Structure And Function Of Olfactory Related Glutathione S-transferase BmGSTD1in Silkworm, Bombyx Mori

Glutathione S-transferases (GSTs, EC2.5.1.18) are a superfamily of multifunctional enzymes ubiquitously distributed in mammals, insects, plants, fungi, and microorganisms. As phase Ⅱ detoxification enzymes, they play a crucial role in metabolizing xenobiotics and/or endobiotics to detoxification. GSTs are also involved in other functions. They exhibit peroxidase activity in protecting against oxidative stress and oxidative damage. In addition, they have been implicated in the isomerization of certain steroids, cellular signal passway and various biosynthetic processes. Additionally, some GSTs can non-catalytically bind numerous endogenous and exogenous ligands.Beyond, olfactory-related GSTs have been identified in cattle, rat, Manduca sexta, Helicoverpa armigera and Papilio xuthus L.. They are proposed to have roles in olfactory system:protecting olfactory system by detoxifying harmful xenobiotics; participating in olfactory process.The silkworm Bombyx mori encodes four isoforms of delta class GSTs, among which BmGSTd1logged in NCBI was cloned from silkworm antenna, and amino acids encoded share similarity of74.4%with antennal-specific GST-msolfl in M. sexta. And BmGSTd1, BmGSTd4and GST-msolfl formed a distinct cluster in phylogenetic analysis reported. Based on these clues, we identified olfactory-related GSTs in GSTs genes of Bombyx mori.In present study, we identified BmGSTdl and BmGSTd4as olfactory-related GSTs candidate genes from delta class GSTs in B. mori based on microarray data combined with RT-PCR. Meanwhile, BmGSTdl was cloned and analyzed. Furthermore, we obtained recombinant BmGSTD1by prokaryotic expression and protein purification. The structures of BmGSTD1apo form and GSH-bound form were resolved through methods of structural biology, and the active site GSH-binding site and hydrophobic substrate binding site were analyzed. Also the functional study of BmGSTD1was performed using spectrophotometry and thin layer chromatography.The main results are as follows:1. Identification of olfactory-related GSTs in B. moriBy doing BlastN in silkworm databes using the full-length cDNA sequences of BmGSTd1, BmGSTd1and BmGSTd3downloaded from GenBank as query sequences, we obtained microarray probe ID. Based on microarray data analysis in various different tissues on day3of the fifth instar larval stage, BmGSTdl shows high levels expression in head and a small amount expression in testis. BmGSTd1expresses in male and female head, fat body, midgut, and hemocyte and the former middle silk gland. BmGSTd3expresses in various tissues except silk gland.Semi-quantitative RT-PCR was carried out to validate the microarray data. In various different tissues on day3of the fifth instar larval stage, BmGSTdl only expresses in head, antennae and maxillary palpus; BmGSTd1and BmGSTd3show expression in all tissues. However the expression of BmGSTd4has not been detected in all tissues. In different tissues of adult moth, BmGSTdl shows high levels expression in antennae and a small amount expression in legs, tail casing and gonad of male, has high and special expression in antennae of female. BmGSTd2and BmGSTd3have the similar expression pattern. BmGSTd4only highly and specific expresses in antennae of male.2. Analysis of B. mori olfactory-related GSTsBmGSTdl and BmGSTd4located on the same scaffold in chromosome6, and have the same transcriptional orientation. The ORF of BmGSTdl is657bp, encoding218amino acids with putative molecular weight is25.2kDa and pI is5.16, and no signal peptide. The ORF of BmGSTd4is738bp, encoding245amino acids with putative molecular weight is27.7kDa and pI is4.63, and containing signal peptide. Domain prediction shows that both of BmGSTDl and BmGSTD4have complete GST N-terminal and C-terminal domains.Amino sequences of BmGSTD1and BmGSTD4show share similarity of61.2%, and each other has similarity in74.4%and51.9%with GST-msolfl in M. sexta, respectively. The amino acid sequences multi-alignment of reported olfactory-related GSTs with BmGSTD1and BmGSTD4shows that the sequences trend to be conservative from the50th residue, especially the50th to190th, some of which are highly conserved. The amino acid sequences multi-alignment of reolved insect delta and epsilon class GSTs with BmGSTD1indicates that they are conserved, especially the residues in active site, including catalytic essential residue. Characteristic analysis of the amino acid sequences of BmGSTD1and BmGSTD4display that both of them have complete secondary structural motif.3. Prokaryotic expression and protein purification of BmGSTD1The ORF fragment of BmGSTD1was subcloned into p28(derived from pET28), and recombinant expression plasmid p28-BmGSTdl was constructed after identification of digestion and being sequenced. The recombinant plasmid was transformed into E. Coli strain Rosetta(DE3) competent cells, a soluble form of expression products were obtained under the induced expression condition of final concentration of0.2mM IPTG, at37℃for4h, and the products showed band on SDS-PAGE, with an approximate size of25kDa, which is consistent with the calculated molecular mass of the BmGSTD1subunit. By Ni-NTA affinity chromatography and gel filtration chromatography, recombinant BmGSTDl were purified. The same way was repeated to recombinant BmGSTD2and BmGSTD3. The purified protein was concentrated and stored at-80°C for subsequent experiments.4. Functions of BmGSTD1GST activity of BmGSTD1was measured using CDNB and GSH as standard substrates with a spectrophotometer. Kinetic parameters Km and Vmax values were0.28mM and456.47μmol/mg/min, respectively. Comparison with kinetic parameters of reported B. mori GSTs, BmGSTDl has well GST activity. BmGSTDl conjugation activities towards odorants trans-2-hexenal and trans-cinnamaldehyde were determined via thin-layer chromatography under condition of100mM NaAc, pH4.5(containg10%DMF). The results clearly show that BmGSTDl can catalyze GSH conjugation to candidate odorant substrate. High and preferential expression in olfactory organs, excellent GST activity, and evident participation in odorant modifications all suggest that BmGSTD1has dual roles in the silkworm olfactory system:protecting olfactory system by detoxifying toxic compounds, and function as a scavenger against certain odorants to maintain the sensitivity and effectiveness of the olfactory system.5. The crystal structures of BmGSTD1The purified recombinant protein was concentrated to10mg/mL via ultrafiltration (Millipore Amicon). A1μL sample for crystallization was mixed with a1μL reservoir solution of0.1M sodium acetate trihydrate (pH4.6) and8%pEG4000. The sample for the apo form contained4mg/mL protein and10mM dithiothreitol. For the GSH-bound form, an extra10mM GSH was added. Crystals were grown in hanging drops equilibrated against the reservoir solution at16℃for1d before being flash-frozen in liquid nitrogen using a reservoir solution with30%glycerol (vol/vol) as cryoprotectant. X-ray diffraction data were collected at100K in a liquid nitrogen stream, using beamline17U with an MX225CCD at the Shanghai Synchrotron Radiation Facility. All diffraction data were indexed, integrated, and scaled using the program HKL2000. Molecular replacement of the BmGSTDl apo form was carried out with MOLREP within the CCP4i suite, using the Anopheles dirus GSTD5monomer (RCSB Protein Data Bank code1R5A) as a search model. REFMAC and WinCoot were employed to perform refinement. The final model quality was checked using MolProbity. The resolved BmGSTD1apo form model was used for the model building of BmGSTD1GSH-bound form. The same refinement procedures were carried out for the apo form. We elucidated on the tertiary structures of BmGSTD1in the apo form (2.51A) and GSH-bound form (2.12A).BmGSTD1exists as a dimer in solution. An asymmetric unit is composed of four monomers, which may be generated during crystal packing. In dimer, the interface of subunits was formed through the residues located on flank of a3, a4, a5, β4and flexible region between a2and a3. The dimeric interface buries a large area of approximately2800A2.BmGSTD1adopts the canonical fold of cytosolic GSTs. Each subunit is obviously organized into two distinct domains linked by a short segment. The N-terminal domain consists of the typical βαβαββα motif of the thioredoxin fold. Five helices (a4-a8) of the C-terminal domain resemble a right-handed a-helical bundle. Other common features in cytosolic GSTs include an equivalent cis conformation Pro58, and the middle interrupting of helix a4induced by equivalent Gly107.The residues formed GSH-binding site are conserved, most of them are are hydrophilic and polar in nature, combine and stable GSH via hydrogen bonds. The thiolate of GSH is located3.65A away from the hydroxyl group of Ser14, which is the putative essential residue for catalysis. The hydrophobic substrate binding site is open and sharps as a shallow hydrophobic channel, which lies adjacent to the G-site. A similar aromatic "zipper" motif in AdGSTD4may exist in H-site of BmGSTDl.