Previous studies on the variable region glycosylation of IgG have been performed mainly on monoclonal antibodies from animals or total IgG from healthy individuals [
37,
38]. To our knowledge, our study is the first investigation on the glycosylation status of the variable region of disease-associated antigen-specific IgGs. Previous studies by Holland et al. [
25] have shown that while the oligosaccharides released from ANCA-containing IgG-Fc were hypogalactosylated and hyposialylated, those released from IgG-Fab were normally galactosylated and sialylated. In this study the authors did not report any difference in variable region glycosylation between ANCA-containing IgG and normal IgG upon analysis of total serum-derived IgG. In the current study, when we compared the variable region glycosylation levels of total IgG in plasma between patients and normal individuals, no significant difference was found either. However, affinity-purified anti-MPO antibodies were found to have a significantly higher level of variable region glycosylation than total IgG, while purified anti-GBM antibody was found to have a significantly lower level of variable region glycosylation than total IgG.
In the current study, incubation with neuraminidase did not significantly change the antigen-binding ability of SNA-binding IgG, while incubation with endoglycosidase F2 significantly changed the antigen-binding ability of SNA-binding IgG. It indicated that the variable region glycan molecule, but not the terminal sialic acid, influenced the antigen-binding ability of autoantibodies It also indicated that electrostatic forces are not a major determinant, because the isoelectric point of α3(IV)NC1 (calculated isoelectric point 8.9 [
39]) is between the isoelectric points of MPO and PR3 (11.0 and 7.9 for MPO and PR3, respectively [
40,
41]). How oligosaccharides in the variable region influence antigen-antibody binding is not clear yet. In a previous study, it was proposed that oligosaccharides in the variable region affect the antigen-binding ability by influencing hydrophilic interactions between the antibody and antigen [
42]. Amino acid sequences of the variable regions of IgG are highly variable and are influenced by a number of factors. Initially, the rearrangement of VH and VL genes occurs in the bone marrow. Then after B cells encounter antigen, the somatic mutation machinery is activated in the germinal center [
43]. Interestingly, sequence analysis has shown that there is no potential N-linked glycosylation site [Asn-X-Ser/Thr (X is any amino acid except Pro, Asp or Glu)] in germline V sequences, suggesting that variable region glycosylation sites mainly arise from somatic mutations [
44‐
46]. Since the N-linked glycosylation site in the variable region is generated during the process of affinity maturation [
47], we speculate that the type of antigen is one of the determinants of oligosaccharide distribution in the variable region of the antibody. Moreover, since the oligosaccharide distribution of the variable region can influence the antigen-antibody binding, it could also influence the process of affinity maturation of the antibody. We speculate that if a variable region sequence, with or without potential glycosylation sites, has higher affinity for an antigen, this sequence will most likely be amplified during affinity maturation.
It has been suggested that hyposialylation of the ANCA antibody Fc part increases the ability of ANCA to induce the respiratory burst in neutrophils [
7]. In the present study, we showed that ANCA IgG with a high level of variable region glycosylation had a higher avidity constant and an increased ability to induce the respiratory burst in neutrophils. Furthermore, the level of variable glycosylation of purified MPO-ANCA positively correlated with disease activity. Moreover, MPO-ANCA from patients in remission demonstrated a significantly lower level of variable region glycosylation compared to MPO-ANCA from patients with active disease. These results suggest that the level of variable region glycosylation of ANCA might be a potent biomarker reflecting disease activity in AAV.