. mori [15]. For reduction of experimental error, rp49 was used as the internal control (Fig. 3 A). bmgcl mRNA was present in all tissues tested. However, lesser amount of the transcript was detected in the silk gland, compared with high levels detected in the other tissues. Moreover, the expression of bmgcl mRNA in the fat body was found to be the highest among the tissues tested on day 3 of the fifth instar. Diazinon, permethrin, and imidacloprid are widely used insecticides globally. However, their frequent use has resulted in emergence of insect species, including the order Lepidoptera, which are highly resistant to them. When the level of mRNAs expression in the fat bodies of silkworm larvae was examined by qPCR, we found that the bmgcl mRNA levels induced after exposure to diazinon, permethrin, and imidacloprid were 2.5-, 2.5-, and 3.5-fold greater, respectively, than those in the control (Fig. 3B). As shown in Fig. 3B, we found that the amount of bmgcl mRNA was 2.8-fold greater when samples were treated with ultraviolet-B (UV-B) irradiation, than in the untreated controls. Changes in bmgcl mRNA were examined in the fat bodies of the fifth-instar silkworms (Fig. 3B). The lowest level was observed on day 7, and then increased to day 11.

Change in GSH content
We examined whether GSH content follows the pattern of expression of bmgcl mRNA in the silkworm fat body after application of various stressors. Figure 4A shows an increase in the GSH expression level in comparison with that in the control group, that received no stress application. The level of GSH was the greatest in response to UV-B irradiation (Fig. 4A), where the second highest expression of bmgcl mRNA was observed (Fig. 3B). We found that fat bodies on days 5 and 7 contained 40-50% of GSH content compared to that on day 1 (Fig. 4B), which correlated with the lower amounts of bmgcl mRNA expressed on the same days (Fig. 3C). Fig. 1 Alignment of Glutamate-cysteine ligase (GCL) amino acid sequences. The sequence of bmGCL was compared to those of Saccharomyces cerevisiae, Homo sapiens, and Rattus norvegicus including the accession number for UniProt and the species name. In this alignment, amino acid residues responsible for binding to glutamate and magnesium are boxed, and written in red letters, respectively. The ATP/ADP-binding, cysteine-binding, and the conserved cysteine are shaded in yellow, green, and orange, respectively

Expression of bmgsto mRNA at various days
Expression of bmgsto mRNA was examined in fat bodies of the fifth-instar silkworms (Fig. 5). The amounts of bmgcl mRNA decreased from days 1 to 7 (the lowest level) and then were elevated on day 11.

Discussion
In this study, we describe a novel GCL, bmGCL, in silkworms. To our knowledge, this is the first report of a B. mori GCL, the study of which is necessary for a comprehensive understanding of the GSH synthesis pathways in silkworms.
There are three groups of GCLs: bacteria (Group 1), nonplant eukaryotic organisms (Group 2), and plants (Group 3). We found that silkworm genome includes only one GCL. This GCL (bmGCL) shares low sequence homology with plant Brassica juncea (19%) and E. coli (17%), while sharing high sequence homologies with non-plant eukaryotic organisms, as shown in in Fig. 1 (41-61%). A phylogenetic tree showed that bmGCL is member of group 2 which is the non-plant eukaryotic GCLs group (Fig. 2).
Glutamate-, cysteine-, Mg 2+ -, and ATP/ADP-binding sites were greatly preserved in the amino acid sequence of bmGCL (Fig. 1). Glutamate and cysteine may act as substrates for bmGCL in the presence of Mg 2+ and ATP. Therefore, we investigated whether bmGCL demonstrated ligase Fig. 2 Phylogenetic analysis of the amino acid sequences of GCLs. The neighbor-joining plot software was used to create phylogenetic tree by including GCL sequences deposited in NCBI (http:// www. ncbi. nlm. nih. gov/) and Swiss-Prot (http:// expasy. org/ sprot/). Each GCL includes the accession number and organism name. The bootstrap values were expressed as 1000 bootstrap replicants on the nodes. The arrow reveals bmGCL. GCL glutamatecysteine ligase, bmGCL Bombyx mori GCL activity with glutamate and cysteine in the presence of ATP and Mg 2+ . In the current study, pTYB21 was used as the expression vector. The N-terminal intein-tag was removed by self-cleavage during purification of the recombinant protein [16][17][18][19][20]. Based on the K m value for glutamate, bmGCL was found to be similar to the enzymes from Drosophila melanogaster, S. cerevisiae, and H. sapiens [16,18,21]. bmGCL showed similar K m values toward cysteine as in D. melanogaster, but different from the values of S. cerevisiae (0.17 mM) [22] and H. sapiens (0.5 mM) [16]. The reason for the difference in the K m value for cysteine remains unclear and further studies are required to establish this difference. Crystallization of the bmGCL-substrate complex is in progress to explore the substrate-binding site.
Real-time PCR showed that bmgcl transcript was located in several organs of B. mori (Fig. 3A), although its level was high in the fat body and low in the silk gland. The fat body is responsible for immune function in insects [23]. This organ is considered equivalent to the vertebrate liver and adipose tissues [24,25]. The mammalian liver contributes are displayed as the average ± standard error of three biological replicates, with technical replicates. Differences between the expression levels observed in each group were determined using a one-way analysis (*P < 0.05). bmGCL: Bombyx mori glutamate-cysteine ligase to in GSH homeostasis [26]. The high level of expression of bmGCL in fat body concurred with previous reports [23][24][25][26], as the production of GSH by bmGCL contributes to the control of cellular redox systems. Interestingly, a low level of bmgcl mRNA was detected in the silk gland. The silk gland generates silk, which is composed of sericin and fibroin. Sericin contains a high content of serine which can be produced by the phosphorylation pathway. In the silk gland, we observed high expression of mRNAs encoding the enzyme components (D-3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase) involved in phosphorylated pathways linked to GSH synthesis [27][28][29]. These could be synthesized by GSH reductase [30,31]. It is necessary to examine the possibility of this enzyme being a key enzyme in the production of GSH in silkworm silk glands.
bmgcl mRNA in the fat body is induced after exposure to various stressors. Our previous data showed that the generation of reactive oxygen species (ROS) increased in the silkworm fat body under environmental stress [32]. Since ROS, such as the superoxide anion and hydrogen peroxide, are likely to impair the structure and function of biomolecules including DNA, proteins, and lipids; maintenance of their redox status is essential for cells. Cytotoxic alkenals such as 4-hydroxy nonenal (4-HNE) are produced via oxidative stress by lipid peroxidation [33]. Presence of 4-HNE has been show to increase the amount of GCL in human cells [34]. B. mori omega-class glutathione transferase (bmGSTO) is able to detoxify 4-HNE and hydrogen peroxide, and the induction of bmGSTO mRNA was also observed with the environmental stresses of diazinon, permethrin, imidacloprid and UV-B [32]. The levels of GSH increased following treatment with the same stressors as above (Fig. 4A). Thus, bmGSTO could be related to increased tolerance to oxidative stress using GSH produced by bmGCL. Similar results have been obtained in mammals [34,35]. Human GCL are induced in response to various stresses to increase GSH biosynthesis.
Oxidative stress promotes pupation in insects, and the level of ROS increases during metamorphosis in the silkworm fat body [36]. It increases around the fourth molt, then increases again on days 9-10 at the fifth instar, just before pupation [37]. Similar patterns were observed for bmgcl mRNA expression, GSH content and bmgsto mRNA expression (Fig. 3C, 4B and 5). Since ROS are toxic due to their high reactivity, causing oxidative damage to macromolecules in cells, GSH produced by bmGCL could be involved in protection against oxidative stress and balancing redox status during this period.
GSH is synthesized by GCL and glutathione synthetase (GS) [1]. We have previously identified a GS in the silkworm genome. We are in the process of characterizing this enzyme. There have been reports on GCL modifiers (GCLM) with low molecular size (~ 31 kDa) that encode different genes in D. melanogaster and H. sapiens [5,21]. GCLM is enzymatically inactive but plays a regulatory role in GCL. We were unable to find GCLM homologs in the Uni-Prot database (https:// www. unipr ot. org) using BLAST search with GCLMs from D. melanogaster and H. sapiens. The GCLM homolog in B. mori may show a very low homology with GCLMs.
In summary, bmGCL, identified in the silkworm B. mori, is a member of group 2 GCL. The K m value of this enzyme revealed that it ligates cysteine and glutamate in the presence of ATP and Mg 2+ , indicating the existence of a GSH synthesis pathway in B. mori. The expression levels of bmgcl mRNA and GSH changed in response to insecticides, UV-B, fourth molt, and pupation. Together with the high ROS content under the same conditions, bmGCL could participate in the protection of oxidative stress via the synthesis of GSH. The identification of other enzymes for the GSH synthesis pathway provides a basis for future studies on the maintenance of redox status in B. mori and other lepidopteran insects.
Author contributions WA and KY collected the data and performed data analysis; WA, SF and KY conceived and designed the study, and wrote the manuscript.

Funding
The work was supported in part by the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University.

Fig. 5
Expression of bmGSTO-encoding transcripts. qPCR was performed to detect bmgsto mRNAs in the fat bodies at various days. The content on day 1 was set to 1. Normalization of data and standard error are the same as those described in the legend of Fig. 3. Comparisons were performed to examine the differences (*P < 0.05). bmG-STO: B. mori omega-class GST; qPCR: real-time PCR