Transsulfuration Is a Significant Source of Sulfur for Glutathione Production in Human Mammary Epithelial Cells

Abstract

The transsulfuration pathway, through which homocysteine from the methionine cycle provides sulfur for cystathionine formation, which may subsequently be used for glutathione synthesis, has not heretofore been identified as active in mammary cells. Primary human mammary epithelial cells (HMEC's) were labeled with ³⁵S-methionine for 24 hours following pretreatment with a vehicle control, the cysteine biosynthesis inhibitor propargylglycine or the gamma-glutamylcysteine synthesis inhibitor buthionine sulfoximine. Cell lysates were prepared and reacted with glutathione-S-transferase and the fluorescent labeling compound monochlorobimane to form a fluorescent glutathione-bimane conjugate. Comparison of fluorographic and autoradiographic images indicated that glutathione had incorporated ³⁵S-methionine demonstrating that functional transsulfuration occurs in mammary cells. Pathway inhibitors reduced incorporation by roughly 80%. Measurement of glutathione production in HMEC's treated with and without hydrogen peroxide and/or pathway inhibitors indicates that the transsulfuration pathway plays a significant role in providing cysteine for glutathione production both normally and under conditions of oxidant stress.

Figure 1

The transsulfuration pathway connects methionine and glutathione biosynthesis. In the methionine cycle, methionine forms S-adenosylmethionine which serves as a methyl donor, generating S-adenosyl homocysteine. This is converted to homocysteine, which is subsequently converted back into methionine. Homocysteine has an alternative fate, however. It can be used to produce cystathionine, which is further converted to cysteine. This latter conversion is catalyzed by gamma-cystathionase and inhibited by propargylglycine. Cysteine can then feed glutathione biosynthesis through production of gamma-glutamylcysteine. This step is catalyzed by gamma-glutamylcysteine synthase and inhibited by buthionine sulfoximine.

Figure 2

Transsulfuration is a significant source of sulfur for glutathione synthesis in human mammary cells. human mammary epithelial cells (HMEC's) were pretreated with vehicle control, pathway inhibitors buthionine sulfoximine (BSO), or propargylglycine (PPG) for 24 hours then labeled with ³⁵S-methionine for 24 hours. Lysate and glutathione bimane conjugates were prepared and analyzed by thin layer chromatography and autoradiography as described under methods. Comparison of the images (fluorescence lanes 1 and 2 with autoradiography lanes 1 and 2) indicates that the fluorescent GSH-MCBi conjugate is radioactive. Incorporation of  ³⁵S-methionine into glutathione (GSH-MCBi bands) demonstrates that functional transsulfuration occurs in mammary cells. PPG inhibitory impact on either glutathione synthesis from all cysteine sources (left panel, fluorescence, compare PPG with control) or the incorporation of  ³⁵S-methionine labeled cysteine (which must be transsulfuration derived) into glutathione demonstrates both transsulfuration and the identity of the TLC spots. BSO predictably inhibited production of glutathione without regard to cysteine source.

Figure 3

Impact of transsulfuration inhibition (PPG) on cellular total glutathione levels in human mammary epithelial cells subjected to oxidative challenge (H₂O₂). Human mammary epithelial cells (HMEC's) were grown in normal mammary epithelial growth medium to 50% confluency and pretreated with a PBS vehicle control or propargylglycine (PPG) for 24 hours, followed by treatment in the same media with vehicle or 300 uM H₂O₂ for two hours. Cell pellets were prepared and analyzed for total glutathione levels as described under methods [45]. Results are expressed as nmol/mg of cell protein (mean ± SEM, N = 5-6). Asterisk indicates a significant difference between PBS/H₂O₂ and PPG/H₂O₂ groups determined by ANOVA (P < 0.002).