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. 1986 Feb;83(3):757–761. doi: 10.1073/pnas.83.3.757

Intracellular transport blockade caused by disruption of the disulfide bridge in the third external domain of major histocompatibility complex class I antigen.

J Miyazaki, E Appella, K Ozato
PMCID: PMC322944  PMID: 3080749

Abstract

The third external domain of major histocompatibility class I antigens has a highly conserved disulfide bridge between cysteine-203 and cysteine-259. To elucidate the functional significance of this disulfide bridge, we have produced a mutant H-2Ld gene by site-directed mutagenesis in which the codon for cysteine-203 is changed to a codon for serine, which is unable to form a disulfide bridge. The mutant H-2Ld gene was introduced into mouse L cells and its expression has been studied. No measurable expression of the H-2Ld antigen was detected on the cell surface of the transformants by antibody-binding assays. However, a large quantity of the mutant H-2Ld antigen was found in the cytoplasm of the transformants as observed by immunoprecipitation of metabolically labeled lysate and by immunocytochemistry of membrane-permeabilized cells, using an antibody specific for the first external domain of the H-2Ld antigen. The mutant antigen was glycosylated and associated, at least in part, with beta 2-microglobulin. Subcellular fractionation experiments indicated that the transport of the antigen was blocked between the endoplasmic reticulum and the plasma membrane. It is concluded that structural integrity of the third external domain is a prerequisite for intracellular transport of class I antigens. On the basis of these findings we suggest that the domain structure containing the disulfide bridge serves as a signal structure necessary for receptor-mediated intracellular transport and that this requirement is the evolutionary basis for high conservation of similar structures present throughout the immunoglobulin supergene family.

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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. Ben-Barak Y., Russell J. T., Whitnall M. H., Ozato K., Gainer H. Neurophysin in the hypothalamo-neurohypophysial system. I. Production and characterization of monoclonal antibodies. J Neurosci. 1985 Jan;5(1):81–97. doi: 10.1523/JNEUROSCI.05-01-00081.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burgert H. G., Kvist S. An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell. 1985 Jul;41(3):987–997. doi: 10.1016/s0092-8674(85)80079-9. [DOI] [PubMed] [Google Scholar]
  5. Camerini-Otero R. D., Zasloff M. A. Nucleosomal packaging of the thymidine kinase gene of herpes simplex virus transferred into mouse cells: an actively expressed single-copy gene. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5079–5083. doi: 10.1073/pnas.77.9.5079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coligan J. E., Kindt T. J., Uehara H., Martinko J., Nathenson S. G. Primary structure of a murine transplantation antigen. Nature. 1981 May 7;291(5810):35–39. doi: 10.1038/291035a0. [DOI] [PubMed] [Google Scholar]
  7. Enquist L. W., Vande Woude G. F., Wagner M., Smiley J. R., Summers W. C. Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus. Gene. 1979 Nov;7(3-4):335–342. doi: 10.1016/0378-1119(79)90052-0. [DOI] [PubMed] [Google Scholar]
  8. Evans G. A., Margulies D. H., Camerini-Otero R. D., Ozato K., Seidman J. G. Structure and expression of a mouse major histocompatibility antigen gene, H-2Ld. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1994–1998. doi: 10.1073/pnas.79.6.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fitting T., Kabat D. Evidence for a glycoprotein "signal" involved in transport between subcellular organelles. Two membrane glycoproteins encoded by murine leukemia virus reach the cell surface at different rates. J Biol Chem. 1982 Dec 10;257(23):14011–14017. [PubMed] [Google Scholar]
  10. Hansen T. H., Ozato K., Sachs D. H. Heterogeneity of H-2D region associated genes and gene products. Adv Immunol. 1983;34:39–70. doi: 10.1016/s0065-2776(08)60376-4. [DOI] [PubMed] [Google Scholar]
  11. Hood L., Kronenberg M., Hunkapiller T. T cell antigen receptors and the immunoglobulin supergene family. Cell. 1985 Feb;40(2):225–229. doi: 10.1016/0092-8674(85)90133-3. [DOI] [PubMed] [Google Scholar]
  12. Hubbard A. L., Cohn Z. A. Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells. J Cell Biol. 1975 Feb;64(2):438–460. doi: 10.1083/jcb.64.2.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ito H., Ike Y., Ikuta S., Itakura K. Solid phase synthesis of polynucleotides. VI. Further studies on polystyrene copolymers for the solid support. Nucleic Acids Res. 1982 Mar 11;10(5):1755–1769. doi: 10.1093/nar/10.5.1755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jones P. P. Analysis of H-2 and Ia molecules by two-dimensional gel electrophoresis. J Exp Med. 1977 Nov 1;146(5):1261–1279. doi: 10.1084/jem.146.5.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  16. Klein J. The major histocompatibility complex of the mouse. Science. 1979 Feb 9;203(4380):516–521. doi: 10.1126/science.104386. [DOI] [PubMed] [Google Scholar]
  17. Kvist S., Roberts L., Dobberstein B. Mouse histocompatibility genes: structure and organisation of a Kd gene. EMBO J. 1983;2(2):245–254. doi: 10.1002/j.1460-2075.1983.tb01413.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Lodish H. F., Kong N., Snider M., Strous G. J. Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates. Nature. 1983 Jul 7;304(5921):80–83. doi: 10.1038/304080a0. [DOI] [PubMed] [Google Scholar]
  20. Maloy W. L., Coligan J. E. Primary structure of the H-2Db alloantigen. II. Additional amino acid sequence information, localization of a third site of glycosylation and evidence for K and D region specific sequences. Immunogenetics. 1982;16(1):11–22. doi: 10.1007/BF00364438. [DOI] [PubMed] [Google Scholar]
  21. Margulies D. H., Evans G. A., Ozato K., Camerini-Otero R. D., Tanaka K., Appella E., Seidman J. G. Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer. J Immunol. 1983 Jan;130(1):463–470. [PubMed] [Google Scholar]
  22. Moore K. W., Sher B. T., Sun Y. H., Eakle K. A., Hood L. DNA sequence of a gene encoding a BALB/c mouse Ld transplantation antigen. Science. 1982 Feb 5;215(4533):679–682. doi: 10.1126/science.7058332. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Nathenson S. G., Uehara H., Ewenstein B. M., Kindt T. J., Coligan J. E. Primary structural: analysis of the transplantation antigens of the murine H-2 major histocompatibility complex. Annu Rev Biochem. 1981;50:1025–1052. doi: 10.1146/annurev.bi.50.070181.005113. [DOI] [PubMed] [Google Scholar]
  25. Orr H. T., Lopez de Castro J. A., Parham P., Ploegh H. L., Strominger J. L. Comparison of amino acid sequences of two human histocompatibility antigens, HLA-A2 and HLA-B7: location of putative alloantigenic sites. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4395–4399. doi: 10.1073/pnas.76.9.4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Owen M. J., Kissonerghis A. M., Lodish H. F. Biosynthesis of HLA-A and HLA-B antigens in vivo. J Biol Chem. 1980 Oct 25;255(20):9678–9684. [PubMed] [Google Scholar]
  27. Ozato K., Evans G. A., Shykind B., Margulies D. H., Seidman J. G. Hybrid H-2 histocompatibility gene products assign domains recognized by alloreactive T cells. Proc Natl Acad Sci U S A. 1983 Apr;80(7):2040–2043. doi: 10.1073/pnas.80.7.2040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Potter T. A., Boyer C., Verhulst A. M., Golstein P., Rajan T. V. Expression of H-2Db on the cell surface in the absence of detectable beta 2 microglobulin. J Exp Med. 1984 Jul 1;160(1):317–322. doi: 10.1084/jem.160.1.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rask L., Lindblom J. B., Peterson P. A. Subunit structure of H-2 alloantigens. Nature. 1974 Jun 28;249(460):833–834. doi: 10.1038/249833a0. [DOI] [PubMed] [Google Scholar]
  30. Reiss C. S., Evans G. A., Margulies D. H., Seidman J. G., Burakoff S. J. Allospecific and virus-specific cytolytic T lymphocytes are restricted to the N or C1 domain of H-2 antigens expressed on L cells after DNA-mediated gene transfer. Proc Natl Acad Sci U S A. 1983 May;80(9):2709–2712. doi: 10.1073/pnas.80.9.2709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reyes A. A., Schöld M., Itakura K., Wallace R. B. Isolation of a cDNA clone for the murine transplantation antigen H-2Kb. Proc Natl Acad Sci U S A. 1982 May;79(10):3270–3274. doi: 10.1073/pnas.79.10.3270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Sege K., Rask L., Peterson P. A. Role of beta2-microglobulin in the intracellular processing of HLA antigens. Biochemistry. 1981 Aug 4;20(16):4523–4530. doi: 10.1021/bi00519a003. [DOI] [PubMed] [Google Scholar]
  34. Severinsson L., Peterson P. A. Beta 2-microglobulin induces intracellular transport of human class I transplantation antigen heavy chains in Xenopus laevis oocytes. J Cell Biol. 1984 Jul;99(1 Pt 1):226–232. doi: 10.1083/jcb.99.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Shiroishi T., Evans G. A., Appella E., Ozato K. Role of a disulfide bridge in the immune function of major histocompatibility class I antigen as studied by in vitro mutagenesis. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7544–7548. doi: 10.1073/pnas.81.23.7544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sly W. S., Fischer H. D. The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J Cell Biochem. 1982;18(1):67–85. doi: 10.1002/jcb.1982.240180107. [DOI] [PubMed] [Google Scholar]
  38. Trägärdh L., Rask L., Wiman K., Fohlman J., Peterson P. A. Complete amino acid sequence of pooled papain-solubilized HLA-A, -B, and -C antigens: relatedness to immunoglobulins and internal homologies. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1129–1133. doi: 10.1073/pnas.77.2.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yokoyama K., Nathenson S. G. Intramolecular organization of Class I H-2 MHC antigens; localization of the alloantigenic determinants and the beta 2 m binding site to different regions of the H-2 Kb glycoprotein. J Immunol. 1983 Mar;130(3):1419–1425. [PubMed] [Google Scholar]
  41. 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]

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