A photoaffinity glycan labeling approach to investigate immunoglobulin glycan binding partners.

Glycans play a pivotal role in biology. However, due to the low-affinity of glycan-protein interactions, many interaction pairs remain unknown. Two important glycoproteins involved in B cell biology are the B cell receptor (BCR) and its secreted counterpart, antibodies (Abs). It has been indicated that glycans expressed by these B cell-specific molecules can modulate immune activation via glycan-binding proteins (GBPs). In several autoimmune diseases, an increased prevalence of variable domain glycosylation of IgG autoantibodies has been observed. Especially, the hallmarking autoantibodies in rheumatoid arthritis (RA), anti-citrullinated protein antibodies (ACPA), carry a substantial amount of variable domain glycans (VDGs). The VDGs expressed by these autoantibodies are N-linked, complex-type, α2-6 sialylated and BCRs carrying VDGs have been hypothesized to promote selection of autoreactive B cells via interactions with GBPs. Here, we use the ACPA response as a prototype to study potential in solution and in situ BCR VDG interactors. We employed SiaDAz, a UV-activatable sialic acid analogue carrying a diazirine moiety that can form covalent bonds with proximal GPBs. We show, using oligosaccharide engineering that SiaDAz can be readily incorporated into VDGs of both Abs and BCRs. Our data show that Ab VDGs are able to interact with inhibitory receptor, CD22. Interestingly, although we did not detect this interaction on the cell surface, we captured CD79 β glycan-BCR interactions. These results show the utility of combining photoaffinity labeling and oligosaccharide engineering for identifying Ab and BCR interactions and indicate that VDGs appear not to be lectin cis ligands in our tested conditions.