Abstract
Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange ("Fab arm exchange"). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.
Highlights
Fab arm exchange requires weak interactions between CH3 domains, such as in human IgG4
CH3-CH3 Interactions in Human Immunoglobulin G (IgG) Subclasses—We investigated the rate of dissociation of CH3 domains for all four human IgG subclasses using a previously developed Förster resonance energy transfer (FRET) assay to monitor Fab arm exchange in real time (Fig. 1B)
These results imply that the inter-heavy chain disulfide bonds of IgG2 and IgG3 will play a crucial role in determining their stability and raise the question if Fab arm exchange might be observed under conditions that mimic the in vivo redox conditions
Summary
Fab arm exchange requires weak interactions between CH3 domains, such as in human IgG4. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/ Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Due to relatively labile disulfide bonds in the hinge as well as weak CH3-CH3 interactions, IgG4 is able to engage in Fab arm exchange [1,2,3,4]; regular IgG antibodies are bivalent, IgG4 becomes effectively monovalent by exchanging half-molecules with other molecules of IgG4, combining random specificities in the second Fab arm for a given specificity of the first Fab arm (Fig. 1A) These antibodies are unable to cross-link antigen to form immune complexes. We systematically investigated non-covalent inter-heavy chain interactions for the different human subclasses as well as polymorphisms in the CH3 domains We found these interactions to vary in strength by more than 6 orders of magnitude, which sheds new light on the role of CH3-CH3 interactions in maintaining inter-heavy chain interactions and modulating Fab arm exchange
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
