Circular Dichroism reveals evidence of coupling between immunoglobulin constant and variable region secondary structure

Antibodies (Ab) are bifunctional molecules with two domains, a constant region (C) that confers effector properties and a variable (V) region responsible of antigen (Ag) binding. Historically the C and V regions were considered to be functionally independent, with Ag specificity being solely determined by the V region. However, recent studies suggest that the C region can affect Ab fine specificity. This has led to the proposal that the CH domain influences the structure of the V region, thus affecting Ab affinity and fine specificity. An inference from this proposal is that V region identical monoclonal Abs (mAbs) differing in C region (eg isotype) would manifest different secondary structures arising from isotype-induced variation in the V–C regions after Ag binding. We hypothesized that such effects could translate into differences in Circular Dichroism (CD) upon Ag–Ab complexes formation. Consequently we studied the interaction of a set of V region identical IgG1, IgG2a, IgG2b and IgG3 mAbs with glucuronoxylomannan (GXM). The native CD spectra of the pairs IgG1/IgG2a and IgG3/IgG2b were strikingly similar, implying similar secondary structure content. GXM binding by IgG1, IgG2a, IgG2b and IgG3 produced different CD changes, with the pairs IgG1/IgG2a and IgG3/IgG2b again manifesting qualitatively similar trends in secondary structure changes. The magnitude of the changes differed among the isotypes with IgG2a > IgG3 > IgG2b > IgG1. These differences in CD changes were interpreted to reflect differences in V–C secondary structures.