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
. 1987 Jul;84(14):5014–5018. doi: 10.1073/pnas.84.14.5014

Site-directed mutagenesis of an HLA-A3 gene identifies amino acid 152 as crucial for major-histocompatibility-complex-restricted and alloreactive cytotoxic-T-lymphocyte recognition.

E P Cowan, M L Jelachich, J E Coligan, W E Biddison
PMCID: PMC305237  PMID: 2440039

Abstract

Major histocompatibility complex-restricted and alloreactive cytotoxic T lymphocytes (CTL) can discriminate between the HLA-A3.1 and HLA-A3.2 antigens. HLA-A3.1 and the rare variant HLA-A3.2 have been shown to differ by two amino acids in the alpha 2 domain at positions 152 (A3.1, glutamic acid; A3.2, valine) and 156 (A3.1, leucine; A3.2, glutamine). To determine the structural basis for the ability of CTL to differentiate A3.1 from A3.2, two site-directed mutants of the HLA-A3.2 gene were produced, 152A3.1-156A3.2 and 152A3.2-156A3.1, that have the indicated codons for positions 152 and 156. These mutated HLA-A3 genes, as well as the nonmutated HLA-A3.1 and HLA-A3.2 genes, were then transfected into the murine cell line P815-HTR and used as targets for human CTL. Influenza virus-specific HLA-A3.1-restricted CTL lysed virus-infected P815 cells transformed with the HLA-A3.1 and 152A3.1-156A3.2 genes, but not P815 cells transformed with the HLA-A3.2 and 152A3.2-156A3.1 genes. HLA-A3.2-allospecific CTL lysed the P815 cells transformed with the HLA-A3.2 and 152A3.2-156A3.1 genes but did not lyse P815 cells transformed with the HLA-A3.1 or 152A3.1-156A3.2 genes. Thus, a single amino acid change at position 152, substituting valine for glutamic acid and thereby introducing a charge difference, produces major structural changes in the epitopes recognized by major histocompatibility complex-restricted and alloreactive CTL.

Full text

PDF
5015

Selected References

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

  1. Arnold B., Burgert H. G., Hamann U., Hämmerling G., Kees U., Kvist S. Cytolytic T cells recognize the two amino-terminal domains of H-2 K antigens in tandem in influenza A infected cells. Cell. 1984 Aug;38(1):79–87. doi: 10.1016/0092-8674(84)90528-2. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Barbosa J. A., Mentzer S. J., Minowada G., Strominger J. L., Burakoff S. J., Biro P. A. Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after gene transfer into human and monkey, but not mouse, cells. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7549–7553. doi: 10.1073/pnas.81.23.7549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berger A. E., Davis J. E., Cresswell P. Monoclonal antibody to HLA-A3. Hybridoma. 1982;1(2):87–90. doi: 10.1089/hyb.1.1982.1.87. [DOI] [PubMed] [Google Scholar]
  5. Biddison W. E., Sharrow S. O., Shearer G. M. T cell subpopulations required for the human cytotoxic T lymphocyte response to influenza virus: evidence for T cell help. J Immunol. 1981 Aug;127(2):487–491. [PubMed] [Google Scholar]
  6. Biddison W. E., Shearer G. M., Shaw S. Influenza virus-specific cytotoxic T cells are restricted by multiple HLA-A3-related self antigens: evidence for recognition of distinct self structures in conjunction with different foreign antigens. J Immunol. 1981 Dec;127(6):2231–2235. [PubMed] [Google Scholar]
  7. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cowan E. P., Coligan J. E., Biddison W. E. Human cytotoxic T-lymphocyte recognition of an HLA-A3 gene product expressed on murine L cells: the only human gene product required on the target cells for lysis is the class I heavy chain. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4490–4494. doi: 10.1073/pnas.82.13.4490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cowan E. P., Jordan B. R., Coligan J. E. Molecular cloning and DNA sequence analysis of genes encoding cytotoxic T lymphocyte-defined HLA-A3 subtypes: the E1 subtype. J Immunol. 1985 Oct;135(4):2835–2841. [PubMed] [Google Scholar]
  10. DeLisi C., Berzofsky J. A. T-cell antigenic sites tend to be amphipathic structures. Proc Natl Acad Sci U S A. 1985 Oct;82(20):7048–7052. doi: 10.1073/pnas.82.20.7048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gomard E., Begue B., Sodoyer S., Maryanski J. L., Jordan B. R., Levy J. P. Murine cells expressing an HLA molecule are specifically lysed by HLA-restricted antiviral human T cells. Nature. 1986 Jan 9;319(6049):153–154. doi: 10.1038/319153a0. [DOI] [PubMed] [Google Scholar]
  12. Kimball E. S., Coligan J. E. Structure of class I major histocompatibility antigens. Contemp Top Mol Immunol. 1983;9:1–63. doi: 10.1007/978-1-4684-4517-6_1. [DOI] [PubMed] [Google Scholar]
  13. Krangel M. S., Biddison W. E., Strominger J. L. Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic T lymphocytes. II. Variant DK1: evidence for a discrete CTL recognition region. J Immunol. 1983 Apr;130(4):1856–1862. [PubMed] [Google Scholar]
  14. Krangel M. S., Taketani S., Biddison W. E., Strong D. M., Strominger J. L. Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic T lymphocytes: variants M7 and DR1. Biochemistry. 1982 Nov 23;21(24):6313–6321. doi: 10.1021/bi00267a042. [DOI] [PubMed] [Google Scholar]
  15. Lopez de Castro J. A., Barbosa J. A., Krangel M. S., Biro P. A., Strominger J. L. Structural analysis of the functional sites of class I HLA antigens. Immunol Rev. 1985 Jul;85:149–168. doi: 10.1111/j.1600-065x.1985.tb01134.x. [DOI] [PubMed] [Google Scholar]
  16. Maryanski J. L., Moretta A., Jordan B., De Plaen E., Van Pel A., Boon T., Cerottini J. C. Human T cell recognition of cloned HLA class I gene products expressed on DNA transfectants of mouse mastocytoma P815. Eur J Immunol. 1985 Nov;15(11):1111–1117. doi: 10.1002/eji.1830151109. [DOI] [PubMed] [Google Scholar]
  17. Mentzer S. J., Barbosa J. A., Strominger J. L., Biro P. A., Burakoff S. J. Species-restricted recognition of transfected HLA-A2 and HLA-B7 by human CTL clones. J Immunol. 1986 Jul 15;137(2):408–413. [PubMed] [Google Scholar]
  18. Miyazaki J., Appella E., Ozato K. Intracellular transport blockade caused by disruption of the disulfide bridge in the third external domain of major histocompatibility complex class I antigen. Proc Natl Acad Sci U S A. 1986 Feb;83(3):757–761. doi: 10.1073/pnas.83.3.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Monos D. S., Tekolf W. A., Shaw S., Cooper H. L. Comparison of structural and functional variation in class I HLA molecules: the role of charged amino acid substitutions. J Immunol. 1984 Mar;132(3):1379–1385. [PubMed] [Google Scholar]
  20. Murre C., Choi E., Weis J., Seidman J. G., Ozato K., Liu L., Burakoff S. J., Reiss C. S. Dissection of serological and cytolytic T lymphocyte epitopes on murine major histocompatibility antigens by a recombinant H-2 gene separating the first two external domains. J Exp Med. 1984 Jul 1;160(1):167–178. doi: 10.1084/jem.160.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Ozato K., Mayer N. M., Sachs D. H. Monoclonal antibodies to mouse major histocompatibility complex antigens. Transplantation. 1982 Sep;34(3):113–120. doi: 10.1097/00007890-198209000-00001. [DOI] [PubMed] [Google Scholar]
  23. Rebaï N., Malissen B. Structural and genetic analyses of HLA class I molecules using monoclonal xenoantibodies. Tissue Antigens. 1983 Aug;22(2):107–117. doi: 10.1111/j.1399-0039.1983.tb01176.x. [DOI] [PubMed] [Google Scholar]
  24. Reiss C. S., Greenstein J. L., Crimmins M. A., Liu L. L., Rao A., Maziaraz R. T., Murre C., Burakoff S. J. Recognition of the alpha-1 and alpha-2 domains of H-2 molecules by allospecific cloned T cells. J Immunol. 1986 Mar 15;136(6):2191–2194. [PubMed] [Google Scholar]
  25. 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]
  26. Schulze D. H., Pease L. R., Geier S. S., Reyes A. A., Sarmiento L. A., Wallace R. B., Nathenson S. G. Comparison of the cloned H-2Kbm1 variant gene with the H-2Kb gene shows a cluster of seven nucleotide differences. Proc Natl Acad Sci U S A. 1983 Apr;80(7):2007–2011. doi: 10.1073/pnas.80.7.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Stroynowski I., Clark S., Henderson L. A., Hood L., McMillan M., Forman J. Interaction of alpha 1 with alpha 2 region in class I MHC proteins contributes determinants recognized by antibodies and cytotoxic T cells. J Immunol. 1985 Sep;135(3):2160–2166. [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. Van Pel A., De Plaen E., Boon T. Selection of highly transfectable variant from mouse mastocytoma P815. Somat Cell Mol Genet. 1985 Sep;11(5):467–475. doi: 10.1007/BF01534840. [DOI] [PubMed] [Google Scholar]
  31. Vega M. A., Bragado R., Ezquerra A., López de Castro J. A. Variability and conformation of HLA class I antigens: a predictive approach to the spatial arrangement of polymorphic regions. Biochemistry. 1984 Feb 28;23(5):823–831. doi: 10.1021/bi00300a007. [DOI] [PubMed] [Google Scholar]
  32. Vega M. A., Wallace L., Rojo S., Bragado R., Aparicio P., López de Castro J. A. Delineation of functional sites in HLA-B27 antigens. Molecular analysis of HLA-B27 variant Wewak I defined by cytolytic T lymphocytes. J Immunol. 1985 Nov;135(5):3323–3332. [PubMed] [Google Scholar]
  33. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  34. Weiss E. H., Mellor A., Golden L., Fahrner K., Simpson E., Hurst J., Flavell R. A. The structure of a mutant H-2 gene suggests that the generation of polymorphism in H-2 genes may occur by gene conversion-like events. Nature. 1983 Feb 24;301(5902):671–674. doi: 10.1038/301671a0. [DOI] [PubMed] [Google Scholar]
  35. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]
  36. van Schravendijk M. R., Biddison W. E., Berger A. E., Coligan J. E. Comparative structural analysis of HLA-A3 antigens distinguishable by cytotoxic T lymphocytes: variant E1. J Immunol. 1985 Jan;134(1):410–416. [PubMed] [Google Scholar]
  37. van de Rijn M., Bernabeu C., Royer-Pokora B., Weiss J., Seidman J. G., de Vries J., Spits H., Terhorst C. Recognition of HLA-A2 by cytotoxic T lymphocytes after DNA transfer into human and murine cells. Science. 1984 Nov 30;226(4678):1083–1085. doi: 10.1126/science.6333726. [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