Effects of Antibody Isotype on Specificities of IgG versus IgE
2Department of Chemistry, University of California, Davis, California, United States
Antibodies (Abs) are crucial for recognizing and binding to specific antigens. It has been widely accepted that Ab specificity and affinity are determined only due to Ab variable region interactions with antigens, in which the V-region variability is a result of processes such as VDJ recombination, and somatic hypermutations. However, recent evidence suggests that the Fc region may also influence Abs specificity and affinity. While this had been demonstrated for several anti-protein Abs, it had not yet been shown for anti-glycan Abs. In the context of food-borne allergies, anti-glycan Abs are frequently cross-reactive. Our lab has previously shown that IgE Abs against the non-human sugar N-glycolylneuraminic acid (Neu5Gc) appears to cross-react with its counterpart, N-acetylneuraminic acid (Neu5Ac), which covers all human cell glycan terminal tips and differs by a single oxygen atom from Neu5Gc. We hypothesize that isotype switching from IgG to IgE affects anti-glycan antibody specificity, particularly for Abs against Neu5Gc that could become cross-reactive with Neu5Ac. For this purpose, we cloned two monoclonal Abs (mAbs) as IgG and IgE. One Ab recognizes Neu5Ac-glycans [RA0; anti-AcSTn (Neu5Aca2-6GalNAc) Ab], and the second Ab recognizes Neu5Gc-glycans [14F7; anti-GcGM3 (Neu5Gca2-3Galb1-4Glc)]. These Abs were cloned into p3BNC plasmids as IgG and IgE isotypes using Gibson assembly and restriction enzymes, then expressed in Expi293 cells and examined first by Western blot. Specificity of these cloned Abs was then examined by ELISA and glycan microarrays against a collection of Neu5Ac-glycans and Neu5Gc-glycans. Glycan microarray analysis revealed that for RA0 mAb, the IgE variants maintained AcSTn specificity (and to its related structures) and did not bind to Neu5Gc-glycans, showing the same specificity pattern as the IgG variants. However, for 14F7 mAb, the IgE variants showed broadened glycan recognition compared to the IgG variants, binding to Neu5Gc-glycans but also binding to Neu5Ac-glycans, suggesting its ability to become cross-reactive as IgE isotype. These studies highlight the potential influence of anti-glycan Ab isotypes on Ab cross-reactivity, which can potentially elucidate the Fc region’s role in modulating Ab specificity, thereby prompting an understanding of the mechanisms by which IgE leads to allergic reactions and the mechanisms underlying autoimmune diseases.