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. 1991 Nov 1;174(5):1111–1120. doi: 10.1084/jem.174.5.1111

Enhanced binding of peptide antigen to purified class II major histocompatibility glycoproteins at acidic pH

PMCID: PMC2118993  PMID: 1940792

Abstract

Helper T lymphocytes recognize peptide antigens stably associated with class II major histocompatibility complex (MHC) glycoproteins on the surface of antigen-presenting cells and serve to regulate a wide variety of immune responses. A previous study from our laboratory had demonstrated that the functional association of various peptide antigens with the antigen-presenting cell membrane was increased at pH 5 as compared to pH 7, consistent with the potential role of acidic endosomal compartments in antigen processing. The mechanism for this effect was not determined. In the present study, assays using purified class II glycoprotein were used to further define this mechanism. The potential requirement for pH-dependent interactions involving non-MHC membrane components was excluded in functional assays with purified class II reconstituted in artificial membranes containing only neutral phospholipids and cholesterol. The association of HEL(104-120) with I- Ed, and OVA(323-339) with I-Ad, was increased at pH 5, as measured by activation of specific T cell hybridomas. An enzyme immunoassay was developed to measure the binding of biotin-labeled peptides to purified class II in detergent micelles. The pH dependence of binding paralleled our previous functional results. Optimum binding of biotin-HEL(104-120) to I-Ed was observed at pH approximately 4.5, whereas maximum binding of biotin-Myo(106-118) to I-Ad occurred at pH approximately 5.5. The latter peptide also bound weakly to I-Ed, but with a pH dependence similar to that observed using HEL(104-120). Further experiments with biotin-HEL(104-120)/I-Ed indicated that both the apparent affinity and the apparent concentration of peptide-binding sites are increased as hydrogen ion concentration is increased from pH 7 to pH 5. The effect of pH in this range was largely reversible and was not associated with a change in peptide dissociation that could be measured with our assay system. Binding was not inhibited in the presence of 1.5 M NaCl, suggesting that electrostatic interactions between HEL(104-120) and I- Ed are not essential for binding. It is proposed that protonation of a critical group(s) in the class II molecule regulates its capacity to form stable complexes with peptide. However, this effect alone does not fully account for the rapid kinetics of peptide binding observed in experiments with intact antigen-presenting cells.

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Selected References

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

  1. Allen P. M., Strydom D. J., Unanue E. R. Processing of lysozyme by macrophages: identification of the determinant recognized by two T-cell hybridomas. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2489–2493. doi: 10.1073/pnas.81.8.2489. [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. Bogen B., Lambris J. D. Minimum length of an idiotypic peptide and a model for its binding to a major histocompatibility complex class II molecule. EMBO J. 1989 Jul;8(7):1947–1952. doi: 10.1002/j.1460-2075.1989.tb03599.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buus S., Sette A., Colon S. M., Grey H. M. Autologous peptides constitutively occupy the antigen binding site on Ia. Science. 1988 Nov 18;242(4881):1045–1047. doi: 10.1126/science.3194755. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Buus S., Sette A., Colon S. M., Miles C., Grey H. M. The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides. Science. 1987 Mar 13;235(4794):1353–1358. doi: 10.1126/science.2435001. [DOI] [PubMed] [Google Scholar]
  7. Chang K. J., Jacobs S., Cuatrecasas P. Quantitative aspects of hormone-receptor interactions of high affinity. Effect of receptor concentration and measurement of dissociation constants of labeled and unlabeled hormones. Biochim Biophys Acta. 1975 Oct 6;406(2):294–303. doi: 10.1016/0005-2736(75)90011-5. [DOI] [PubMed] [Google Scholar]
  8. Demotz S., Grey H. M., Appella E., Sette A. Characterization of a naturally processed MHC class II-restricted T-cell determinant of hen egg lysozyme. Nature. 1989 Dec 7;342(6250):682–684. doi: 10.1038/342682a0. [DOI] [PubMed] [Google Scholar]
  9. Germain R. N. Immunology. The ins and outs of antigen processing and presentation. Nature. 1986 Aug 21;322(6081):687–689. doi: 10.1038/322687a0. [DOI] [PubMed] [Google Scholar]
  10. Gorga J. C., Horejsí V., Johnson D. R., Raghupathy R., Strominger J. L. Purification and characterization of class II histocompatibility antigens from a homozygous human B cell line. J Biol Chem. 1987 Nov 25;262(33):16087–16094. [PubMed] [Google Scholar]
  11. Jensen P. E. Protein synthesis in antigen processing. J Immunol. 1988 Oct 15;141(8):2545–2550. [PubMed] [Google Scholar]
  12. Jensen P. E. Regulation of antigen presentation by acidic pH. J Exp Med. 1990 May 1;171(5):1779–1784. doi: 10.1084/jem.171.5.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jensen P. E. Stable association of processed antigen with antigen-presenting cell membranes. J Immunol. 1989 Jul 15;143(2):420–425. [PubMed] [Google Scholar]
  14. Kappler J. W., Skidmore B., White J., Marrack P. Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas. Lack of independent antigen and H-2 recognition. J Exp Med. 1981 May 1;153(5):1198–1214. doi: 10.1084/jem.153.5.1198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kappler J. W., Skidmore B., White J., Marrack P. Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas. Lack of independent antigen and H-2 recognition. J Exp Med. 1981 May 1;153(5):1198–1214. doi: 10.1084/jem.153.5.1198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kovac Z., Schwartz R. H. The molecular basis of the requirement for antigen processing of pigeon cytochrome c prior to T cell activation. J Immunol. 1985 May;134(5):3233–3240. [PubMed] [Google Scholar]
  17. Lakey E. K., Margoliash E., Pierce S. K. Identification of a peptide binding protein that plays a role in antigen presentation. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1659–1663. doi: 10.1073/pnas.84.6.1659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lark L. R., Berzofsky J. A., Gierasch L. M. T-cell antigenic peptides from sperm whale myoglobin fold as amphipathic helices: a possible determinant for immunodominance? Pept Res. 1989 Sep-Oct;2(5):314–321. [PubMed] [Google Scholar]
  19. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  20. Nowell J., Quaranta V. Chloroquine affects biosynthesis of Ia molecules by inhibiting dissociation of invariant (gamma) chains from alpha-beta dimers in B cells. J Exp Med. 1985 Oct 1;162(4):1371–1376. doi: 10.1084/jem.162.4.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ozato K., Epstein S. L., Henkart P., Hansen T. H., Sachs D. H. Studies on monoclonal antibodies to mouse MHC products. Transplant Proc. 1981 Mar;13(1 Pt 2):958–962. [PubMed] [Google Scholar]
  22. Ozato K., Sachs D. H. Monoclonal antibodies to mouse MHC antigens. III. Hybridoma antibodies reacting to antigens of the H-2b haplotype reveal genetic control of isotype expression. J Immunol. 1981 Jan;126(1):317–321. [PubMed] [Google Scholar]
  23. Quill H., Carlson L., Fox B. S., Weinstein J. N., Schwartz R. H. Optimization of antigen presentation to T cell hybridomas by purified Ia molecules in planar membranes. Ia molecule polymorphism determines the antigenic fine specificity of the response to cytochrome c peptides. J Immunol Methods. 1987 Apr 2;98(1):29–41. doi: 10.1016/0022-1759(87)90432-7. [DOI] [PubMed] [Google Scholar]
  24. Rodbard D. Mathematics of hormone-receptor interaction. I. Basic principles. Adv Exp Med Biol. 1973;36(0):289–326. doi: 10.1007/978-1-4684-3237-4_14. [DOI] [PubMed] [Google Scholar]
  25. Sadegh-Nasseri S., McConnell H. M. A kinetic intermediate in the reaction of an antigenic peptide and I-Ek. Nature. 1989 Jan 19;337(6204):274–276. doi: 10.1038/337274a0. [DOI] [PubMed] [Google Scholar]
  26. Schmid S. L., Fuchs R., Male P., Mellman I. Two distinct subpopulations of endosomes involved in membrane recycling and transport to lysosomes. Cell. 1988 Jan 15;52(1):73–83. doi: 10.1016/0092-8674(88)90532-6. [DOI] [PubMed] [Google Scholar]
  27. Schmid S., Fuchs R., Kielian M., Helenius A., Mellman I. Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants. J Cell Biol. 1989 Apr;108(4):1291–1300. doi: 10.1083/jcb.108.4.1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sette A., Buus S., Colon S., Miles C., Grey H. M. Structural analysis of peptides capable of binding to more than one Ia antigen. J Immunol. 1989 Jan 1;142(1):35–40. [PubMed] [Google Scholar]
  29. Shimonkevitz R., Colon S., Kappler J. W., Marrack P., Grey H. M. Antigen recognition by H-2-restricted T cells. II. A tryptic ovalbumin peptide that substitutes for processed antigen. J Immunol. 1984 Oct;133(4):2067–2074. [PubMed] [Google Scholar]
  30. Shimonkevitz R., Kappler J., Marrack P., Grey H. Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing. J Exp Med. 1983 Aug 1;158(2):303–316. doi: 10.1084/jem.158.2.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sipe D. M., Murphy R. F. High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7119–7123. doi: 10.1073/pnas.84.20.7119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Weiland G. A., Molinoff P. B. Quantitative analysis of drug-receptor interactions: I. Determination of kinetic and equilibrium properties. Life Sci. 1981 Jul 27;29(4):313–330. doi: 10.1016/0024-3205(81)90324-6. [DOI] [PubMed] [Google Scholar]
  33. Yamashiro D. J., Tycko B., Fluss S. R., Maxfield F. R. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell. 1984 Jul;37(3):789–800. doi: 10.1016/0092-8674(84)90414-8. [DOI] [PubMed] [Google Scholar]
  34. Yewdell J. W., Bennink J. R. The binary logic of antigen processing and presentation to T cells. Cell. 1990 Jul 27;62(2):203–206. doi: 10.1016/0092-8674(90)90356-j. [DOI] [PubMed] [Google Scholar]
  35. Ziegler H. K., Unanue E. R. Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells. Proc Natl Acad Sci U S A. 1982 Jan;79(1):175–178. doi: 10.1073/pnas.79.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]

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