Structural Studies of the Fc Region of Murine Immunoglobulin G Antibodies using Single Molecule FRET

Abstract

The glycosylation of antibodies is known to influence their ability to instigate an immune response. The biantennary sugars present in the crystallizable fragment (Fc) region of antibodies are thought to stabilize the conformation of the Fc region so it can successfully bind to its receptors, thereby initiating an immune response. Prior studies have suggested that sugar removal leads to an increase in the flexibility of the Fc region. Other results show a collapse of the Fc region upon sugar removal. Either of these conformational alterations would impact receptor binding, thereby explaining the decreased immune response observed upon sugar removal. To examine the structure of the Fc region of murine immunoglobulin G (IgG) antibodies, click chemistry was used to attach dye molecules to azide-modified sugars in the Fc region. In addition, the enzyme EndoS was used to cleave the majority of the sugars from the Fc region. This enzyme leaves behind a single N-acetylglucosamine moiety, which was modified with an azide group and then reacted via click chemistry with dye molecules. These dye-labeled glycosylated and deglycosylated fully intact IgG antibodies were then examined using single molecule FRET to observe the structural impact of sugar removal.