Evidence that Immunosuppression is an Intrinsic Property of the Alpha-Fetoprotein Molecule

  • D. J. Semeniuk
  • R. Boismenu
  • J. Tam
  • W. Weissenhofer
  • R. A. Murgita
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 383)


Among the proteins that comprise the albumin family, alpha-fetoprotein (AFP) is the only member which exhibits immunoregulatory properties. However, some investigations have argued that AFP-mediated immunosuppression is not an inherent property of the molecule itself, but is instead, hypothesized to be either a function of a low molecular weight inhibitor bound to AFP or to a post-translational modification of the protein. AFP cannot be isolated from natural sources in quantities sufficient for the detailed biochemical and functional analyses required to resolve these issues. We have therefore produced recombinant forms of the protein (rAFP) by cloning the cDNA’s for mouse and human AFP in both eukaryotic and prokaryotic expression systems. As described in this report, we were able to abundantly express rAFP’s in bacterial, baculovirus and yeast expression systems. Recombinant proteins derived from each expression system were recognized by polyclonal and monoclonal anti-AFP antibodies as determined by immunoblot analysis. Pure recombinant protein samples, as characterized by polyacrylamide gel analyses, N-terminal sequencing and FPLC and HPLC chromatography, were evaluated for their immunoregulatory properties in murine and human in vitro immunological assays. The results of these studies establish that rAFP is functionally equivalent to natural fetal derived AFP molecules. Importantly, the data reported here demonstrate that AFP-mediated immunoregulation is an activity intrinsic to the molecule itself and cannot be attributed to either putative non-covalently bound moieties or to post-translational modifications such as glycosylation and sialylation. These studies provide a basis for initiating detailed investigations into the potential clinical usefulness of AFP as an immunotherapeutic agent.


Human Serum Albumin Sialic Acid Recombinant Baculovirus Yeast Expression System Prokaryotic Expression System 
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. Altman, J.D., Schneider, S.L., Thompson, D.A., Cheng, H.-L., Tomasi, T.B., 1990, A Transforming Growth Factor β2 (TGF- (β2)-like Immunosuppressive Factor in Amniotic Fluid and Localization of TGF-β2 mRNA in the Pregnant Uterus, J. Exp. Med. 172:1391–1401.15.PubMedCrossRefGoogle Scholar
  2. Attardi, B., and Ruosiahti, E.. 1976. Foetoneonatal oestradiol-binding protein in mouse brain cytosol is a foetoprotein, Nature 263:685–687.PubMedCrossRefGoogle Scholar
  3. Aoyagi, Y., Ikenaka, T., and lchida, F., 1978, Copper(II)-binding ability of human AFP, Cancer Res. 38:3483–3486.PubMedGoogle Scholar
  4. Boismenu, R., Semeniuk, D., and Murgita, R. A., 1995a, Expression cloning of mouse alpha-fetoprotein cDNA: Recovery of full-length recombinant AFP molecules and peptide domain fragments in a form suitable for structure-function analysis, Submitted for publication: Protein Expression and Purification. Google Scholar
  5. Boismenu, R., Semeniuk, D., Bennett, J.A., Jacobson, H., and Murgita, R.A., 1995b, Expression cloning of Human Alpha-fetoprotein and its growth regulatory properties on autoreactive lymphocytes and estrogen-stimulated tissues, Submitted for publication: Cancer Research. Google Scholar
  6. Brown, J.R., 1976, Structural origins of mammalian albumin, Fed. Proc. 35:2141–2144. PubMedGoogle Scholar
  7. Deutsch, H.F., 1991, Chemistry and Biology of a-fetoprotein. Adv. Cancer Res. 56:253–312.PubMedCrossRefGoogle Scholar
  8. Gorin, M.B., Cooper, D.L., Eiferman, F., van de Rijn, P., and Tilghman, S.M., 1981, The evolution of alpha-fetoprotein and albumin. A comparison of the primary amino acid sequences of mammalian alpha-fetoprotein and albumin. J. Biol. Chem. 256:1954–1959.PubMedGoogle Scholar
  9. Haourigui, M., Thobie, N., Martin, M.E., Benassayag, C., and Nunez, E.A., 1992, In vivo transient rise in plasma free fatty acids alters the functional properties of a-Fetoprotein, Biochimica et Biophysica Acta. 25:157–165.CrossRefGoogle Scholar
  10. Hooper, D.C., and Murgita, R.A., 1981, Regulation of murine T-cell responses to autologous antigens by alpha-fetoprotein, Cell. Immunol 63:417–425.PubMedCrossRefGoogle Scholar
  11. Hooper, D.C., O’Neill, G., Gronvik, K.-O., Gold, P., and Murgita, R.A., 1989, Human AFP inhibits cell proliferation and NK-like cytotoxic activity generated in autologous, but not in allogeneic mixed lymphocyte reactions, in Biological Activities of Alpha-Fetoprotein. Vol 11, CRC Press, Boca Raton, FL, 184–197.Google Scholar
  12. Hoskin, D., and Murgita, R. A., 1989, Specific maternal anti-fetal lymphoproliferative responses and their regulation by natural immunosuppressive factors. Clin. Exp. Immunol. 76:262–267.PubMedGoogle Scholar
  13. Kerhl, J.H., Wakefield, L.M., Roberts, A.B., Jakowlew, S., Alvarez-Mon, M., Derynck, R., Sporn, M.B., and Fauci, A. S., 1986, The production of TGF-β by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp. Med. 163:1037–1050.CrossRefGoogle Scholar
  14. Kioussis, D., Eiferman, F., van de Rijn, P., Gorin, M.B., Ingram, R.S., and Tilghman, S.M., 1981, The evolution of the alpha-fetoprotein and albumin genes in the mouse. J. Biol Chem. 256:1960–1967.PubMedGoogle Scholar
  15. Lang, A.K., and Searle, R.F., 1994, The immunomodulatory activity of human amniotic fluid can be correlated with transforming growth factor (TGF-β1) and β2 activity, Clin. Exp. Immunol. 97:158–163.PubMedCrossRefGoogle Scholar
  16. Lawrence, D.A., Pircher, R., Kryceve-Martiniere, C., and Julien, P., 1984, Normal embryo fibroblasts release transforming growth factors in a latent form. J. Cell. Physiol. 121:184–188.PubMedCrossRefGoogle Scholar
  17. Lester, E.P., Miller, J.B., and Yachnin, S., 1976, Human alphafetoprotein as a modulator of human lymphocyte transformation: Correlation of biological potency with electrophoretic variants. Proc. Natl. Acad. Sci. USA 73:4645–4648.CrossRefGoogle Scholar
  18. Lester, E.P., Miller, J.B., and Yachnin, S., 1977, A post-synthetic modification of human a-fetoprotein controls its immunosuppressive potency, Proc. Natl. Acad. Sci. USA 74:3988–3992.PubMedCrossRefGoogle Scholar
  19. Lichenstein, H.S., Lyons, D.E., Wurfel, M.M., Johnson, D.A., McGinley, M.D., Leidli, J.C., Trollinger, D.B., Mayer, J.P., Wright, S.D., and Zukowski, M.M., 1994, Afamin is a new member of the albumin, a-fetoprotein, and vitamin D-binding protein gene family, J. Biol. Chem. 269(27): 18149–18154.PubMedGoogle Scholar
  20. Luft, A.J. and Lorscheider, F. L., 1983, Structural analysis of human and bovine alpha-fetoprotein by electron microscopy, image processing, and circular dichroism. Biochemistry. 22:5978–5981.PubMedCrossRefGoogle Scholar
  21. Morinaga, T., Sakai, M., Wegmann, T.G., and Tamaoki, T., 1983, Primary structures of human alpha-fetoprotein and its mRNA. Proc. Natl. Acad. Sci. USA 80:4604–4608.PubMedCrossRefGoogle Scholar
  22. Nishi, S., Koyama, Y., Sakamoto, T., Soda, M., and Kairiyama, C.B., 1988, Expression of rat a-fetoprotein cDNA in Escherichia coli and in yeast. J. Biochem. 104:968–972.PubMedGoogle Scholar
  23. O’Connor-McCourt, M. D., and Wakefield, L.M., 1987, Latent Transforming Growth Factor-β in Serum. A specific complex with α2-Macroglobulin, J. Biol Chem. 262 (29): 14090–14099.PubMedGoogle Scholar
  24. Olsen, M.K., Rockenbach, S.K., Curry, K.A., and Tomich, C-S.C., 1989, Enhancement of heterologous polypeptide expression by alterations in the ribosome binding site sequence. J Biotechnology 9:179–180.CrossRefGoogle Scholar
  25. O’Reilly, D., Miller, L.K., and Luckow, V. A., 1992, Post-translational modification in Baculovirus expression vectors. W.H. Freeman and Company. 216–236.Google Scholar
  26. Philip, A., and O’Connor-McCourt, M. D., 1991, Interaction of transforming growth factor-β1 with a α2-macroglobulin. Role in transforming growth factor-β clearance. J. Biol Chem. 266:22290–22296.PubMedGoogle Scholar
  27. Ruoslahti, E. and Engvall E., 1976, Immunological crossreaction between alpha-fetoprotein and albumin. Proc. Natl. Acad. Sci. 73:4641–4644.PubMedCrossRefGoogle Scholar
  28. Ruoslahti, E., and Terry, W.D., 1976, α-fetoprotein and serum albumin show sequence homology, Nature 260:804–805.PubMedCrossRefGoogle Scholar
  29. Savu, L., Benassayag, C., Vallette, G., Christeff, N., and Nunez, E., 1981, Mouse a-Fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands. J. Biol. Chem. 256:9414–9418.PubMedGoogle Scholar
  30. Semeniuk, D.J., Boismenu, R., and Murgita, R.A., 1995a, Recombinant alpha-fetoprotein produced by a baculovirus expression system exhibits immunoregulatory properties that are functionally equivalent to its authentic fetal-derived counterpart. Submitted for publication: J. Exp. Med. Google Scholar
  31. Semeniuk, D. J., Boismenu, R., Bennett, J. A., Jacobson, H.I., and Murgita, R. A., 1995b, Recombinant human alpha-fetoprotein expressed in insect cells and Escherichia coli demonstrate equivalent biological activities. Submitted for publication: Proc. Natl. Acad. Sci. USA. Google Scholar
  32. Schoentgen, F., Metz-Boutigue, M.-H., Jolies, J., Constans, J., and Joiles, P., 1986, Complete amino acid sequence of human vitamin D-binding protein (group specific component): evidence of a three-fold internal homology as in serum albumin and α-fetoprotein, Biochimica et Biophysica Acta 871:189198.Google Scholar
  33. Tessier, D.C., Thomas, D.Y., Khouri, H.E., Laliberte, F., and Vernet, T., 1991, Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide. Gene 98:177–183.Google Scholar
  34. van Oers, N.S.C., Cohen, B.L., and Murgita, R. A., 1989, Isolation and characterization of a distinct immunoregulatory isoform of α-fetoprotein produced by the normal fetus, J. Exp. Med. 170:811–825.PubMedCrossRefGoogle Scholar
  35. van Oers, N.S.C., Powell, W.S., Semeniuk, D.J., Weissenhofer, W., and Murgita, R.A., 1995, The capacity of natural fetal-derived alphafetoprotein to regulate immune responses is independent of ligand-binding by fatty acids. Submitted for publication: Biochemistry. Google Scholar
  36. Wahl, S.M., 1994, Transforming growth factor β: The good, the bad, and the ugly, J. Exp. Med. 180:1587–1590.PubMedCrossRefGoogle Scholar
  37. Yachnin, S., 1976, Demonstration of the inhibitory effects of human alpha-fetoprotein on in vitro transformation of human lymphocytes, Proc. Natl. Acad. Sci. USA 73:2857–2861.PubMedCrossRefGoogle Scholar
  38. Yamamoto, R., Sakamoto, T., Nishi, S., Sakai, M., Morinaga, T., and Tamaoki, T., 1990, Expression of human a-fetoprotein in yeast. Life Sciences 46:1679–1686.PubMedCrossRefGoogle Scholar
  39. Zimmerman, E. F., Voorting-Hawking, M., and Michael, J. G., 1977, Immunosuppression by mouse sialylated alpha-fetoprotein. Nature. 26:354–356.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • D. J. Semeniuk
    • 1
    • 2
  • R. Boismenu
    • 1
    • 2
  • J. Tam
    • 1
    • 2
  • W. Weissenhofer
    • 1
    • 2
  • R. A. Murgita
    • 1
    • 2
  1. 1.Department of Microbiology and ImmunologyMcGill UniversityMontrealCanada
  2. 2.RudolfinerhausDepartment of SurgeryViennaAustria

Personalised recommendations