MONOCLONAL-ANTIBODY BW835 DEFINES A SITE-SPECIFIC THOMSEN-FRIEDENREICH DISACCHARIDE LINKED TO THREONINE WITHIN THE VTSA MOTIF OF MUC1 TANDEM REPEATS

mAb BW835 (IgG1) has been generated to breast cancer cell lines by alt ernating injections of MCF-7 or SW-613 cells and has been demonstrated to be of value in the serodiagnosis of mammary carcinoma. BW835 defin es a carbohydrate epitope on integrated or secreted MUC1 glycoforms fr om carcinoma cells and human milk. To identify BW835-reactive glycopep tides on MUC1, proteolgtic fragments of the mucin obtained by digestio n with the Gly-C-specific endopeptidase IV from papaya corresponding t o low molecular mass fragments (<10 kilodaltons) of the tandem repeat domain were screened. A glycosylated fragment (glycopeptide 17) contai ning the mAb HMFG-2-defined epitope was highly reactive to BW835 antib ody, while nonglycosylated tandem repeat peptide TAP25 or its in vitro -glycosylated N-acetylgalactosamine (GalNAc) derivatives were unreacti ve. Glycopeptide 17 bound to peanut agglutinin and to a Thomsen-Friede nreich antigen (TF alpha)-specific mAb (A78-G/A7). Binding of BW835 to glycopeptide 17 or to MUC1 was competitively inhibited by peanut aggl utinin and by the synthetic glycopeptides TF alpha Ser or TF alpha Thr but not by their beta-anomers. Evidence for site specificity of bindi ng by BW835 to glycopeptide 17 was revealed by demonstrating nonreacti vity of the antibody to other TF alpha-expressing glycoproteins with p eptide moieties lacking MUC1-specific motifs at putative glycosylation sites. The epitope of BW835 was localized to threonine within the VTS A-peptide moth by site-specific enzymatic beta-galactosylation of the synthetic tandem repeat peptide TAP25-GalNAc(1) TAPPAHGVT(-O-alpha Gal NAc)SAPDTRPAPGSTAPPA. This is the first report on a TF alpha-specific mAb that shows a strict peptide sequence dependency of binding.