Surface Ig variable domain glycosylation affects autoantigen binding and acts as threshold for human autoreactive B cell activation

Abstract

The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.

Glycobiology of rheumatic diseases

“…Thus, Fab glycans could facilitate the escape of B cells from important checkpoints that control their activation and their ability to expand. Consistent with the idea of antigenic redemption, ACPA IgG Fab glycans were found to reduce binding to certain lower affinity (auto)antigens, while maintaining binding to other, higher affinity antigens, compared with ACPA IgG lacking Fab glycans 105 . Structural and crystallographic analyses suggest that antigen binding is probably affected by steric repulsion between the spatially demanding Fab glycans terminating with negatively charged sialic acids and the cognate antigens, and/or by competition between the antigen and the Fab glycan for the ACPA binding pocket (Fig.…”

“…Galactosylation of Fc glycans enhances hexamerization and subsequent C1q binding and complement-dependent cytotoxicity (CDC). b , Processed disialylated IgG Fab glycans can influence antigen binding via steric or charge-induced repulsion or by competing with the antigen for the binding pocket, as evidenced by dynamic simulations on Fab crystal structures 105 . Fab glycans can enhance B cell receptor (BCR) signalling while downregulating BCR internalization and antigen uptake.…”

“…In experiments involving the expression of membrane IgG BCRs derived from patients with RA on a human Burkitt lymphoma-derived (Ramos) model B cell line, knocked out for its endogenous BCR and the enzyme activation-induced cytidine deaminase, BCRs with Fab glycosylation had longer surface expression after antigenic triggering and more intense activation than their non-Fab-glycosylated counterparts 105 . These observations substantiate the hypothesis that N-glycans in the Fab region provide a selective advantage for autoreactive B cells by affecting antigen binding and the threshold of B cell activation (Fig.…”

“…For example, sialylated trophoblast glycans can engage CD22 inhibitory signalling in antigen-specific follicular B cells in mice, and as B cells present trophoblast antigens to CD4 + T cells, this B cell suppression in turn suppresses T cell responses, promoting fetomaternal tolerance 106 . However, in ACPA-expressing B cells, no influence of the negative regulator CD22 on the Fab glycan-mediated effects on BCR signalling is apparent 105 . Therefore, a precise mechanistic understanding of how Fab glycans affect B cell activation and biology is presently lacking.…”

“…Part b is adapted from ref. 105 , CC BY-NC 4.0 (https:// creativecommons.org/licenses/by-nc/4.0/). most prominent genetic risk factor for ACPA-positive RA, the human leukocyte antigen shared epitope alleles 102,103 .…”