Effect of disulfide bonding and multimerization on proteoglycan 4’s cartilage boundary lubricating ability and adsorption

Abstract

Purpose The objectives of this study were to assess the cartilage boundary lubricating ability of (1) non reduced (NR) disulfide-bonded proteoglycan 4 (PRG4) multimers versus PRG4 monomers, (2) NR versus reduced and alkylated (R/A) PRG4 monomers, and (3) assess the ability of NR PRG4 multimers versus monomers to adsorb to an articular cartilage surface. Materials and Methods PRG4 was separated into two preparations, PRG4 multimer enriched (PRG4Multi+) and PRG4 multimer deficient (PRG4Multi−), using size exclusion chromatography (SEC) and characterised by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cartilage boundary lubricating ability of PRG4Multi+ and PRG4Multi− was compared at a physiological concentration (450 μg/mL) and assessed over a range of concentrations (45, 150 and 450 μg/mL). R/A and NR PRG4Multi− were evaluated at 450 μg/mL. Immunohistochemistry with anti-PRG4 antibody 4D6 was performed to visualise the adsorption of PRG4 preparations to the surface of articular cartilage explants. Results Separation into enriched populations of PRG4Multi+ and PRG4Multi− was achieved using SEC and was confirmed by SDS-PAGE. PRG4Multi+ and PRG4Multi− both functioned as effective friction-reducing cartilage boundary lubricants at 450 μg/mL; with PRG4Multi+ being more effective than PRG4Multi−. PRG4Multi+ lubricated in a dose-dependent manner, however PRG4Multi− did not. R/A PRG4Multi− lubricated similar to NR PRG4Multi−. PRG4 containing solutions showed 4D6 immunoreactivity at the articular surface; the immunoreactive intensity of PRG4Multi+ appeared to be similar to SF, whereas PRG4Multi− appeared to have less intensity. Conclusions These results demonstrate that the inter-molecular disulfide-bonded multimeric structure of PRG4 is important for its ability to adsorb to a cartilage surface and function as a boundary lubricant. These findings contribute to a greater understanding of the molecular basis of cartilage boundary lubrication of PRG4. Elucidating the PRG4 structure-lubrication function relationship will further contribute to the understanding of PRG4's role in diarthrodial joint homeostasis and disease.

Proteoglycan 4: From Mere Lubricant to Regulator of Tissue Homeostasis and Inflammation

“…As stated earlier, much of lubricating ability is thought to be driven PRG4's ability to bind to biological surfaces and through the abundance of negatively charged sugars in O ‐linked glycosylated mucin rich domain . This has been empirically demonstrated as alterations in glycosylation significantly reduce PRG4's lubricating ability . Systems wide comparison, using shotgun proteomics, of wild‐type versus Prg4 −/− mice in models of joint inflammation (e.g., collagen‐induced arthritis or DMM injury model) may reveal global protein differences altered by PRG4 expression.…”

“…However, it remains unknown if these different protein conformations of PRG4 (monomers or covalent complexes) have overlapping or distinct biological function. While in vitro studies have examined the importance of PRG4's higher order structure on its lubricating properties, additional in vivo experiments are needed to elucidate if the quaternary structures of PRG4 have physiological roles or only the monomer is sufficient. PRG4 dimers have been detected but a better characterization is needed and whether or not they impact lubrication or cell signaling will need to be demonstrated as well in vivo.…”

“…Hence, our knowledge of PRG4 function remains highly biased toward its mechanical roles, some of which are discussed here. PRG4 has been predominantly investigated for its lubricating functions as it is necessary for proper lubrication of articulating cartilage within joints . In the synovial joint, PRG4 is synthesized and secreted into the synovial fluid by chondrocytes and synovial fibroblasts acting primarily as a boundary lubricant between the articulating cartilage–cartilage surfaces of the joint .…”

“…Isoform lacking exon 3, which is believed to be critical to disulfide bond, has recently been reported and interaction of PRG4 with different cell surface receptors and extracellular components may be impaired due to alternative splicing. Tertiary and quaternary folding structures of PRG4 are necessary for its biological function although additional experiments are needed in in vivo to determine whether only the monomeric form or also the covalent forms of PRG4 play a physiological role . Isoforms lacking exon 4 or 5 shows an alteration in binding efficiency to heparin or the ECM .…”

Lubricin/proteoglycan 4 detected in vocal folds of humans and five other mammals

“…Initial tests showed faint staining unless antigen retrieval was used. Slides were blocked using 3% bovine serum albumin and incubated overnight at 4°C with mouse monoclonal antibodies: mAb 9G3 (Cat# MABT401; MilliporeSigma, Burlington, MA), anti‐PRG4 mAb 4D6 (noncommercial mAb provided by Phillip B. Messersmith, University of California, Berkeley, Berkeley, CA) or MilliporeSigma clone Ci4 IgG1 (Cat# MABC002) isotype control. The 9G3 antibody was used at dilutions ranging from 1:1000 to 1:2000.…”