Studies on the structure and immunological activity of carcinoembryonic antigen - the role of disulphide bonds

Abstract

Carcinoembryonic antigen (CEA) has been shown to contain no free cysteine thiol groups but 6 cystine disulphide bonds. 5'5-Dithiobis-(2-nitrobenzoic acid) (DTNB) will react with CEA only after reduction of the disulphide bonds with dithioerythritol. Reduction-alkylation of CEA using dithioerythritol and bromo-[1-14C] acetic acid confirmed the presence of 6 disulphide bonds, as did oxidation of the glycoprotein with performic acid. The products from the DTNB and reduction-alkylation treatments of CEA had less capacity to inhibit the binding of [125I]-CEA to anti-CEA in a radioimmunoassay than the original CEA but could, in sufficient quantities, totally inhibit the binding. Removal, using mercaptoethanol, of the thiol blocking groups from the DTNB-treated CEA resulted in a 55% recovery of antigenic activity. The product from the performic acid oxidation could only inhibit approximately 50% of the binding. Treatment of CEA with 0.533M sodium periodate (NaIO4) greatly reduced its antigenic activity, presumably a result of the oxidative cleavage of the disulphide bonds. No loss in activity, however, was observed when 5.33mM NaIO4 was used, and one Smith degradation (i.e. treatment in sequence with periodate, borohydride and mild acid) of CEA removed approximately 50% of the carbohydrate, including all of the fucose, sialic acid and 2-acetamido-2-deoxygalactose but did not change the antigenic activity.