COMPREHENSIVE CHARACTERIZATION OF THREE GLUTATHIONE S-TRANSFERASE FAMILY PROTEINS (GST ω, GST ρ AND GST θ) FROM BLACK ROCKFISH (Sebastes schlegeli)

Abstract

Glutathione-S-transferases (GSTs) 는 매우 다양한 다양한 촉매활성을 가지며 여러방 면으로 중요한 기능을 수행하는 phase II enzymes에 속한다 . 구핵성의 성향을 가지 고 있는 GSTs는 글루타티온 글루타티온 (GSH) 을 음이온의 형태인 티올레트 티올레트 형태 (GS-) 로 바꾸고 전자친화성 생체이물을 생체이물을 티올레이트와 복합체를 형성하는 반응을 촉진시킨 다. GSTs는 다양한 전사와 전사후 변형을 통하여 특정 생체이물에 대한 내성을 조절하는 능력을 갖는데 갖는데 이러한 GSTs의 해독능력은 해독능력은 마약류 , 항생물질 항생물질 , 살충제 그 리고 농약 개발에서 중요한 요소로서 알려져왔었다 . Cytosolic GST인 GST omega (GSTω) 는 n-말단 연장선상에 연장선상에 Proline (19-20 residues) 이 풍부하고 풍부하고 , GSH와 이황화 결합하는 활성부위에서 독특한 시스테인 잔기를 소유하고 있으 며 이 부분에서 다 른 GSTs들과 뚜렷하게 뚜렷하게 차이가 보인다 . 또 다른 cytosolic GST인 GST rho (GSTρ)는 많은 연구에서 어류에서만 발견되는 GST로 밝혀져왔었지만 , 최근 연체동물에서 도 발견되었다고 보고되었다 . Theta class GST (GSTθ) 는 또 다른 cytosolic GST로서 alpha, mu, Pi GST 등 다른 GST들과 구조뿐만 아니라 기능적으로도 다른 형태를 가 지고 있다 . GSTθ는 N-말단에 티로신 대신 세린잔기 가 존재하며 , 기질 1-chloro-2,4-dinitrobenzene (CDNB)에 반응하여 거의 검출되지 검출되지 않을 정도의 극미량으로 활성을 띈다 . 현재 , 경골어류에서의 GSTω, GSTρ, GSTθ에 대한 연구가 미미한 실정이다 . 본 연구에서는 조피볼락 (Sebastes schlegeli)으로부터 GST계열의 3가지 paralogs (GSTω, GSTρ, GSTθ)를 이미 확립된 cDNA 데이터베이스와 데이터베이스와 NCBI-BLAST 도구를 이용하여 확인하였고 확인하였고 , 분자적 특징은 특징은 molecular biological software and web-based servers 이용하였다 이용하였다 . 생체이물 스트레스로부터 스트레스로부터 보호하기 위해 발생하는 항산 화 정도와 생화학적 특성을 조사하기 위해 3개의 GST를 각각 pMAL-c5X vector에 닝하였고 , Escherichia coli ER2523 cells에 형질 전환하여 다양한 assay를 실시하 였다 . 재조합 단백질은 과발현시켜 정제하였고 , 다양한 기질에 대해 글루타티온 (GST) 을 감소시키는 활성을 조사하기 위해 분광광도분석을 실시하였다 실시하였다 . 각 기질 에 대해 온도별 (10 °C – 60 °C) 과 pH별(3 – 12 pH) 의 영향에 따른 효능 평가를 수 행하였고 , Michaelis–Menten constant (Km)와 maximum reaction velocity (Vmax)를 조사 하기 위해 Kinetic assays을 실시하였다 . 상업적으로 상업적으로 이용가능한 두 개의 GST 억제 제인 cibacron blue (CB) (0.001–100 μM)와 hematin (0.001–100 μM)를 사용하여 억제제 의 영향에 대한 GST 촉매활성을 연구하고자 사용하였다 . 또한 , H2O2와 중금속 (Cd, Zn, Cu) 에 의해 발생되는 산화스트레스에 대한 생존효능을 측정하기 위해 디 스크확산법을 실시하였다 . 뿐만 아니라 , 박테리아와 박테리아와 바이러스 자극제에 자극제에 대한 반응 을 보기 위해 조피볼락에 살아있는 살아있는 Streptococcus iniae 박테리아와 polyinosinic:polycytidylic acid (poly I:C)를 접종하여 접종하여 공격실험을 실시하였다 . 공격실 험 후, quantitative real time PCR (qPCR)을 이용하여 이용하여 세 GST 유전자의 시간별 mRNA 발현양상을 조사하였다 . 세 개의 GST 유전자는 유전자는 모두 일반적인 도메인 도메인 구조를 가지고 있으며 , 활성 , 활성모티브 , GSH binding pocket 구조에 따라 조금씩 구별되었다 . 각 GST의 효소활 성은 두 개의 기질을 통한 활성과 온도별 , pH별 활성 , 동력학적 계수를 통한 활성 과 GST 억제제 에 대한 실험을 통해 잠재적 억제 효능에 따라 각각의 GST는 서 로 다른 활성을 나타내었고 나타내었고 이에 따른 기능적 기능적 차이를 보였다 . 또한 , 디스크 확산 법은 통해 in vitro상에서 세 GST가 산화적 스트레스와 Cd, Cu, Zn 와 같은 중금속 으로부터 보호해주는 것을 확인하였다 . 세 유전자는 모두 면역 관련 조직에서 높 은 발현을 보이고 있으며 있으며 , 병원체 스트레스로 스트레스로 인한 RfGSTω와 RfGSTθ의 mRNA발현 증가는 GST가 초기면역에 활발히 관여하고 있음을 보여준다 . 종합해보면 , 이 연구 결과는 경골어류로부터 경골어류로부터 GSTω, GSTρ, GSTθ 의 폭넓은 활성 범위를 보여주고 있으며 앞으로의 연구에 기반이 될 수 있을 것으로 보인다 .

GSTs are coming under the phase II enzymes which are an important multifunctional family accompanied with a wide variety of catalytic activities. GSTs catalyze the reduced glutathione (GSH) to form thiolate (GS-), the anion and form a complex with GST which nucleophilically attacks the close by electrophilic xenobiotics. GSTs undergo different transcriptional and posttranscriptional modifications and gain the ability to modulate resistance to specific xenobiotics. Due to their role in detoxification, GSTs have been identified as an important category in development of drugs, antibiotics, insecticides and pesticides. A cytosolic GST, GST omega (GSTω) distinctly differs from the other GSTs with the presence of N-terminal extension rich in Proline (19-20 residues) and possessing a unique cysteine residue at the active sites that can form a disulfide bond with GSH. GST rho (GSTρ), a cytosolic GST, has been identified as a fish specific GST in numerous studies but, recently it has been reported to be present in mollusks as well. Theta class GST (GSTθ), another cytosolic GST which is different from the other related GSTs such as Alpha, Mu and Pi class not only in struc ture but also in function. GSTθ demonstrated minute or non-detectable activity towards the model substrate of the GSTs; 1-chloro-2,4-dinitrobenzene (CDNB) and in the place of N-terminal tyrosine, GSTθ possesses a serine residue. However, very limited reports are available in GSTω, GSTρ and GSTθ in accordance with teleosts. In the present study, three paralogs of GST family; GSTω, GSTρ, and GSTθ from black rockfish (Sebastes schlegeli) was identified using a previously established cDNA database and with the help of NCBI-BLAST tool and molecularly characterized using molecular biological software and web-based servers. In order to investigate their biochemical characteristics and antioxidant dimensions to protect the fish from the xenobiotic stress, the three paralogs were independently cloned into pMAL-c5X vector, transformed into Escherichia coli ER2523 cells and subjected to various assays. Recombinant proteins were over expressed and purified. Spectrophotometric analysis was carried out to determine the specific activities towards various substrates with reduced Glutathione (GSH). The effect of temperature (10 °C – 60 °C) and pH (3 – 12 pH) were assayed towards respective substrate. Kinetic assays were conducted to determine the Michaelis–Menten constant (Km) and maximum reaction velocity (Vmax). Two commercially available GST inhibitors cibacron blue (CB) (0.001–100 μM) and hematin (0.001–100 μM) were used to study the effect of inhibitors on the catalytic activity. Additionally, to determine the survival efficacy upon oxidative stress generated by H2O2 and heavy metals (Cd, Zn, Cu), disk diffusion assay was conducted. Furthermore, the response upon bacterial and viral stimuli, black rockfish were undergone challenge experiments with live Streptococcus iniae bacteria and polyinosinic:polycytidylic acid (poly I:C). The temporal transcriptional modulation of these three genes upon challenge experiments was investigated using quantitative real time PCR (qPCR). The open reading frame (ORF) of RfGSTω was 717 bp and the putative protein encoded by the ORF was 239 amino acids in length and 28 kD in molecular weight. RfGSTρ consisted with an ORF of 678 bp and the putative protein was 226 amino acids in length with a molecular weight of 26 kD. Putative protein of RfGSTθ was 240 amino acids in length and 28 kD in molecular weight which was encoded by an ORF of 720 bp. The ExPASy prosite database revealed that the RfGSTω consisted of a GST N-terminal domain (Asn20 - Lys99) where GST binding site (G-site) lies within and, a C-terminal domain (Ser104 - Val229) where the substrate binding pocket (H-site) lies within. According to the results generated by the Pfam, the N-terminal domain of RfGSTρ was located at Gln3 - Gly85 while C-terminal domain was located at Ser92 - Gln220. In the instance of RfGSTθ, the N-terminal domain was lied at Met1 - Asp80 while C-terminal domain was at Glu86 - Leu228. The ORFs of each three paralogs were cloned into pMAL-c5X expression vector, respectively and overexpressed in E. coli ER2523. The fused proteins with maltose binding protein (MBP); rRfGSTω, rRfGSTρ and rRfGSTθ were purified separately and confirmed the purity using the SDS-PAGE analysis. The biochemical properties of each protein and MBP protein were analyzed using standard functional assays. The rRfGSTω protein exhibited a detectable activity only towards DHA (0.36 ± 0.01 μmol min-1 mg-1, n = 3), while both rRfGSTρ and rRfGSTθ showed detectable activities only towards CDNB (1.56 ± 0.01 μmol min-1 mg-1 and 2.05 ± 0.01 μmol min-1 mg-1, n = 3). Subsequently, CDNB was selected as the respective substrate for both rRfGSTρ and rRfGSTθ while DHA was selected in the instance of rRfGSTω for further analysis. When the GSH concentration was fixed, the Km and Vmax values were 33.62 ± 2.05 mM and 18.04 ± 0.4 mM, respectively in the kinetic assay conducted with rRfGSTω where DHA was used as the substrate. For the same assay conducted with rRfGSTρ using CDNB as the substrate, the Km and Vmax values were 3.02 ± 0.08 mM and 3.40 ± 0.09 mM, respectively, while with rRfGSTθ the Km and Vmax values were 2.63 ± 0.04 mM and 15.80 ± 1.00 mM, respectively. The Km and Vmax values of rRfGSTω were 0.97 ± 0.14 mM and 2.02 ± 0.14 mM, respectively when the substrate concentration (DHA) was fixed. For the same assay conducted with CDNB as the substrate, the Km and Vmax values were 14.10 ± 0.25 mM and 11.28 ± 0.70 mM, respectively for rRfGSTρ, while with rRfGSTθ the Km and Vmax values were 1.40 ± 0.15 mM and 10.01 ± 0.75 mM, respectively. The optimum temperature and the optimum pH for the DHAR activity with rRfGSTω were 40 °C and 8.5, respectively. Optimum temperatures for the CDNB conjugation activity with rRfGSTρ and rRfGSTθ were 40 °C and 20 °C, respectively while the optimum pH value was 7.5 in both of the instances. In the inhibition assay, the IC50 (half maximal inhibitory concentration) values of the inhibition were 0.11 M and 0.004 M for CB and Hematin, respectively in the instance of rRfGSTω. In rRfGSTρ, the IC50 values of the inhibition were 7M and 0.1 M for CB and Hematin, respectively. In the instance of CDNB conjugation activity of rRfGSTθ, the IC50 values of the inhibition were 0.52 M and 0.049 M for CB and Hematin, respectively. Clearance zone was observed around the disks treated with 5 μL of H2O2 and the heavy metal treated disks in different magnitudes after incubation at 37 °C overnight. The rRfGSTω and rRfGSTθ transformed E. coli cells showed significance survival efficacy towards the oxidative stress developed by H2O2. All three genes implicated their protective potential towards the oxidative stress developed by heavy metals in different magnitudes. All three paralogs (RfGSTω, RfGSTρ and RfGSTθ) were ubiquitously expressed in blood cells and all the tissues analyzed in the study namely; head kidney, spleen, liver, gill, intestine, kidney, heart, testis and ovary. The highest mRNA expression of RfGSTω was detected in blood, liver and ovary tissues. The RfGSTρ paralog was depicted its higher expression level in the liver, testes and ovary. Liver, testes and gill showed higher mRNA expression levels in the instance of RfGSTθ. Upon the S. iniae bacterial challenge, RfGSTω mRNA level in blood was significantly elevated at 3 hours post injection (h.p.i) and 6 h.p.i., respectively. RfGSTρ mRNA expression level in liver tissue was significantly down-regulated at the early phase of the bacterial infection and. Significant up-regulation of RfGSTθ mRNA expression in liver tissue was observed at all the time points except at 24 h.p.i. Poly I:C challenge experiment results revealed that, RfGSTω mRNA level could be significantly up-regulated by viral stimuli. The expression level of RfGSTρ mRNA in liver tissue showed a fluctuated pattern with significant down-regulations and control levels. RfGSTθ mRNA expression in liver tissue achieved its highest mRNA level at 48 h.p.i. All three paralogs revealed their common domain architecture though they were slightly diverged from their corresponding orthologs in respect to activities, active moieties and GSH binding pocket architectures. Their enzymatic activities towards respective substrates, activities under different temperatures and pH values, kinetic parameters as well as inhibitory