ANALYSIS OF A HETEROGENEOUS GROUP OF HUMAN BREAST-CARCINOMA ASSOCIATED GLYCOPROTEINS BEARING THE TN DETERMINANT

The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A mu rine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. I n the present study we characterized the glycoprotein antigen identifi ed by 83D4 in the human breast carcinoma cell line MCF-7. We further s howed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m-periodate tr eatment after desialylation. Western-blot analysis resolved the 83D4 a ntigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE rev ealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typica l densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but p oor in threonine and proline, confirmed the divergence from other muci n-like carcinoma-associated glycoproteins. Dicarboxylic amino acids we re abundant, accounting in part for the acidic nature of the molecules . ELISA and Western-blot analysis of the subcellular fractions from MC F-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (s low and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-gl ycosylation in tumor cells.