The influence of glycosylation on the thermal stability and effector function expression of human IgG1-Fc: properties of a series of truncated glycoforms.

Antibodies are multifunctional molecules that following the formation of antibody antigen complexes, may activate mechanisms to effect the clearance and destruction of the antigen (pathogen). The IgG molecule is comprised of three globular protein moieties (2Fab+Fc) linked through a flexible hinge region. While the Fabs bind antigens, the Fc triggers effector mechanisms through interactions with specific ligands, e.g. cellular receptors (FcgammaR), and the C1 component of complement. Glycosylation of IgG-Fc has been shown to be essential for efficient activation of FcgammaR and C1. We report the generation of a series of truncated glycoforms of IgG-Fc, and the analysis of the contribution of the residual oligosaccharide to IgG-Fc function and thermal stability. Differential scanning microcalorimetry has been used to compare the stabilities of the homogeneous glycoforms of IgG1-Fc. The results show that all truncated oligosaccharides confer a degree of functional activity, and thermodynamic stability to the IgG1-Fc, in comparison with deglycosylated IgG1-Fc. The same truncated glycoforms of an intact IgG1 anti-MHC Class II antibody are shown to exhibit differential functional activity for FcgammaRI and C1 ligands, relative to deglycosylated IgG1. The minimal glycoform investigated had a trisaccharide attached to each heavy chain and can be expected to influence protein structure primarily in the proximity of the N-terminal region of the C(H)2 domain, implicated as a binding site for multiple effector ligands. These data provide a thermodynamic rationale for the modulation of antibody effector functions by different glycoforms.