Glycans play a pivotal role in biology. However, because of the low-affinity of glycan-protein interactions, many interaction pairs remain unknown. Two important glycoproteins involved in B-cell... Show moreGlycans play a pivotal role in biology. However, because of 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 and its secreted counterpart, antibodies. It has been indicated that glycans expressed by these B-cell-specific molecules can modulate immune activation via glycan-binding proteins. In several autoimmune diseases, an increased prevalence of variable domain glycosylation of IgG autoantibodies has been observed. Especially, the hallmarking autoantibodies in rheumatoid arthritis, anti-citrullinated protein antibodies, carry a substantial amount of variable domain glycans. The variable domain glycans expressed by these autoantibodies are N-linked, complex-type, and alpha 2-6 sialylated, and B-cell receptors carrying variable domain glycans have been hypothesized to promote selection of autoreactive B cells via interactions with glycan-binding proteins. Here, we use the anti-citrullinated protein antibody response as a prototype to study potential in solution and in situ B-cell receptor-variable domain glycan interactors. We employed SiaDAz, a UV-activatable sialic acid analog carrying a diazirine moiety that can form covalent bonds with proximal glycan-binding proteins. We show, using oligosaccharide engineering, that SiaDAz can be readily incorporated into variable domain glycans of both antibodies and B-cell receptors. Our data show that antibody variable domain glycans are able to interact with inhibitory receptor, CD22. Interestingly, although we did not detect this interaction on the cell surface, we captured CD79 beta glycan-B-cell receptor interactions. These results show the utility of combining photoaffinity labeling and oligosaccharide engineering for identifying antibody and B-cell receptor interactions and indicate that variable domain glycans appear not to be lectin cis ligands in our tested conditions. Show less
Multivalent scaffolds that carry multiple molecules with immunophenotyping or immunomodulatory properties areinvaluable tools for studying and modulating specific functions ofhuman immune responses... Show moreMultivalent scaffolds that carry multiple molecules with immunophenotyping or immunomodulatory properties areinvaluable tools for studying and modulating specific functions ofhuman immune responses. So far, streptavidin-biotin-basedtetramers have been widely used for B-cell immunophenotypingpurposes. However, the utility of these tetramers is limited by theirtetravalency, the inherent immunogenicity of streptavidin (abacterial protein that can potentially be recognized by B cells),and the limited feasibility to functionalize these reagents. This has rendered tetramers suboptimal for studying rare, in particular,antigen-specific B-cell populations in the context of clinical applications. Here, we used polyisocyanopeptides (PICs), multivalentpolymeric scaffolds functionalized with around 50 peptide antigens, to detect autoreactive B cells in the peripheral blood of patientswith rheumatoid arthritis. To explore the potential immunomodulatory functionalities, we functionalized PICs with autoantigenicpeptides and a trisaccharide CD22 ligand to inhibit autoreactive B-cell activation through interference with the B-cell receptoractivation pathway, as evidenced by reduced phospho-Syk expression upon PIC binding. Given the possibilities to functionalizePICs, our data demonstrate that the modular and versatile character of PIC scaffolds makes them promising candidates for futureclinical applications in B-cell-mediated diseases Show less
