Biophysical characterization of recombinant human Proteoglycan 4 (rhPRG4): Effect of Mg²⁺ and high pH

Abstract

Purpose : Proteoglycan 4 (PRG4, or lubricin) is a mucin-like glycoprotein present on the ocular surface and in tears, with both lubricating and anti-inflammatory properties. Biological processes occur in solution; therefore, studying proteins’ biophysical properties in solution contribute to understanding their function. Here, we biophysically characterize recombinant human PRG4 (rhPRG4), previously shown to be clinically effective in reducing signs and symptoms of dry eye, and determine the effect of the cation Mg²⁺ and high pH on rhPRG4 size in solution.

Methods : rhPRG4 (Lubris BioPharma) at pH7, and in some cases native human PRG4, was assessed in solution for hydrodynamic diameter (Dh, by dynamic light scattering), charge (zeta potential), structure (circular dichroism, CD), density (analytic ultracentrifugation, AUC), and molecular fingerprint (raman spectroscopy). The kinetics of rhPRG4 binding to type 1 Collagen (Col1) was assessed (quartz crystal microbalance). Finally, the effects of 1-5mM MgCl₂ or pH9-10 on Dh was assessed.

Results : rhPRG4 had a main (~85%) peak at Dh=~170nm (with a minor at ~30nm, z-avg Dh=~110nm) and a -21mV zeta potential. Native PRG4 Dh and charge were similar to rhPRG4. CD indicated rhPRG4 had a main extended structure peak ~202nm and no discernible tryptophan peak at ~290nm. AUC indicated a dominant species at ~4S. Raman spectroscopy showed the ratio of normalized (to 891cm-1, C-C) peaks at 980cm^-1 and at 550cm^-1, associated with β-sheets and disulfide bonds respectively, was ~2.9. rhPRG4 rapidly bound to Col1, with a t~8sec. MgCl₂ caused a decrease of the main Dh peak in a dose dependent manner, reducing to ~140nm at 5mM. Finally, pH9 caused a decrease in the main peak and an emergence of an intermediate peak at ~120nm, and pH10 even more so.

Conclusions : The clinically effective rhPRG4 assessed here had similar size and charge to native PRG4, and quickly adhered to Col1 (which is present on the ocular surface). The size, charge, and density for rhPRG4 are larger than those published for a different version of recombinant human lubricin (ECF843: Dh=~60nm, ~0mV, and a more compact 6.5S major peak), which recently did not produce supportive data in a dry eye clinical trial. Reduced Dh of rhPRG4 caused by Mg²⁺ and pH9-10 may be due to osmotic deswelling of the mucin domain and disulfide bond reshuffling, respectively, which are likely to affect function as well.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.