Author contributions: SA planned and executed the glycomic analysis, KT and LA planned and executed the glycoproteomic analysis, SH performed ELISA analysis, SR performed SPR analysis, YM and JH designed the recombinant expression and experiments executed by YM. OR, LB, RK, TS and TE were responsible for control and patient sample collection. TS and GJ contributed with design and provision of rhPRG4 for the analysis, MS and AK participated in planning and supervision of galectin-3 experiments. SF, KT and NK performed the writing with input from all contributors. NK coordinated and directed the research. All authors signed off on the final version.ABSTRACT Synovial fluid lubricin (proteoglycan 4) is a mucin-type O-linked glycosylated (60% of the mass) biological lubricant involved in osteoarthritis (OA) development. Lubricin has been reported to be cross-linked by synovial galectin-3 on the lubricating articular surface. Here, we confirm that binding to galectin-3 depended on core-2 O-linked glycans, where surface plasmon resonance of a recombinant lubricin (rhPRG4) devoid of core-2 structures lacked binding capacity to recombinant galectin-3. Both galectin-3 levels and interactions with synovial lubricin were found to be decreased in late-stage OA patients coinciding with an increase of truncated and less sialylated core 1 O-glycans. These data suggest a defect cross-linking of surface active molecules in OA and provides novel insights into OA molecular pathology.