Glycosylation Encodes Selective Galectin Recognition and Signaling by Osteopontin

Abstract Post-translational modifications critically regulate extracellular molecular recognition, yet their roles in intrinsically disordered proteins remain poorly defined. Here, we integrate glycoproteomics, NMR, and cellular assays to reveal how glycosylation shapes the interaction network of osteopontin (OPN), a highly glycosylated intrinsically disordered protein. We show that OPN displays a heterogeneous repertoire of predominantly sialylated O-glycans that drive selective lectin binding: galectin-3 preferentially binds terminal LacNAc-containing epitopes, whereas galectin-8 engages multiple sialylated and sulfated glycan motifs, promoting cross-linking of OPN. In contrast, binding to CD44 receptor is largely glycosylation-independent and is mediated primarily through the polypeptide backbone. This dual recognition mechanism enables OPN to act as a molecular scaffold that couples glycan-mediated galectin binding with CD44 receptor engagement. Consistent with this proposed mechanistic model, OPN/galectin complexes are recruited to CD44-expressing cells where glycosylated OPN suppresses galectin-8-induced apoptosis, whereas deglycosylated OPN does not. Together, our results reveal that glycosylation encodes interaction specificity and downstream signaling outcomes in an intrinsically disordered extracellular protein, establishing OPN as a paradigm for how glycans organize extracellular recognition networks.