No abstract

In order to determine the structure of the tumor-specific immunodominant group of the carcinoembryonic antigen ( C E A ) of human gastro-intestinal tumors, immunologically active fragments from this glycoprotein were prepared by partial enzymatic hydrolysis using the proteolytic enzyme nagase. The fragments were purified by sequential molecular sieve, adsorption and ion exchange chromatography, and were analysed for carbohydrate and amino-acid composition by gas-liquid chromatography. The antigenic activity of each fragment was tested using both the Z-gel and Farr radioimmunoassay techniques for CEA. Immunologically active glycopeptides with molecular weights of 1,000-5,000 Daltons, which contained the tumor antigenic determinant of CEA, had relatively high contents of N-acetyl-D-ghcosamine, aspartic acid or asparagine, and glutamic acid or glutamine.The carcinoembryonic antigen (CEA) is found in adenocarcinomas of the human digestive tract and in digestive organs of the human fetus during the first two trimesters of gestation (Gold and Freedman 1965a, b). The CEA molecule is a glycoprotein with a usual carbohydrate/protein ratio of 2-3/1 (Banjo et al., 1972), although greater variation has on occasion been seen. The molecule contains no lipid (Banjo et al., in press). In a previous report from this laboratory (Banjo et al., 1972) data were presented which indicated that a heterosaccharide grouping with a relatively high concentration of N-acetylglucosamine (GlcNac) may be of major importance in the core structure or the immunologic determinant of the tumor-specific site of the CEA molecule.The purpose of the present investigation was to prepare immunologically active glycopeptides from CEA by enzymatic degradation of this material in an attempt to elucidate the chemical structure of the carbohydrate chain(s) responsible for the tumor-specific antigenicity of the CEA molecule. MATERIAL AND METHODS Chemical reagentsAll solutions were prepared in deionized water. Reagents were of analytical grade. Neuraminidase (3.3.1.18) Preparation of CEA and anti-CEA antisera Purified CEA was prepared from the hepatic metastases of a colonic carcinoma, arising in a patient of blood group A, according to the method of Krupey et a/. (1972). The anti-CEA antiserum was raised in a goat by repeated intramuscular injections of 1 mg aliquots of purified CEA emulsified in Freund's Complete Adjuvant. To assure monospecificity against the tumor-specific grouping of the CEA molecule, the antiserum was thoroughly absorbed with large quantities of normal human serum, normal human liver and normal human bowel, which had been extracted in 1.0 M perchloric acid, and subsequently dialyzed against distilled water and lyophilized.Neuraminidase treatment of CEA A 325mg aliquot of purified CEA was incubated with 10mg of neuraminidase in 4 m l of sodium acetate buffer (PH 5.1, 0.5 M) for 20 h at 37" C (Burton, 1963). After incubation, the reaction mixture was applied to a Sephadex G-200 column (95 cm x 5 cm) previously equilibrated with 0.05 M a...