Molecular chaperone function for myocilin

Abstract

Purpose: Myocilin is thought to be a stress response protein, but its exact molecular functions have not been established. Studies were conducted to see whether myocilin can act as a general molecular chaperone.

Methods: Myocilin was isolated and purified from porcine trabecular meshwork (TM) cell culture media. Its ability to protect citrate synthase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the restriction endonuclease DrdI from thermal inactivation was evaluated. Light scattering was used to evaluate thermally induced aggregation of citrate synthase. Myocilin induction was assessed after exposure of TM cells to several types of stress treatments.

Results: Levels of extracellular myocilin expressed by TM cells were increased in response to mechanical stretch, heat shock, TNFα, or IL-1α. Myocilin protected citrate synthase activity against thermal inactivation for 5 minutes at 55°C in a concentration-dependent manner, with nearly full protection of 1.5 μM citrate synthase in the presence of 650 nM myocilin. Myocilin significantly reduced thermal aggregation of citrate synthase to levels 36% to 44% of control levels. Myocilin also protected GAPDH from thermal inactivation for 10 minutes at 45°C. Myocilin at 18 nM was more effective than 1 μM bovine serum albumin at protecting DrdI from thermal inactivation.

Conclusions: Myocilin is induced in response to several cellular stresses and displays general molecular chaperone activity by protecting DrdI, citrate synthase, and GAPDH from thermal inactivation. Myocilin also suppresses the thermal aggregation of citrate synthase. One function of myocilin may be to serve as a molecular chaperone.

Figure 1.

Stress induction of myocilin. Myocilin levels in culture medium were analyzed by Western immunoblots. Porcine TM cells were treated by (A) unstretched compared with mechanically stretched control for 24 hours. (B) Control (37°C) compared with heat shock (44°C) incubation for 45 minutes, both followed by incubation at 37°C for 24 hours. (C) Treatment with 10 ng/mL TNFα compared with vehicle control for 48 hours. (D) Treatment with 25 ng/mL IL-1α compared with vehicle control for 48 hours. Data shown represent mean band densities of the 55-/57-kDa doublet, added together and plotted with SEM, where n = 3 to 5. The t-test significance is as indicated above each graph.

Figure 2.

Immunoaffinity-purified myocilin from porcine TM cell medium. (A) Western immunoblots were probed with rabbit anti-human myocilin polyclonal antibody comparing 40 μL of 10× concentrated media (lane 1) and 10 μL affinity-purified column eluent (lane 2). (B) Silver stain of SDS-PAGE of a similar affinity-purified column eluent. The positions of two molecular weight markers are shown on the left side of the gel. Arrows depict myocilin bands at approximately of 66 kDa and 55 to 57 kDa.

Figure 3.

HPLC purification of myocilin from porcine TM cell medium. (A) HPLC elution profile showing protein absorbance at 280 nm (asterisk) indicating the position of the myocilin peak. Step elution gradient for NaCl is as shown. (B) Gels of material (asterisk) peak are shown after silver staining (lane 1) or immunostaining with myocilin antibody (lane 2). Lane 3 shows molecular weight markers with kilodalton migration, as indicated. Arrow indicates dominant myocilin immunostaining band at approximately 55 kDa.

Figure 4.

Protection against thermal inactivation of citrate synthase activity. Solutions containing 1.50 μM citrate synthase and the indicated concentrations of either myocilin (A) or BSA (B) were heat inactivated at 55°C for 5 minutes before monitoring activity at 25°C for 20 minutes. One control (solid lines) was not heat inactivated, and another control (dashed line) was heat inactivated with no added myocilin or BSA. The data shown represent typical runs. (C) Solutions containing 1.5 μM citrate synthase were not heated (1.5 μM control) or were heated without additions (heat control) or with 0.65 μM myocilin, 60 μM BSA, 1.5 μM α-crystallin, or 0.043 mg/mL RP1 (the anti-myocilin antibody used for immunoaffinity purification of myocilin). (D) Solutions containing 0.5 μM citrate synthase were not heated (0.5 μM control) or were heated without additions (heat control) or 2.0 μM HSP90. Heat inactivation was at 48°C for 15 minutes before monitoring activity at 25°C for 20 minutes. (C, D) Mean values from experiments, where n ≥ 3. *P < 0.01 compared with heat control, as determined by one-way ANOVA with Dunnett's multiple comparison test.

Figure 5.

Protection of citrate synthase against heat-induced aggregation. Solutions containing 0.5 μM citrate synthase were heated for 5 minutes at 55°C with no addition (control), 0.5 μM BSA, 30 μM BSA, or 0.22 μM myocilin. Light scattering was then assessed using excitation and emission wavelengths of 488 nm. Means and standard errors are shown, where n ≥ 3. *P < 0.01 compared with heated control using one-way ANOVA with Dunnett's multiple comparison test.

Figure 6.

Protection of GAPDH activity against thermal inactivation. Solutions containing 0.1 μM GAPDH were not heated (control) or were heated for 10 minutes at 45°C (A) or 42°C (B) with no addition (labeled 45°C 10 minutes or 42°C 10 minutes) or with the addition of 0.4 μM HSP90, 0.2 μM myocilin, 0.2 μM α-crystallin, or 60 μM BSA, as indicated. GAPDH enzyme activity is shown as means with standard errors for n ≥ 3. *P < 0.01 compared with heated with no additional activities, as determined by one-way ANOVA with Dunnett's multiple comparison posttest.

Figure 7.

Protection of DrdI restriction endonuclease activity from heat inactivation. The ability of DrdI to cleave 2 μg λDNA was assessed after heat inactivation for 0, 20, or 30 minutes, as indicated in the table above the lane. Control had no addition, and, as indicated, other lanes had 18 nM myocilin or 1 μM BSA. A typical ethidium bromide–stained gel is shown with the uncleaved band at approximately 5.1 kb, the cleaved doublet at approximately 4 kb, and a single band at approximately 2 kb. Values in the table represent means of band fraction distributions determined from scans of gel lanes, where experiments were performed in triplicate.