Elucidating how N-glycosylation impacts immunoglobulin G Fc structure and receptor binding
N-linked glycans are post-translational modifications that link an oligosaccharide to an asparagine residue. The attached oligosaccharide then undergoes extensive modification in the Golgi apparatus prior to protein secretion. This type of modification helps with protein folding, resistance to protease activity, immunogenicity, and imparting biological activity. Antibodies, such as IgG1, offer an idealized system to study proteins containing an N-linked glycan.
IgG1 is a homodimeric protein with a molecular weight of 150 kDa. It is composed of two heavy and two light chains, the heavy chain encompassing the Fab (fragment antigen binding) and Fc (fragment constant) and the light chain only being part of the Fab region. The two regions of IgG1, Fab and Fc, are linked by a hinge which contains several disulfide bonds creating a flexible linker allowing the Fab and Fc domains freedom to orient and bind their targets. The Fab region binds antigen and the Fc region binds immune receptors. Under the proper conditions these receptors trigger a cellular response to an antigen.
In this work we seek to understand how this cellular response is modulated by the N-linked glycans present on the Fc region. The Fc contains two N-linked glycans, which are attached in a region that is critical for immune receptor binding. It is thought that these glycans, through contacts across a beta sheet, restrict the motion of a loop region allowing for a high affinity interaction between the Fc region and immune receptors. Through NMR and X–ray crystallography studies we show that one specific loop becomes mobile when the glycan is truncated and that when a full glycan is present the loops moves by more than 5Ã Â . Fc with the truncated N-glycan binds receptor with 100-fold less affinity that Fc with a full glycan and thus this reorganization is incredibly important to understanding complex formation.
Additionally, the immune receptors that interact with IgG molecules are themselves glycosylated. In this work we study the interaction of IgG1 Fc with CD16A, a receptor present on natural killer cells (NK cells). This receptor is responsible for the antibody dependent cell-mediated cytotoxicity, (ADCC), response from NK cells and which is a major tumor clearing mechanism in the body. Through several SPR and ITC experiments we show that one of the glycans present on CD16A can interact with the glycan on IgG1 Fc, which in turn can modulate the affinity of the complex. One such modification of the IgG1 Fc, the addition of a monosaccharide called fucose, disrupts the IgG1 Fc / CD16A complex. An analysis of extensive all-atom molecular dynamics simulations show that this modification causes a shift in conformation in the CD16A glycan interacting with IgG1 Fc potentially biasing it into an energetically unfavorable area leading to affinity loss. Both of these studies seek to better understand the role N-glycans play in the interaction of Fc regions and their immune receptors to design better monoclonal antibody therapy treatments.