Altered IgG N-Glycosylation at Onset of Type 1 Diabetes in Children Is Predominantly Driven by Changes in the Fab N-Glycans.

Abstract

Background: N-glycosylation is a post-translational modification involving the attachment of oligosaccharides to proteins and is known to influence immunoglobulin G (IgG) effector functions and even antigen binding. IgG contains an evolutionarily conserved N-glycosylation site in its fragment crystallizable (Fc) region, while during V-D-J recombination and somatic hypermutation processes it can also obtain N-glycosylation sites in its antigen binding fragment (Fab). Our previous study demonstrated altered IgG N-glycosylation in children at type 1 diabetes (T1D) onset, with the most prominent changes involving sialylated glycans, hypothesized to mainly come from the Fab region, however, the analytical method used could not distinguish between Fc and Fab. Methods: IgG was isolated from plasma from 118 children with T1D and 98 healthy controls from the Danish Registry of Childhood and Adolescent Diabetes. Isolated IgG was cleaved into Fc and Fab fragments using IdeS enzyme. N-glycans were enzymatically released from each fragment, fluorescently labelled with procainamide, and analyzed separately using the UPLC-MS method. Structural annotation of resulting chromatograms was performed using MS/MS. Results: T1D related N-glycosylation changes were more pronounced in the Fab glycans compared to Fc glycans, with five Fab glycans (Man5, Man7, FA2BG1S1, A2G2S2, FA2BG2S1) being significantly altered compared to only one in the Fc region (FA2[3]BG1). Comparing Fc and Fab glycosylation overall reveals stark differences in the types of glycans on each region, with a more diverse and complex repertoire being present in the Fab region. Conclusions: These findings suggest that N-glycosylation changes in early onset T1D predominantly originate from the Fab region, underscoring their potential role in modulating (auto)immunity and highlighting distinct glycosylation patterns between Fc and Fab.