Skip to main content
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1988 Nov 1;168(5):1839–1854. doi: 10.1084/jem.168.5.1839

Isolation and sequence analysis of serine protease cDNAs from mouse cytolytic T lymphocytes

PMCID: PMC2189106  PMID: 3053963

Abstract

Three new cDNA clones (designated MCSP-1, MCSP-2, and MCSP-3) encoding mouse serine proteases were isolated from cloned cytolytic T lymphocytes (CTL) by a modified differential screening procedure. The putative mature proteins of MCSP-2 and MCSP-3 are each composed of 228 amino acids with molecular weights of 25,477 and 25,360, respectively. NH2-terminal amino acids of MCSP-2- and MCSP-3-predicted proteins were identical to those reported for granzyme E and F, respectively. The third species, MCSP-1, was closely related to the two other cDNA species but approximately 30 amino acids equivalents of the NH2- terminal portion of the cDNA were not cloned. The amino acids forming the active sites of serine proteases were well conserved among the three predicted proteins. The active site pocket residue positioned six residues before the active-site Ser184 is alanine in MCSP-1, threonine in MCSP-2, and serine in MCSP-3, indicating that both MCSP-2 and MCSP-3 may have chymotrypsin-like specificity. There are three potential asparagine-linked glycosylation sites in MCSP-1 and MCSP-3, and four in MCSP-2-deduced amino acid sequences. Amino acid comparison of MCSP-1 with four other reported serine proteases whose active site pocket residue is alanine revealed that MCSP-1 was substantially different from the other molecules, indicating that MCSP-1 may be a new member of mouse T cell serine protease family. Antibodies made against a MCSP-1 lacZ gene fusion protein stain granules of CTL and react on immunoblots with two distinct granule protein bands of 29 and 35-40 kD. Only the 35- kD species labels with [3H]DFP. Since a protease cascade may play a key role in cytolytic lymphocyte activation, our isolation of cDNAs representative of unique serine esterases should help to investigate such a cascade process.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

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

  1. Benfey P. N., Yin F. H., Leder P. Cloning of the mast cell protease, RMCP II. Evidence for cell-specific expression and a multi-gene family. J Biol Chem. 1987 Apr 15;262(11):5377–5384. [PubMed] [Google Scholar]
  2. 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]
  3. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brunet J. F., Dosseto M., Denizot F., Mattei M. G., Clark W. R., Haqqi T. M., Ferrier P., Nabholz M., Schmitt-Verhulst A. M., Luciani M. F. The inducible cytotoxic T-lymphocyte-associated gene transcript CTLA-1 sequence and gene localization to mouse chromosome 14. Nature. 1986 Jul 17;322(6076):268–271. doi: 10.1038/322268a0. [DOI] [PubMed] [Google Scholar]
  5. Chang T. W., Eisen H. N. Effects of N alpha-tosyl-L-lysyl-chloromethylketone on the activity of cytotoxic T lymphocytes. J Immunol. 1980 Mar;124(3):1028–1033. [PubMed] [Google Scholar]
  6. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  7. Gershenfeld H. K., Weissman I. L. Cloning of a cDNA for a T cell-specific serine protease from a cytotoxic T lymphocyte. Science. 1986 May 16;232(4752):854–858. doi: 10.1126/science.2422755. [DOI] [PubMed] [Google Scholar]
  8. Glasebrook A. L., Fitch F. W. Alloreactive cloned T cell lines. I. Interactions between cloned amplifier and cytolytic T cell lines. J Exp Med. 1980 Apr 1;151(4):876–895. doi: 10.1084/jem.151.4.876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goldfarb R. H. Cell-mediated cytotoxic reactions. Hum Pathol. 1986 Feb;17(2):138–145. doi: 10.1016/s0046-8177(86)80286-6. [DOI] [PubMed] [Google Scholar]
  10. Gross-Bellard M., Oudet P., Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem. 1973 Jul 2;36(1):32–38. doi: 10.1111/j.1432-1033.1973.tb02881.x. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Jenne D., Rey C., Haefliger J. A., Qiao B. Y., Groscurth P., Tschopp J. Identification and sequencing of cDNA clones encoding the granule-associated serine proteases granzymes D, E, and F of cytolytic T lymphocytes. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4814–4818. doi: 10.1073/pnas.85.13.4814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jenne D., Rey C., Masson D., Stanley K. K., Herz J., Plaetinck G., Tschopp J. cDNA cloning of granzyme C, a granule-associated serine protease of cytolytic T lymphocytes. J Immunol. 1988 Jan 1;140(1):318–323. [PubMed] [Google Scholar]
  14. Kraut J. Serine proteases: structure and mechanism of catalysis. Annu Rev Biochem. 1977;46:331–358. doi: 10.1146/annurev.bi.46.070177.001555. [DOI] [PubMed] [Google Scholar]
  15. Kwon B. S., Kim G. S., Prystowsky M. B., Lancki D. W., Sabath D. E., Pan J. L., Weissman S. M. Isolation and initial characterization of multiple species of T-lymphocyte subset cDNA clones. Proc Natl Acad Sci U S A. 1987 May;84(9):2896–2900. doi: 10.1073/pnas.84.9.2896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Lobe C. G., Finlay B. B., Paranchych W., Paetkau V. H., Bleackley R. C. Novel serine proteases encoded by two cytotoxic T lymphocyte-specific genes. Science. 1986 May 16;232(4752):858–861. doi: 10.1126/science.3518058. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Masson D., Tschopp J. A family of serine esterases in lytic granules of cytolytic T lymphocytes. Cell. 1987 Jun 5;49(5):679–685. doi: 10.1016/0092-8674(87)90544-7. [DOI] [PubMed] [Google Scholar]
  20. Mehra V., Sweetser D., Young R. A. Efficient mapping of protein antigenic determinants. Proc Natl Acad Sci U S A. 1986 Sep;83(18):7013–7017. doi: 10.1073/pnas.83.18.7013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neurath H. Evolution of proteolytic enzymes. Science. 1984 Apr 27;224(4647):350–357. doi: 10.1126/science.6369538. [DOI] [PubMed] [Google Scholar]
  23. Pasternack M. S., Eisen H. N. A novel serine esterase expressed by cytotoxic T lymphocytes. 1985 Apr 25-May 1Nature. 314(6013):743–745. doi: 10.1038/314743a0. [DOI] [PubMed] [Google Scholar]
  24. Pasternack M. S., Verret C. R., Liu M. A., Eisen H. N. Serine esterase in cytolytic T lymphocytes. Nature. 1986 Aug 21;322(6081):740–743. doi: 10.1038/322740a0. [DOI] [PubMed] [Google Scholar]
  25. Redelman D., Hudig D. The mechanism of cell-mediated cytotoxicity. I. Killing by murine cytotoxic T lymphocytes requires cell surface thiols and activated proteases. J Immunol. 1980 Feb;124(2):870–878. [PubMed] [Google Scholar]
  26. Reid K. B. Immune system. Complement-like cytotoxicity? Nature. 1986 Aug 21;322(6081):684–684. doi: 10.1038/322684a0. [DOI] [PubMed] [Google Scholar]
  27. Salvesen G., Farley D., Shuman J., Przybyla A., Reilly C., Travis J. Molecular cloning of human cathepsin G: structural similarity to mast cell and cytotoxic T lymphocyte proteinases. Biochemistry. 1987 Apr 21;26(8):2289–2293. doi: 10.1021/bi00382a032. [DOI] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Segal D. M., Powers J. C., Cohen G. H., Davies D. R., Wilcox P. E. Substrate binding site in bovine chymotrypsin A-gamma. A crystallographic study using peptide chloromethyl ketones as site-specific inhibitors. Biochemistry. 1971 Sep 28;10(20):3728–3738. doi: 10.1021/bi00796a014. [DOI] [PubMed] [Google Scholar]
  30. Simon M. M., Hoschützky H., Fruth U., Simon H. G., Kramer M. D. Purification and characterization of a T cell specific serine proteinase (TSP-1) from cloned cytolytic T lymphocytes. EMBO J. 1986 Dec 1;5(12):3267–3274. doi: 10.1002/j.1460-2075.1986.tb04638.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  32. Trinchieri G., Perussia B. Human natural killer cells: biologic and pathologic aspects. Lab Invest. 1984 May;50(5):489–513. [PubMed] [Google Scholar]
  33. Tschopp J., Jongeneel C. V. Cytotoxic T lymphocyte mediated cytolysis. Biochemistry. 1988 Apr 19;27(8):2641–2646. doi: 10.1021/bi00408a001. [DOI] [PubMed] [Google Scholar]
  34. Tschopp J., Nabholz M. The role of cytoplasmic granule components in cytolytic lymphocyte-mediated cytolysis. Ann Inst Pasteur Immunol. 1987 Mar-Apr;138(2):290–295. doi: 10.1016/s0769-2625(87)80081-8. [DOI] [PubMed] [Google Scholar]
  35. Vujanovic N. L., Herberman R. B., Maghazachi A. A., Hiserodt J. C. Lymphokine-activated killer cells in rats. III. A simple method for the purification of large granular lymphocytes and their rapid expansion and conversion into lymphokine-activated killer cells. J Exp Med. 1988 Jan 1;167(1):15–29. doi: 10.1084/jem.167.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Woodbury R. G., Katunuma N., Kobayashi K., Titani K., Neurath H., Anderson W. F., Matthews B. W. Covalent structure of a group-specific protease from rat small intestine. Appendix: crystallographic data for a group specific protease from rat intestine. Biochemistry. 1978 Mar 7;17(5):811–819. doi: 10.1021/bi00598a010. [DOI] [PubMed] [Google Scholar]
  37. Young J. D., Cohn Z. A. Cellular and humoral mechanisms of cytotoxicity: structural and functional analogies. Adv Immunol. 1987;41:269–332. doi: 10.1016/s0065-2776(08)60033-4. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES