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 alternating injections of MCF-7 or SW-613 cells and has been demonstrated to be of value in the serodiagnosis of mammary carcinoma. BW835 defines a carbohydrate epitope on integrated or secreted MUC1 glycoforms from carcinoma cells and human milk. To identify BW835-reactive glycopeptides on MUC1, proteolytic fragments of the mucin obtained by digestion with the Gly-C-specific endopeptidase IV from papaya corresponding to low molecular mass fragments (<10 kilodaltons) of the tandem repeat domain were screened. A glycosylated fragment (glycopeptide 17) containing the mAb HMFG-2-defined epitope was highly reactive to BW835 antibody, while nonglycosylated tandem repeat peptide TAP25 or its in vitro -glycosylated N -acetylgalactosamine (GalNAc) derivatives were unreactive. Glycopeptide 17 bound to peanut agglutinin and to a Thomsen-Friedenreich antigen (TFα)-specific mAb (A78-G/A7). Binding of BW835 to glycopeptide 17 or to MUC1 was competitively inhibited by peanut agglutinin and by the synthetic glycopeptides TFαSer or TFαThr but not by their β-anomers. Evidence for site specificity of binding by BW835 to glycopeptide 17 was revealed by demonstrating nonreactivity of the antibody to other TFα-expressing glycoproteins with peptide moieties lacking MUC1-specific motifs at putative glycosylation sites. The epitope of BW835 was localized to threonine within the VTSA-peptide motif by site-specific enzymatic β-galactosylation of the synthetic tandem repeat peptide TAP25-GalNAc1 TAPPAHGVT(-O-αGalNAc)SAPDTRPAPG-STAPPA. This is the first report on a TFα-specific mAb that shows a strict peptide sequence dependency of binding.