Characterization of Recombinant Human PRG4 as an Ocular Surface Boundary Lubricant

Authors

  • N. Masala University of Calgary
  • S. Morrison University of Calgary
  • M. Samsom University of Calgary
  • H. Sheardown McMaster University
  • T. Schmidt University of Calgary

Keywords:

PRG4, Ocular Surface Boundary Lubricant, Dry-eye disease

Abstract

Introduction: Dry-eye disease involves tear film instability that can result in surface-to-surface contact between the cornea and eyelid or contact lens, where boundary lubrication can be dominant1. Motivated by the recent discovery that proteoglycan 4 (PRG4, a mucin-like glycoprotein originally discovered in synovial fluid as a boundary lubricant2), functions as an ocular surface boundary lubricant3, advances in recombinant protein expression technology4, and PRG4’s potential use as a friction-reducing contact lens coating, the objectives of this study were to: 1) biochemically characterize recombinant human PRG4 (rh- PRG4); and 2) assess the boundary lubricating properties of rh-PRG4, both before and after autoclave sterilization, at a cornea-contact lens material (PDMS) biointerface.

Methods: SDS-PAGE western blot analysis using a variety of anti-PRG4 antibodies and lectins was performed on native PRG4 (nPRG4) and rh-PRG4 samples, both nonreduced and reduced, with and without enzymatic removal of O-linked glycosylations. Human corneas and PDMS were articulated against each other, subject to physiological loads of 8-25 kPa, at effective sliding velocities of 0.3-30 mm/s. Test lubricant sequences were A) saline, rh-PRG4 @300μg/mL, nPRG4 @300μg/mL, and saline; and B) saline, autoclaved rh-PRG4 @300μg/mL, rh-PRG4 @300μg/mL, and saline. Static and kinetic coefficients of friction were calculated.

Results: rh-PRG4 demonstrated similar immunoreactivity to nPRG4, and effectively lowered friction at the cornea-PDMS biointerface. Western blotting indicated immunoreactive rh-PRG4 bands had a similar apparent molecular weight (MW) to nPRG4, and decreased appropriately upon reduction as well as enzymatic removal of glycosylations. Kinetic friction coefficients, which were highest in saline (0.31±0.06 to 0.40±0.06, mean±SEM), were similar in rh-PRG4 (0.12±0.01 to 0.25±0.03) and nPRG4 (0.19±0.02 to 0.28±0.03) across all velocities. Autoclaved rh-PRG4 had similar values to rh-PRG4 as well (0.19±0.02 to 0.26±0.04, 0.16±0.02 to 0.26±0.02, respectively).

Conclusions: rh-PRG4 demonstrates similar biochemical and ocular surface lubricating properties to nPRG4, and may function as an effective friction-reducing contact lens coating.

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References

1. Holly FJ and Holly TF. “Advances in Ocular Tribology” in Lacrimal Gland, Tear Film, and Dry Eye Syndromes. Sullivan DA, Ed. New York: Plenum Press, 1994, pp. 275-283.

2. Swann DA, Slayter HS, Silver FH, “The molecular structure of lubricating glycoprotein-I, the boundary lubricant for articular cartilage”, J Biol Chem, vol. 256, no. 11, 1981. pp. 5921-5925.

3. Morrison S, Sullivan DA, Sullivan BD, Sheardown H, Schmidt TA, “Dose-dependent & synergistic effects of proteoglycan 4 (PRG4) on boundary lubrication at a human cornea – contact lens biointerface”, Eye & Contact Lens (in revision).

4. Girod P, Nguyen D, Calabrese D, Puttini S, Grandjean M, Martinet D, Regamey A, Saugy D, Beckmann JS, Bucher P, Mermod N, “Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells”, Nature Methods, vol. 4, no. 9, 2007. pp. 747-753.

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Published

2011-10-14

Issue

Vol. 1 No. 1 (2011)

Section

Articles

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