Convergent synthesis of a hexadecavalent heterobifunctional ABO blood group glycoconjugate.

Naturally occurring glycans are often found in a multivalent presentation. Cell surface receptors that recognize these displays may form clusters, which can lead to signalling or endocytosis. One of the challenges in generating synthetic displays of multivalent carbohydrates is providing high valency as well as access to heterofunctional conjugates to allow attachment of multiple antigens or payloads. We designed a strategy based on a set of bifunctional linkers to generate a heterobifunctional multivalent display of two carbohydrate antigens to bind BCR and CD22 with four and twelve antigen copies, respectively. We confirmed that the conjugates were able to engage both CD22 and BCR on cells by observing receptor clustering. The strategy is modular and would allow for alternative carbohydrate antigens to be attached bearing amine and alkyne groups and should be of interest for the development of immunomodulators and vaccines.

NEU1 and NEU3 enzymes alter CD22 organization on B cells.

The B cell membrane expresses sialic-acid-binding immunoglobulin-like lectins, also called Siglecs, that are important for modulating immune response. Siglecs have interactions with sialoglycoproteins found on the same membrane (cis-ligands) that result in homotypic and heterotypic receptor clusters. The regulation and organization of these clusters, and their effect on cell activation, is not clearly understood. We investigated the role of human neuraminidase enzymes NEU1 and NEU3 on the clustering of CD22 on B cells using confocal microscopy. We observed that native NEU1 and NEU3 activity influence the cluster size of CD22. Using single-particle tracking, we observed that NEU3 activity increased the lateral mobility of CD22, which was in contrast to the effect of exogenous bacterial NEU enzymes. Moreover, we show that native NEU1 and NEU3 activity influenced cellular Ca2+ levels, supporting a role for these enzymes in regulating B cell activation. Our results establish a role for native NEU activity in modulating CD22 organization and function on B cells.