Decrease of core 2 O-glycans on synovial lubricin in osteoarthritis reduces galectin-3 mediated crosslinking

Abstract

The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin’s ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens, core-1 and core-2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core-2 O-linked glycans mediate this lubricin-galectin 3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core-2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary (CHO) cells with the core 2 GlcNAc transferase acting on a mucin type O-glycoprotein displayed increased galectin-3 binding. Both the level of galectin-3 and the galectin-3 interactions with synovial lubricin were found to be decreased in late-stage OA patients, coinciding with an increase in truncated and less sialylated core 1 O-glycans and Tn-antigens. These data suggest a defect in cross-linking of surface-active molecules in OA and provides novel insights into OA molecular pathology.

The level of synovial human VEGFA, IL-8 and MIP-1α correlate with truncation of lubricin glycans in osteoarthritis

“…Using ELISA on SF from 29 OA and 19 non-OA control we could infer that SF lubricin on our OA patient cohort contained significant more Tn antigens compared to T-antigens (Figure 6B). This showed that the oligosaccharides on OA lubricin are shorter than the controls, in line with previous publication(13).…”

“…TNF-α as well as IL-6 have been shown to up-regulate Tn levels in gingival fibroblasts, probably by downregulation of the COSMC chaperon required for activation of the core-1 galactosyl transferase that allows O -linked glycans to be elongated. This latest example would provide a the type of truncation of the glycosylation profile consistent with what has been observed in on OA lubricin in this report and previous(13). MIP-1α is a chemokine with limited information about its association with OA.…”

“…Hence, synovial cells has to be able to regulate its own cytokine/chemokine environment, and as such, crosstalk between glycosylation of lubricin and cytokines/chemokines is regulated localy in the joint. In this paper and in a previous paper(13) we showed that O -linked glycans are significantly shortened on synovial lubricin in late stage OA, increasing the number of exposed Tn-antigens. This exposure would allow OA synovial lubricin to establish different type of interaction with synovial cells.…”

“…Since our data suggested that lubricin glycans became truncated in OA, displaying more Tn-antigens, we set out to test the hypothesis that lubricin with truncated glycans are able to stimulate cytokine expression in cells from the synovial area. We generated glycoforms of lubricin by treating recombinant lubricin containing sialylated core-1 structures(13) using sialidase and/or β-galactosidase. Treating FLS cells isolated from healthy individuals and OA patients with these lubricin glycovariants, we could detect that the cytokines that were identified correlating with increased truncation(MIP-1α, MCP-1, Il-8,VEGFA, and MDC) were effected (Figure 7G-L).…”

“…O -linked glycosylation changes due to inflammation are less frequently reported; in ulcerative colitis, sialyl Tn expression is upregulated(11) and pulmonary inflammation is associated with decreased expression of blood group antigens, altered sulfation, a switch of core types and increased expression of sialyl Lewis x(12). We have identified that O -linked glycans in OA become less sialylated and are more truncated with increased level of T- and Tn-antigens(13).…”