Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1983;2(4):493–497. doi: 10.1002/j.1460-2075.1983.tb01452.x

Antigenic, receptor-binding and mitogenic activity of proteolytic fragments of human growth hormone.

R Aston, J Ivanyi
PMCID: PMC555050  PMID: 6194985

Abstract

Analysis of the subtilisin-digested, two-chain form of human growth hormone (hGH) and its constituent polypeptide fragments has been aided by the use of monoclonal antibodies which bind specifically to four distinct epitopes on the native hormone. Using the SDS-polyacrylamide immunoblotting technique, it was shown that one epitope (shared with human chorionic somatomammotropin) detected by EB1 (or EB3) antibody was expressed to a similar extent by both the N-terminal (15 K) and C-terminal (7 K) polypeptides. This epitope is unique in that it represents a repeating determinant within the single chain structure of the hormone. Another three epitopes detected by monoclonal antibodies QA68/NA27, NA71 or NA39/EB2 were absent from the 7-K fragment but were expressed on the 15-K fragment to a similar extent to that on unmodified growth hormone. Binding of NA71 antibody was demonstrated only by radioimmunoassay since this, presumably conformational epitope could not be detected by immunoblotting. The functional importance of the 15-K peptide was demonstrated by its ability to bind specifically to hormone receptors on IM9 human lymphoblastoid cells and by its retention of mitogenicity for the NB2 rat lymphoma cell line. However, all tested monoclonal antibodies inhibited the binding of [125I]15-K to IM9 cell receptors by either steric hindrance or by an allosteric mechanism and therefore could not be further related topographically to the receptor-binding moiety of hGH.

Full text

PDF
493

Images in this article

494Fig. 1.
on p.494
494Fig. 3.
on p.494

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Cadman H. F., Wallis M., Ivanyi J. The effects of monoclonal antibodies against human growth hormone on hormone--receptor interactions. FEBS Lett. 1982 Jan 11;137(1):149–152. doi: 10.1016/0014-5793(82)80335-9. [DOI] [PubMed] [Google Scholar]
  2. Clarke W. C., Hayashida T., Li C. H. Human pituitary growth hormone: immunochemical investigations of biologically active fragments. Arch Biochem Biophys. 1974 Oct;164(2):571–574. doi: 10.1016/0003-9861(74)90068-x. [DOI] [PubMed] [Google Scholar]
  3. Fahey J. L., Buell D. N., Sox H. C. Proliferation and differentiation of lymphoid cells: studies with human lymphoid cell lines and immunoglobulin synthesis. Ann N Y Acad Sci. 1971 Dec 31;190:221–234. doi: 10.1111/j.1749-6632.1971.tb13537.x. [DOI] [PubMed] [Google Scholar]
  4. Gout P. W., Beer C. T., Noble R. L. Prolactin-stimulated growth of cell cultures established from malignant Nb rat lymphomas. Cancer Res. 1980 Jul;40(7):2433–2436. [PubMed] [Google Scholar]
  5. Gráf L., Li C. H., Cheng C. H., Jibson M. D. Two contiguous thrombin fragments of human somatotropin form a functionally active recombinant, but the two homologous fragments from sheep hormone do not. Biochemistry. 1981 Dec 8;20(25):7251–7258. doi: 10.1021/bi00528a031. [DOI] [PubMed] [Google Scholar]
  6. Gráf L., Li C. H. Isolation and properties of two biologically active fragments from limited tryptic hydrolysis of bovine and ovine pituitary growth hormones. Biochemistry. 1974 Dec 17;13(26):5408–5415. doi: 10.1021/bi00723a026. [DOI] [PubMed] [Google Scholar]
  7. Ivanyi J., Davies P. Monoclonal antibodies against human growth hormone. Mol Immunol. 1980 Feb;17(2):287–290. doi: 10.1016/0161-5890(80)90082-6. [DOI] [PubMed] [Google Scholar]
  8. Ivanyi J. Study of antigenic structure and inhibition of activity of human growth hormone and chorionic somatomammotropin by monoclonal antibodies. Mol Immunol. 1982 Dec;19(12):1611–1618. doi: 10.1016/0161-5890(82)90272-3. [DOI] [PubMed] [Google Scholar]
  9. Jones R. L., Benker G., Salacinski P. R., Lloyd T. J., Lowry P. J. Large-scale preparation of highly purified pyrogen-free human growth hormone for clinical use. J Endocrinol. 1979 Jul;82(1):77–86. doi: 10.1677/joe.0.0820077. [DOI] [PubMed] [Google Scholar]
  10. Kostyo J. L. The search for the active core of pituitary growth hormone. Metabolism. 1974 Sep;23(9):885–899. doi: 10.1016/0026-0495(74)90122-x. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Lesniak M. A., Gorden P., Roth J., Gavin J. R., 3rd Binding of 125I-human growth hormone to specific receptors in human cultured lymphocytes. Characterization of the interaction and a sensitive radioreceptor assay. J Biol Chem. 1974 Mar 25;249(6):1661–1667. [PubMed] [Google Scholar]
  13. Lewis U. J., Pence S. J., Singh R. N., VanderLaan W. P. Enhancement of the growth promoting activity of human growth hormone. Biochem Biophys Res Commun. 1975 Nov 17;67(2):617–624. doi: 10.1016/0006-291x(75)90857-8. [DOI] [PubMed] [Google Scholar]
  14. Lewis U. J., Singh R. N., Tutwiler G. F., Sigel M. B., VanderLaan E. F., VanderLaan W. P. Human growth hormone: a complex of proteins. Recent Prog Horm Res. 1980;36:477–508. doi: 10.1016/b978-0-12-571136-4.50019-x. [DOI] [PubMed] [Google Scholar]
  15. Lewis U. J., Singh R. N., Vanderlaan W. P., Tutwiler G. F. Enhancement of the hyperglycemic activity of human growth hormone by enzymic modification. Endocrinology. 1977 Nov;101(5):1587–1603. doi: 10.1210/endo-101-5-1587. [DOI] [PubMed] [Google Scholar]
  16. Li C. H., Gráf L. Human pituitary growth hormone: isolation and properties of two biologically active fragments from plasmin digests. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1197–1201. doi: 10.1073/pnas.71.4.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liberti J. P., Miller M. S. Somatomedin-like effects of biologically active bovine growth hormone fragments. Endocrinology. 1978 Jul;103(1):29–34. doi: 10.1210/endo-103-1-29. [DOI] [PubMed] [Google Scholar]
  18. Linde S., Hansen B., Lernmark A. Stable iodinated polypeptide hormones prepared by polyacrylamide gel electrophoresis. Anal Biochem. 1980 Sep 1;107(1):165–176. doi: 10.1016/0003-2697(80)90507-2. [DOI] [PubMed] [Google Scholar]
  19. Mittra I. A novel "cleaved prolactin" in the rat pituitary: Part II. In vivo mammary mitogenic activity of its N-terminal 16K moiety. Biochem Biophys Res Commun. 1980 Aug 29;95(4):1760–1767. doi: 10.1016/s0006-291x(80)80102-1. [DOI] [PubMed] [Google Scholar]
  20. Mittra I. A novel "cleaved prolactin" in the rat pituitary: part I. Biosynthesis, characterization and regulatory control. Biochem Biophys Res Commun. 1980 Aug 29;95(4):1750–1759. doi: 10.1016/s0006-291x(80)80101-x. [DOI] [PubMed] [Google Scholar]
  21. Niall H. D., Hogan M. L., Sauer R., Rosenblum I. Y., Greenwood F. C. Sequences of pituitary and placental lactogenic and growth hormones: evolution from a primordial peptide by gene reduplication. Proc Natl Acad Sci U S A. 1971 Apr;68(4):866–870. doi: 10.1073/pnas.68.4.866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Noble R. L., Beer C. T., Gout P. W. Evidence in vivo and in vitro of a role for the pituitary in the growth of malignant lymphomas in Nb rats. Cancer Res. 1980 Jul;40(7):2437–2440. [PubMed] [Google Scholar]
  23. Reagan C. R., Mills J. B., Kostyo J. L., Wilhelmi A. E. Isolation and biological characterization of fragments of human growth hormone produced by digestion with plasmin. Endocrinology. 1975 Mar;96(3):625–636. doi: 10.1210/endo-96-3-625. [DOI] [PubMed] [Google Scholar]
  24. Retegui L. A., Milne R. W., Cambiaso C. L., Masson P. L. The recognition by monoclonal antibodies of various portions of a major antigenic site of human growth hormone. Mol Immunol. 1982 Jul;19(7):865–875. doi: 10.1016/0161-5890(82)90352-2. [DOI] [PubMed] [Google Scholar]
  25. Tanaka T., Shiu R. P., Gout P. W., Beer C. T., Noble R. L., Friesen H. G. A new sensitive and specific bioassay for lactogenic hormones: measurement of prolactin and growth hormone in human serum. J Clin Endocrinol Metab. 1980 Nov;51(5):1058–1063. doi: 10.1210/jcem-51-5-1058. [DOI] [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wallis M., Ivanyi J., Surowy T. K. Binding specificity of monoclonal antibodies towards the size variants of human growth hormone. Mol Cell Endocrinol. 1982 Nov-Dec;28(3):363–372. doi: 10.1016/0303-7207(82)90133-2. [DOI] [PubMed] [Google Scholar]
  28. Yadley R. A., Chrambach A. Isohormones of human growth hormone. II. Plasmin-catalyzed transformation and increase in prolactin biological activity. Endocrinology. 1973 Oct;93(4):858–865. doi: 10.1210/endo-93-4-858. [DOI] [PubMed] [Google Scholar]
  29. Zakin M. M., Peña C., Poskus E., Stewart J. M., Paladini A. C. Immunological properties of two related fragments from human and equine growth hormones. Eur J Immunol. 1977 Oct;7(10):701–704. doi: 10.1002/eji.1830071010. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES