Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1997 Feb;6(2):407–415. doi: 10.1002/pro.5560060217

Intrachain disulfide bond in the core hinge region of human IgG4.

J W Bloom 1, M S Madanat 1, D Marriott 1, T Wong 1, S Y Chan 1
PMCID: PMC2143633  PMID: 9041643

Abstract

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571 (S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded half-IgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP571 and CDP571(gamma 1) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgG1 molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgG1 and thus may permit formation of a stable intrachain disulfide bond more readily.

Full Text

The Full Text of this article is available as a PDF (2.9 MB).

Selected References

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

  1. Angal S., King D. J., Bodmer M. W., Turner A., Lawson A. D., Roberts G., Pedley B., Adair J. R. A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody. Mol Immunol. 1993 Jan;30(1):105–108. doi: 10.1016/0161-5890(93)90432-b. [DOI] [PubMed] [Google Scholar]
  2. Bardwell J. C., McGovern K., Beckwith J. Identification of a protein required for disulfide bond formation in vivo. Cell. 1991 Nov 1;67(3):581–589. doi: 10.1016/0092-8674(91)90532-4. [DOI] [PubMed] [Google Scholar]
  3. Burton D. R. Immunoglobulin G: functional sites. Mol Immunol. 1985 Mar;22(3):161–206. doi: 10.1016/0161-5890(85)90151-8. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Colcher D., Milenic D., Roselli M., Raubitschek A., Yarranton G., King D., Adair J., Whittle N., Bodmer M., Schlom J. Characterization and biodistribution of recombinant and recombinant/chimeric constructs of monoclonal antibody B72.3. Cancer Res. 1989 Apr 1;49(7):1738–1745. [PubMed] [Google Scholar]
  6. Dhainaut J. F., Vincent J. L., Richard C., Lejeune P., Martin C., Fierobe L., Stephens S., Ney U. M., Sopwith M. CDP571, a humanized antibody to human tumor necrosis factor-alpha: safety, pharmacokinetics, immune response, and influence of the antibody on cytokine concentrations in patients with septic shock. CPD571 Sepsis Study Group. Crit Care Med. 1995 Sep;23(9):1461–1469. doi: 10.1097/00003246-199509000-00004. [DOI] [PubMed] [Google Scholar]
  7. Dorrington K. J., Tanford C. Molecular size and conformation of immunoglobulins. Adv Immunol. 1970;12:333–381. doi: 10.1016/s0065-2776(08)60173-x. [DOI] [PubMed] [Google Scholar]
  8. Dorrington K. J. The structural basis for the functional versatility of immunoglobulin G1. Can J Biochem. 1978 Dec;56(12):1087–1101. doi: 10.1139/o78-172. [DOI] [PubMed] [Google Scholar]
  9. Grauschopf U., Winther J. R., Korber P., Zander T., Dallinger P., Bardwell J. C. Why is DsbA such an oxidizing disulfide catalyst? Cell. 1995 Dec 15;83(6):947–955. doi: 10.1016/0092-8674(95)90210-4. [DOI] [PubMed] [Google Scholar]
  10. Hawkins H. C., de Nardi M., Freedman R. B. Redox properties and cross-linking of the dithiol/disulphide active sites of mammalian protein disulphide-isomerase. Biochem J. 1991 Apr 15;275(Pt 2):341–348. doi: 10.1042/bj2750341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Holmgren A., Söderberg B. O., Eklund H., Brändén C. I. Three-dimensional structure of Escherichia coli thioredoxin-S2 to 2.8 A resolution. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2305–2309. doi: 10.1073/pnas.72.6.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hög J. O., Jörnvall H., Holmgren A., Carlquist M., Persson M. The primary structure of Escherichia coli glutaredoxin. Distant homology with thioredoxins in a superfamily of small proteins with a redox-active cystine disulfide/cysteine dithiol. Eur J Biochem. 1983 Oct 17;136(1):223–232. doi: 10.1111/j.1432-1033.1983.tb07730.x. [DOI] [PubMed] [Google Scholar]
  13. Kamitani S., Akiyama Y., Ito K. Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme. EMBO J. 1992 Jan;11(1):57–62. doi: 10.1002/j.1460-2075.1992.tb05027.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. King D. J., Adair J. R., Angal S., Low D. C., Proudfoot K. A., Lloyd J. C., Bodmer M. W., Yarranton G. T. Expression, purification and characterization of a mouse-human chimeric antibody and chimeric Fab' fragment. Biochem J. 1992 Jan 15;281(Pt 2):317–323. doi: 10.1042/bj2810317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klintrot I. M., Hög J. O., Jörnvall H., Holmgren A., Luthman M. The primary structure of calf thymus glutaredoxin. Homology with the corresponding Escherichia coli protein but elongation at both ends and with an additional half-cystine/cysteine pair. Eur J Biochem. 1984 Nov 2;144(3):417–423. doi: 10.1111/j.1432-1033.1984.tb08481.x. [DOI] [PubMed] [Google Scholar]
  16. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  18. Lin T. Y., Kim P. S. Urea dependence of thiol-disulfide equilibria in thioredoxin: confirmation of the linkage relationship and a sensitive assay for structure. Biochemistry. 1989 Jun 13;28(12):5282–5287. doi: 10.1021/bi00438a054. [DOI] [PubMed] [Google Scholar]
  19. Luthman M., Holmgren A. Rat liver thioredoxin and thioredoxin reductase: purification and characterization. Biochemistry. 1982 Dec 21;21(26):6628–6633. doi: 10.1021/bi00269a003. [DOI] [PubMed] [Google Scholar]
  20. Lyles M. M., Gilbert H. F. Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer. Biochemistry. 1991 Jan 22;30(3):613–619. doi: 10.1021/bi00217a004. [DOI] [PubMed] [Google Scholar]
  21. Missiakas D., Georgopoulos C., Raina S. The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation. EMBO J. 1994 Apr 15;13(8):2013–2020. doi: 10.1002/j.1460-2075.1994.tb06471.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Missiakas D., Schwager F., Raina S. Identification and characterization of a new disulfide isomerase-like protein (DsbD) in Escherichia coli. EMBO J. 1995 Jul 17;14(14):3415–3424. doi: 10.1002/j.1460-2075.1995.tb07347.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morrison S. L., Canfield S., Porter S., Tan L. K., Tao M. H., Wims L. A. Production and characterization of genetically engineered antibody molecules. Clin Chem. 1988 Sep;34(9):1668–1675. [PubMed] [Google Scholar]
  24. Petersen J. G., Dorrington K. J. An in vitro system for studying the kinetics of interchain disulfide bond formation in immunoglobulin G. J Biol Chem. 1974 Sep 10;249(17):5633–5641. [PubMed] [Google Scholar]
  25. Stephens S., Emtage S., Vetterlein O., Chaplin L., Bebbington C., Nesbitt A., Sopwith M., Athwal D., Novak C., Bodmer M. Comprehensive pharmacokinetics of a humanized antibody and analysis of residual anti-idiotypic responses. Immunology. 1995 Aug;85(4):668–674. [PMC free article] [PubMed] [Google Scholar]
  26. Tan L. K., Shopes R. J., Oi V. T., Morrison S. L. Influence of the hinge region on complement activation, C1q binding, and segmental flexibility in chimeric human immunoglobulins. Proc Natl Acad Sci U S A. 1990 Jan;87(1):162–166. doi: 10.1073/pnas.87.1.162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Virella G., Parkhouse R. M. Sensitivity to reduction of human immunoglobulin G of different heavy chain sub-classes. Immunochemistry. 1973 Apr;10(4):213–217. doi: 10.1016/0019-2791(73)90197-3. [DOI] [PubMed] [Google Scholar]
  28. Weber U., Hartter P. Synthese und Stabilität von cyclischen Disulfiden des Typs Cys-(X)n-Cys. Hoppe Seylers Z Physiol Chem. 1974 Feb;355(2):189–199. [PubMed] [Google Scholar]
  29. Zapun A., Bardwell J. C., Creighton T. E. The reactive and destabilizing disulfide bond of DsbA, a protein required for protein disulfide bond formation in vivo. Biochemistry. 1993 May 18;32(19):5083–5092. doi: 10.1021/bi00070a016. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES