Mucin-type <i>O</i> -glycans regulate proteoglycan stability and chondrocyte maturation

<i>O</i>-glycosylation is a ubiquitous post-translational modification essential for protein stability, cell signaling, and tissue organization, yet how distinct <i>O</i>-glycan subclasses coordinate tissue development remains unclear. Here, we identify functional crosstalk between extended mucin-type <i>O</i>-glycans and heparan sulfate proteoglycans (HSPGs). Genetic ablation of β-1,3-galactosyltransferase 1 (C1GALT1) or its chaperone COSMC in human chondrocytes reduced cell surface HSPGs and fibroblast growth factor (FGF) binding, leading to impaired MAPK/ERK signaling, which was recapitulated in hypomorphic <i>COSMC-CDG</i> patient cells. Transcriptomic and secretome analyses revealed selective loss of proteoglycan expression and broader extracellular matrix remodeling in COSMC- and C1GALT1-deficient cells. Mechanistically, truncation of <i>O</i>-GalNAc glycans reduced Syndecan-1 levels at the cell surface via enhancing its lysosome-dependent degradation and diminished CD44v3-mediated FGF1 binding. Functionally, loss of complex <i>O</i>-GalNAc glycans disrupted chondrogenesis of growth plate-like chondroprogenitors. Overall, these findings reveal previously unrecognized roles for mucin-type <i>O</i>-glycans in maintaining proteoglycan stability and function, highlighting cross-regulatory mechanisms of glycosylation crucial for normal development that likely contribute to the pathophysiology of glycosylation-related disorders.