Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1994 Sep 1;180(3):1087–1096. doi: 10.1084/jem.180.3.1087

Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse-human immunoglobulin G1

PMCID: PMC2191655  PMID: 8064227

Abstract

Immunoglobulin G (IgG) molecules are glycosylated in CH2 at Asn297; the N-linked carbohydrates attached there have been shown to contribute to antibody (Ab) stability and various effector functions. The carbohydrate attached to the IgG constant region is a complex biantennary structure. Alterations in the structure of oligosaccharide have been associated with human diseases such as rheumatoid arthritis and osteoarthritis. To study the effects of altered carbohydrate structure on Ab effector function, we have used gene transfection techniques to produce mouse-human chimeric IgG1 Abs in the Chinese hamster ovary (CHO) cell line Lec 1, which is incapable of processing the high-mannose intermediate through the terminal glycosylation steps. We also produced IgG1 Abs in Pro-5, the wild-type CHO cell line that is the parent of Lec 1. The Pro-5-produced Ab (IgG1-Pro-5) was similar to IgG1-My 1, a myeloma-produced IgG1 Ab of the same specificity, in its biologic properties such as serum half-life, ability to effect complement-mediated cytolysis, and affinity for Fc gamma RI. Although the Lec 1-produced Ab, IgG1-Lec 1, was properly assembled and retained antigen specificity, it was incapable of complement-mediated hemolysis and was substantially deficient in complement consumption, C1q binding, and C1 activation. IgG1-Lec 1 also showed reduced but significant affinity for Fc gamma R1 receptors. The in vivo half-life of IgG1-Lec 1 was shorter than that of either the myeloma- or Pro-5-produced counterpart, with more being cleared during the alpha-phase and with more rapid clearance during the beta-phase. Clearance of IgG1-Lec 1 could be inhibited by the administration of yeast-derived mannan. Thus the uptake of IgG1-Lec 1 appears to be accelerated by the presence of terminally mannosylated oligosaccharide. Therefore, certain Ab functions as well as the in vivo fate of the protein are dramatically affected by altered carbohydrate structure. Expression of Igs in cell lines with defined glycosylation mutations is shown to be a useful technique for investigating the contribution of carbohydrate structure to Ab function.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  1. Axford J. S., Sumar N., Alavi A., Isenberg D. A., Young A., Bodman K. B., Roitt I. M. Changes in normal glycosylation mechanisms in autoimmune rheumatic disease. J Clin Invest. 1992 Mar;89(3):1021–1031. doi: 10.1172/JCI115643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bindon C. I., Hale G., Brüggemann M., Waldmann H. Human monoclonal IgG isotypes differ in complement activating function at the level of C4 as well as C1q. J Exp Med. 1988 Jul 1;168(1):127–142. doi: 10.1084/jem.168.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bindon C. I., Hale G., Waldmann H. Complement activation by immunoglobulin does not depend solely on C1q binding. Eur J Immunol. 1990 Feb;20(2):277–281. doi: 10.1002/eji.1830200208. [DOI] [PubMed] [Google Scholar]
  4. Bourrie B. J., Casellas P., Blythman H. E., Jansen F. K. Study of the plasma clearance of antibody--ricin-A-chain immunotoxins. Evidence for specific recognition sites on the A chain that mediate rapid clearance of the immunotoxin. Eur J Biochem. 1986 Feb 17;155(1):1–10. doi: 10.1111/j.1432-1033.1986.tb09451.x. [DOI] [PubMed] [Google Scholar]
  5. Bulens F., Vandamme A. M., Bernar H., Nelles L., Lijnen R. H., Collen D. Construction and characterization of a functional chimeric murine-human antibody directed against human fibrin fragment-D dimer. Eur J Biochem. 1991 Jan 1;195(1):235–242. doi: 10.1111/j.1432-1033.1991.tb15699.x. [DOI] [PubMed] [Google Scholar]
  6. Canfield S. M., Morrison S. L. The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region. J Exp Med. 1991 Jun 1;173(6):1483–1491. doi: 10.1084/jem.173.6.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Channing C. P., Sakai C. N., Bahl O. P. Role of the carbohydrate residues of human chorionic gonadotropin in binding and stimulation of adenosine 3',5'-monophosphate accumulation by porcine granulosa cells. Endocrinology. 1978 Aug;103(2):341–348. doi: 10.1210/endo-103-2-341. [DOI] [PubMed] [Google Scholar]
  8. Crowe J. S., Hall V. S., Smith M. A., Cooper H. J., Tite J. P. Humanized monoclonal antibody CAMPATH-1H: myeloma cell expression of genomic constructs, nucleotide sequence of cDNA constructs and comparison of effector mechanisms of myeloma and Chinese hamster ovary cell-derived material. Clin Exp Immunol. 1992 Jan;87(1):105–110. doi: 10.1111/j.1365-2249.1992.tb06421.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deisenhofer J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry. 1981 Apr 28;20(9):2361–2370. [PubMed] [Google Scholar]
  10. Dorai H., Mueller B. M., Reisfeld R. A., Gillies S. D. Aglycosylated chimeric mouse/human IgG1 antibody retains some effector function. Hybridoma. 1991 Apr;10(2):211–217. doi: 10.1089/hyb.1991.10.211. [DOI] [PubMed] [Google Scholar]
  11. Duncan A. R., Winter G. The binding site for C1q on IgG. Nature. 1988 Apr 21;332(6166):738–740. doi: 10.1038/332738a0. [DOI] [PubMed] [Google Scholar]
  12. Gross V., Heinrich P. C., vom Berg D., Steube K., Andus T., Tran-Thi T. A., Decker K., Gerok W. Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins. Eur J Biochem. 1988 May 2;173(3):653–659. doi: 10.1111/j.1432-1033.1988.tb14048.x. [DOI] [PubMed] [Google Scholar]
  13. Horwitz A. H., Chang C. P., Better M., Hellstrom K. E., Robinson R. R. Secretion of functional antibody and Fab fragment from yeast cells. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8678–8682. doi: 10.1073/pnas.85.22.8678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jefferis R., Lund J., Mizutani H., Nakagawa H., Kawazoe Y., Arata Y., Takahashi N. A comparative study of the N-linked oligosaccharide structures of human IgG subclass proteins. Biochem J. 1990 Jun 15;268(3):529–537. doi: 10.1042/bj2680529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Keene J. L., Matzuk M. M., Boime I. Expression of recombinant human choriogonadotropin in Chinese hamster ovary glycosylation mutants. Mol Endocrinol. 1989 Dec;3(12):2011–2017. doi: 10.1210/mend-3-12-2011. [DOI] [PubMed] [Google Scholar]
  16. Kindberg G. M., Magnusson S., Berg T., Smedsrød B. Receptor-mediated endocytosis of ovalbumin by two carbohydrate-specific receptors in rat liver cells. The intracellular transport of ovalbumin to lysosomes is faster in liver endothelial cells than in parenchymal cells. Biochem J. 1990 Aug 15;270(1):197–203. doi: 10.1042/bj2700197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kuiper J., Otter M., Rijken D. C., van Berkel T. J. Characterization of the interaction in vivo of tissue-type plasminogen activator with liver cells. J Biol Chem. 1988 Dec 5;263(34):18220–18224. [PubMed] [Google Scholar]
  18. Kukuruzinska M. A., Bergh M. L., Jackson B. J. Protein glycosylation in yeast. Annu Rev Biochem. 1987;56:915–944. doi: 10.1146/annurev.bi.56.070187.004411. [DOI] [PubMed] [Google Scholar]
  19. Leatherbarrow R. J., Rademacher T. W., Dwek R. A., Woof J. M., Clark A., Burton D. R., Richardson N., Feinstein A. Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor. Mol Immunol. 1985 Apr;22(4):407–415. doi: 10.1016/0161-5890(85)90125-7. [DOI] [PubMed] [Google Scholar]
  20. Lund J., Tanaka T., Takahashi N., Sarmay G., Arata Y., Jefferis R. A protein structural change in aglycosylated IgG3 correlates with loss of huFc gamma R1 and huFc gamma R111 binding and/or activation. Mol Immunol. 1990 Nov;27(11):1145–1153. doi: 10.1016/0161-5890(90)90103-7. [DOI] [PubMed] [Google Scholar]
  21. Mattes M. J. Biodistribution of antibodies after intraperitoneal or intravenous injection and effect of carbohydrate modifications. J Natl Cancer Inst. 1987 Oct;79(4):855–863. [PubMed] [Google Scholar]
  22. Michaelsen T. E., Garred P., Aase A. Human IgG subclass pattern of inducing complement-mediated cytolysis depends on antigen concentration and to a lesser extent on epitope patchiness, antibody affinity and complement concentration. Eur J Immunol. 1991 Jan;21(1):11–16. doi: 10.1002/eji.1830210103. [DOI] [PubMed] [Google Scholar]
  23. Mizuochi T., Taniguchi T., Shimizu A., Kobata A. Structural and numerical variations of the carbohydrate moiety of immunoglobulin G. J Immunol. 1982 Nov;129(5):2016–2020. [PubMed] [Google Scholar]
  24. 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]
  25. Morton H. C., Atkin J. D., Owens R. J., Woof J. M. Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells. J Immunol. 1993 Nov 1;151(9):4743–4752. [PubMed] [Google Scholar]
  26. Neumaier M., Shively L., Chen F. S., Gaida F. J., Ilgen C., Paxton R. J., Shively J. E., Riggs A. D. Cloning of the genes for T84.66, an antibody that has a high specificity and affinity for carcinoembryonic antigen, and expression of chimeric human/mouse T84.66 genes in myeloma and Chinese hamster ovary cells. Cancer Res. 1990 Apr 1;50(7):2128–2134. [PubMed] [Google Scholar]
  27. Nose M., Wigzell H. Biological significance of carbohydrate chains on monoclonal antibodies. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6632–6636. doi: 10.1073/pnas.80.21.6632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Parekh R. B., Dwek R. A., Sutton B. J., Fernandes D. L., Leung A., Stanworth D., Rademacher T. W., Mizuochi T., Taniguchi T., Matsuta K. Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature. 1985 Aug 1;316(6027):452–457. doi: 10.1038/316452a0. [DOI] [PubMed] [Google Scholar]
  29. Parekh R. B., Roitt I. M., Isenberg D. A., Dwek R. A., Ansell B. M., Rademacher T. W. Galactosylation of IgG associated oligosaccharides: reduction in patients with adult and juvenile onset rheumatoid arthritis and relation to disease activity. Lancet. 1988 Apr 30;1(8592):966–969. doi: 10.1016/s0140-6736(88)91781-3. [DOI] [PubMed] [Google Scholar]
  30. Rademacher T. W., Homans S. W., Parekh R. B., Dwek R. A. Immunoglobulin G as a glycoprotein. Biochem Soc Symp. 1986;51:131–148. [PubMed] [Google Scholar]
  31. Rademacher T. W., Parekh R. B., Dwek R. A., Isenberg D., Rook G., Axford J. S., Roitt I. The role of IgG glycoforms in the pathogenesis of rheumatoid arthritis. Springer Semin Immunopathol. 1988;10(2-3):231–249. doi: 10.1007/BF01857227. [DOI] [PubMed] [Google Scholar]
  32. Shin S. U., Morrison S. L. Production and properties of chimeric antibody molecules. Methods Enzymol. 1989;178:459–476. doi: 10.1016/0076-6879(89)78034-4. [DOI] [PubMed] [Google Scholar]
  33. Smedsrød B., Melkko J., Risteli L., Risteli J. Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells. Biochem J. 1990 Oct 15;271(2):345–350. doi: 10.1042/bj2710345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stanley P. Glycosylation mutants of animal cells. Annu Rev Genet. 1984;18:525–552. doi: 10.1146/annurev.ge.18.120184.002521. [DOI] [PubMed] [Google Scholar]
  35. Stanley P., Siminovitch L. Complementation between mutants of CHO cells resistant to a variety of plant lectins. Somatic Cell Genet. 1977 Jul;3(4):391–405. doi: 10.1007/BF01542968. [DOI] [PubMed] [Google Scholar]
  36. Sutton B. J., Phillips D. C. The three-dimensional structure of the carbohydrate within the Fc fragment of immunoglobulin G. Biochem Soc Trans. 1983 Apr;11(Pt 2):130–132. [PubMed] [Google Scholar]
  37. Tao M. H., Morrison S. L. Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J Immunol. 1989 Oct 15;143(8):2595–2601. [PubMed] [Google Scholar]
  38. Tao M. H., Smith R. I., Morrison S. L. Structural features of human immunoglobulin G that determine isotype-specific differences in complement activation. J Exp Med. 1993 Aug 1;178(2):661–667. doi: 10.1084/jem.178.2.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Thornburg R. W., Day J. F., Baynes J. W., Thorpe S. R. Carbohydrate-mediated clearance of immune complexes from the circulation. A role for galactose residues in the hepatic uptake of IgG-antigen complexes. J Biol Chem. 1980 Jul 25;255(14):6820–6825. [PubMed] [Google Scholar]
  40. Tsuchiya N., Endo T., Matsuta K., Yoshinoya S., Aikawa T., Kosuge E., Takeuchi F., Miyamoto T., Kobata A. Effects of galactose depletion from oligosaccharide chains on immunological activities of human IgG. J Rheumatol. 1989 Mar;16(3):285–290. [PubMed] [Google Scholar]
  41. Walker M. R., Lund J., Thompson K. M., Jefferis R. Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors. Biochem J. 1989 Apr 15;259(2):347–353. doi: 10.1042/bj2590347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weidle U. H., Borgya A., Mattes R., Lenz H., Buckel P. Reconstitution of functionally active antibody directed against creatine kinase from separately expressed heavy and light chains in non-lymphoid cells. Gene. 1987;51(1):21–29. doi: 10.1016/0378-1119(87)90470-7. [DOI] [PubMed] [Google Scholar]
  43. Zuckier L. S., Georgescu L., Chang C. J., Scharff M. D., Morrison S. L. The use of severe combined immunodeficiency mice to study the metabolism of human immunoglobulin G. Cancer. 1994 Feb 1;73(3 Suppl):794–799. doi: 10.1002/1097-0142(19940201)73:3+<794::aid-cncr2820731308>3.0.co;2-a. [DOI] [PubMed] [Google Scholar]

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

RESOURCES