Guy W. MAINWARING, John R. FOSTER and Trevor GREEN1. Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10-4TJ,. U.K..
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Biochem. J. (1998) 329, 431–432 (Printed in Great Britain)
Nuclear and cellular immunolocalization of Theta-class glutathione S-transferase GSTT1-1 in the liver and lung of the mouse The Theta family of soluble glutathione S-transferases (GSTs) has been recognized in rats, mice and humans [1,2] and, at present, three rat Theta enzymes are known (rGSTT1-1, rGSTT22 and rGSTT3-3), two mouse enzymes (mGSTT1-1 and mGSTT2-2) and two human enzymes (hGSTT1-1 and hGSTT22). Sequencing has revealed that rat GSTT1-1, mouse GSTT1-1 and human T1-1 are orthologous proteins, as are rat GSTT2-2, mouse T2-2 and human T2-2. In a recent publication, we used in situ hybridization to show that the distribution of Theta glutathione S-transferase GSTT1-1 mRNA in mouse tissues differed markedly from that in rat or human tissues [3]. Specifically, the mRNA in mouse tissues was present in much higher concentrations, and showed a preferential distribution in certain cell types and in nuclei, which was not seen in rat or human tissues. The specific cellular and nuclear localization of GSTT1-1 was considered to be a major factor in the mouse-specific carcinogenicity of methylene chloride [4], a substrate for this enzyme. The high nuclear concentrations of mRNA were an unusual finding, and it was uncertain whether high mRNA concentrations in nuclei reflected high enzyme-protein concentrations, since the nucleus is not a major site of protein synthesis. This question has now been addressed by raising an antibody to this enzyme and studying the distribution of the GSTT1-1 protein in the same tissues. Two antibodies were generated to two different peptides
MM (kDa)
derived from mGSTT1-1 ²VA1 (228–240) [C]KQKLMPRVLTMIQ and VA2 (150–161) [C]KFLQDKDFLVGP´. The peptides were synthesized and conjugated to keyhole-limpet haemocyanin by Cambridge Research Biochemicals (Gadbrook Park, Northwich, Cheshire, U.K.) and the antibodies raised in rabbits. Western blots of mouse and rat liver cytosol gave single bands of the right molecular mass and showed clearly that GSTT1-1 is far more abundant in mouse liver cytosol than in rat liver cytosol (Figure 1). This is consistent with the 10-fold higher activity measured in mouse liver cytosol with the specific GSTT1-1 substrate methylene chloride. Immunocytochemistry, which was performed using the same methodology as that previously described [3], demonstrated exactly the same distribution of GSTT1-1 protein as that seen for
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Figure 1 Western blot of GSTT1-1 in mouse and rat liver cytosol using an antibody raised to peptide sequence VA1 Lane 1, 5 g of mouse liver cytosol ; lane 2, 5 g of rat liver cytosol ; lane 3, 10 g of mouse liver cytosol ; lane 4, 10 g of rat liver cytosol ; lane MMS, molecular-mass (MM) standards.
Figure 2 Immunolocalization of GSTT1-1 in mouse liver (a) and lung (b) sections using an antibody raised to peptide sequence VA1 The presence of the enzyme is indicated by brown staining. In the liver, the limiting-plate hepatocytes around the central vein, and some nuclei, have high concentrations of GSTT1-1. In the lung, the Clara cells in the bronchiolar epithelium contain the highest amount of enzyme.
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GSTT1-1 mRNA in the livers and lungs of mice, rats and humans [3]. Both antibodies (VA1 and VA2) also showed the same distribution, although the titre with VA2 was lower than that of VA1. Figure 2 illustrates the distribution of GSTT1-1 protein (with VA1) in male B C F mouse liver and lung. In the ' $ " livers, the highest concentrations are found in the limiting plate hepatocytes of the central vein and in the nuclei of some, but not all, cells. The periportal hepatocytes remained largely unstained (Figure 2a). Interlobular-bile-duct epithelial cells also showed immunostaining at levels comparable with those seen in the central-vein hepatocytes. The distribution of GSTT1-1 protein in the lungs of mice is also shown (Figure 2b). As was seen previously for mRNA [3], the GSTT1-1 protein was localized in the bronchiolar epithelium and in the type II alveolar cells of the lung parenchyma. Within the bronchiolar epithelium itself the apical cytoplasm of Clara cells contained the highest concentrations of reaction product, with lesser amounts also present in the ciliated bronchiolar cells. The present study, using an antibody to GSTT1-1, has confirmed that the cellular and species-specific distribution of GSTT1-1 mRNA [3] does indeed reflect the distribution of GSTT1-1 protein. Although nuclear GSTs are well known [5–7], the role of GSTT1-1 in the nuclei of mouse cells remains unclear, as does the role of high nuclear concentrations of mRNA in the synthesis of these enzymes. The presence of this enzyme in some, but not all, nuclei, and in only one of the two nuclei of binucleate
cells, has also been a consistent finding which has yet to be explained. Thus, in addition to the activation of mouse carcinogens such as methylene chloride, it would appear that a much more fundamental role for this nuclear GST has yet to be discovered.
Guy W. MAINWARING, John R. FOSTER and Trevor GREEN1 Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10-4TJ, U.K. 1
To whom correspondence should be addressed.
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Hayes, J. D. and Pulford, D. J. (1995) Crit. Rev. Biochem. Mol. Biol. 30, 445–600 Mainwaring, G. W., Nash, J. A., Davidson, M. and Green, T. (1996) Biochem. J. 314, 445–448 Mainwaring, G. W., Williams, S. M., Foster, J. R., Tugwood, J. and Green, T. (1996) Biochem. J. 318, 297–303 National Toxicology Program (1986) Natl. Toxicol. Program Tech. Rep. Ser. no. 306 Tan, K. H., Meyer, D. J., Gillies, N. and Ketterer, B. (1988) Biochem. J. 254, 841–845 Abei, M., Harad, S., Tanaka, N., McNeil, M. and Osuga, T. (1989) Biochim. Biophys. Acta 995, 279–284 Tirmenstein, M. A. and Reed, D. J. (1989) Biochim. Biophys. Acta 995, 174–180
Received 11 September 1997
