A cellular platform for the evaluation of the effect of O-glycan modification on B cell receptor signaling

Abstract The importance of N-glycosylation, a ubiquitous post-translational modification (PTM) of cell-surface and secreted proteins in vertebrates, in the regulation of B cell receptor (BCR) signaling has been well documented. On the other hand, the importance of O-glycosylation, another type of ubiquitous PTM, in B cell function is getting recognized only recently. A simplified system to study the effects of O-glycan modifications in BCR signaling would facilitate the efforts to understand the mechanism of B cell regulation by O-glycans. To this end, we reconstituted BCR components and CD45 with orthogonal self-labeling protein tags in the mouse myeloma J558L cell line, which is deficient in the components of BCR and several co-receptors. The enzymes that modify O-glycans were further introduced, and the cells were subjected to imaging-based quantitative analysis of the distance between BCR and CD45, as well as the evaluation of BCR signaling by Western blotting. Gene expression profiles among the cells were also compared by RNA sequencing. Our study revealed that core 2 branching of O-glycans has minimal effect on the distance between BCR and CD45, whereas the addition of an α2–8-linked sialic acid by ST8Sia-VI reduces the distance between them, facilitating the downstream BCR signaling. Our system may be adapted for the functional evaluation of other PTMs in BCR signaling. Supported by a grant from the Ministry of Science and Technology (MOST)/National Science and Technology Council (NSTC) of Taiwan (MOST 109-2311-B-001-009-MY3)