Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1987 May 1;165(5):1371–1382. doi: 10.1084/jem.165.5.1371

Dissociation of membrane binding and lytic activities of the lymphocyte pore-forming protein (perforin)

PMCID: PMC2188309  PMID: 3494808

Abstract

Granules isolated from CTL and NK cells contain a cytolytic pore- forming protein (PFP/perforin). At low temperatures (on ice), PFP binds to erythrocyte membranes without producing hemolysis. Hemolysis occurs when the PFP-bound erythrocytes are warmed up to 37 degrees C, which defines a temperature-dependent, lytic (pore-formation) step distinct from the membrane-binding event. Ca2+ and neutral pH are required for both membrane binding and pore formation by PFP. Serum, LDL, HDL, and heparin inhibit the hemolytic activity of PFP by blocking its binding to lipid membranes. Lysis by PFP that has bound to erythrocyte membranes is no longer susceptible to the effect of these inhibitors. The hemolytic activities associated with intact granules and solubilized PFP show different requirements for Ca2+ and pH, indicating that cytolysis produced by isolated granules may involve an additional step, possibly fusion of granules with membranes. It is suggested that three distinct Ca2+- and pH-dependent events may be involved during cell killing by CTL and NK cells: fusion of cytoplasmic granules of effector cells with their plasma membrane, releasing PFP from cells; binding of the released PFP to target membranes; and insertion of monomers and the subsequent formation of lytic pores in the target membrane. The serum-mediated inhibition of membrane binding by PFP could prevent the accidental injury of bystander cells by cell-released PFP, but would allow cytolysis to proceed to completion once PFP has bound to the target membrane.

Full Text

The Full Text of this article is available as a PDF (782.3 KB).

Selected References

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

  1. Berke G. Cytotoxic T-lymphocytes. How do they function? Immunol Rev. 1983;72:5–42. doi: 10.1111/j.1600-065x.1983.tb01071.x. [DOI] [PubMed] [Google Scholar]
  2. Blumenthal R., Millard P. J., Henkart M. P., Reynolds C. W., Henkart P. A. Liposomes as targets for granule cytolysin from cytotoxic large granular lymphocyte tumors. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5551–5555. doi: 10.1073/pnas.81.17.5551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Criado M., Lindstrom J. M., Anderson C. G., Dennert G. Cytotoxic granules from killer cells: specificity of granules and insertion of channels of defined size into target membranes. J Immunol. 1985 Dec;135(6):4245–4251. [PubMed] [Google Scholar]
  4. Dvorak A. M., Galli S. J., Marcum J. A., Nabel G., der Simonian H., Goldin J., Monahan R. A., Pyne K., Cantor H., Rosenberg R. D. Cloned mouse cells with natural killer function and cloned suppressor T cells express ultrastructural and biochemical features not shared by cloned inducer T cells. J Exp Med. 1983 Mar 1;157(3):843–861. doi: 10.1084/jem.157.3.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Henkart P. A. Mechanism of lymphocyte-mediated cytotoxicity. Annu Rev Immunol. 1985;3:31–58. doi: 10.1146/annurev.iy.03.040185.000335. [DOI] [PubMed] [Google Scholar]
  6. Henkart P. A., Millard P. J., Reynolds C. W., Henkart M. P. Cytolytic activity of purified cytoplasmic granules from cytotoxic rat large granular lymphocyte tumors. J Exp Med. 1984 Jul 1;160(1):75–93. doi: 10.1084/jem.160.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Klompmakers A. A., Hendriks T. A spectrophotometric microassay for sulfated glycosaminoglycans using a laser densitometer. Anal Biochem. 1986 Feb 15;153(1):80–84. doi: 10.1016/0003-2697(86)90064-3. [DOI] [PubMed] [Google Scholar]
  8. MacDermott R. P., Schmidt R. E., Caulfield J. P., Hein A., Bartley G. T., Ritz J., Schlossman S. F., Austen K. F., Stevens R. L. Proteoglycans in cell-mediated cytotoxicity. Identification, localization, and exocytosis of a chondroitin sulfate proteoglycan from human cloned natural killer cells during target cell lysis. J Exp Med. 1985 Dec 1;162(6):1771–1787. doi: 10.1084/jem.162.6.1771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Martz E. Mechanism of specific tumor-cell lysis by alloimmune T lymphocytes: resolution and characterization of discrete steps in the cellular interaction. Contemp Top Immunobiol. 1977;7:301–361. doi: 10.1007/978-1-4684-3054-7_9. [DOI] [PubMed] [Google Scholar]
  10. Masson D., Corthésy P., Nabholz M., Tschopp J. Appearance of cytolytic granules upon induction of cytolytic activity in CTL-hybrids. EMBO J. 1985 Oct;4(10):2533–2538. doi: 10.1002/j.1460-2075.1985.tb03967.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Masson D., Nabholz M., Estrade C., Tschopp J. Granules of cytolytic T-lymphocytes contain two serine esterases. EMBO J. 1986 Jul;5(7):1595–1600. doi: 10.1002/j.1460-2075.1986.tb04401.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Masson D., Tschopp J. Isolation of a lytic, pore-forming protein (perforin) from cytolytic T-lymphocytes. J Biol Chem. 1985 Aug 5;260(16):9069–9072. [PubMed] [Google Scholar]
  13. Millard P. J., Henkart M. P., Reynolds C. W., Henkart P. A. Purification and properties of cytoplasmic granules from cytotoxic rat LGL tumors. J Immunol. 1984 Jun;132(6):3197–3204. [PubMed] [Google Scholar]
  14. Montgomery R. R., Nathan C. F., Cohn Z. A. Effects of reagent and cell-generated hydrogen peroxide on the properties of low density lipoprotein. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6631–6635. doi: 10.1073/pnas.83.17.6631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Podack E. R., Konigsberg P. J. Cytolytic T cell granules. Isolation, structural, biochemical, and functional characterization. J Exp Med. 1984 Sep 1;160(3):695–710. doi: 10.1084/jem.160.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Podack E. R., Young J. D., Cohn Z. A. Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8629–8633. doi: 10.1073/pnas.82.24.8629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schmidt R. E., MacDermott R. P., Bartley G., Bertovich M., Amato D. A., Austen K. F., Schlossman S. F., Stevens R. L., Ritz J. Specific release of proteoglycans from human natural killer cells during target lysis. Nature. 1985 Nov 21;318(6043):289–291. doi: 10.1038/318289a0. [DOI] [PubMed] [Google Scholar]
  18. Tschopp J., Masson D., Schäfer S. Inhibition of the lytic activity of perforin by lipoproteins. J Immunol. 1986 Sep 15;137(6):1950–1953. [PubMed] [Google Scholar]
  19. Young J. D., Cohn Z. A. Role of granule proteins in lymphocyte-mediated killing. J Cell Biochem. 1986;32(2):151–167. doi: 10.1002/jcb.240320207. [DOI] [PubMed] [Google Scholar]
  20. Young J. D., Hengartner H., Podack E. R., Cohn Z. A. Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity. Cell. 1986 Mar 28;44(6):849–859. doi: 10.1016/0092-8674(86)90007-3. [DOI] [PubMed] [Google Scholar]
  21. Young J. D., Leong L. G., DiNome M. A., Cohn Z. A. A semiautomated hemolysis microassay for membrane lytic proteins. Anal Biochem. 1986 May 1;154(2):649–654. doi: 10.1016/0003-2697(86)90042-4. [DOI] [PubMed] [Google Scholar]
  22. Young J. D., Leong L. G., Liu C. C., Damiano A., Cohn Z. A. Extracellular release of lymphocyte cytolytic pore-forming protein (perforin) after ionophore stimulation. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5668–5672. doi: 10.1073/pnas.83.15.5668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Young J. D., Leong L. G., Liu C. C., Damiano A., Wall D. A., Cohn Z. A. Isolation and characterization of a serine esterase from cytolytic T cell granules. Cell. 1986 Oct 24;47(2):183–194. doi: 10.1016/0092-8674(86)90441-1. [DOI] [PubMed] [Google Scholar]
  24. Young J. D., Nathan C. F., Podack E. R., Palladino M. A., Cohn Z. A. Functional channel formation associated with cytotoxic T-cell granules. Proc Natl Acad Sci U S A. 1986 Jan;83(1):150–154. doi: 10.1073/pnas.83.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Young J. D., Podack E. R., Cohn Z. A. Properties of a purified pore-forming protein (perforin 1) isolated from H-2-restricted cytotoxic T cell granules. J Exp Med. 1986 Jul 1;164(1):144–155. doi: 10.1084/jem.164.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES