The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds

Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma h ave been reported to be functionally monovalent. In a previous paper, we sh owed that the apparent monovalency of circulating IgG4 antibodies is caused by asymmetry of plasma IgG4 - a large fraction has two antigen-binding sit es resulting in bispecificity. We postulated that the generation of bispeci fic antibodies was caused by a post-secretion mechanism, involving the exch ange of IgG4 half-molecules (i.e. one heavy and one light chain). This hypo thesis was based on the observed instability of the inter-heavy chain disul fide bonds of IgG4. To investigate this instability, we constructed IgG4 mu tants and analyzed the covalent interaction between the heavy chains by sod ium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) under no n-reducing conditions. The mutation to serine of one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, resulted in a mo re 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 IgG 1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent inte raction between the heavy-chains. These two observations suggested an expla nation for the observed instability of the inter-heavy chain disulfide bond s: the formation of an alternative, intra-chain cystine. Obviously, this in tra-chain cystine cannot be formed in the mutant where Cys226 is replaced b y Set, and cannot easily be formed in the mutant with the IgG1 hinge sequen ce (Cys-Pro-Pro-Cys) due to the restricted torsional freedom of prolines. W e, therefore, postulate that the lack of a covalent heavy-chain interaction in a subpopulation of IgG4 reflects an equilibrium between inter- and intr a-chain cystines. Based upon the published structure of the IgG4-related hi nge-deleted IgG1 myeloma protein Meg, 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. (C) 2001 Elsevier Science Ltd. All rights reserved.