Influence of the immunoglobulin heavy chain in antibodies to Cryptococcus neoformans glucuronoxylomannan on binding affinity, specificity and immunoglobulin structure

Abstract

The immunological effector functions of an Ab are believed to be determined by its associated CH region, whereas the V-region is responsible for the affinity and specificity of the binding to the Ag. This notion is supported by a vast amount of immunological, biochemical, and structural evidence. Nevertheless, several lines of evidence indicate that the C H region could affect Ab binding for multivalent Ags.;Thus, we investigate the effect of isotype on the binding affinity and specificity of mAbs to Cryptococcus neoformans capsular GXM. Previous work indicated that the varying functional efficacies of these mAbs were dependent on isotype, affinity and fine specificity.;We began our studies by examining the binding characteristics of mouse-human chAbs. Collectively, our results suggest that the C-region can affect the fine specificity of an Ab towards a multivalent Ag by affecting segmental flexibility, altering idiotype, or by the ability of certain isotypes to associate. The possibility that these changes were a result of creating an artificial molecule could not be excluded since the synthesis of chAbs involve joining of a V-region to a C-region from two different species. Thus, we re-evaluated the concept that C-region can affect V-region fine specificity and idiotype by evaluating the reactivity of two families of murine IgG switch variants that differ in V-region usage for C. neoformans GXM. Our studies revealed isotype-related differences in fine specificity, idiotype and IF patterns for these switch variants validating our observations with two sets of V-region identical mAbs. We further demonstrated that the differences in fine specificity of the IgG switch variants were due to different associated C-regions. For these studies, we used SPR analysis to measure the kinetic and thermodynamics properties of binding of these mAbs, IgG2a and IgG3 Fab molecules to a peptide mimetic of GXM. The results obtained from the SPR analysis combined with structural data obtained from homology modeling indicate that the differences in the kinetic and thermodynamic parameters of the Ag-Ab interactions of the IgG subclasses may be due to changes in electrostatic and hydrophobic forces of the CH region, and not to gross conformational changes.