Constant Region Effects on Antibody-Antigen Binding

Abstract

Antibodies (Abs), are products of the adaptive immune response that function as antimicrobial proteins composed of two regions, a constant (C) and a variable (V) region. The C region confers effector properties such as interaction with Fc receptors, half-life, and complement actions. The V region directly binds antigens (Ags) through a unique sequence that is selected based Ag specificity and affinity. Early Ab studies gave functional and structural reasons for the functional independence of the C and V regions. In recent decades, however, a number of independent groups have identified Ab isotypes which show differences in binding properties to the same Ag. The focus of this thesis is to further characterize how C region structure may be influencing the binding surface (paratope) of the V region by using a biochemical approach and a family of murine IgG isotypes.;By measuring changes in secondary structural properties using Circular Dichroism (CD) spectroscopy of a family of murine 3E5 IgG mAb isotypes raised against glucuronoxylomannan (GXM), we have identified a structural relationship between the C and V regions. Tryptophan (Trp) fluorescence studies comparing the emission spectra of Trp residues in the paratopes of the 3E5 mAbs before and after Ag binding revealed significant differences in the electronic properties of their binding surfaces. NMR studies done subsequently to probe the chemical properties of the 3E5 paratopes, revealed that all of the 3E5 IgGs hydrolyzed a peptide mimetic (P1) of the native Ag at 37°C with different rates, except IgG3; indicating that the chemistry and temperature-dependent modulation of the energy landscapes of the 3E5 paratopes differ.;We further solved the X-ray structure of the 3E5 IgG3 Fab and used Small Angle X-ray Spectroscopy (SAXS) to identify differences in 3E5 mAb hinge angles as well as overall solution structures. These studies provide a mechanism for prior observations that IgG isotypes differ in Ag specificity, and show that the presence of different C regions confers new secondary structure, electronic, chemical, and catalytic properties to the V region. We also provide a new example of Ab catalysis as an intrinsic characteristic of Ab function.