小鼠θ类谷胱甘肽转移酶mGSTT1-1的基因结构、表达及染色体定位
Gene structure, expression and chromosomal localization of murine theta class glutathione transferase mGSTT1-1.
作者信息
Whittington A, Vichai V, Webb G, Baker R, Pearson W, Board P
机构信息
Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, Australian Capital Territory, 2601 Australia.
出版信息
Biochem J. 1999 Jan 1;337 ( Pt 1)(Pt 1):141-51.
We have isolated and characterized a cDNA and partial gene encoding a murine subfamily 1 Theta class glutathione transferase (GST). The cDNA derived from mouse GSTT1 has an open reading frame of 720 bp encoding a peptide of 240 amino acids with a calculated molecular mass of 27356 Da. The encoded protein shares only 51% deduced amino acid sequence identity with mouse GSTT2, but greater than 80% deduced amino acid sequence identity with rat GSTT1 and human GSTT1. Mouse GSTT1-1 was expressed in Escherichia coli as an N-terminal 6x histidine-tagged protein and purified using immobilized-metal affinity chromatography on nickel-agarose. The yield of the purified recombinant protein from E. coli cultures was approx. 14 mg/l. Recombinant mouse GSTT1-1 was catalytically active towards 1, 2-epoxy-3-(p-nitrophenoxy)propane, 4-nitrobenzyl chloride and dichloromethane. Low activity towards 1-menaphthyl sulphate and 1-chloro-2,4-dinitrobenzene was detected, whereas mouse GSTT1-1 was inactive towards ethacrynic acid. Recombinant mouse GSTT1-1 exhibited glutathione peroxidase activity towards cumene hydroperoxide and t-butyl hydroperoxide, but was inactive towards a range of secondary lipid-peroxidation products, such as the trans-alk-2-enals and trans,trans-alka-2,4-dienals. Mouse GSTT1 mRNA is most abundant in mouse liver and kidney, with some expression in intestinal mucosa. Mouse GSTT1 mRNA is induced in liver by phenobarbital, but not by butylated hydroxyanisole, beta-napthoflavone or isosafrole. The structure of mouse GSTT1 is conserved with that of the subfamily 2 Theta class GST genes mouse GSTT2 and rat GSTT2, comprising five exons interrupted by four introns. The mouse GSTT1 gene was found, by in situ hybridization, to be clustered with mouse GSTT2 on chromosome 10 at bands B5-C1. This region is syntenic with the location of the human Theta class GSTs clustered on chromosome 22q11.2. Similarity searches of a mouse-expressed sequence tag database suggest that there may be two additional members of the Theta class that share 70% and 88% protein sequence identity with mouse GSTT1, but less than 55% sequence identity with mouse GSTT2.
我们已经分离并鉴定了一个编码小鼠1类Theta型谷胱甘肽S-转移酶（GST）的cDNA和部分基因。从小鼠GSTT1获得的cDNA具有720 bp的开放阅读框，编码一个240个氨基酸的肽段，计算分子量为27356 Da。该编码蛋白与小鼠GSTT2的推导氨基酸序列同一性仅为51%，但与大鼠GSTT1和人GSTT1的推导氨基酸序列同一性大于80%。小鼠GSTT1-1在大肠杆菌中作为N端带有6x组氨酸标签的蛋白表达，并使用镍琼脂糖上的固定金属亲和色谱法进行纯化。从大肠杆菌培养物中纯化的重组蛋白产量约为14 mg/l。重组小鼠GSTT1-1对1,2-环氧-3-（对硝基苯氧基）丙烷、4-硝基苄氯和二氯甲烷具有催化活性。检测到对1-萘基硫酸盐和1-氯-2,4-二硝基苯的活性较低，而小鼠GSTT1-1对依他尼酸无活性。重组小鼠GSTT1-1对氢过氧化异丙苯和叔丁基过氧化氢表现出谷胱甘肽过氧化物酶活性，但对一系列二级脂质过氧化产物，如反式-alk-2-烯醛和反式，反式-alka-2,4-二烯醛无活性。小鼠GSTT1 mRNA在小鼠肝脏和肾脏中最为丰富，在肠黏膜中也有一些表达。小鼠GSTT1 mRNA在肝脏中可被苯巴比妥诱导，但不能被丁基羟基茴香醚、β-萘黄酮或异黄樟素诱导。小鼠GSTT1的结构与2类Theta型GST基因小鼠GSTT2和大鼠GSTT2的结构保守，由五个外显子和四个内含子间隔组成。通过原位杂交发现，小鼠GSTT1基因在10号染色体的B5-C1带与小鼠GSTT2聚集在一起。该区域与人22q11.2染色体上聚集的Theta型GSTs的位置是同线的。对小鼠表达序列标签数据库的相似性搜索表明，Theta类可能还有另外两个成员，它们与小鼠GSTT1的蛋白序列同一性分别为70%和88%，但与小鼠GSTT2的序列同一性小于55%。
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