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. 1994 May 1;125(3):595–605. doi: 10.1083/jcb.125.3.595

Separation of subcellular compartments containing distinct functional forms of MHC class II

PMCID: PMC2119994  PMID: 7909813

Abstract

Antigen processing in B lymphocytes entails initial binding of antigen to the surface Ig and internalization of the antigen into acidic compartments where the antigen is degraded, releasing peptides for binding to major histocompatibility complex class II molecules. Using subcellular fractionation techniques we show that functional, processed antigen-class II complexes capable of activating antigen-specific T cells in vitro are first formed in dense vesicles cosedimenting with lysosomes which are distinct from early endosomes and the bulk of late endosomes. With time, processed antigen-class II complexes appear in vesicles sedimenting with early endosomes and finally cofractionate with plasma membrane. A separate compartment is identified which contains major histocompatibility complex class II receptive to peptide binding but which does not have access to processed antigen in the B cell. These class II molecules are in the so-called "floppy" form in contrast to the class II molecules in the very dense vesicles which are in the "compact" form. These results demonstrate a correlation between the floppy and compact forms of class II molecules and their association with processed antigen and show that floppy and compact forms of class II reside in distinct and physically separable subcellular compartments.

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Selected References

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  1. Adams C. J., Maurey K. M., Storrie B. Exocytosis of pinocytic contents by Chinese hamster ovary cells. J Cell Biol. 1982 Jun;93(3):632–637. doi: 10.1083/jcb.93.3.632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson M. S., Miller J. Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2282–2286. doi: 10.1073/pnas.89.6.2282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bakke O., Dobberstein B. MHC class II-associated invariant chain contains a sorting signal for endosomal compartments. Cell. 1990 Nov 16;63(4):707–716. doi: 10.1016/0092-8674(90)90137-4. [DOI] [PubMed] [Google Scholar]
  5. Beaudet A. L., Manschreck A. A. Metabolism of sphingomyelin by intact cultured fibroblasts: differentiation of Niemann-Pick disease type A and B. Biochem Biophys Res Commun. 1982 Mar 15;105(1):14–19. doi: 10.1016/s0006-291x(82)80004-1. [DOI] [PubMed] [Google Scholar]
  6. Brodsky F. M., Guagliardi L. E. The cell biology of antigen processing and presentation. Annu Rev Immunol. 1991;9:707–744. doi: 10.1146/annurev.iy.09.040191.003423. [DOI] [PubMed] [Google Scholar]
  7. Brown J. H., Jardetzky T. S., Gorga J. C., Stern L. J., Urban R. G., Strominger J. L., Wiley D. C. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature. 1993 Jul 1;364(6432):33–39. doi: 10.1038/364033a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Casten L. A., Lakey E. K., Jelachich M. L., Margoliash E., Pierce S. K. Anti-immunoglobulin augments the B-cell antigen-presentation function independently of internalization of receptor-antigen complex. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5890–5894. doi: 10.1073/pnas.82.17.5890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Casten L. A., Pierce S. K. Receptor-mediated B cell antigen processing. Increased antigenicity of a globular protein covalently coupled to antibodies specific for B cell surface structures. J Immunol. 1988 Jan 15;140(2):404–410. [PubMed] [Google Scholar]
  11. Chavrier P., Parton R. G., Hauri H. P., Simons K., Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990 Jul 27;62(2):317–329. doi: 10.1016/0092-8674(90)90369-p. [DOI] [PubMed] [Google Scholar]
  12. Chen J. W., Murphy T. L., Willingham M. C., Pastan I., August J. T. Identification of two lysosomal membrane glycoproteins. J Cell Biol. 1985 Jul;101(1):85–95. doi: 10.1083/jcb.101.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chicz R. M., Urban R. G., Lane W. S., Gorga J. C., Stern L. J., Vignali D. A., Strominger J. L. Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size. Nature. 1992 Aug 27;358(6389):764–768. doi: 10.1038/358764a0. [DOI] [PubMed] [Google Scholar]
  14. Coleman R., Bell R. M. Phospholipid synthesis in isolated fat cells. Studies of microsomal diacylglycerol cholinephosphotransferase and diacylglycerol ethanolaminephosphotransferase activities. J Biol Chem. 1977 May 10;252(9):3050–3056. [PubMed] [Google Scholar]
  15. Cresswell P. Intracellular class II HLA antigens are accessible to transferrin-neuraminidase conjugates internalized by receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8188–8192. doi: 10.1073/pnas.82.23.8188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Davidson H. W., Reid P. A., Lanzavecchia A., Watts C. Processed antigen binds to newly synthesized MHC class II molecules in antigen-specific B lymphocytes. Cell. 1991 Oct 4;67(1):105–116. doi: 10.1016/0092-8674(91)90575-j. [DOI] [PubMed] [Google Scholar]
  17. Demotz S., Grey H. M., Sette A. The minimal number of class II MHC-antigen complexes needed for T cell activation. Science. 1990 Aug 31;249(4972):1028–1030. doi: 10.1126/science.2118680. [DOI] [PubMed] [Google Scholar]
  18. Domanico S. Z., DeNagel D. C., Dahlseid J. N., Green J. M., Pierce S. K. Cloning of the gene encoding peptide-binding protein 74 shows that it is a new member of the heat shock protein 70 family. Mol Cell Biol. 1993 Jun;13(6):3598–3610. doi: 10.1128/mcb.13.6.3598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Dornmair K., Rothenhäusler B., McConnell H. M. Structural intermediates in the reactions of antigenic peptides with MHC molecules. Cold Spring Harb Symp Quant Biol. 1989;54(Pt 1):409–416. doi: 10.1101/sqb.1989.054.01.050. [DOI] [PubMed] [Google Scholar]
  20. Germain R. N., Hendrix L. R. MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding. Nature. 1991 Sep 12;353(6340):134–139. doi: 10.1038/353134a0. [DOI] [PubMed] [Google Scholar]
  21. Germain R. N., Rinker A. G., Jr Peptide binding inhibits protein aggregation of invariant-chain free class II dimers and promotes surface expression of occupied molecules. Nature. 1993 Jun 24;363(6431):725–728. doi: 10.1038/363725a0. [DOI] [PubMed] [Google Scholar]
  22. Gillis S., Ferm M. M., Ou W., Smith K. A. T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol. 1978 Jun;120(6):2027–2032. [PubMed] [Google Scholar]
  23. Guagliardi L. E., Koppelman B., Blum J. S., Marks M. S., Cresswell P., Brodsky F. M. Co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment. Nature. 1990 Jan 11;343(6254):133–139. doi: 10.1038/343133a0. [DOI] [PubMed] [Google Scholar]
  24. Harding C. V., Collins D. S., Slot J. W., Geuze H. J., Unanue E. R. Liposome-encapsulated antigens are processed in lysosomes, recycled, and presented to T cells. Cell. 1991 Jan 25;64(2):393–401. doi: 10.1016/0092-8674(91)90647-h. [DOI] [PubMed] [Google Scholar]
  25. Harding C. V., Geuze H. J. Class II MHC molecules are present in macrophage lysosomes and phagolysosomes that function in the phagocytic processing of Listeria monocytogenes for presentation to T cells. J Cell Biol. 1992 Nov;119(3):531–542. doi: 10.1083/jcb.119.3.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Harding C. V., Geuze H. J. Immunogenic peptides bind to class II MHC molecules in an early lysosomal compartment. J Immunol. 1993 Oct 15;151(8):3988–3998. [PubMed] [Google Scholar]
  27. Harding C. V., Roof R. W., Allen P. M., Unanue E. R. Effects of pH and polysaccharides on peptide binding to class II major histocompatibility complex molecules. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2740–2744. doi: 10.1073/pnas.88.7.2740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Haughton G., Arnold L. W., Bishop G. A., Mercolino T. J. The CH series of murine B cell lymphomas: neoplastic analogues of Ly-1+ normal B cells. Immunol Rev. 1986 Oct;93:35–51. doi: 10.1111/j.1600-065x.1986.tb01501.x. [DOI] [PubMed] [Google Scholar]
  29. Hunt D. F., Michel H., Dickinson T. A., Shabanowitz J., Cox A. L., Sakaguchi K., Appella E., Grey H. M., Sette A. Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad. Science. 1992 Jun 26;256(5065):1817–1820. doi: 10.1126/science.1319610. [DOI] [PubMed] [Google Scholar]
  30. Jelachich M. L., Grusby M. J., Clark D., Tasch D., Margoliash E., Pierce S. K. Synergistic effects of antigen and soluble T-cell factors in B-lymphocyte activation. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5537–5541. doi: 10.1073/pnas.81.17.5537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Jensen P. E. Enhanced binding of peptide antigen to purified class II major histocompatibility glycoproteins at acidic pH. J Exp Med. 1991 Nov 1;174(5):1111–1120. doi: 10.1084/jem.174.5.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Koch N., Koch S., Hämmerling G. J. Ia invariant chain detected on lymphocyte surfaces by monoclonal antibody. Nature. 1982 Oct 14;299(5884):644–645. doi: 10.1038/299644a0. [DOI] [PubMed] [Google Scholar]
  33. Kvist S., Wiman K., Claesson L., Peterson P. A., Dobberstein B. Membrane insertion and oligomeric assembly of HLA-DR histocompatibility antigens. Cell. 1982 May;29(1):61–69. doi: 10.1016/0092-8674(82)90090-3. [DOI] [PubMed] [Google Scholar]
  34. Lakey E. K., Casten L. A., Niebling W. L., Margoliash E., Pierce S. K. Time dependence of B cell processing and presentation of peptide and native protein antigens. J Immunol. 1988 May 15;140(10):3309–3314. [PubMed] [Google Scholar]
  35. Lotteau V., Teyton L., Peleraux A., Nilsson T., Karlsson L., Schmid S. L., Quaranta V., Peterson P. A. Intracellular transport of class II MHC molecules directed by invariant chain. Nature. 1990 Dec 13;348(6302):600–605. doi: 10.1038/348600a0. [DOI] [PubMed] [Google Scholar]
  36. Louvard D., Morris C., Warren G., Stanley K., Winkler F., Reggio H. A monoclonal antibody to the heavy chain of clathrin. EMBO J. 1983;2(10):1655–1664. doi: 10.1002/j.1460-2075.1983.tb01640.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Machamer C. E., Cresswell P. Biosynthesis and glycosylation of the invariant chain associated with HLA-DR antigens. J Immunol. 1982 Dec;129(6):2564–2569. [PubMed] [Google Scholar]
  38. Madden E. A., Storrie B. The preparative isolation of mitochondria from Chinese hamster ovary cells. Anal Biochem. 1987 Jun;163(2):350–357. doi: 10.1016/0003-2697(87)90235-1. [DOI] [PubMed] [Google Scholar]
  39. Marsh E. W., Dalke D. P., Pierce S. K. Biochemical evidence for the rapid assembly and disassembly of processed antigen-major histocompatibility complex class II complexes in acidic vesicles of B cells. J Exp Med. 1992 Feb 1;175(2):425–436. doi: 10.1084/jem.175.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. McCoy K. L., Miller J., Jenkins M., Ronchese F., Germain R. N., Schwartz R. H. Diminished antigen processing by endosomal acidification mutant antigen-presenting cells. J Immunol. 1989 Jul 1;143(1):29–38. [PubMed] [Google Scholar]
  41. Neefjes J. J., Ploegh H. L. Inhibition of endosomal proteolytic activity by leupeptin blocks surface expression of MHC class II molecules and their conversion to SDS resistance alpha beta heterodimers in endosomes. EMBO J. 1992 Feb;11(2):411–416. doi: 10.1002/j.1460-2075.1992.tb05069.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Neefjes J. J., Ploegh H. L. Intracellular transport of MHC class II molecules. Immunol Today. 1992 May;13(5):179–184. doi: 10.1016/0167-5699(92)90123-O. [DOI] [PubMed] [Google Scholar]
  43. Neefjes J. J., Stollorz V., Peters P. J., Geuze H. J., Ploegh H. L. The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route. Cell. 1990 Apr 6;61(1):171–183. doi: 10.1016/0092-8674(90)90224-3. [DOI] [PubMed] [Google Scholar]
  44. Niebling W. L., Pierce S. K. Antigen entry into early endosomes is insufficient for MHC class II processing. J Immunol. 1993 Apr 1;150(7):2687–2697. [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. Peters P. J., Neefjes J. J., Oorschot V., Ploegh H. L., Geuze H. J. Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature. 1991 Feb 21;349(6311):669–676. doi: 10.1038/349669a0. [DOI] [PubMed] [Google Scholar]
  48. Pieters J., Horstmann H., Bakke O., Griffiths G., Lipp J. Intracellular transport and localization of major histocompatibility complex class II molecules and associated invariant chain. J Cell Biol. 1991 Dec;115(5):1213–1223. doi: 10.1083/jcb.115.5.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Roche P. A., Cresswell P. Invariant chain association with HLA-DR molecules inhibits immunogenic peptide binding. Nature. 1990 Jun 14;345(6276):615–618. doi: 10.1038/345615a0. [DOI] [PubMed] [Google Scholar]
  50. Rudensky AYu, Preston-Hurlburt P., Hong S. C., Barlow A., Janeway C. A., Jr Sequence analysis of peptides bound to MHC class II molecules. Nature. 1991 Oct 17;353(6345):622–627. doi: 10.1038/353622a0. [DOI] [PubMed] [Google Scholar]
  51. Sadegh-Nasseri S., Germain R. N. A role for peptide in determining MHC class II structure. Nature. 1991 Sep 12;353(6340):167–170. doi: 10.1038/353167a0. [DOI] [PubMed] [Google Scholar]
  52. Sadegh-Nasseri S., Germain R. N. How MHC class II molecules work: peptide-dependent completion of protein folding. Immunol Today. 1992 Feb;13(2):43–46. doi: 10.1016/0167-5699(92)90131-P. [DOI] [PubMed] [Google Scholar]
  53. Salamero J., Humbert M., Cosson P., Davoust J. Mouse B lymphocyte specific endocytosis and recycling of MHC class II molecules. EMBO J. 1990 Nov;9(11):3489–3496. doi: 10.1002/j.1460-2075.1990.tb07557.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Srinivasan M., Marsh E. W., Pierce S. K. Characterization of naturally processed antigen bound to major histocompatibility complex class II molecules. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):7928–7932. doi: 10.1073/pnas.88.18.7928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Stern L. J., Wiley D. C. The human class II MHC protein HLA-DR1 assembles as empty alpha beta heterodimers in the absence of antigenic peptide. Cell. 1992 Feb 7;68(3):465–477. doi: 10.1016/0092-8674(92)90184-e. [DOI] [PubMed] [Google Scholar]
  56. Sullivan P. C., Ferris A. L., Storrie B. Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles. J Cell Physiol. 1987 Apr;131(1):58–63. doi: 10.1002/jcp.1041310110. [DOI] [PubMed] [Google Scholar]
  57. Teyton L., O'Sullivan D., Dickson P. W., Lotteau V., Sette A., Fink P., Peterson P. A. Invariant chain distinguishes between the exogenous and endogenous antigen presentation pathways. Nature. 1990 Nov 1;348(6296):39–44. doi: 10.1038/348039a0. [DOI] [PubMed] [Google Scholar]
  58. Tulsiani D. R., Hubbard S. C., Robbins P. W., Touster O. alpha-D-Mannosidases of rat liver Golgi membranes. Mannosidase II is the GlcNAcMAN5-cleaving enzyme in glycoprotein biosynthesis and mannosidases Ia and IB are the enzymes converting Man9 precursors to Man5 intermediates. J Biol Chem. 1982 Apr 10;257(7):3660–3668. [PubMed] [Google Scholar]
  59. Watts T. H., McConnell H. M. High-affinity fluorescent peptide binding to I-Ad in lipid membranes. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9660–9664. doi: 10.1073/pnas.83.24.9660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Weissman A. M., Klausner R. D., Rao K., Harford J. B. Exposure of K562 cells to anti-receptor monoclonal antibody OKT9 results in rapid redistribution and enhanced degradation of the transferrin receptor. J Cell Biol. 1986 Mar;102(3):951–958. doi: 10.1083/jcb.102.3.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wettstein D. A., Boniface J. J., Reay P. A., Schild H., Davis M. M. Expression of a class II major histocompatibility complex (MHC) heterodimer in a lipid-linked form with enhanced peptide/soluble MHC complex formation at low pH. J Exp Med. 1991 Jul 1;174(1):219–228. doi: 10.1084/jem.174.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Witt S. N., McConnell H. M. A first-order reaction controls the binding of antigenic peptides to major histocompatibility complex class II molecules. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8164–8168. doi: 10.1073/pnas.88.18.8164. [DOI] [PMC free article] [PubMed] [Google Scholar]

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