Abstract

Given the pivotal role of the Eph-Ephrin signaling pathway in tumor progression, agonists or antagonists targeting Eph/Ephrin have emerged as promising anticancer strategies. However, the implications of glycosylation modifications within Eph/Ephrin and their targeted protein therapeutics remain elusive. Here, we identify that N-glycosylation within the receptor-binding domain (RBD) of ephrin B1 (EFNB1) is indispensable for its functional repertoire. Notably, compared to wild-type EFNB1, the glycosylation-deficient N139D mutant drastically diminishes the sensitivity of tumor cells to chemotherapeutic agents, suggesting the existence of both glycosylation-dependent and independent effects mediated by EFNB1. Transcriptomic analysis highlights immune response and oxidative phosphorylation as the primary signaling pathways modulated by glycosylation modifications. In coculture systems, the EFNB1-RBD-Fc recombinant protein, while inhibiting B-lymphoma cells, also exerts differential impacts on stromal cells depending on their glycosylation status. Furthermore, the efficacy of both glycosylated and non-glycosylated EFNB1-RBD-Fc is influenced by the endogenous EFNB1 levels within tumor cells. Taking together, this study demonstrates the complexity and multifaceted roles of glycosylation in modulating EFNB1 function. These findings underscore the need for a nuanced understanding of glycosylation patterns in Eph/Ephrin-targeted therapies to optimize their therapeutic potential.