Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1994 Aug 1;180(2):577–586. doi: 10.1084/jem.180.2.577

Defective secretion of an immunoglobulin caused by mutations in the heavy chain complementarity determining region 2

PMCID: PMC2191617  PMID: 8046334

Abstract

We have investigated four secretion-deficient antibodies (Abs) derived from a panel of 46 mutant T15 anti-phosphocholine Abs, all of which have point mutations in the heavy chain (H) complementarity determining region 2 (CDR2). The level of secretion for these four Abs was < 10% of wild type when expressed together with the T15 light chain (L) in either SP2/0 or P3X63Ag8.653 myeloma cells although normal levels of H and L chain mRNA were produced. Moreover, abundant intracellular H and L chain proteins were detected. Three of the four mutants had little or no assembled H and L complexes intracellularly whereas one had a significant amount of intracellular immunoglobulin (Ig) which was shown to be capable of binding Ag. Thus, we demonstrate for the first time that point mutations confined to CDR2 of the H chain variable (V) region can impede Ab assembly and secretion. We then introduced the same CDR2 mutations into a related H chain which is encoded by the same T15 VH gene but different diversity (D) and joining (J) genes. When these H chains were expressed with a non-T15 L chain, the resulting Abs were secreted normally. The results thus suggest that the effects of the CDR2 mutations on Ab secretion are dependent on their interactions with L and/or H chain D-J sequences. These results also reveal a novel mechanism that could contribute to B cell wastage.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Alberini C. M., Bet P., Milstein C., Sitia R. Secretion of immunoglobulin M assembly intermediates in the presence of reducing agents. Nature. 1990 Oct 4;347(6292):485–487. doi: 10.1038/347485a0. [DOI] [PubMed] [Google Scholar]
  2. Baumal R., Potter M., Scharff M. D. Synthesis, assembly, and secretion of gamma globulin by mouse myeloma cells. 3. Assembly of the three subclasses of IgG. J Exp Med. 1971 Nov 1;134(5):1316–1334. doi: 10.1084/jem.134.5.1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buenafe A. C., Makowski F. F., Rittenberg M. B. Molecular analysis and fine specificity of antibodies against an organophosphorus hapten. J Immunol. 1989 Jul 15;143(2):539–545. [PubMed] [Google Scholar]
  5. Cerny J., Wallich R., Hammerling G. J. Analysis of T15 idiotopes by monoclonal antibodies: variability of idiotopic expression on phosphorylcholine-specific lymphocytes from individual inbred mice. J Immunol. 1982 Apr;128(4):1885–1891. [PubMed] [Google Scholar]
  6. Chen C., Roberts V. A., Rittenberg M. B. Generation and analysis of random point mutations in an antibody CDR2 sequence: many mutated antibodies lose their ability to bind antigen. J Exp Med. 1992 Sep 1;176(3):855–866. doi: 10.1084/jem.176.3.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen C., Stenzel-Poore M. P., Rittenberg M. B. Natural auto- and polyreactive antibodies differing from antigen-induced antibodies in the H chain CDR3. J Immunol. 1991 Oct 1;147(7):2359–2367. [PubMed] [Google Scholar]
  8. Chien N. C., Pollock R. R., Desaymard C., Scharff M. D. Point mutations cause the somatic diversification of IgM and IgG2a antiphosphorylcholine antibodies. J Exp Med. 1988 Mar 1;167(3):954–973. doi: 10.1084/jem.167.3.954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chothia C., Lesk A. M., Tramontano A., Levitt M., Smith-Gill S. J., Air G., Sheriff S., Padlan E. A., Davies D., Tulip W. R. Conformations of immunoglobulin hypervariable regions. Nature. 1989 Dec 21;342(6252):877–883. doi: 10.1038/342877a0. [DOI] [PubMed] [Google Scholar]
  10. Chothia C., Novotný J., Bruccoleri R., Karplus M. Domain association in immunoglobulin molecules. The packing of variable domains. J Mol Biol. 1985 Dec 5;186(3):651–663. doi: 10.1016/0022-2836(85)90137-8. [DOI] [PubMed] [Google Scholar]
  11. Coffino P., Baumal R., Laskov R., Scharff M. D. Cloning of mouse myeloma cells and detection of rare variants. J Cell Physiol. 1972 Jun;79(3):429–440. doi: 10.1002/jcp.1040790313. [DOI] [PubMed] [Google Scholar]
  12. Desaymard C., Giusti A. M., Scharff M. D. Rat anti-T15 monoclonal antibodies with specificity for VH- and VH-VL epitopes. Mol Immunol. 1984 Oct;21(10):961–967. doi: 10.1016/0161-5890(84)90154-8. [DOI] [PubMed] [Google Scholar]
  13. Dul J. L., Argon Y. A single amino acid substitution in the variable region of the light chain specifically blocks immunoglobulin secretion. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8135–8139. doi: 10.1073/pnas.87.20.8135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dul J. L., Burrone O. R., Argon Y. A conditional secretory mutant in an Ig L chain is caused by replacement of tyrosine/phenylalanine 87 with histidine. J Immunol. 1992 Sep 15;149(6):1927–1933. [PubMed] [Google Scholar]
  15. Early P., Huang H., Davis M., Calame K., Hood L. An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH. Cell. 1980 Apr;19(4):981–992. doi: 10.1016/0092-8674(80)90089-6. [DOI] [PubMed] [Google Scholar]
  16. French D. L., Laskov R., Scharff M. D. The role of somatic hypermutation in the generation of antibody diversity. Science. 1989 Jun 9;244(4909):1152–1157. doi: 10.1126/science.2658060. [DOI] [PubMed] [Google Scholar]
  17. Gardner A. M., Aviel S., Argon Y. Rapid degradation of an unassembled immunoglobulin light chain is mediated by a serine protease and occurs in a pre-Golgi compartment. J Biol Chem. 1993 Dec 5;268(34):25940–25947. [PubMed] [Google Scholar]
  18. George A. J., Foreman R. C., Stevenson F. K. Characterisation of a light chain loss variant of the BCL1 lymphoma. Mol Immunol. 1991 Jul;28(7):789–799. doi: 10.1016/0161-5890(91)90122-z. [DOI] [PubMed] [Google Scholar]
  19. Grey H. M., Mannik M. Specificity of recombination of H and L chains from human gamma-G-myeloma proteins. J Exp Med. 1965 Sep 1;122(3):619–632. doi: 10.1084/jem.122.3.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hamel P. A., Klein M. H., Dorrington K. J. The role of the VL- and VH-segments in the preferential reassociation of immunoglobulin subunits. Mol Immunol. 1986 May;23(5):503–510. doi: 10.1016/0161-5890(86)90113-6. [DOI] [PubMed] [Google Scholar]
  21. Hamers-Casterman C., Atarhouch T., Muyldermans S., Robinson G., Hamers C., Songa E. B., Bendahman N., Hamers R. Naturally occurring antibodies devoid of light chains. Nature. 1993 Jun 3;363(6428):446–448. doi: 10.1038/363446a0. [DOI] [PubMed] [Google Scholar]
  22. Harpold M. M., Evans R. M., Salditt-Georgieff M., Darnell J. E. Production of mRNA in Chinese hamster cells: relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences. Cell. 1979 Aug;17(4):1025–1035. doi: 10.1016/0092-8674(79)90341-6. [DOI] [PubMed] [Google Scholar]
  23. Hendershot L., Bole D., Köhler G., Kearney J. F. Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein. J Cell Biol. 1987 Mar;104(3):761–767. doi: 10.1083/jcb.104.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hurtley S. M., Helenius A. Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:277–307. doi: 10.1146/annurev.cb.05.110189.001425. [DOI] [PubMed] [Google Scholar]
  25. Knittler M. R., Haas I. G. Interaction of BiP with newly synthesized immunoglobulin light chain molecules: cycles of sequential binding and release. EMBO J. 1992 Apr;11(4):1573–1581. doi: 10.1002/j.1460-2075.1992.tb05202.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kranz D. M., Voss E. W., Jr Restricted reassociation of heavy and light chains from hapten-specific monoclonal antibodies. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5807–5811. doi: 10.1073/pnas.78.9.5807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kwan S. P., Rudikoff S., Seidman J. G., Leder P., Scharff M. D. Nucleic acid and protein sequences of phosphocholine-binding light chains. J Exp Med. 1981 May 1;153(5):1366–1370. doi: 10.1084/jem.153.5.1366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morrison S. L., Johnson M. J., Herzenberg L. A., Oi V. T. Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6851–6855. doi: 10.1073/pnas.81.21.6851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Morrison S. L. Murine heavy chain disease. Eur J Immunol. 1978 Mar;8(3):194–199. doi: 10.1002/eji.1830080311. [DOI] [PubMed] [Google Scholar]
  30. Morrison S. L., Scharff M. D. Heavy chain-producing variants of a mouse myeloma cell line. J Immunol. 1975 Feb;114(2 Pt 1):655–659. [PubMed] [Google Scholar]
  31. Mosmann T. R., Williamson A. R. Structural mutations in a mouse immunoglobulin light chain resulting in failure to be secreted. Cell. 1980 Jun;20(2):283–292. doi: 10.1016/0092-8674(80)90614-5. [DOI] [PubMed] [Google Scholar]
  32. Oi V. T., Morrison S. L., Herzenberg L. A., Berg P. Immunoglobulin gene expression in transformed lymphoid cells. Proc Natl Acad Sci U S A. 1983 Feb;80(3):825–829. doi: 10.1073/pnas.80.3.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pepe V. H., Sonenshein G. E., Yoshimura M. I., Shulman M. J. Gene transfer of immunoglobulin light chain restores heavy chain secretion. J Immunol. 1986 Oct 1;137(7):2367–2372. [PubMed] [Google Scholar]
  34. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  35. Rice D., Baltimore D. Regulated expression of an immunoglobulin kappa gene introduced into a mouse lymphoid cell line. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7862–7865. doi: 10.1073/pnas.79.24.7862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roholt O. A., Radzimski G., Pressman D. Preferential recombination of antibody chains to form effective binding sites. J Exp Med. 1965 Oct 1;122(4):785–798. doi: 10.1084/jem.122.4.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rudikoff S., Pumphrey J. G. Functional antibody lacking a variable-region disulfide bridge. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7875–7878. doi: 10.1073/pnas.83.20.7875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Seligmann M., Mihaesco E., Preud'homme J. L., Danon F., Brouet J. C. Heavy chain diseases: current findings and concepts. Immunol Rev. 1979;48:145–167. doi: 10.1111/j.1600-065x.1979.tb00302.x. [DOI] [PubMed] [Google Scholar]
  39. Sharon J., Gefter M. L., Wysocki L. J., Margolies M. N. Recurrent somatic mutations in mouse antibodies to p-azophenylarsonate increase affinity for hapten. J Immunol. 1989 Jan 15;142(2):596–601. [PubMed] [Google Scholar]
  40. Sharon J. The invariant tryptophan in an H chain V region is not essential to antibody binding. J Immunol. 1988 Apr 15;140(8):2666–2669. [PubMed] [Google Scholar]
  41. Shlomchik M. J., Aucoin A. H., Pisetsky D. S., Weigert M. G. Structure and function of anti-DNA autoantibodies derived from a single autoimmune mouse. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9150–9154. doi: 10.1073/pnas.84.24.9150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sitia R., Neuberger M., Alberini C., Bet P., Fra A., Valetti C., Williams G., Milstein C. Developmental regulation of IgM secretion: the role of the carboxy-terminal cysteine. Cell. 1990 Mar 9;60(5):781–790. doi: 10.1016/0092-8674(90)90092-s. [DOI] [PubMed] [Google Scholar]
  43. Sonenshein G. E., Siekevitz M., Siebert G. R., Gefter M. L. Control of immunoglobulin secretion in the murine plasmacytoma line MOPC 315. J Exp Med. 1978 Jul 1;148(1):301–312. doi: 10.1084/jem.148.1.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stenzel-Poore M. P., Bruderer U., Rittenberg M. B. The adaptive potential of the memory response: clonal recruitment and epitope recognition. Immunol Rev. 1988 Oct;105:113–136. doi: 10.1111/j.1600-065x.1988.tb00769.x. [DOI] [PubMed] [Google Scholar]
  45. Stenzel-Poore M. P., Rittenberg M. B. Clonal diversity, somatic mutation, and immune memory to phosphocholine-keyhole limpet hemocyanin. J Immunol. 1989 Dec 15;143(12):4123–4133. [PubMed] [Google Scholar]
  46. Wilkinson M. RNA isolation: a mini-prep method. Nucleic Acids Res. 1988 Nov 25;16(22):10933–10933. doi: 10.1093/nar/16.22.10933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Winberry L., Marks A., Baumal R. Immunoglobulin production and secretion by variant clones of the MOPC 315 mouse myeloma cell line. J Immunol. 1980 Mar;124(3):1174–1182. [PubMed] [Google Scholar]
  48. Wu G. E., Hozumi N., Murialdo H. Secretion of a lambda 2 immunoglobulin chain is prevented by a single amino acid substitution in its variable region. Cell. 1983 May;33(1):77–83. doi: 10.1016/0092-8674(83)90336-7. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES