Molecular Analysis of Epitopic Heterogeneity of the Breast Mucin
The breast mucin is a highly glycosylated large molecular weight glycoprotein on the surface of breast epithelial cells, in breast carcinoma cells, and is a major component of the human milk fat globule membrane. By epitope mapping we have determined that 5 different monoclonal antibodies (Mc1, Mc5, BrEl, BrE2, BrE3) raised against human milk fat globule and selected for breast specificity recognize 4 distinct but overlapping linear amino acid sequences encompassing the most hydrophilic region of the 20 amino acid tandem repeat that makes up a large part of polypeptide core of the breast mucin. Although these MoAbs bind to overlapping polypeptide epitopes they have different tissue and tumor specificities in histopathology, differ quantitatively in the binding to breast carcinoma cell lines by flow cytometry, and they have distinct competition patterns for bind-ng to the native antigen on breast carcinoma cells. Even though BrE2 nd BrE3 have the same polypeptide epitope, they differ in their relative binding to breast carcinoma cell as determined by flow cytometry and binding to fusion proteins containing mimotopes produced by cDNA clone isolated from a breast cDNA library. There is considerable variation in the breast mucin mRNA, but the expression of the different epitopes shows little correlation with mRNA levels. Surface expression of an epitope and detection of the epitope in secreted material is associated with its presence on a larger size molecular specie than the exclusively cytoplasmic expression of an epitope. This detailed analysis of the epitopic heterogeneity of an immunodominant region of the tandem repeat segment of the core polypeptide of the breast mucin, possibly involving altered glycosylation, reveals an epitopic heterogenity that may have functional significance and suggests approaches for preparing new monoclonal antibodies with improved qualities for breast cancer diagnosis and/or therapy.
KeywordsTandem Repeat Breast Epithelial Cell Epitope Mapping Breast Carcinoma Cell Line Tandem Repeat Sequence
Unable to display preview. Download preview PDF.
- 1.Taylor-Papadimitriou, J., Peterson, J.A., Arklie, J., Burchell, J., Ceriani, R.L., and Bodmer, W.F. Monoclonal antibodies to epithelium-specific components of the human milk fat globule membrane: production and reaction with cells in culture. Int. J. Cancer, 28: 17–21, 1981.PubMedCrossRefGoogle Scholar
- 3.Peterson, J.A., Zava, D.T., Duwe, A.K., Blank, E.W., Battifora, H., and Ceriani, R.L. Biochemical and histological characterization of antigens preferentially expressed on the surface and cytoplasm of breast carcinoma cells identified by monoclonal antibodies against the human milk fat globule. Hybridoma, 9: 221–235, 1990.PubMedCrossRefGoogle Scholar
- 5.Hull, S.R., Bright, A., Carraway, K.L., Abe, M., Hayes, D.F., and Kufe, D.W. Oligosaccharide differences in the DF3 sialomucin antigen from normal human milk and the BT-20 human breast carcinoma cell line. Cancer Comm., 1: 261–267, 1989.Google Scholar
- 9.Heyderman, E., Strudley, I., Powell, G., Richardson, T.C., Cordell, J.L., and Mason, D.Y. A new monoclonal antibody to epithelial membrane antigen (EMA)-E29. A comparison of its immunocytochemical reactivity with polyclonal anti-EMA antibodies and with another monoclonal antibody, HMFG-2. Br. J. of Cancer, 52: 355–361, 1985.CrossRefGoogle Scholar
- 11.Ligtenberg, M.J.L., Vos, H.L., Gennissen, A.M.C., and Hilkens, J. Episialin, a carcinoma-associated mucin, is generated by a polymorphic gene encoding splice variants with alternative amino termini. J. of Biol. Chem., 265: 5573–5578, 1990.Google Scholar
- 14.Ceriani, R.L., Hill, D.L., Osvaldo, L., Kandell, C., and Blank, E.W. Immunohistochemical studies in breast cancer using monoclonal antibodies against breast epithelial cell components and with lectins. In: J. Russo (ed.), Immunocytochemistry in Tumor Diagnosis, pp. 233–263, Boston: Martinus Nijhoff Publications. 1985.Google Scholar
- 21.Ceriani, R.L. Solid phase identification and molecular weight determination of cell membrane antigens with monoclonal antibodies. In: K.B. Bechtol, T.J. McKern and R. Kennett (eds.), Monoclonal Antibodies and Functional Cell Lines. Progress and Applications, pp. 398–402, New York: Plenum Press. 1984.Google Scholar
- 25.Larocca, D., Peterson, J.A., Walkup, G., and Ceriani, R.L. High level expression in E. coil of an alternate reading frame of pS2 mRNA that encodes a mimotope of human breast epithelial mucin tandem repeat. 1990. (Submitted for publication)Google Scholar
- 26.Gendler, S.J., Burchell, J.M., Duhig, T., Lamport, D., White, R., Parker, M., and Taylor-Papadimitriou, J. Cloning of partial cDNA encoding differentiation and tumor-associated mucin glycoproteins expressed by human mammary epithelium. Proc. Natl. Acad. Sci. USA, 84: 6060–6064, 1987.PubMedCrossRefGoogle Scholar
- 30.Ceriani, R.L., Larocca, D., Peterson, J.A., Enloe, S., Amiya, R., Enloe, S., and Blank, E.W. A novel serum assay using recombinant breast epithelial mucin antigen. (see this volume)Google Scholar
- 31.Xing, P.X., Tjandca, J.J., Reynolds, K., McLaughlin, P.J., Purcell, D.F.J., and Mckenzie, I.F.C. Reactivity of anti-human milk fat globule antibodies with synthetic peptides. J. Immunol., 142: 3503–3509, 1990.Google Scholar
- 37.Hareuveni, M., Tsarfaty, I., Zaretsky, J., Kotkes, P., Horev, J., Zrihan, S., Weiss, M., Green, S., Lathe, R., Keydar, I., and Wreschner, D.H. A transcribed gene, containing a variable number of tandem repeats, codes for a human epithelial tumor antigen. Eur. J. Biochem., 189: 475–486, 1990.PubMedCrossRefGoogle Scholar