Molecular Analysis of Epitopic Heterogeneity of the Breast Mucin

  • Jerry A. Peterson
  • David Larocca
  • Gary Walkup
  • Richard Amiya
  • Roberto L. Ceriani


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.


Tandem Repeat Breast Epithelial Cell Epitope Mapping Breast Carcinoma Cell Line Tandem Repeat Sequence 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 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
  2. 2.
    Ceriani, R.L., Peterson, J.A., Lee, J.Y., Moncada, R., and Blank, E.W. Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule. Somat. Cell Genet., 9: 415–427, 1983.PubMedCrossRefGoogle Scholar
  3. 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
  4. 4.
    Hilkens, J., Buijs, F., and Ligtenberg, M. Complexity of MAM-6, an epithelial sialomucin associated with carcinomas. Cancer Res., 49: 786–793, 1989.PubMedGoogle Scholar
  5. 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
  6. 6.
    Shimizu, M., Yamauchi, K., Miyauchi, Y., Sakurai, T., Tokugawa, K., and McIlhinney, R.A.J. High-Mr glycoprotein profiles in human milk serum and fat-globule membrane. Biochem. J., 233: 725–730, 1986PubMedGoogle Scholar
  7. 7.
    Gendler, S., Taylor-Papadimitriou, J., Duhig, T., Rothbard, J., and Burchell, J. A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J. Biol. Chem., 263: 12820–12823, 1988.PubMedGoogle Scholar
  8. 8.
    Johnson, V.G., Schlom, J., Paterson, A.J., Bennett, J., Magnani, J.L., and Colcher, D. Analysis of a human tumor-associated glycoprotein (TAG-72) identified by monoclonal antibody 872.3. Cancer Res., 46: 850–857, 1986.PubMedGoogle Scholar
  9. 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
  10. 10.
    Wreschner, D.H., Hareuveni, M., Tsarfaty, I., Smorodinsky, N., Horev, J., Zaretsky, J., Kotkes, P., Weiss, M., Lathe, R., Dion, A., and Keydar, I. Human epithelial tumor antigen CDNA sequences. Eur. J. Biochem., 189: 463–473, 1990.PubMedCrossRefGoogle Scholar
  11. 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
  12. 12.
    Gendler, S.J., Lancaster, C.A., Taylor-Papadimitriou, J., Duhig, T., Peat, N., Burchell, J., Pemberton, L., Lalani, E.N., and Wilson, D. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J. Biol. Chem., 265: 15286–15293, 1990.PubMedGoogle Scholar
  13. 13.
    Lan, M.S., Batra, S.K., Qi, W.N., Metzgar, R.S., and Hollingsworth, M.A. Cloning and sequencing of a human pancreatic tumor mucin cDNA. J. Biol. Chem., 265: 15294–15299, 1990.PubMedGoogle Scholar
  14. 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
  15. 15.
    Ceriani, R.L., Sasaki, M., Sussman, H., Wara, W.M., and Blank, E.W. Circulating human mammary epithelial antigens in breast cancer. Proc. Natl. Acad. Sci. USA, 79: 5420–5424, 1982.PubMedCrossRefGoogle Scholar
  16. 16.
    Ceriani, R.L., Blank, E.W., and Peterson, J.A. Experimental immunotheraphy of human breast carcinomas implanted in nude mice with a mixture of monoclonal antibodies against human milk fat globule components. Cancer Res., 47: 532–540, 1987.PubMedGoogle Scholar
  17. 17.
    Ceriani, R.L. and Blank, E.W. Experimental therapy of human breast tumors with 1311-labeled monoclonal antibodies prepared against the human milk fat globule [published erratum appears in Cancer Res 1989 Sep 15;49(18): 5236]. Cancer Res., 48: 4664–4672, 1988.PubMedGoogle Scholar
  18. 18.
    Burchell, J., Gendler, S., Taylor-Papadimitriou, J., Girling, A., Lewis, A., Millis, R., and Lamport, D. Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin. Cancer Res., 47: 5476–5482, 1987.PubMedGoogle Scholar
  19. 19.
    Geysen, H.M., Meloen, R.H., and Barteling, S.J. Use of peptide synthesis to probe vital antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA, 81: 3998–4002, 1984.PubMedCrossRefGoogle Scholar
  20. 20.
    Abe, M. and Kufe, D.W. Identification of a family of high molecular weight tumor-associated glycoproteins. J. Immunol., 139: 257–261, 1987.PubMedGoogle Scholar
  21. 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
  22. 22.
    Peterson, J.A., Bartholomew, J.C., Stampfer, M., and Ceriani, R.L. Analysis of expression of human mammary epithelial antigens in normal and malignant breast cells at the single cell level by flow cytofluorimetry. Exp. Cell Biol., 49: 1–14, 1981.PubMedGoogle Scholar
  23. 23.
    Kearney, J.F. and Lawton, A.R. B lymphocyte differentiation induced by lipopolysaccharide. J. Immunol., 115: 671–676, 1975.PubMedGoogle Scholar
  24. 24.
    Webb, N.R., Madoulet, C., Pierre-Francois, T., Broussard, D.R., Sneed, L., Nicolau, C., and Summers, M.D. Cell-surface expression and purification of human CD4 produced in baculovirus-infected insect cells. Proc. Natl. Acad. Sci. USA, 86: 7731–7735, 1989.PubMedCrossRefGoogle Scholar
  25. 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. 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
  27. 27.
    Siddiqui, J., Abe, M., Hayes, D, Shani, E., Yunis, E., and Kufe, D.W. Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen. Proc. Natl. Acad. Sci. USA, 85: 2320–2323, 1988.PubMedCrossRefGoogle Scholar
  28. 28.
    Laver, W.G., Air, G.M.G, Webster, R.G., and Smith-Gill, S.J. Epitopes on protein antigens: Misconceptions and Realities. Cell, 61: 553–556, 1990.PubMedCrossRefGoogle Scholar
  29. 29.
    Jakowlew, S.B., Breathnach, R., Jeltsch, J.M., Masiakowski, P., and Chambon, P. Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line MCF-7. Nucleic Acids Res., 12: 2861–2878, 1984.PubMedCrossRefGoogle Scholar
  30. 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. 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
  32. 32.
    Abe, M. and Kufe, D.W. Structural analysis of the DF3 human breast carcinoma-associated protein. Cancer Res., 49: 2834–2839, 1989.PubMedGoogle Scholar
  33. 33.
    Burchell, J., Taylor-Papadimitriou, J., Boshell, M., Gendler, S., and Duhig, T. A short sequence, within the amino acid tandem repeat of a cancer-associated mucin, contains immunodominant epitopes. Int. J. Cancer, 44: 691–696, 1989.PubMedCrossRefGoogle Scholar
  34. 34.
    Shimizu, M. and Yamauchi, K. Isolation and characterization of mucin-like glycoprotein in human milk fat globule membrane. J. Biochem., 91: 515–524, 1982.PubMedGoogle Scholar
  35. 35.
    Lan, M.S., Khorrami, A., Kaufman, B., and Metzgar, R.S. Molecular characterization of a mucin-type antigen associated with human pancreatic cancer. The DU-PAN-2 antigen. J. Biol. Chem., 262: 12863–12870, 1987.PubMedGoogle Scholar
  36. 36.
    Parodi, A.J., Blank, E.W., Peterson, J.A., and Ceriani, R.L. Glycosyl transferases in mouse and human milk fat globule membranes. Mol. Cell Biochem., 58: 157–163, 1984.PubMedCrossRefGoogle Scholar
  37. 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
  38. 38.
    Barnd, D.L., Lan, M.S., Metzgar, R.S., and Finn, O.J. Specific, major histocompatibility complex-unrestricted recognition of tumor-associated mucins by human cytotoxic T cells. Proc. Natl. Acad. Sci. USA, 86: 7159–7163, 1989.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1991

Authors and Affiliations

  • Jerry A. Peterson
    • 1
  • David Larocca
    • 1
  • Gary Walkup
    • 1
  • Richard Amiya
    • 1
  • Roberto L. Ceriani
    • 1
  1. 1.John Muir Cancer and Aging Research InstituteWalnut CreekUSA

Personalised recommendations