Abstract
BackgroundThe objective of this study was to use confocal fluorescence recovery after photobleaching (FRAP) to examine the specific and dose-dependent effect of proteoglycan 4 (PRG4) on hyaluronan (HA) solutions of different molecular weight; and assess the effect of reduction and alkylation (R/A) of PRG4 on its effects on HA solutions.MethodsConfocal FRAP was used to determine the diffusion coefficient of fluorescein isothiocyanate (FITC)-dextran tracer (Dt) through 1500 kDa and 500 kDa HA solutions (0–3.3 mg/ml) ± PRG4 or a control protein, bovine serum albumin (BSA), at physiological (450 μg/ml) or pathophysiological (45 μg/ml) concentrations. The effect of PRG4 or R/A PRG4 on 1500 kDa HA solutions was also investigated. Empirical constants obtained from fitting data to the universal scaling equation were used to calculate the average distribution of apparent mesh sizes.ResultsPRG4 at both 45 and 450 μg/ml slowed the diffusion of the FITC-dextran tracer for all concentrations of HA and caused a decrease in the apparent mesh size within the HA solution. This effect was specific to PRG4, not observed with BSA, but not dependent on its tertiary/quaternary structure as the effect remained after R/A of PRG4.ConclusionsThese results demonstrate that PRG4 can significantly alter the solution properties of HA; PRG4 essentially reduced the permeability of the HA network. This effect may be due to PRG4 entangling HA molecules through binding and/or HA crowding PRG4 molecules into a self-assembled network. Collectively these findings contribute to the understanding of PRG4 and HA interaction(s) in solution and therefore the function of SF in diarthroidal joints.
Highlights
The objective of this study was to use confocal fluorescence recovery after photobleaching (FRAP) to examine the specific and dose-dependent effect of proteoglycan 4 (PRG4) on hyaluronan (HA) solutions of different molecular weight; and assess the effect of reduction and alkylation (R/A) of PRG4 on its effects on HA solutions
HA ± PRG4 PRG4 at 450 μg/ml slowed the diffusion of the fluorescein isothiocyanate (FITC)-dextran tracer for all concentrations of HA and had an effect on the HA solution network
The addition of PRG4 at 450 μg/ml caused an average decrease in dextran tracer (Dt) of 1.592 × 10−8cm2 s−1, in 1500 kDa HA, and 1.068 × 10−8cm2 s−1, in 500 kDa HA, both of which were significant (p < 0.05)
Summary
The objective of this study was to use confocal fluorescence recovery after photobleaching (FRAP) to examine the specific and dose-dependent effect of proteoglycan 4 (PRG4) on hyaluronan (HA) solutions of different molecular weight; and assess the effect of reduction and alkylation (R/A) of PRG4 on its effects on HA solutions. Proteoglycan 4 (PRG4) is a mucin like glycoprotein, with extensive O-linked glycosylation and an apparent MW of ~ 460 kDa. Proteoglycan 4 (PRG4) is a mucin like glycoprotein, with extensive O-linked glycosylation and an apparent MW of ~ 460 kDa It is present in SF and covers the surface of articulating cartilage [6, 7]. PRG4 has been reported at an average concentration of 287 +/− 31.8 μg/ml in healthy human SF, though it can vary from 129 to 450 μg/ml [10]. PRG4 effectively reduces friction in a dose-dependent manner at a cartilage-cartilage biointerface under boundary lubrication, [4] as well as at cartilage-glass and latex-glass surfaces [6, Bloom et al BMC Musculoskeletal Disorders (2019) 20:93
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