Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1984 Jul 1;160(1):116–124. doi: 10.1084/jem.160.1.116

Immunochemical analysis of the released Leu-2 (T8) molecule

PMCID: PMC2187423  PMID: 6203997

Abstract

We recently have found that the human T cell antigen Leu-2 was specifically released from Leu-2-bearing cells. The preliminary study showed that the released Leu-2 (RLeu-2) from HPB-ALL cells was composed of a single polypeptide chain of 27,000 molecular weight (mol wt), which was smaller than the subunit of the homodimeric molecule found on the cell surface. In the present study, RLeu-2 was further characterized and compared with cellular Leu-2 (CLeu-2). Metabolically radiolabeled Leu-2 was released from HPB-ALL cells and this released Leu-2 molecule had a mol wt of 27,000. Cell surface radioiodinated HPB- ALL cells were found to release radioactive Leu-2 molecules and this antigen also had the same mol wt of 27,000. In both experiments, the CLeu-2 was reconfirmed to be composed of a 33,000-mol wt subunit under reducing conditions. These experiments establish that the 27,000-mol wt single polypeptide chain of Leu-2 released from the cell is derived directly from the homodimeric Leu-2 molecule on the cell surface, presumably by a specific proteolytic cleavage. Two-dimensional gel analysis showed that CLeu-2 exhibited extensive charge heterogeneity with predominantly basic isoelectric points, whereas RLeu-2 was a group of more acidic proteins with less charge heterogeneity. Although CLeu-2 and RLeu-2 showed several different immunochemical characteristics, the homology between these two antigens was confirmed by the following results: CLeu-2 and RLeu-2 were found to share at least three different antigenic determinants, Leu-2a and Leu-2b, and those which were detected by a polyvalent rabbit antiserum. Significant similarities between CLeu-2 and RLeu-2 were demonstrated by peptide mapping analysis of these antigens. Therefore, RLeu-2 appears to be the specific, physiological product of the CLeu-2 protein.

Full Text

The Full Text of this article is available as a PDF (958.4 KB).

Selected References

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

  1. Cantor H., Boyse E. A. Lymphocytes as models for the study of mammalian cellular differentiation. Immunol Rev. 1977 Jan;33:105–124. doi: 10.1111/j.1600-065x.1977.tb00364.x. [DOI] [PubMed] [Google Scholar]
  2. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  3. Engleman E. G., Benike C. J., Glickman E., Evans R. L. Antibodies to membrane structures that distinguish suppressor/cytotoxic and helper T lymphocyte subpopulations block the mixed leukocyte reaction in man. J Exp Med. 1981 Jul 1;154(1):193–198. doi: 10.1084/jem.154.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Engleman E. G., Benike C. J., Grumet F. C., Evans R. L. Activation of human T lymphocyte subsets: helper and suppressor/cytotoxic T cells recognize and respond to distinct histocompatibility antigens. J Immunol. 1981 Nov;127(5):2124–2129. [PubMed] [Google Scholar]
  5. Evans R. L., Wall D. W., Platsoucas C. D., Siegal F. P., Fikrig S. M., Testa C. M., Good R. A. Thymus-dependent membrane antigens in man: inhibition of cell-mediated lympholysis by monoclonal antibodies to TH2 antigen. Proc Natl Acad Sci U S A. 1981 Jan;78(1):544–548. doi: 10.1073/pnas.78.1.544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fujimoto J., Levy S., Levy R. Spontaneous release of the Leu-2 (T8) molecule from human T cells. J Exp Med. 1983 Sep 1;158(3):752–766. doi: 10.1084/jem.158.3.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goding J. W., Harris A. W. Subunit structure of cell surface proteins: disulfide bonding in antigen receptors, Ly-2/3 antigens, and transferrin receptors of murine T and B lymphocytes. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4530–4534. doi: 10.1073/pnas.78.7.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Haustein D. Effective radioiodination by lactoperoxidase and solubilisation of cell-surface proteins of cultured murine T lymphoma cells. J Immunol Methods. 1975 Apr;7(1):25–38. doi: 10.1016/0022-1759(75)90127-1. [DOI] [PubMed] [Google Scholar]
  9. Jay G., Palladino M. A., Khoury G., Old L. J. Mouse Lyt-2 antigen: evidence for two heterodimers with a common subunit. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2654–2657. doi: 10.1073/pnas.79.8.2654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kress M., Cosman D., Khoury G., Jay G. Secretion of a transplantation-related antigen. Cell. 1983 Aug;34(1):189–196. doi: 10.1016/0092-8674(83)90149-6. [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. Ledbetter J. A., Evans R. L., Lipinski M., Cunningham-Rundles C., Good R. A., Herzenberg L. A. Evolutionary conservation of surface molecules that distinguish T lymphocyte helper/inducer and cytotoxic/suppressor subpopulations in mouse and man. J Exp Med. 1981 Feb 1;153(2):310–323. doi: 10.1084/jem.153.2.310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ledbetter J. A., Seaman W. E., Tsu T. T., Herzenberg L. A. Lyt-2 and lyt-3 antigens are on two different polypeptide subunits linked by disulfide bonds. Relationship of subunits to T cell cytolytic activity. J Exp Med. 1981 Jun 1;153(6):1503–1516. doi: 10.1084/jem.153.6.1503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Meuer S. C., Schlossman S. F., Reinherz E. L. Clonal analysis of human cytotoxic T lymphocytes: T4+ and T8+ effector T cells recognize products of different major histocompatibility complex regions. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4395–4399. doi: 10.1073/pnas.79.14.4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Natori T., Tanigaki N., Pressman D. A mouse plasma substance carrying beta2-microglobulin activity and lacking in H-2 alloantigenic activity. J Immunogenet. 1976 Apr;3(2):123–134. doi: 10.1111/j.1744-313x.1976.tb00563.x. [DOI] [PubMed] [Google Scholar]
  16. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  17. Reilly E. B., Auditore-Hargreaves K., Hämmerling U., Gottlieb P. D. Lyt-2 and Lyt-3 alloantigens: precipitation with monoclonal and conventional antibodies and analysis on one- and two-dimensional polyacrylamide gels. J Immunol. 1980 Nov;125(5):2245–2251. [PubMed] [Google Scholar]
  18. Reinherz E. L., Hussey R. E., Fitzgerald K., Snow P., Terhorst C., Schlossman S. F. Antibody directed at a surface structure inhibits cytolytic but not suppressor function of human T lymphocytes. Nature. 1981 Nov 12;294(5837):168–170. doi: 10.1038/294168a0. [DOI] [PubMed] [Google Scholar]
  19. Reinherz E. L., Meuer S. C., Schlossman S. F. The human T cell receptor: analysis with cytotoxic T cell clones. Immunol Rev. 1983;74:83–112. doi: 10.1111/j.1600-065x.1983.tb01085.x. [DOI] [PubMed] [Google Scholar]
  20. Rothenberg E., Triglia D. Lyt-2 glycoprotein is synthesized as a single molecular species. J Exp Med. 1983 Jan 1;157(1):365–370. doi: 10.1084/jem.157.1.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Snow P., Spits H., De Vries J., Terhorst C. Comparison of target antigens of monoclonal reagents OKT5, OKT8, and Leu2A, which inhibit effector function of human cytotoxic T lymphocytes. Hybridoma. 1983;2(2):187–199. doi: 10.1089/hyb.1983.2.187. [DOI] [PubMed] [Google Scholar]
  22. Terhorst C., van Agthoven A., Reinherz E., Schlossman S. Biochemical analysis of human T lymphocyte differentiation antigens T4 and T5. Science. 1980 Jul 25;209(4455):520–521. doi: 10.1126/science.6967228. [DOI] [PubMed] [Google Scholar]

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

RESOURCES