Abstract
The cytolytic pore-forming protein (PFP, perforin) of lymphocyte granules has recently been isolated and characterized. The lytic activities expressed by both the isolated granules and the purified PFP require the presence of Ca2+. Here, we report on the extracellular release of PFP after stimulation of lymphocytes with the Ca2+ ionophore A23187, which degranulates the cells. The secreted protein associates with lipid to form structural and functional channels and supramolecular complexes that partially resist dissociation by sodium dodecyl sulfate and reducing agents. Immunoblots of the released material reveal positive identification with antibodies specific for mouse PFP and human complement component C9, indicating cross-reactivity between these two molecules. By using these specific antibodies as immunoadsorbents, the lymphocyte PFP has been affinity purified from the supernatant of stimulated cells. The extracellular release of PFP is associated with simultaneous formation of functional ion-nonselective channels with conductances of 550-600 pS in 0.15 M NaCl, as measured in planar model bilayers. In the absence of extracellular Ca2+, 15% of the maximal release activity is observed. Ca2+ appears to be required to elicit both secretion by lymphocytes and the assembly of the released PFP into tubular polymers. Similar secretion of PFP may occur during cell killing by lymphocytes, resulting in its assembly on target membranes to form tubular transmembrane lesions.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- 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]
- 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]
- 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]
- Kupfer A., Dennert G. Reorientation of the microtubule-organizing center and the Golgi apparatus in cloned cytotoxic lymphocytes triggered by binding to lysable target cells. J Immunol. 1984 Nov;133(5):2762–2766. [PubMed] [Google Scholar]
- 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]
- Lynch E. C., Rosenberg I. M., Gitler C. An ion-channel forming protein produced by Entamoeba histolytica. EMBO J. 1982;1(7):801–804. doi: 10.1002/j.1460-2075.1982.tb01250.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Montal M., Mueller P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3561–3566. doi: 10.1073/pnas.69.12.3561. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Palladino M. A., Obata Y., Stockert E., Oettgen H. F. Characterization of interleukin 2-dependent cytotoxic T-cell clones: specificity, cell surface phenotype, and susceptibility to blocking by Lyt antisera. Cancer Res. 1983 Feb;43(2):572–576. [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Yannelli J. R., Sullivan J. A., Mandell G. L., Engelhard V. H. Reorientation and fusion of cytotoxic T lymphocyte granules after interaction with target cells as determined by high resolution cinemicrography. J Immunol. 1986 Jan;136(2):377–382. [PubMed] [Google Scholar]
- Young J. D., Blake M., Mauro A., Cohn Z. A. Properties of the major outer membrane protein from Neisseria gonorrhoeae incorporated into model lipid membranes. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3831–3835. doi: 10.1073/pnas.80.12.3831. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Young J. D., Cohn Z. A. Molecular mechanisms of cytotoxicity mediated by Entamoeba histolytica: characterization of a pore-forming protein (PFP). J Cell Biochem. 1985;29(4):299–308. doi: 10.1002/jcb.240290404. [DOI] [PubMed] [Google Scholar]
- Young J. D., Cohn Z. A., Podack E. R. The ninth component of complement and the pore-forming protein (perforin 1) from cytotoxic T cells: structural, immunological, and functional similarities. Science. 1986 Jul 11;233(4760):184–190. doi: 10.1126/science.2425429. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Young J. D., Peterson C. G., Venge P., Cohn Z. A. Mechanism of membrane damage mediated by human eosinophil cationic protein. Nature. 1986 Jun 5;321(6070):613–616. doi: 10.1038/321613a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Young J. D., Unkeless J. C., Kaback H. R., Cohn Z. A. Macrophage membrane potential changes associated with gamma 2b/gamma 1 Fc receptor-ligand binding. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1357–1361. doi: 10.1073/pnas.80.5.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Young J. D., Unkeless J. C., Young T. M., Mauro A., Cohn Z. A. Role for mouse macrophage IgG Fc receptor as ligand-dependent ion channel. Nature. 1983 Nov 10;306(5939):186–189. doi: 10.1038/306186a0. [DOI] [PubMed] [Google Scholar]
- Young J. D., Young T. M., Lu L. P., Unkeless J. C., Cohn Z. A. Characterization of a membrane pore-forming protein from Entamoeba histolytica. J Exp Med. 1982 Dec 1;156(6):1677–1690. doi: 10.1084/jem.156.6.1677. [DOI] [PMC free article] [PubMed] [Google Scholar]