title: The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds.
abstract: Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma have been reported to be functionally monovalent. In a previous paper, we showed that the apparent monovalency of circulating IgG4 antibodies is caused by asymmetry of plasma IgG4-a large fraction has two antigen-binding sites resulting in bispecificity. We postulated that the generation of bispecific antibodies was caused by a post-secretion mechanism, involving the exchange of IgG4 half-molecules (i.e. one heavy and one light chain). This hypothesis was based on the observed instability of the inter-heavy chain disulfide bonds of IgG4. To investigate this instability, we constructed IgG4 mutants and analyzed the covalent interaction between the heavy chains by sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The mutation to serine of one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, resulted in a more stable rather than a more labile inter-heavy chain linkage. Moreover, we confirmed that mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent interaction between the heavy-chains. These two observations suggested an explanation for the observed instability of the inter-heavy chain disulfide bonds: the formation of an alternative, intra-chain cystine. Obviously, this intra-chain cystine cannot be formed in the mutant where Cys226 is replaced by Ser, and cannot easily be formed in the mutant with the IgG1 hinge sequence (Cys-Pro-Pro-Cys) due to the restricted torsional freedom of prolines. We, therefore, postulate that the lack of a covalent heavy-chain interaction in a subpopulation of IgG4 reflects an equilibrium between inter- and intra-chain cystines. Based upon the published structure of the IgG4-related hinge-deleted IgG1 myeloma protein Mcg, we propose a model for the two forms of IgG4 and for the half-molecule exchange reaction, which might result in the formation of bispecific IgG4 antibodies.
authors.affiliation: Laboratory for Experimental and Clinical Immunology, Department of Immunopathology of the CLB, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. j.schuurman@azu.nl
authors.forename: J
authors.initials: J
authors.lastname: Schuurman
authors.forename: G J
authors.initials: GJ
authors.lastname: Perdok
authors.forename: A D
authors.initials: AD
authors.lastname: Gorter
authors.forename: R C
authors.initials: RC
authors.lastname: Aalberse
mesh_terms: D000818:Animals; D004220:Disulfides; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics; D051379:Mice
chemical_list: D004220:Disulfides; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains