Glutathione (GSH) contents and capacity to increase GSH synthesis in response to stress are important in cellular protection. Premature infants have low plasma GSH concentrations, suggesting a possible functional deficit. GSH synthesis begins with the rate-limiting, ATP-dependent addition of cysteine to glutamate by GGCS, which exists as a dimer of non-identical subunits. The heavy chain subunit (GGCS-Hc) contains the active site and is feedback inhibited by GSH, while the light subunit (GGCS-Lc) is important in regulation of enzyme activity. In an effort to augment antioxidant defense functions by enhancing GSH synthetic capacity, we used liposome-mediated transfer of the cDNAs for rat GGCS-Hc or GGCS-Lc to transfect CHO cells. GGCS activities were increased from 0.50±0.09 (mean±SD) mU/mg pro to 10.63±2.96 by transfection with the GGCS-Hc cDNA, and to 0.96±0.12 with the GGCS-Lc cDNA (n=4-6). CHO cell lines with stably enhanced GGCS activities (GGCS-h) were developed, and Southern and northern analyses indicate the presence of the transcript in genomic DNA, and increased transcription of the GGCS-Hc gene, respectively. Treated and selected CHO cells that showed no increases in GGCS activities were used as controls. Northern analyses of the stable cell lines revealed GGCS-Lc mRNA levels to be comparable to the native CHO cell line. To determine whether the enhanced GGCS activities of the GGCS-h cells could be further augmented by increased expression of the GGCS-Lc subunit, GGCS-u and GGCS-h cells were transfected with either 4 or 8 μg of a plasmid containing the GGCS-Lc cDNA. GGCS activities of the GGCS-h cells were increased by more than 200% following transfection with the GGCS-Lc cDNA (22.56±1.22 mU/mg pro in the 8 μg group vs. 6.17±0.35 in the basal GGCS-h cells); similar transfections in stably transfected cell lines that displayed unchanged GGCS activities(GGCS-u) resulted in minimal change in GGCS activities (0.44±0.01 vs. 0.35±0.06 respectively)(n=3).
Conclusion: These results demonstrate that increases in GGCS activities in CHO cells achieved by stable transfection with the GGCS-Hc cDNA can be enhanced further by transient transfection with the cDNA for the GGCS-Lc regulatory subunit. Enhanced expression of GGCS-Hc alone offers significant protection against oxidant stresses, and the present data suggest that increases in GGCS-Lc expression may provide even greater GSH synthetic capacities and protection against oxidant stress.
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(Supported by Wyeth Pediatric Grant Program and HD27823).
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Fernandes, C., Rogers, L., McMicken, H. et al. Transient Transfection with γ-Glutamylcysteine synthetase (GGCS) Light chain cDNA augments stably enhanced GGCS activities of Chinese Hamster Ovary(CHO) cells † 1005. Pediatr Res 43 (Suppl 4), 173 (1998). https://doi.org/10.1203/00006450-199804001-01026
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DOI: https://doi.org/10.1203/00006450-199804001-01026