Introduction
The redlip mullet ( Liza haematocheilus) is a saltwater fish which holds paramount importance as food fish worldwide as well as in Korea. Present study, we characterized the GST kappa (LzGSTκ) and omega (LhGSTω) from redlip mullet . Functional studies were carried out with the recombinant protein to determine its enzymatic and antioxidant properties . In addition, the transcriptional levels of LzGSTκ , LzGSTω were determined under normal physiological conditions and immunologically challenged conditions.
Materials and methods
The transcriptome database of mullet cDNA sequences was developed using de novo assembly. Mullet fish (~100g, length 24 cm) purchased from Sangdeok fish farm in Hadong, Korea . The fish were acclimatized in the laboratory aquarium tanks at 20 °C for a week prior to the experiment. Total RNA was extracted by using RNAiso plus (TaKaRa, Japan) and cleaned with RNeasy spin column (Qiagen, Germany). Quantitative real-time PCR (qPCR), with specifically designed primers were performed to analyze the expression profile of LzGSTκ , LzGSTω. The coding sequence (CDS) of the cDNA fragment was amplified by using gene-specific cloning primers. The size of the amplicon were 687 bp (LzGSTκ) and 720 bp (LzGSTω) respectively . D igested PCR products were ligated into the pMAL-c5X vector. The ligated product was then transformed into Escherichia coli (E.coli) DH5α and the coding sequence was confirmed by sequencing. To express the recombinant LzGSTκ , LzGSTω protein (rLzGSTκ , rLzGSTω), the pMal-c5X/ LzGSTκ , LzGSTω construct was transformed into E. coli BL21 and incubated at 37 °C in LB broth medium containing 100 μg/mL ampicillin, until the OD600 reach 0.6. Isopropyl-β-galactoside (IPTG) was then added to the culture (final concentration 0.5 mM) and incubated for 24 h at 15 °C to induce the expression of the recombinant protein. After incubation, the cells were harvested by centrifugation. The rLzGSTκ , rLzGSTω protein was purified from the supernatant using maltose affinity chromatography. Enzyme activity was measured separately using CDNB as substrates. The absorbance of the reaction was measured, immediately and 5 min after addition of the substrate.
Result
To understand the potential endogenous functions of LzGSTκ , LzGSTω , its relative expression was examined in different tissues. Analysis of the expression profiles from the redlip mullet revealed that LzGSTκ was strongly expressed in the heart, while the lowest expression was observed in the head kidney (Fig.1.A). LzGSTω was strongly expressed in intestine , whereas the lowest expression was observed in the head kidney. (Fig.2.A) Activities of rLzGSTκ , rLzGSTω and MBP against different substrates, including CDNB(2,4-Dinitrochlorobenzene), DCNB(2,4-Dinitrochlorobenzene), 4-NPB(4-nitrophenethyl bromide), 4-NBC(4-nitrobenzyl chloride), and ECA(ethacrynic acid) were then measured. Only detectable activity was observed, when CDNB used as the substrate. No significant activity was detected for MBP against any of the substrates and therefore it was treated as a control. rLzGSTκ was shown to have GSH:CDNB conjugating activity at pH range 6 to 8 (Fig.1.B ). Its highest activity was observed at pH 7. For RLzGSTω, GSH:CDNB conjugating activity observed at pH range 6 to 9 (Fig.2.B) , wherein the highest activities were observed on pH 8.
B
A
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Fig. 1. (A) Relative mRNA expression level of GSTκ in different tissues. The tissues were collected from healthy mullet fish, and expression levels in each tissue were analysed using real-time qPCR. (B) Effect of pH on the GSH conjugating activity of LzGSTκ.
Relative mRNA expression level of GSTω in different tissues. The tissues were collected from healthy mullet fish, and expression levels in each tissue were analysed using real-time qPCR. (B) Effect of pH on the GSH conjugating activity of LzGSTω.
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