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. 1992 Jan 1;175(1):191–202. doi: 10.1084/jem.175.1.191

Disparate interaction of peptide ligand with nascent versus mature class I major histocompatibility complex molecules: comparisons of peptide binding to alternative forms of Ld in cell lysates and the cell surface

PMCID: PMC2119095  PMID: 1730917

Abstract

To determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular Ld molecules can be detected in an alternative conformation designated Ldalt. Ldalt molecules are non- ligand associated and show weak if any beta 2-microglobulin (beta 2m) association. We report here that Ld molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs Ld surface half-life. By contrast, Ldalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with Ld, live cells were incubated with iodinated peptides and Ld molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface Ld molecules was found not to depend upon exchange with exogenous beta 2m, but did correlate with the level of beta 2m association. To study the intracellular interaction of peptide with Ld, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded Ld. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to Ld was found to affect almost exclusively immature (Endo Hs) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Ldalt molecules are precursors awaiting constituents such as peptide and beta 2m that influence folding, whereas surface Ldalt molecules appear refractory to association with peptide, beta 2m, and consequent folding.

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Selected References

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  1. Allen H., Fraser J., Flyer D., Calvin S., Flavell R. Beta 2-microglobulin is not required for cell surface expression of the murine class I histocompatibility antigen H-2Db or of a truncated H-2Db. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7447–7451. doi: 10.1073/pnas.83.19.7447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cerundolo V., Alexander J., Anderson K., Lamb C., Cresswell P., McMichael A., Gotch F., Townsend A. Presentation of viral antigen controlled by a gene in the major histocompatibility complex. Nature. 1990 May 31;345(6274):449–452. doi: 10.1038/345449a0. [DOI] [PubMed] [Google Scholar]
  3. Ciavarra R., Forman J. H-2L-restricted recognition of viral antigens In the H-2d haplotype, anti-vesicular stomatitis virus cytotoxic T cells are restricted solely by H-2L. J Exp Med. 1982 Sep 1;156(3):778–790. doi: 10.1084/jem.156.3.778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cullen S. E., Schwartz B. D. An improved method for isolation of H-2 and Ia alloantigens with immunoprecipitation induced by protein A-bearing staphylococci. J Immunol. 1976 Jul;117(1):136–142. [PubMed] [Google Scholar]
  5. Elliott T., Cerundolo V., Elvin J., Townsend A. Peptide-induced conformational change of the class I heavy chain. Nature. 1991 May 30;351(6325):402–406. doi: 10.1038/351402a0. [DOI] [PubMed] [Google Scholar]
  6. Gorka J., McCourt D. W., Schwartz B. D. Automated synthesis of a C-terminal photoprobe using combined Fmoc and t-Boc synthesis strategies on a single automated peptide synthesizer. Pept Res. 1989 Nov-Dec;2(6):376–380. [PubMed] [Google Scholar]
  7. Krangel M. S., Orr H. T., Strominger J. L. Assembly and maturation of HLA-A and HLA-B antigens in vivo. Cell. 1979 Dec;18(4):979–991. doi: 10.1016/0092-8674(79)90210-1. [DOI] [PubMed] [Google Scholar]
  8. Kvist S., Hamann U. A nucleoprotein peptide of influenza A virus stimulates assembly of HLA-B27 class I heavy chains and beta 2-microglobulin translated in vitro. Nature. 1990 Nov 29;348(6300):446–448. doi: 10.1038/348446a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Lie W. R., Myers N. B., Connolly J. M., Gorka J., Lee D. R., Hansen T. H. The specific binding of peptide ligand to Ld class I major histocompatibility complex molecules determines their antigenic structure. J Exp Med. 1991 Feb 1;173(2):449–459. doi: 10.1084/jem.173.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lie W. R., Myers N. B., Gorka J., Rubocki R. J., Connolly J. M., Hansen T. H. Peptide ligand-induced conformation and surface expression of the Ld class I MHC molecule. Nature. 1990 Mar 29;344(6265):439–441. doi: 10.1038/344439a0. [DOI] [PubMed] [Google Scholar]
  12. Ljunggren H. G., Stam N. J., Ohlén C., Neefjes J. J., Höglund P., Heemels M. T., Bastin J., Schumacher T. N., Townsend A., Kärre K. Empty MHC class I molecules come out in the cold. Nature. 1990 Aug 2;346(6283):476–480. doi: 10.1038/346476a0. [DOI] [PubMed] [Google Scholar]
  13. Lurquin C., Van Pel A., Mariamé B., De Plaen E., Szikora J. P., Janssens C., Reddehase M. J., Lejeune J., Boon T. Structure of the gene of tum- transplantation antigen P91A: the mutated exon encodes a peptide recognized with Ld by cytolytic T cells. Cell. 1989 Jul 28;58(2):293–303. doi: 10.1016/0092-8674(89)90844-1. [DOI] [PubMed] [Google Scholar]
  14. Myers N. B., Lie W. R., Nett M., Rubocki R. J., Hansen T. H. The conformation of Ld induced by beta 2-microglobulin is fixed during de novo synthesis and irreversible by exchange or dissociation. J Immunol. 1989 Apr 15;142(8):2751–2758. [PubMed] [Google Scholar]
  15. Nuchtern J. G., Bonifacino J. S., Biddison W. E., Klausner R. D. Brefeldin A implicates egress from endoplasmic reticulum in class I restricted antigen presentation. Nature. 1989 May 18;339(6221):223–226. doi: 10.1038/339223a0. [DOI] [PubMed] [Google Scholar]
  16. Oi V. T., Jones P. P., Goding J. W., Herzenberg L. A., Herzenberg L. A. Properties of monoclonal antibodies to mouse Ig allotypes, H-2, and Ia antigens. Curr Top Microbiol Immunol. 1978;81:115–120. doi: 10.1007/978-3-642-67448-8_18. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Ortiz-Navarrete V., Hämmerling G. J. Surface appearance and instability of empty H-2 class I molecules under physiological conditions. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3594–3597. doi: 10.1073/pnas.88.9.3594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ozato K., Hansen T. H., Sachs D. H. Monoclonal antibodies to mouse MHC antigens. II. Antibodies to the H-2Ld antigen, the products of a third polymorphic locus of the mouse major histocompatibility complex. J Immunol. 1980 Dec;125(6):2473–2477. [PubMed] [Google Scholar]
  20. Ozato K., Mayer N., Sachs D. H. Hybridoma cell lines secreting monoclonal antibodies to mouse H-2 and Ia antigens. J Immunol. 1980 Feb;124(2):533–540. [PubMed] [Google Scholar]
  21. Reddehase M. J., Bühring H. J., Koszinowski U. H. Cloned long-term cytolytic T-lymphocyte line with specificity for an immediate-early membrane antigen of murine cytomegalovirus. J Virol. 1986 Jan;57(1):408–412. doi: 10.1128/jvi.57.1.408-412.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reddehase M. J., Rothbard J. B., Koszinowski U. H. A pentapeptide as minimal antigenic determinant for MHC class I-restricted T lymphocytes. Nature. 1989 Feb 16;337(6208):651–653. doi: 10.1038/337651a0. [DOI] [PubMed] [Google Scholar]
  23. Rock K. L., Gramm C., Benacerraf B. Low temperature and peptides favor the formation of class I heterodimers on RMA-S cells at the cell surface. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4200–4204. doi: 10.1073/pnas.88.10.4200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rock K. L., Rothstein L. E., Gamble S. R., Benacerraf B. Reassociation with beta 2-microglobulin is necessary for Kb class I major histocompatibility complex binding of exogenous peptides. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7517–7521. doi: 10.1073/pnas.87.19.7517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rubocki R. J., Connolly J. M., Hansen T. H., Melvold R. W., Kim B. S., Hildebrand W. H., Martinko J. Mutation at amino acid position 133 of H-2Dd prevents beta 2m association and immune recognition but not surface expression. J Immunol. 1991 Apr 1;146(7):2352–2357. [PubMed] [Google Scholar]
  26. Rötzschke O., Falk K., Deres K., Schild H., Norda M., Metzger J., Jung G., Rammensee H. G. Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells. Nature. 1990 Nov 15;348(6298):252–254. doi: 10.1038/348252a0. [DOI] [PubMed] [Google Scholar]
  27. Schumacher T. N., Heemels M. T., Neefjes J. J., Kast W. M., Melief C. J., Ploegh H. L. Direct binding of peptide to empty MHC class I molecules on intact cells and in vitro. Cell. 1990 Aug 10;62(3):563–567. doi: 10.1016/0092-8674(90)90020-f. [DOI] [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. Smith M. H., Parker J. M., Hodges R. S., Barber B. H. The preparation and characterization of anti-peptide heteroantisera recognizing subregions of the intracytoplasmic domain of class I H-2 antigens. Mol Immunol. 1986 Oct;23(10):1077–1092. doi: 10.1016/0161-5890(86)90006-4. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Townsend A., Elliott T., Cerundolo V., Foster L., Barber B., Tse A. Assembly of MHC class I molecules analyzed in vitro. Cell. 1990 Jul 27;62(2):285–295. doi: 10.1016/0092-8674(90)90366-m. [DOI] [PubMed] [Google Scholar]
  32. Townsend A., Ohlén C., Bastin J., Ljunggren H. G., Foster L., Kärre K. Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature. 1989 Aug 10;340(6233):443–448. doi: 10.1038/340443a0. [DOI] [PubMed] [Google Scholar]
  33. Van Bleek G. M., Nathenson S. G. Isolation of an endogenously processed immunodominant viral peptide from the class I H-2Kb molecule. Nature. 1990 Nov 15;348(6298):213–216. doi: 10.1038/348213a0. [DOI] [PubMed] [Google Scholar]
  34. Vitiello A., Potter T. A., Sherman L. A. The role of beta 2-microglobulin in peptide binding by class I molecules. Science. 1990 Dec 7;250(4986):1423–1426. doi: 10.1126/science.2124002. [DOI] [PubMed] [Google Scholar]
  35. Yewdell J. W., Bennink J. R. Brefeldin A specifically inhibits presentation of protein antigens to cytotoxic T lymphocytes. Science. 1989 Jun 2;244(4908):1072–1075. doi: 10.1126/science.2471266. [DOI] [PubMed] [Google Scholar]

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