Single-nucleotide polymorphic variants of human glutathione transferase T1-1 differ in stability and functional properties.
We have previously expressed hexa-histidine-tagged human glutathione transferase GST T1-1 at very high levels in an Escherichia colilacZ mutagenicity assay strain. Ethylene dibromide (EDB), which is activated by GST T1-1, produces a potent response in the mutation assay. We have now constructed and expressed two SNP variants of wild-type GST T1-1:D141N and E173K. The EDB activation activities of both variant enzymes, as measured by the lacZ mutagenicity assay, are greatly reduced The D141N variant behaved similarly to the wild-type enzyme, in terms of expression level and specific activities for conjugation of glutathione with 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), ethylene diiodide (EDI), and 4-nitrobenzyl chloride (NBCl), and for peroxidative detoxication of cumene hydroperoxide (CuOOH). In contrast, variant E173K is poorly expressed, has no detectable activity with EPNP, NBCl, or CuOOH, and has EDI activity much lower than that of the wild-type enzyme. The circular dichroism (CD) thermal denaturation profiles of the wild-type protein and variant D141N show a sharp two-state transition between native and denatured states. Variant E173K showed a very different profile, consistent with improper or incomplete protein folding. Our results show that SNP variants can give rise to GSTT1-1 proteins with significantly altered properties.
Josephy
P David
PD
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ont., Canada N1G 2W1. djosephy@uoguelph.ca
Kent
Meredith
M
Mannervik
Bengt
B
D001555:Benzene Derivatives / Q000493:pharmacokinetics*; D004795:Enzyme Stability / Q000235:genetics*; D004852:Epoxy Compounds / Q000378:metabolism; D014644:Genetic Variation; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006847:Hydrocarbons, Iodinated / Q000378:metabolism; D008658:Inactivation, Metabolic / Q000235:genetics; D009578:Nitrobenzenes / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide
D001555:Benzene Derivatives; D004852:Epoxy Compounds; D006847:Hydrocarbons, Iodinated; D009578:Nitrobenzenes; D009596:Nitrophenols; C027562:4-nitrobenzyl chloride; C007606:1,2-epoxy-3-(p-nitrophenoxy)propane; D005982:Glutathione Transferase; C495790:glutathione transferase T1-1, human; C007164:cumene hydroperoxide