Covalent modification of proteins by green tea polyphenol (-)-epigallocatechin-3-gallate through autoxidation

Green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has various beneficial properties including chemopreventive, anticarcinogenic, and antioxidant actions. The interaction with proteins known as EGCG-binding targets may be related to the anticancer effects. However, the binding mechanisms for this activity remain poorly understood. Using mass spectrometry and chemical detection methods, We found that EGCG forms covalent adducts With cysteinyl thiol residues in proteins through autoxidation. To investigate the functional Modulation Caused by binding of EGCC., we examined the interaction between EGCG and a thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Concentration-dependent covalent binding of EGCG to GAPDH was found to be Coupled to the irreversible inhibition of GAPDH activity. Mutation experiments revealed that EGCG is primarily bound to the cysteinyl thiol group of the active center, indicating that the irreversible inhibition of GAPDH is Clue to the covalent attachment of EGCG to tire active-center cysteine. Moreover, Using EGCG-treated cancer cells, we identified GAPDH as a target of EGCG covalent binding through specific interactions between catechols and aminophenyl boronate agarose resin. Based oil these findings, we propose that the covalent modification of proteins by EGCG may be a novel pathway related to the biological activity of EGCG. (C) 2008 Elsevier Inc. All rights reserved.