Abstract 1780 Effects of Oxygen on Lung Oxidant/Antioxidant Balance Poster Symposium, Sunday, 5/2

Human glutathione reductase (hGR) is a homodimeric protein that catalyzes the reduction of glutathione disulfide (GSSG) to GSH, and thereby contributes to the primary cellular antioxidant defense system. Chinese hamster ovary (CHO) cells transfected with a hGR expression vector (pRC-hGR) acquired increased cytosolic GR activities, and demonstrated greater resistance to tert-butyl hydroperoxide than did non-transfected control cells. We hypothesized that GR activities could be attenuated by transfecting cells with hGR expression constructs containing the mutations C58A and H467L, which substantially alter essential components of the GSSG binding catalytic domain. CHO cells transfected with the C58A/H467L double mutant construct expressed 10-fold more transgene than endogenous GR, but demonstrated only modest decreases in GR activities. In cells co-transfected with the pRC-hGR and the mutant constructs, the dominant negative construct was much more effective in attenuating the increased GR activities obtained by transfection with pRC-hGR. These observations suggested that the human GR monomer did not dimerize efficiently with the native hamster protein, a hypothesis we tested by transfecting human H441 cells with the C58A/H467L double mutant. H441 cells transiently transfected with 10 µg of the mutant construct had 15% lower GR activities than control cells (18.0 ± 1.33 vs. 21.3 ± 2.67 mU/mg protein), while cells transfected with 20 µg of the construct retained only 50% (10.4 ± 5.15 mU/mg protein) of endogenous GR activities. These observations indicate that the mutant hGR gene product can dimerize more effectively with the endogenous human protein and diminish intracellular GR activities. The greater attenuations of GR activities seen in cells transfected with 20 µg of DNA were probably caused by greater transfection efficiencies in that group. This implies that a range of GR hypomorphs with mild, moderate or severe deficiencies in GR function may be attainable by generating stable cell lines containing varying copy numbers of the dominant negative transgene. Although the GR peptide sequence is highly conserved across species, it appears that GR homodimer formation is a very species-specific process. For this reason, we anticipate that construction of dominant negative mutants of the murine GR cDNA will be an essential prerequisite for use of this strategy to produce transgenic hypomorphic GR mice.

NIH U10 HL52637 and GM44263.