Purification and composition of a novel gastrointestinal tumor-associated glycoprotein expressing sialylated lacto-N-fucopentaose II (CA 19-9).
Monoclonal antibody 1116NS 19-9 (Mab 19-9) exhibits selective reactivity with human gastrointestinal carcinomas and recognizes a carbohydrate determinant (CA 19-9) defined as a sialylated lacto-N-fucopentaose II. A scheme was devised for the purification of a human gastrointestinal tumor-associated glycoprotein antigen expressing CA 19-9 from colorectal carcinoma cell line SW1116 culture media in high yield. The key steps in the purification were immunoaffinity column chromatography with Mab 19-9 followed by reduction and alkylation of the specifically bound proteins in the presence of 6 M guanidine hydrochloride and a second Mab 19-9 immunoaffinity fractionation. The purified CA 19-9 containing glycoprotein ran as a single band on sodium dodecyl sulfate-polyacrylamide gradient gels with an apparent molecular mass of 210 kilodaltons. In the absence of detergents, this purified glycoprotein apparently reassociated to form aggregates of 600-2000 kilodaltons molecular mass as determined by size-exclusion chromatography. Amino acid analysis of CA 19-9 containing glycoprotein revealed that serine, threonine, and proline together accounted for greater than 35% of the amino acid residues, consistent with a mucin-like structure for the protein. Carbohydrate compositional analysis, however, was in contrast to a typical mucin with a fucose:mannose:galactose:N-acetylgalactosamine: N-acetylglucosamine:N-acetylneuraminic acid molar ratio of 4:1:12:2.5:5:5. The presence of both N-acetylgalactosamine and mannose suggested that both O- and N-linked oligosaccharides may exist on CA 19-9 containing glycoprotein. Protein and carbohydrate analyses indicated that this novel tumor-associated glycoprotein was 85% carbohydrate by weight. This purification procedure may be applicable to the isolation of other epithelial tumor-associated antigens.
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000032:analysis / Q000302:isolation & purification*; D015295:Antigens, Tumor-Associated, Carbohydrate; D002241:Carbohydrates / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis / Q000302:isolation & purification*; D005770:Gastrointestinal Neoplasms / Q000276:immunology*; D006023:Glycoproteins / Q000032:analysis / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D002241:Carbohydrates; D000939:Epitopes; D006023:Glycoproteins