Abstract
We have characterized a new selenium-dependent glutathione peroxidase, GSHPx-GI, by expressing a GSHPx-GI cDNA isolated from human hepatoma HepG2 cells in human mammary carcinoma MCF-7 cells, which have virtually undetectable expression of either the classical cellular enzyme, GSHPx-1, or GSHPx-GI at the protein level. One of the G418-resistant clones, neo-D1, expresses the transfected GSHPx-GI cDNA. This is based on 1) the presence of an additional GSHPx-GI DNA restriction fragment detected by Southern analysis; 2) the presence of a 1.9-kilobase (kb) GSHPx-GI mRNA in addition to the 1.0-kb endogenous mRNA by Northern analysis; and 3) the appearance of a 22-kDa 75Se-labeled protein which is absent in parental MCF-7 cells revealed by SDS-polyacrylamide gel electrophoresis. GSHPx-GI expressed in neo-D1 is a tetrameric protein localized in cytosol. GSHPx-GI does not cross-react with antisera against human GSHPx-1 or human plasma glutathione peroxidase (GSHPx-P). Similar substrate specificities are found for GSHPx-1 and GSHPx-GI; they both catalyze the reduction of H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and linoleic acid hydroperoxide with glutathione, but not of phosphatidylcholine hydroperoxide. GSHPx-GI mRNA was readily detected in human liver and colon, and occasionally in human breast samples, but not other human tissues including kidney, heart, lung, placenta, or uterus. In rodent tissues, GSHPx-GI mRNA is only detected in the gastrointestinal tract, and not in other tissues including liver. In fact, GSHPx-GI appears to be the major glutathione-dependent peroxidase activity in rodent GI tract. This finding suggests that GSHPx-GI could play a major role in protecting mammals from the toxicity of ingested lipid hydroperoxides. In conclusion, we have demonstrated that GSHPx-GI is the fourth member in the selenium-dependent glutathione peroxidase family, in addition to GSHPx-1, GSHPx-P, and phospholipid hydroperoxide glutathione peroxidase (PHGPX).
Highlights
From the Department of Medical Oncology and Therapeutics Research, City of Hope Natwml Medical Center, Duarte, California 91010
Sequence discrepancies were found at five locations between our full length HepG2 clone and the sequence published by Akasaka et al, and at four locations between our HepGZ clone and our liver clone
The only place that all threeclones have different sequences is at nucleotides 143-145, which results in three different codons
Summary
It has a UGA codon for selenocysteine a t nucleotide positions 152-154. We have transfected the full length HepG2 GSHPx-GI cDNA in pRSV, a mammalian expression vector, into a human mammary carcinoma cell line, MCF-7 cells. Sixteen transfectants which were resistant t o 400 pg/ml G418,and six transfectants which were resistant t o 8 ng/ml colchicine were isolated as individual clones. All of these transfectantswere analyzed by SDS-PAGE for [7sSe] selenoproteins, Northern and Southern analyses.
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