Purpose
The mechanism for increased resistance to aqueous humor outflow at the trabecular meshwork (TM) in patients with primary open angle glaucoma is currently unknown. Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein known to regulate extracellular matrix (ECM) turnover in many tissues and is highly expressed in the TM. Overexpression of SPARC in human TM cells results in increased levels of ECM proteins without an increase in mRNA. We hypothesize that SPARC regulates ECM equilibrium in the TM by acting as a chaperone to stabilize ECM proteins.
Methods
Matrix metalloproteinase (MMP) digestion assays were performed for collagens (COLs) in the presence or absence of SPARC. The assays were performed using a 2.0 mM calcium buffer in a native condition, an oxidative denaturing condition (H2O2), and a thermal denaturing condition (45°C for 1hr). The chaperone activity of SPARC was compared to two controls: H2O and bovine serum albumin (BSA), a weak chaperone. Final reaction concentrations were 2.5 ng/μL COL, 300 μM H2O2, 1.5 μM SPARC, 1.5 μM BSA, and 1 ng/μL MMP. All reactions were performed at 37°C for 6 hours. Immunoblot analysis was performed to analyze the amount of COL for each reaction.
Results
1.5 μM SPARC led to increased recovery of COL 1 following MMP-9 mediated degradation (Fig. 1). Under native condition, the relative amount of COL 1 protein retained was 0.05 ± 0.04 for H2O, 0.16 ± 0.05 for BSA, and 0.69 ± 0.07 for SPARC, (p= 0.001, n=3, H2O vs. SPARC), (p=0.004, n=3, BSA vs. SPARC). Under oxidative stress, the amount of COL 1 retained was 0.05 ± 0.01 for H2O, 0.20 ± 0.04 for BSA, and 0.39 ± 0.08 for SPARC, (p=0.013, n=3, H2O vs. SPARC), (p=0.099, n=3, BSA vs. SPARC). No significant change in COL 1 was found under heat denaturation. SPARC led to increased recovery of COL 1 following MMP-2 mediated degradation (Fig. 2). Under native condition, the amount of COL 1 protein retained was 0.72 ± 0.08 for H2O, 0.61 ± 0.04 for BSA, and 1.08 ± 0.00 for SPARC, (p= 0.040, n=2, H2O vs. SPARC), (p=0.005, n=2, BSA vs. SPARC). No significant change in COL 1 was found under oxidative and heat denaturing conditions.
Conclusions
Our results suggest that SPARC acts as a chaperone for MMP-2 and MMP-9 mediated COL 1 digestion under native conditions. We will investigate SPARC’s possible chaperone activity for other ECM proteins to gain further insight in to how SPARC regulates ECM equilibrium.