Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Sep 1;101(3):1107–1114. doi: 10.1083/jcb.101.3.1107

Selective destruction of nerve growth factor receptor-bearing cells in vitro using a hybrid toxin composed of ricin A chain and a monoclonal antibody against the nerve growth factor receptor

PMCID: PMC2113716  PMID: 2993314

Abstract

A hybrid toxin composed of ricin A chain and a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor (192-IgG) was prepared using the heterobifunctional cross-linking agent N- succinimidyl-3-(2-pyridyldithio)-propionate and purified by affinity chromatography. Characterization studies showed that the hybrid, 192-s- s-A, displaced bound 125I-labeled 192-IgG from rat superior cervical ganglion (SCG) membranes with an IC50 3-5 times lower than that of unconjugated 192-IgG. When incubated with cultured rat SCG neurons, 192- s-s-A inhibited protein synthesis in a concentration-dependent fashion. The effect of 192-s-s-A on these neurons was reversed by coincubation with an excess of 192-IgG. The IC50 of 192-s-s-A on protein synthesis in rat SCG neurons was 4 nM. Intact ricin and ricin A chain inhibited protein synthesis in these neurons with IC50 values of 5 pM and 500 nM, respectively. The 192-s-s-A hybrid had no effect on mouse SCG neurons or a human melanoma cell line known to have NGF receptors. This is consistent with the finding that 192-IgG recognizes only the rat NGF receptor. Also, 192-s-s-A did not inhibit protein synthesis in primary cultures of rat skeletal muscle or Vero cells, which do not have cell surface receptors for NGF. 192-s-s-A was able to inhibit protein synthesis in PC12 cells but the potency was 10-100 times less in these cells compared to rat SCG neurons. Ricin and A chain were also 10-100 times less potent in PC12 cells than neurons. Rat SCG neurons exposed to 192-s-s-A lost their refractile appearance under phase-contrast optics, showed granular degeneration of neurites, and died. Thus the decreased protein synthesis caused by the hybrid toxin correlated with the morphological destruction of the neurons. 192-s-s-A represents a potentially powerful tool by which to selectively destroy NGF receptor- bearing cells in vitro. The hybrid toxin may prove useful as an in vivo toxin.

Full Text

The Full Text of this article is available as a PDF (844.5 KB).

Selected References

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

  1. Axén R., Porath J., Ernback S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature. 1967 Jun 24;214(5095):1302–1304. doi: 10.1038/2141302a0. [DOI] [PubMed] [Google Scholar]
  2. Bacha P., Murphy J. R., Reichlin S. Thyrotropin-releasing hormone-diphtheria toxin-related polypeptide conjugates. Potential role of the hydrophobic domain in toxin entry. J Biol Chem. 1983 Feb 10;258(3):1565–1570. [PubMed] [Google Scholar]
  3. Banerjee S. P., Snyder S. H., Cuatrecasas P., Greene L. A. Binding of nerve growth factor receptor in sympathetic ganglia. Proc Natl Acad Sci U S A. 1973 Sep;70(9):2519–2523. doi: 10.1073/pnas.70.9.2519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cawley D. B., Herschman H. R., Gilliland D. G., Collier R. J. Epidermal growth factor-toxin A chain conjugates: EGF-ricin A is a potent toxin while EGF-diphtheria fragment A is nontoxic. Cell. 1980 Nov;22(2 Pt 2):563–570. doi: 10.1016/0092-8674(80)90366-9. [DOI] [PubMed] [Google Scholar]
  5. Chandler C. E., Parsons L. M., Hosang M., Shooter E. M. A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells. J Biol Chem. 1984 Jun 10;259(11):6882–6889. [PubMed] [Google Scholar]
  6. Edwards D. C. Targeting potential of antibody conjugates. Pharmacol Ther. 1983;23(1):147–177. doi: 10.1016/0163-7258(83)90029-3. [DOI] [PubMed] [Google Scholar]
  7. Krolick K. A., Uhr J. W., Slavin S., Vitetta E. S. In vivo therapy of a murine B cell tumor (BCL1) using antibody-ricin A chain immunotoxins. J Exp Med. 1982 Jun 1;155(6):1797–1809. doi: 10.1084/jem.155.6.1797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Lawrence J. C., Jr, Salsgiver W. J. Levels of enzymes of energy metabolism are controlled by activity of cultured rat myotubes. Am J Physiol. 1983 May;244(5):C348–C355. doi: 10.1152/ajpcell.1983.244.5.C348. [DOI] [PubMed] [Google Scholar]
  10. Marchalonis J. J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem J. 1969 Jun;113(2):299–305. doi: 10.1042/bj1130299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Miskimins W. K., Shimizu N. Genetics of cell surface receptors for bioactive polypeptides: variants of Swiss/3T3 fibroblasts resistant to a cytotoxic chimeric insulin. Proc Natl Acad Sci U S A. 1981 Jan;78(1):445–449. doi: 10.1073/pnas.78.1.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Oeltmann T. N., Heath E. C. A hybrid protein containing the toxic subunit of ricin and the cell-specific subunit of human chorionic gonadotropin. II. Biologic properties. J Biol Chem. 1979 Feb 25;254(4):1028–1032. [PubMed] [Google Scholar]
  13. Olsnes S., Pihl A. Chimeric toxins. Pharmacol Ther. 1981;15(3):355–381. doi: 10.1016/0163-7258(81)90050-4. [DOI] [PubMed] [Google Scholar]
  14. Sherwin S. A., Minna J. D., Gazdar A. F., Todaro G. J. Expression of epidermal and nerve growth factor receptors and soft agar growth factor production by human lung cancer cells. Cancer Res. 1981 Sep;41(9 Pt 1):3538–3542. [PubMed] [Google Scholar]
  15. Simmons B. M., Russell J. H. A single affinity column step method for the purification of ricin toxin from castor beans (Ricinus communis). Anal Biochem. 1985 Apr;146(1):206–210. doi: 10.1016/0003-2697(85)90417-8. [DOI] [PubMed] [Google Scholar]
  16. Taniuchi M., Johnson E. M., Jr Characterization of the binding properties and retrograde axonal transport of a monoclonal antibody directed against the rat nerve growth factor receptor. J Cell Biol. 1985 Sep;101(3):1100–1106. doi: 10.1083/jcb.101.3.1100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Thoenen H., Barde Y. A. Physiology of nerve growth factor. Physiol Rev. 1980 Oct;60(4):1284–1335. doi: 10.1152/physrev.1980.60.4.1284. [DOI] [PubMed] [Google Scholar]
  18. Wiley R. G., Blessing W. W., Reis D. J. Suicide transport: destruction of neurons by retrograde transport of ricin, abrin, and modeccin. Science. 1982 May 21;216(4548):889–890. doi: 10.1126/science.6177039. [DOI] [PubMed] [Google Scholar]
  19. Yamamoto T., Iwasaki Y., Konno H. Experimental sensory ganglionectomy by way of suicide axoplasmic transport. J Neurosurg. 1984 Jan;60(1):108–114. doi: 10.3171/jns.1984.60.1.0108. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES