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

PURPOSE: The objectives of this study were to assess the cartilage boundary lubricating ability of (1) nonreduced (NR) disulfide-bonded proteoglycan 4 (PRG4) multimers versus PRG4 monomers and (2) NR versus reduced and alkylated (R/A) PRG4 monomers and to assess (3) 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 characterized 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 visualize 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 intermolecular 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.