Background
Hyaluronan (HA) is a vital macromolecular component of synovial fluid (SF) with several important functions. HA is a negatively charged biopolymer composed of alternating D-glucuronic acid and N-acetylglucosamine that forms dynamic networks in solution [
1]. HA exists in SF at molecular weights (MW) between 0.2 to 6 MDa, and concentrations of 1–4 mg/ml [
2,
3]. A major role of HA in SF is to impart fluid viscosity and elasticity to help transfer loads across the cartilage within the articulating joint. HA has also been shown to effectively reduce friction in dose-dependent manner at a cartilage-cartilage biointerface under boundary mode lubrication [
4,
5].
Proteoglycan 4 (PRG4) is a mucin like glycoprotein, with extensive O-linked glycosylation and an apparent MW of ~ 460 kDa. It is also present in SF and covers the surface of articulating cartilage [
6,
7]. PRG4 is a flexible rod ~200 nm in length and 1-2 nm in width, and its hydrodynamic diameter as measured by light scattering has been reported to be ~ 200 nm as well [
8,
9]. 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,
11,
12]. PRG4 is capable of dimerization via intermolecular disulfide bonds and exists in SF in both monomeric and dimeric forms [
13]. Reduction and alkylation (R/A) of PRG4, causing disruption of intra and inter molecular disulfide bonds, has been shown to reduce multimers into monomers and release small fragments from the PRG4 structure (~ 70 kDa) [
13]. This results in a significant reduction in binding of PRG4 to the surface of articular cartilage and an associated reduction in its cartilage boundary lubrication [
7,
12,
14].
PRG4 and HA function synergistically as lubricants at the cartilage surface, and possibly in solution within SF. When combined in solution at physiological concentrations PRG4 and HA reduce friction at a cartilage-cartilage biointerface under boundary lubrication to lower levels than either alone, approaching the lubrication of healthy SF [
4,
5,
15]. Additionally PRG4 has been shown to enhance/alter the viscosity of HA solutions [
16]. This functional synergism has been demonstrated with various MW HA without any significant variation in lubricating ability [
5]. This synergism suggests a functional interaction between HA and PRG4 at the cartilage surface, and possibly in solution as well. However interactions of macromolecules may not be the same at a surface and in solution.
Previous studies have attempted to elucidate the mode of interaction of PRG4 and HA in solution [
2,
5]. An electrophoretic mobility shift assay provided evidence of a weak PRG4 + HA interaction [
5]. Additionally, a multiple-particle-tracking microrheology technique has been used to study the effect PRG4 has on the biophysical properties of SF (a semi dilute HA solution) and provide evidence for an interaction in solution [
2]. Experimentation performed on healthy as well as PRG4-deficient SF suggested that PRG4 creates a network of “entanglements” within HA-containing SF, resulting in an increased relaxation time for SF [
2]. However, the specific mechanism and concentration dependence of this interaction has yet to be determined. A more detailed understanding of HA and PRG4 interaction in solution could help explain the molecular basis of the biophysical properties of normal and pathological SF. Accordingly, an experimental technique which allows us to probe the biophysical properties of complex HA and PRG4 solutions would be valuable.
Confocal fluorescence recovery after photobleaching (FRAP) is a microscopic technique that has been used to investigate solution properties and molecular networks of HA. Confocal FRAP provides a powerful tool for studying concentrated and complex polymer solutions in the absence of shear stress [
17]. Gribbon et al. [
18] used FRAP to determine how electrolyte concentration and pH effect the hydrogen and electrostatic intramolecular bonds within the repeat sugar subunits of HA. The resulting change in intramolecular bonding was shown to change the stiffness, and contraction of the HA molecules, and thus network formation. Due to confocal FRAP’s ability to reveal details about the structure HA networks it is an ideal method to evaluate the specific and dose-dependent effect PRG4 has on HA solution properties.
The objectives of this study were to 1) use confocal FRAP to examine the specific and dose-dependent effect of PRG4 on HA solution networks by analyzing the diffusion of a fluorescein isothiocyanate (FITC)-dextran tracer through HA solutions of different MW, and 2) assess the effect of an altered tertiary/quaternary PRG4 structure, through R/A, on the observed effects on HA solutions at different concentrations.
Discussion
These results demonstrate that PRG4, at physiological concentrations, can significantly alter the solution properties of 1500 and 500 kDa HA; PRG4 significantly decreased the tracer diffusion at all HA concentrations tested here. The physical implication of this finding was characterized by a decreased in the apparent mesh size distribution for each mixture of HA, calculated from the empirical constants (β and ν) from the universal scaling equations successfully fit to the concentration series of HA. This effect was specific to PRG4 and was not observed with BSA, indicating it was not a result of a protein simply being in solution with HA. Interestingly, the reduced permeability observed appeared similar in magnitude for both 450 and 45 μg/ml, and was also not dependent on its tertiary/quaternary structure as the effect remained after R/A of PRG4. As PRG4 and HA are key SF macromolecular constituents that play functional roles in various SF properties (e.g. viscosity, lubrication, solution meshwork), collectively these findings contribute to the understanding of their interaction(s) in solution as well as the function of SF in diarthroidal joints.
The HA and PRG4 used in this study are representative of those in native SF and have been used in other studies. Both the HA MW and concentration range used is relevant to physiological and pathological conditions [
10,
22]. The MW of HA used was found to be polydisperse, which may explain the lack of observed difference in tracer diffusion between the 1500 and 500 kDa HA. Due to the unique properties of PRG4, it was difficult to choose the perfect protein control for the tracer diffusion studies. BSA, whose hydrodynamic diameter is ~ 7 nm, was chosen as a practical and relevant protein control that would not interact with HA at the pH employed here, and as it is abundant within SF [
23]. Future work could potentially address this limitation by examining other (glyco)protein proteins as potential controls proteins that do not interact with HA. Measurements for D
t were shown to have a significant variance between experimental sets, with a standard deviation between measured D
t values of 1.4 × 10
−8cm2 s−1. As such, to reduce and control for the effect of the variations between prepared samples, all comparisons of experimental sets (e.g. one replicate of HA vs. HA+ 450 μg/ml PRG4) were performed on the same day from the same prepared HA solutions. Thus, while the day to day variance between experiments can give a sizable variance between identical measurements made on separate days, all analysis of HA vs HA with an additive (PRG4 or BSA) were made between identical sample preparations. The mean calculated diffusion coefficient from all data points (from all experiments) along with the standard error of the mean for the tracer through 1500 kDa HA at 0, 0.1, 0.3, 1.0 and 3.3 mg/ml were 19.29 ± 0.39, 19.57 ± 0.80, 18.27 ± 0.68, 15.12 ± 0.60, 11.51 ± 0.63 × 10
−8cm2 s−1, respectively. Those for 500kD HA at 0, 0.1, 0.3, 1.0 and 3.3 mg/ml were 19.41 ± 0.48, 19.05 ± 0.36, 18.27 ± 0.38, 16.18 ± 0.73, 11.81 ± 0.82 × 10
−8cm2 s−1, respectively. We did not normalize our measurements, so we could accurately show the day to day variance between experiments. Another limitation of the proposed study was the assumption of a strictly 2D diffusing system. This 2D assumption has been shown to be appropriate when using a NA lens, and a large confocal aperture setting [
17]. Additionally, any diffusion within the Z plane would be consistent between measurements.
The results from this study agree with those from Gribbon et al. [
18], and extend them to include the effect of PRG4. While the results presented here show the same negative exponential trend in tracer diffusion constants in relation to HA concentration, the reported D
t0 of the 2000 kDa FITC-dextran tracer are slightly higher. This could be due variations in data analysis and FRAP parameters (e.g. higher bleaching times, different objective lens), but nevertheless the results from this study are in good agreement with previous research [
18,
24,
25]. Furthermore, the predicted trend in tracer diffusion through increasing HA concentration was observed here and the results demonstrate a clear and specific effect of PRG4. Lastly, while future studies could potentially examine direct measurement of mesh size in HA solutions, those calculated here are in good agreement with those previously reported by Gribbon et al. [
18]
The mechanism of the PRG4 + HA interaction in solution observed here, and in other studies [
2,
5], remains to be completely elucidated. It has been speculated to be one of physical interaction involving non-covalent entanglements, [
2] potentially mediated through the hemopexin like and somatomedin B like domains on the C- and N-terminus, [
12,
26] respectively, of PRG4. Indeed, hemopexin is able to bind to HA, supporting the plausible mechanism for PRG4 entangling HA molecules through this domain [
26]. This highly entangled HA matrix is therefore a conceivable explanation for the observed decrease in tracer diffusion and smaller observed mesh sizes when PRG4 is present. The similar effect observed between native PRG4 and R/A PRG4 was somewhat unexpected since if PRG4 interacts with HA through its globular domains, they would be unfolded by R/A. A potential explanation for this is while PRG4 no longer makes entanglements with HA, it is capable of producing its own solution networks or gels independent (which is well documented in mucins [
27]). It would then be these PRG4 networks within the space not occupied by HA, and not entanglements with HA, which cause the denser less permeable networks thus is impeding the diffusion of the FITC-dextran tracers. Indeed, R/A can cause the protein end units of mucins to unfold and expose large hydrophobic domains resulting in aggregation into dense networks [
28]. A recent study demonstrating PRG4, but not R/A PRG4, can enhance the viscosity of HA solutions is also consistent with the above interpretation [
16]. The hydrodynamic radius of R/A PRG4 has not been reported, however the elution time on a size exclusion column remained similar compared to non-reduced PRG4 suggesting the two have similar hydrodynamic radii [
11]. Unfortunately the relative ratio of multimers/monomers in a PRG4 solution has not been quantified, but based on size exclusion chromatographs [
7] it seems reasonable to assume they are present in similar (order of magnitude) quantifies. The effect of concentration on this ratio is currently unknown as well. Future studies are required, likely with more than one technique, to further define and validate the model where PRG4 interactions with HA through the hemopexin and somatomedin B domains, entangling HA molecules and creating a tighter and solution matrix with altered rheological properties.