Osteopontin O-glycosylation contributes to its phosphorylation and cell-adhesion properties

OPN (osteopontin) is a multiphosphorylated extracellular glycoprotein, which has important roles in bone remodelling, inflammation and cancer metastasis. OPN regulates cell spreading and adhesion primarily through its association with several integrins such as alpha v beta 3, and its phosphorylation affects these processes. However, the mechanism by which OPN O-glycosylation affects these processes is not completely understood. In the present study, we demonstrated that OPN O-glycosylation self-regulates its biological activities and also affects its phosphorylation status. We prepared two recombinant OPNs, WT (wild-type)-OPN and mutant OPN (Delta O-OPN), which lacks five O-glycosylation sites at a threonine/proline-rich region. O-glycan defects in OPN increased its phosphorylation level, as observed by dephosphorylation assays. Moreover, compared with WT-OPN, Delta O-OPN exhibited enhanced cell spreading and adhesion activities and decreased associations with beta 1 integrins. This suggested that defects in O-glycans in OPN altered these activities, and that beta 1. integrins have a less important role in adhesion to Delta O-OPN. The cell-adhesion activity of dephosphorylated Delta O-OPN was higher than the cell-adhesion activities of Delta O-OPN and dephosphorylated WT-OPN. This suggested that some of the phosphorylation in Delta O-OPN caused by O-glycan defects and O-glycans of OPN suppressed the OPN cell-adhesion activity. Thus functional activities of OPN can be determined by the combined glycosylation and phosphorylation statuses and not by either status alone.