Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 May;85(10):3535–3539. doi: 10.1073/pnas.85.10.3535

Saturation mutagenesis of a major histocompatibility complex protein domain: identification of a single conserved amino acid important for allorecognition.

R Murray 1, C A Hutchison 3rd 1, J A Frelinger 1
PMCID: PMC280247  PMID: 3285344

Abstract

The interactive association between T lymphocytes and their target cells is an important system of cell-cell interactions. Major histocompatibility complex class I molecules are the cell surface structures recognized by cytolytic T lymphocytes. To define the molecular structures recognized by cytotoxic T lymphocytes, we have saturated the 270-base-pair alpha 1 exon of the H-2Dp gene with point mutations, rapidly producing a "library" of 2.5 x 10(3) independent mutants. The library contains enough recombinant clones (each clone encoding approximately one amino acid replacement mutation) to predict a mutation at each nucleotide position of the alpha 1 exon. The functional analysis of the first five transfected gene products tested has shown that mutation of a conserved tyrosine at position 27 to asparagine destroys recognition of the H-2Dp gene product by polyclonal alloreactive cytotoxic T lymphocytes. Recognition of the same mutant molecule by three monoclonal antibodies and H-2-restricted lymphocytic choriomenengitis virus-specific cytotoxic T lymphocytes is unaffected.

Full text

PDF
3535

Images in this article

3537Image
on p.3537

Selected References

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

  1. Arnold B., Horstmann U., Kuon W., Burgert H. G., Hämmerling G. J., Kvist S. Alloreactive cytolytic T-cell clones preferentially recognize conformational determinants on histocompatibility antigens: analysis with genetically engineered hybrid antigens. Proc Natl Acad Sci U S A. 1985 Oct;82(20):7030–7034. doi: 10.1073/pnas.82.20.7030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babbitt B. P., Allen P. M., Matsueda G., Haber E., Unanue E. R. Binding of immunogenic peptides to Ia histocompatibility molecules. 1985 Sep 26-Oct 2Nature. 317(6035):359–361. doi: 10.1038/317359a0. [DOI] [PubMed] [Google Scholar]
  3. Benzer S. FINE STRUCTURE OF A GENETIC REGION IN BACTERIOPHAGE. Proc Natl Acad Sci U S A. 1955 Jun 15;41(6):344–354. doi: 10.1073/pnas.41.6.344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bjorkman P. J., Saper M. A., Samraoui B., Bennett W. S., Strominger J. L., Wiley D. C. Structure of the human class I histocompatibility antigen, HLA-A2. Nature. 1987 Oct 8;329(6139):506–512. doi: 10.1038/329506a0. [DOI] [PubMed] [Google Scholar]
  5. Bjorkman P. J., Saper M. A., Samraoui B., Bennett W. S., Strominger J. L., Wiley D. C. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature. 1987 Oct 8;329(6139):512–518. doi: 10.1038/329512a0. [DOI] [PubMed] [Google Scholar]
  6. Buus S., Sette A., Colon S. M., Jenis D. M., Grey H. M. Isolation and characterization of antigen-Ia complexes involved in T cell recognition. Cell. 1986 Dec 26;47(6):1071–1077. doi: 10.1016/0092-8674(86)90822-6. [DOI] [PubMed] [Google Scholar]
  7. Darsley M. J., Takahashi H., Macchi M. J., Frelinger J. A., Ozato K., Appella E. New family of exon-shuffled recombinant genes reveals extensive interdomain interactions in class I histocompatibility antigens and identifies residues involved. J Exp Med. 1987 Jan 1;165(1):211–222. doi: 10.1084/jem.165.1.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Evans G. A., Margulies D. H., Shykind B., Seidman J. G., Ozato K. Exon shuffling: mapping polymorphic determinants on hybrid mouse transplantation antigens. Nature. 1982 Dec 23;300(5894):755–757. doi: 10.1038/300755a0. [DOI] [PubMed] [Google Scholar]
  9. Geier S. S., Zeff R. A., McGovern D. M., Rajan T. V., Nathenson S. G. An approach to the study of structure-function relationships of MHC class I molecules: isolation and serologic characterization of H-2Kb somatic cell variants. J Immunol. 1986 Aug 15;137(4):1239–1243. [PubMed] [Google Scholar]
  10. Guillet J. G., Lai M. Z., Briner T. J., Buus S., Sette A., Grey H. M., Smith J. A., Gefter M. L. Immunological self, nonself discrimination. Science. 1987 Feb 20;235(4791):865–870. doi: 10.1126/science.2433769. [DOI] [PubMed] [Google Scholar]
  11. Harmon R. C., Stein N., Frelinger J. A. Monoclonal antibodies reactive with H-2 determinants. Immunogenetics. 1983;18(5):541–545. doi: 10.1007/BF00364395. [DOI] [PubMed] [Google Scholar]
  12. Hasenkrug K. J., Cory J. M., Stimpfling J. H. Monoclonal antibodies defining mouse tissue antigens encoded by the H-2 region. Immunogenetics. 1987;25(2):136–139. doi: 10.1007/BF00364282. [DOI] [PubMed] [Google Scholar]
  13. Hutchison C. A., 3rd, Nordeen S. K., Vogt K., Edgell M. H. A complete library of point substitution mutations in the glucocorticoid response element of mouse mammary tumor virus. Proc Natl Acad Sci U S A. 1986 Feb;83(3):710–714. doi: 10.1073/pnas.83.3.710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Judd B. H., Young M. W. An examination of the one cistron: one chromomere concept. Cold Spring Harb Symp Quant Biol. 1974;38:573–579. doi: 10.1101/sqb.1974.038.01.061. [DOI] [PubMed] [Google Scholar]
  15. Kanda T., LaPan K., Takahashi H., Appella E., Frelinger J. A. The alpha 1 and alpha 2 domains of H-2 class I molecules interact to form unique epitopes. Immunogenetics. 1987;25(2):110–115. doi: 10.1007/BF00364276. [DOI] [PubMed] [Google Scholar]
  16. MacDonald H. R., Glasebrook A. L., Bron C., Kelso A., Cerottini J. C. Clonal heterogeneity in the functional requirement for Lyt-2/3 molecules on cytolytic T lymphocytes (CTL): possible implications for the affinity of CTL antigen receptors. Immunol Rev. 1982;68:89–115. doi: 10.1111/j.1600-065x.1982.tb01061.x. [DOI] [PubMed] [Google Scholar]
  17. Macchi M. J., Woodward J. G., McLaughlin-Taylor E., Griffin J., Hood L., Frelinger J. A. Cloning and identification of the H-2Dp gene. Immunogenetics. 1984;19(3):195–204. doi: 10.1007/BF00364763. [DOI] [PubMed] [Google Scholar]
  18. McLaughlin-Taylor E., Woodward J. G., McMillan M., Frelinger J. A. Distinct epitopes are recognized by cytolytic T lymphocyte clones on the same class I molecule: direct demonstration using DNA-transfected targets and long-term cytolytic T cell clones. Eur J Immunol. 1984 Nov;14(11):969–974. doi: 10.1002/eji.1830141102. [DOI] [PubMed] [Google Scholar]
  19. Nathenson S. G., Geliebter J., Pfaffenbach G. M., Zeff R. A. Murine major histocompatibility complex class-I mutants: molecular analysis and structure-function implications. Annu Rev Immunol. 1986;4:471–502. doi: 10.1146/annurev.iy.04.040186.002351. [DOI] [PubMed] [Google Scholar]
  20. Orn A., Goodenow R. S., Hood L., Brayton P. R., Woodward J. G., Harmon R. C., Frelinger J. A. Product of a transferred H-2Ld gene acts as restriction element for LCMV-specific killer T cells. Nature. 1982 Jun 3;297(5865):415–417. doi: 10.1038/297415a0. [DOI] [PubMed] [Google Scholar]
  21. Pious D., Krangel M. S., Dixon L. L., Parham P., Strominger J. L. HLA antigen structural gene mutants selected with an allospecific monoclonal antibody. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7832–7836. doi: 10.1073/pnas.79.24.7832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Porter S. D., Smith M. Homoeo-domain homology in yeast MAT alpha 2 is essential for repressor activity. Nature. 1986 Apr 24;320(6064):766–768. doi: 10.1038/320766a0. [DOI] [PubMed] [Google Scholar]
  23. Potter T. A., Bluestone J. A., Rajan T. V. A single amino acid substitution in the alpha 3 domain of an H-2 class I molecule abrogates reactivity with CTL. J Exp Med. 1987 Oct 1;166(4):956–966. doi: 10.1084/jem.166.4.956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Salter R. D., Clayberger C., Lomen C. E., Krensky A. M., Parham P. In vitro mutagenesis at a single residue introduces B and T cell epitopes into a class I HLA molecule. J Exp Med. 1987 Jul 1;166(1):283–288. doi: 10.1084/jem.166.1.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  26. Santos-Aguado J., Biro P. A., Fuhrmann U., Strominger J. L., Barbosa J. A. Amino acid sequences in the alpha 1 domain and not glycosylation are important in HLA-A2/beta 2-microglobulin association and cell surface expression. Mol Cell Biol. 1987 Mar;7(3):982–990. doi: 10.1128/mcb.7.3.982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schepart B. S., Takahashi H., Cozad K. M., Murray R., Ozato K., Appella E., Frelinger J. A. The nucleotide sequence and comparative analysis of the H-2Dp class I H-2 gene. J Immunol. 1986 May 1;136(9):3489–3495. [PubMed] [Google Scholar]
  28. Shiroishi T., Evans G. A., Appella E., Ozato K. In vitro mutagenesis of a mouse MHC class I gene for the examination of structure-function relationships. J Immunol. 1985 Jan;134(1):623–629. [PubMed] [Google Scholar]
  29. Stroynowski I., Orn A., Goodenow R. S., McMillan M., Forman J., Brayton P. R., Frelinger J., Hood L. Cytotoxic T lymphocytes recognize determinants on the BALB/c-H-2Ld molecule controlled by alpha 1 and alpha 2 but not alpha 3 external domains. Immunogenetics. 1984;20(2):141–154. doi: 10.1007/BF00364486. [DOI] [PubMed] [Google Scholar]
  30. Stuart G. W., Searle P. F., Chen H. Y., Brinster R. L., Palmiter R. D. A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7318–7322. doi: 10.1073/pnas.81.23.7318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Townsend A. R., Rothbard J., Gotch F. M., Bahadur G., Wraith D., McMichael A. J. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell. 1986 Mar 28;44(6):959–968. doi: 10.1016/0092-8674(86)90019-x. [DOI] [PubMed] [Google Scholar]
  32. Woodward J. G., Orn A., Harmon R. C., Goodenow R. S., Hood L., Frelinger J. A. Specific recognition of the product of a transferred major histocompatibility complex gene by cytotoxic T lymphocytes. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3613–3617. doi: 10.1073/pnas.79.11.3613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yunis E. J., Amos D. B. Three closely linked genetic systems relevant to transplantation. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3031–3035. doi: 10.1073/pnas.68.12.3031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zinkernagel R. M., Doherty P. C. MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function, and responsiveness. Adv Immunol. 1979;27:51–177. doi: 10.1016/s0065-2776(08)60262-x. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES