Preparation and isolation of immunologically active glycopeptides from carcinoembryonic antigen (CEA)

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...

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

“…Evidence for the involvement of 2-acetamido-2-deoxyglucose in the immunodominant grouping has been presented by Banjo et al (1972) and is well supported by further work from the same laboratories (Banjo et al, 1974). In addition, Watanabe and Hakomori (1973) have shown that purified glycosphingolipids from tumour tissue or erythrocytes give a strong precipitin reaction with wheat germ lectin and with anti-CEA.…”

“…In addition, Watanabe and Hakomori (1973) have shown that purified glycosphingolipids from tumour tissue or erythrocytes give a strong precipitin reaction with wheat germ lectin and with anti-CEA. It is known that sialic acid does not form part of the immunological determinant (Banjo et al, 1972;Coligan et al, 1973), nor, according to recent work on the treatment of CEA with periodate, does fucose (see Terry et al, 1974). Recently, Neveu et al (1975) proposed that CEA and the " nonspecific crossreacting antigen ", NCA, have common antigenic determinants on both the carbohydrate and polypeptide parts of the molecule, while Hammarstrom et al (1975) conclude, tentatively, that the carbohydrate moiety of CEA does not contain the tumour associated determinant(s).…”

“…The latter statement concurs with our report (Westwood, Thomas and Foster, 1974b) and that of Coligan and Todd (1975) that a large quantity of carbohydrate in CEA can be destroyed using periodate, without causing any loss in antigenic activity. Whichever parts of the molecule constitute the immunological determinants, carbohydrate or protein or both, it is obvious that disorientation of the determinant groups by cleavage of the protein chain using either proteolytic enzymes (Banjo et al, 1974) or dilute acid or alkali (Westwood et al, 1974a) produces a marked reduction in the ability of the molecule to bind to anti-CEA antiserum.…”

Study of the antigenic cross reactivity between carcinoembryonic antigen and “nonspecific cross reacting antigens” (NCA and NCA 2)

“…It cannot be excluded that this is due to similarity between the protein moieties of molecules but it seems more probable that the glucidic moieties were involved. These moieties play an important role in the reactivity of CEA (and logically NCA) with specific antibody, as judged by the work of Banjo et al (1974); these authors demonstrated that acetylglucosamine and asparagine played an important role in the main antigenic determinant of CEA.…”

Carcinoembryonic antigen: characterization of binding with insoluble lectins

“…No other carbohydrates were detected. In a subsequent report, digestion of CEA with the proteolytic enzyme nagase resulted in immunologically active glycopeptides (Banjo et al, 1974). N-acetyl D-glucosamine was present on all the fragments which were antigenic.…”