ISSN : 1225-3480
본 연구에서는 북방전복 (Haliotis discus hannai) 의 대용량 염기서열 분석을 통해 GST유전자의 전장 cDNA를 동정하였다. 북방전복 GST 유전자의 총 길이는 1669 bp로 672 bp의 ORF는 총 223개의 아미노산을 코딩하고 있으며 등전점은 5.69, 분자량은 25.8 kDa으로 예측되었다. 북방전복 GST 아미노산 서열은 둥근전복과 지중해 담치와 같은 패류의GSTA와 가장 유사성이 높았으며 계통수 분석을 통해 GSTA 와 하나의 그룹을 이루었다. 북방전복 GST에는 GSTA의 특징을 갖는 두 site (N- 말단의 G-site, C-말단의 H-site)가 보존되어 있었고 효소활성과 구조 유지에 중요한 잔기가 종간에매우 보존되어 있었다. 북방전복 GST 유전자의 mRNA는 관찰된 모든 조직에서 발현하고 있었으며, 특히 외투막, 아가미, 간췌장, 소화관에서 높은 발현이 확인되었다. 북방전복의 GST 는 비브리오균을 인위감염 시킨 전복의 간췌장에서 감염 후 1 시간 뒤 발현이 급격히 증가했다가 3시간까지 증가한 뒤 감소하였고, 혈구세포에서는 감염 3시간 경과 후 발현 정도가 최고로 증가했다가 감소하였다. 따라서 북방전복 GST는 alpha class GST의 특징을 가지며 병원체 감염에 따른 면역반응 조절에 관여할 것이라 생각되며 병원균 감염에 따른 바이오마커로 활용가능 할 것이라 예상된다.
Glutathione S-transferases (GSTs) are a superfamily of detoxification enzymes that primarily catalyze the nucleophilic addition of reduced glutathione to both endogenous and exogenous electrophiles. In this study, we isolated and characterized a full-length of alpha class GST cDNA from the abalone (Haliotis discus hannai). The abalone GST cDNA encodes a 223-amino acid polypeptide with a calculated molecular mass of 25.8 kDa and isoelectric point of 5.69. Multiple alignments and phylogenetic analysis with the deduced abalone GST protein revealed that it belongs to the alpha class GSTs and showed strong homology with disk abalone (Haliotis discus discus) putative alpha class GST. Abalone GST mRNA was ubiquitously detected in all tested tissues. GST mRNA expression was comparatively high in the mantle, gill, liver, and digestive duct, however, lowest in the hemocytes. Expression level of abalone GST mRNA in the mantle, gill, liver, and digestive duct was 182.7-fold, 114.8-fold, 4675.8-fold, 406.1-fold higher than in the hemocytes, respectively. Expression level of abalone GST mRNA in the liver was peaked at 6 h post-infection with Vibrio parahemolyticus and decreased at 12 h post-infection. While the expression level of abalone GST mRNA in the hemocytes was drastically increased at 3 h post-infection with Vibrio parahemolyticus. These results suggest that abalone GST is conserved through evolution and may play roles similar to its mammalian counterparts.
Aceto, A., Dragani, B., Bucciarelli, T., Sacchetta, P., Martini, F., Angelucci., S., Amicarelli, F., Miranda, M. and Di Ilio, C. (1993) Purification and characterization of the major glutathione transferase from adult toad (Bufo bufo) liver. The Biochemical Journal, 289: 417-422.
Adewale, I.O. and Afolayan, A. (2005) Organ distribution and kinetics of Glutathione transferase from African river prawn, Macrobrachium vollenhovenii (Herklots). Aquatic Toxicology, 71: 193-202.
Adeyemi, J.A. (2014) Oxidative stress and antioxidant enzymes activities in the African catfish, Clarias gariepinus, experimentally challenged with Escherichia coli and Vibrio fischeri. Fish Physiology and Biochemistry, 40: 347-354.
Armstrong, R.N. (1997) Structure, catalytic mechanism, and evolution of the glutathione transferases. Chemical Research in Toxicology, 10: 2-18
Atkinson, H.J. and Babbitt, P.C. (2009) Glutathione transferases are structural and functional outliers in the thioredoxin fold. Biochemistry, 48: 11108-11116.
Beckett, G.J., Chapman, B.J., Dyson, E.H. and Hayes, J.D. (1985) Plasma glutathione S-transferase measurements after paracetamol overdose: evidence for early hepatocellular damage. Gut, 26: 26-31.
Blanchette, B., Feng, X. and Singh, B.R. (2007) Marine glutathione S-transferases. Marine Biotechnology (New York,N.Y.), 9: 513-542.
Cairrao, E., Couderchet, M., Soares, A.M.V.M. and Guilhermino, L. (2004) Glutathione-S-transferase activity of Fucus spp. as a biomarker of environmental contamination. Aquatic Toxicology, 70:277-286.
Donaghy, L., Lambert, C., Choi, K. S. and Soudant, P. (2009) Hemocytes of the carpet shell clam (Ruditapes decussatus) and the Manila clam (Ruditapes philippinarum), current knowledge and future prospects. Aquaculture, 297: 10-24.
Duan, Y., Liu, P., Li, J., Li, J. and Chen, P. (2013)Expression profiles of selenium dependent glutathione peroxidase and glutathione S-transferase from Exopalaemon carinicauda in response to Vibrio anguillarum and WSSV challenge. Fish and Shellfish Immunology, 35: 661-670.
Hamed, R.R., Farid, N.M., Elowa, Sh,.E. and Abdalla, A.M. (2003) Glutathione related enzyme levels of freshwater fish as bioindicators of pollution. The Environmentalist, 23: 313-322.
Hansson, T., Schiedek, D., Lehtonen, K.K., Vuorinen, P.J., Liewenborg, Noaksson, E., Tjarnlund, U., Hansson, M. and Balk L. (2006) Biochemical biomarkers in adult female perch (Perca fluviatilis)in a chronically polluted gradient in the Stockholm recipient (Sweden). Marine Pollution Bulletin, 53:451-468.
Hayes, J.D. and Pulford, D.J. (1995) The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemprotection and drug resistance. Critical Reviews in Biochemistry and Molecular Biology., 30: 445-600.
Hayes, J. D., Flanagan, J. U. and Jowsey, I. R. (2005)Glutathione transferases. Annual Review of Pharmacology and Toxicology, 45: 51-88.
Hotard, S. and Zou, E. (2008) Activity of Glutathione S-transferase in the hepatopancreas is not influenced by the molting cycle in the Fidder crab Uca pugilator. Bulletin of Environmental Contamination and Toxicology, 81: 242-244.
Hughes, V.F., Trull, A.K., Gimson, A., Friend, P.J., Jamieson, N., Duncan, A., Wight, D.G., Prevost, A.T. and Alexander, G.J. (1997) Randomized trial to evaluate the clinical benefits of serum alpha-glutathione S-transferase concentration monitoring after liver transplantation. Transplantation, 64: 1446-1452.
Ivarsson, Y., Mackey, A.J., Edalat, M., Pearson,W.R. and Mannervik, B. (2003) Identification of residues in glutathione transferase capable of driving functional diversification in evolution. A novel approach to protein redesign. The Journal of Biological Chemistry, 278: 8733-8738.
Johansson, A.S. and Mannervik, B. (2002) Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3. The Journal of biological chemistry, 277: 16648-16654.
Jeong, J.E. and Lee, Y.S. (2013) Identification, sequence characterization and expression analysis of the arginine kinase gene in response to laminarin challenge from the Oriental land snail, Nesiohelix samarangae. Korean Journal of Malacology, 29:71-179.
Kanaoka, Y., Ago, H., Inagaki, E., Nanayama, T., Miyano, M., Kikuno, R., Fujii, Y., Eguchi, N., Toh, H., Urade, Y., Hayaishi, O. (1997) Cloning and crystal structure of hematopoietic prostaglandin D synthase. Cell, 90: 1085-1095.
Lau, P.S. and Wong, H.L. (2003) Effect of size, tissue and location on six biochemical markers in the greenlipped mussel, Perna viridis. Marine Pollution Bulletin, 46: 1563-1572.
Liao, W.Q., Liang, X.F., Wang, L., Lei, L.M. and Han, B.P. (2006) Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes. Journal of Biochemical and Molecular Toxicology, 20: 114-126.
Livak, K.J. and Schmittgen, T.D. (2007) Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods, 25: 402-408.
Loguercio, C., Caporaso, N., Tuccillo, C., Morisco, F., Del Vecchio Blanco, G. and Del Vecchio Blanco, C. (1998)Alpha-glutathione transferases in HCV-related chronic hepatitis: a new predictive index of response to interferon therapy? Journal of Hepatology, 28:390-395.
Luca-Abbott, S.B., Richardson, B.J., McClellan, K.E., Zheng, G.J., Martin, M. and Lam, P.K.S. (2005)Field validation of antioxidant enzyme biomarkers in mussels (Perna viridis) and clams (Ruditapes philippinarum) transplanted in Hong Kong coastal waters. Marine Pollution Bulletin, 51: 694-707.
Mannervik, B. and Danielson, U.H. (1988) Glutathione transferases-transferase and catalytic activity. CRC Critical Reviews in Biochemistry, 23: 283-337.
Mannervik, B., Board, P.G., Hayes, J.D., Listowsky, I. and Pearson, W.R. (2005) Nomenclature for mammalian soluble glutathione transferases. Methods in Enzymology, 401: 1-8.
Oakley, A. (2011) Glutathione transferases: a structural perspective. Drug Metabolism Reviews, 43: 138-151.
Polekhina, G., Board, P.G., Blackburn, A.C. and Parker, M.W. (2001) Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the molecular basis for its remarkable catalytic promiscuity. Biochemistry, 40: 1567-1576.
Ren, Q., Sun, R.R., Zhao, X.F. and Wang, J.X. (2009) A selenium-dependent glutathione peroxidase (Se-GPx)and two glutathione S-transferases (GSTs) from Chinese shrimp (Fenneropenaeus chinensis). Comparative Biochemistry and Physiology. C, Toxicology and Pharmacology, 149: 613-623.
Rushmore, T.H. and Pickett, C.B. (1993) Glutathione S-transferase,structure, regulation and therapeutic implications. The Journal of Biological Chemistry, 268: 11475-11478.
Sinning, I., Kleywegt, G.J., Cowan, S.W., Reinemer, P., Dirr, H.W., Huber, R., Gilliland, G.L,, Armstrong, R.N., Ji, X., Board, P.G., Olin, B., Mannervik, B. and Jones, T.A. (1993) Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the mu and pi class enzymes. Journal of Molecular Biology, 232:192-212.
Sun, Y., Yu, H., Zhang, J., Yin, Y., Shen, H., Liu, H. and Wang, X. (2006) Bioaccumulation and antioxidant responses in goldfish Carassius auratus under HC Orange No 1 exposure. Ecotoxicology and Environmental Safety, 63: 430-437.
Sun, Y.J., Kuan, I.C., Tam, M.F. and Hsiao, C.D. (1998)The three-dimensional structure of an avian class-mu glutathione-S-transferase, cGSTM1-1 at 1.94 A resolution. Journal of Molecular Biology, 278:239-252.
Wan, Q., Whang, I. and Lee, J. (2008) Molecular characterization of mu class glutathione-S-transferase from disk abalone (Haliotis discus discus), a potential biomarker of endocrine-disrupting chemicals. Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 150:187-99.
Wan, Q., Whang, I. and Lee, J. (2008) Molecular cloning and characterization of three sigma glutathione S-transferases from disk abalone (Haliotis discus discus). Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology, 151:257-267.
