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Monoclonal Antibody BW835 Defines a Site-specific Thomsen-Friedenreich Disaccharide Linked to Threonine within the VTSA Motif of MUC1 Tandem Repeats¹

Institute of Immunobiology, University Clinic Cologne, Kerpener Str. 15, 50924 Cologne [F-G. H., T. S.], and Behringwerke AG, P.O. Box 1140, 35001 Marburg [K. B.], Germany

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 (TF)-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 TFSer or TFThr but not by their ß-anomers. Evidence for site specificity of binding by BW835 to glycopeptide 17 was revealed by demonstrating nonreactivity of the antibody to other TF-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 ß-galactosylation of the synthetic tandem repeat peptide TAP25-GalNAc₁ TAPPAHGVT(-O-GalNAc)SAPDTRPAPG-STAPPA. This is the first report on a TF-specific mAb that shows a strict peptide sequence dependency of binding.

¹ The present study was supported by the Deutsche Forschungsgemeinschaft Grant Uh8/16-3.

² To whom requests for reprints should be addressed, at Institute of Immunobiology, University Clinic Cologne, Kerpener Str. 15, 50937 Cologne, Germany.

Received 2/27/95. Accepted 7/12/95.

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