The constant region affects antigen binding of anti-DNA antibodies by altering secondary structure (P4002)

Abstract

Abstract We previously demonstrated an important role of the constant (C) region in the pathogenicity of anti-DNA antibodies (Ab) through modulation of immunoglobulin affinity and specificity. However, the mechanisms by which the C region affects the binding of autoantibodies to nuclear antigens (Ag) have yet to be elucidated. From a parent IgG3 anti-nuclear Ab, PL9-11, we generated IgG1, IgG2b, and IgG2a variants that share identical variable regions but that differ in their C region. The affinity of these Abs for histone antigens was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine the wavelength shift of the Ab panel upon the addition of DNA and histone Ags. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure of the PL9-11 panel corresponding to Ab-Ag interactions. SPR analysis revealed significant differences of histone 2A/2B binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the Ab isotype, while increasing salt concentrations lowered KD values. Circular dichroism analysis determined that changes in Ab secondary structure content differed between the IgG isotypes upon Ag binding. In conclusion, the Ag-binding affinity of anti-nuclear Abs is dependent on the particular C region. Ag binding effects on Trp fluorescence, as well as secondary structure shifts, were also C region dependent. Alteration of secondary structures influenced by C regions may explain differences in fine specificity of anti-DNA Abs between Abs with similar variable regions, as well as cross-reactivity of anti-DNA Abs with non-DNA Ags.