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

Abstract

Summary.-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 reductionalkylation treatments of CEA had less capacity to inhibit the binding of [1251]-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 550o recovery of antigenic activity. The product from the performic acid oxidation could only inhibit approximately 5o0°/ 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%O of the carbohydrate, including all of the fucose, sialic acid and 2-acetamido-2-deoxygalactose but did not change the antigenic activity.

Localization of human breast-carcinoma xenografts using antibodies to carcinoembryonic antigen

“…The antibody and iodine labelling.-The CEA used as immunogen and for coupling of Sepharose 4B (see below) was isolated from hepatic metastases of a human colonic carcin-oma and purified to satisfy the criteria previously described (Westwood & Thomas, 1975;Westwood, 1978). Anti-CEA sera were raised by monthly s.c. injections of a goat with 100 jug of CEA, emulsified with Freund's adjuvant.…”

Chemical modifications of the protein of carcinoembryonic antigen: associated changes in immunological activity and conformation

“…Our slight modifications to the method of isolation and criteria of purity of the material have been reported previously (Westwood and Thomas, 1975).…”

“…Tryptophan was determined spectrophotometrically. Details of our use of these methods have been previously described (WestwNood and Thomas, 1975). Sialic acid was determined using Warren's (1959) thiobarbituric acid method.…”

“…Inhibitory activities in the radioimmunoassay were measured. In a similar experiment to the one described above, performic-acid-oxidized CEA (Westwood and Thomas, 1975) was treated with acetyl imidazole and the amount of substitution of the tyrosine residues determined.…”

“…OUR results have so far shown that the antibodies in goat anti-CEA sera § are directed predominantly, if not totally, against sections of the protein part of the molecule (Westwood and Thomas, 1975). No evidence is yet available which gives information about the precise nature of the antigenic determinants.…”