Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Design of stable biologically active recombinant lutropin analogs

Nature Biotechnology volume 15, pages 663–667 (1997)Cite this article

Abstract

Glycoprotein hormones are noncovalent heterodimers comprised of a common α subunit and a hormone-specific β subunit. Secretion and biologic action of these hormones are dependent on the formation of the heterodimer. The human LHβ subunit is unique among the other β subunits in that it assembles inefficiently with the α subunit. To bypass this rate-limiting step, we constructed the LH single chains where the carboxy terminus of β was fused to the amino terminus of α subunit through a linker. Compared to the human LH heterodimer, the extent of secretion was greater for the tethers although the rate was dependent on the nature of the linker. The LH single chains were biologically active even though there was loss of recognition by a LH-specific monoclonal antibody. This suggests that receptor binding of the single chains is not impaired by changes in the heterodimeric configuration resulting from tethering the subunits. In addition, single chains exhibited a remarkably greater in vitro stability than the heterodimer, implying that these analogs will be useful as diagnostic reagents and that their purification will be facilitated.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Pierce, J. and Parsons, T. 1981. Glycoprotein hormones: Structure and function.Annu. Rev. Biochem. 50: 465–495.

    Article  CAS  PubMed  Google Scholar 

  2. Catt, K.J. and Pierce J.G. 1986. Gonadotropin hormones of the adenohypophysis. Reproductive Endocrinology. 75–114 Yen, S.S.C. and Jaffe, R.B. (eds.). W.B. Saunders Co., Philadelphia.

    Google Scholar 

  3. Talmadge, K., Vamvakopoulos, N.C. and Fiddes, J.C. 1984. Evolution of the genes for the β subunits of human chorionic gonadotropin and luteinizing hormone. Nature 307: 37–40.

    Google Scholar 

  4. Corless, C.L., Matzuk, M.M., Ramabhadran, T.V., Krichevsky, A. and Boime, I. 1987. LH and hCG beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells. J. Cell Biol. 104: 1173–1181.

    Article  CAS  PubMed  Google Scholar 

  5. Matzuk, M.M., Spangler, M.M., Camel, M., Suganuma, N. and Boime, I. 1989. Mutagenesis and chimeric genes define determinants in the β subunits of human chorionic gonadotropin and lutropin for secretion and assembly. J. Cell Biol. 109: 1429–1438.

    Article  CAS  PubMed  Google Scholar 

  6. Keene, J., Matzuk, M., Otani, T., Fauser, B., Galway, A., Hsueh, A. and Boime, I. 1989. Expression of biologically active human follitropin in Chinese hamster ovary cells. J. Cell Biol. 264: 4769–4775.

    CAS  Google Scholar 

  7. Matzuk, M., Kornmeier, C., Whitfield, G., Kourides, I. and Boime, I. 1988. The glycoprotein a subunit is critical for secretion and stability of the thyrotropin β subunit. Mol. Endocrinol. 2: 95–100.

    Article  CAS  PubMed  Google Scholar 

  8. Kesner, U.S., Knecht, E.A. and Krieg, E.F. Jr. 1995. Stability of urinary female reproductive hormones stored under various conditions. Mol. Endocrinol. 9: 239–244.

    CAS  Google Scholar 

  9. Livesey, J.H., Roud, H.K., Metcalf, M.G. and Donald, R.A. 1983. Glycerol prevents loss of immunoreactive follicle-stimulating horone and luteinizing hormone from frozen urine. J. Endocrinol. 98: 381–384.

    Article  CAS  PubMed  Google Scholar 

  10. Livesey, J.H., Hodgkinson, S.C., Roud, H.R. and Donald, R.A. 1980. Effect of time, temperature and freezing on the stability of immunoreactive LH, FSH, TSH, growth hormone, prolactin and insulin in plasma. Clin. Biochem. 13: 151–155.

    Article  CAS  PubMed  Google Scholar 

  11. deMedeiros, S.F., Amato, R. and Norman, R.J. 1991. Stability of immunoreactive β-core fragment of hCG. Obst. & Gyn. 77: 53–59.

    CAS  Google Scholar 

  12. Saketos, M., Sharma, N., Tovaghol, A., Raghuwanshi, M. and Santoro, N. 1994. Time resolved immunofluometric assay and specimen conditions for measuring urinary gonadotropins. Clin. Chem. 40: 749–753.

    CAS  PubMed  Google Scholar 

  13. Sugahara, T., Pixley, M.R., Minami, S., Perias, E., PBen-Menahem, D., Hsueh, A.J.W. 1995. Biosynthesis of a biologically active single peptide chain containing the human common α and chorionic gonadotropin β subunits in tandem. Proc. Nail. Acad. Sci. USA 92: 2041–2045.

    Article  CAS  Google Scholar 

  14. Narayan, R., Wu, C. and Puett, D. 1995. Functional expression of yoked human chorionic gonadotropin in baculovirus-infected insect cells. Mol. Endocrinol. 9: 1720–1726.

    CAS  PubMed  Google Scholar 

  15. Muyan, M., Furuhashi, M., Suguhara, T. and Boime, I. 1996. The carboxy-terminal region of the β-subunits of luteinizing hormone and chorionic gonadotropin differentially influence secretion and assembly of the heterodimer. Mol. Endocrinol. 10: 1678–1687.

    CAS  PubMed  Google Scholar 

  16. Sugahara, T., Grootenhuis, P., Sato, A., Kudo, M., Ben-Menahem, D., Pixley, M. et al 1996. Expression of biologically active fusion genes encoding the common α subunit and either 1996. CGβ or the FSHβ subunits: Role of a linker sequence. Mol. Cell Endocrinol. 125: 71–77.

    Article  PubMed  Google Scholar 

  17. Matzuk, M.M., Krieger, M., Corless, C.L. and Boime, I. 1987. Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA 84: 1173–1181.

    Article  Google Scholar 

  18. Harikrishna, J.A., Black, S.M., Szklarz, G.D. and Miller, W.L. 1993. Construction and function of fusion enzymes of the human cytochrome P450scc system. DNA Cell Biol. 12: 371–379.

    Article  CAS  PubMed  Google Scholar 

  19. Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.S., Novotny, J., Margolies, M.N., et al 1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85: 5879–5883.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Pomerantz, J.L., Sharp, P.A. and Pabo, C.O., 1995. Structure-based design of transcription factors. Science 267: 93–96.

    Article  CAS  PubMed  Google Scholar 

  21. Sano, T., Glazer, A.N. and Cantor, C.N., 1992. A streptavidin-metallothionein chimera that allows specific labeling of biological materials with many different heavy metal ions. Proc. Natl. Acad. Sci. USA 89: 1534–1538.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schimmel, P., Shepard, A. and Shiba, K., 1992. Intron locations and functional deletions in relation to the design and evolution of a subgroup of class I tRNA synthetases. Protein Sci. 1: 1387–1391.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Somoza, J.R., Jiang, F., Tong, L., Kang, C-H., Cho, J.M. and Kirn, S-H., 1993. Two crystal structures of a potently sweet protein: natural monellin at 2.75 Å resolution and single-chain monellin at 1.7 Å resolution. J. Mol. Biol. 204: 390–404.

    Article  Google Scholar 

  24. Toth, M.J. and Schimmel, P., 1986. Internal structural features of E. coli glycyl-tRNA synthetase examined by subunit polypeptide chain fusions. J. Biol. Chem. 261: 6643–6646.

    CAS  PubMed  Google Scholar 

  25. Whitlow, M. and Filpula, D., 1991. Single-chain Fv proteins and their fusion proteins. Methods 2: 97–105.

    Article  CAS  Google Scholar 

  26. Yeh, P., Landais, D., Lemaitre, M., Maury, I., Crenne, J.Y., Becquart, J., et al. 1992. Design of yeast-secreted albumin derivatives for human therapy: Biological and antiviral properties of a serum albumin-CD4 genetic conjugate. Proc. Natl. Acad. Sci. USA 89: 1904–1908.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hallewell, R.A., Laria, T., Tabrizi, A., Carlin, G., Getzoff, E.D., Tainer, J.A., et al. 1989. Genetically engineered polymers of human CuZn superoxide dismutase. J. Biol. Chem. 264: 5260–5269.

    CAS  PubMed  Google Scholar 

  28. Arora, N., Williamson, L.C., Leppla, S.H. and Halpern, J.L., 1994. Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non-neuronal cells. J. Biol. Chem. 269: 26,165–26,171.

    CAS  Google Scholar 

  29. Suganuma, N., Matzuk, M.M. and Boime, I, 1989. Elimination of disulfide bonds affects assembly and secretion of the human chorionic gonadotropin β subunit. J. Biol. Chem. 264: 19,302–19,307.

    CAS  Google Scholar 

  30. Huth, J.R., Mountjoy, K., Perini, F. and Ruddon, R.W., 1992. Intraeellular folding pathway of human chorionic gonadotropin beta subunit. J. Biol. Chem. 267: 8870–8879.

    CAS  PubMed  Google Scholar 

  31. Lapthorn, A., Harris, D., Littlejohn, A., Machin, R., et al. 1994. Crystal structure of human chorionic gonadotropin. Nature 369: 455–461.

    Article  CAS  PubMed  Google Scholar 

  32. Wu, H., Lustbader, J.W., Liu, Y., Canfield, R.E. and Hendricksond, W.A. 1994. Structure of human chorionic gonadotropin at 2.6 Å resolution from MAD analysis of the selenomethionyl protein. Structure 2: 545–558.

    Article  CAS  PubMed  Google Scholar 

  33. Argos, P. 1994. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J. Mol. Bbl. 211: 943–948.

    Article  Google Scholar 

  34. Sugahara, T., Sato, A., Kudo, M., Ben-Menahem, D., Pixley, M.R., Hsueh, A.J.W. et al. 1996. Expression of biologically active fusion genes encoding the common α subunit and the follicle-stimulating hormone β subunit. Role of a linker sequence. J. Biol. Chem. 271: 10445–10448.

    Article  CAS  PubMed  Google Scholar 

  35. Matzuk, M.M. and Boime, I., 1988. The role of asparagine-linked oligosaccharides of the a subunit in secretion and assembly of human chorionic gonadotropin. J. Cell Biol. 106: 1049–1059.

    Article  CAS  PubMed  Google Scholar 

  36. Jia, X-C., Oikawa, M., Bo, M., Tanaka, T., Ny, T., Boime, I., et al 1991. Expression of human luteinizing hormone (LH) receptor: interaction with LH and chorionic gonadotropin from human but not equine, rat and ovine species. Mol. Endocrinol. 5: 759–768.

    Article  CAS  PubMed  Google Scholar 

  37. Thorell, J.I. and Johansson, B.G., 1971. Enzymatic iodination of polypeptides with 125! of high specific activity. Biochem. Biophys. Acta 251: 363–369.

    CAS  PubMed  Google Scholar 

  38. Rao, M.C., Richards, J.J., Midgley, A.R., Jr., and Reichert, L.E., Jr. 1977. Regulation of gonadotropin receptors by luteinizing hormone in granulosa cells. Endocrinology 101: 512–523.

    Article  CAS  PubMed  Google Scholar 

  39. Davoren, J.B. and Hsueh, A.J.W., 1985. Vasoactive intestinal peptide: a novel stimulator of steroidogenesis by cultured rat granulosa cells. Biol. Reprod. 33: 37–52.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8103, St. Louis, MO, 63110

    Burkhard Hirsch & Aaron J.W. Hsueh

  2. Division of Reproductive Biology, Department of Gynecology/Obstetrics, Stanford University Medical Center, Stanford, CA, 94305-5317

    Vicenta Garcia-Campayo, Asomi Sato, Tadashi Sugahara, Mesut Muyan & Irving Boime

Authors
  1. Vicenta Garcia-Campayo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Asomi Sato
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Burkhard Hirsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tadashi Sugahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mesut Muyan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Aaron J.W. Hsueh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Irving Boime
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Garcia-Campayo, V., Sato, A., Hirsch, B. et al. Design of stable biologically active recombinant lutropin analogs. Nat Biotechnol 15, 663–667 (1997). https://doi.org/10.1038/nbt0797-663

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0797-663

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing