Analysis of the covalent high molecular weight protein complex containing optineurin and its relationship with glaucoma

Abstract

Purpose: We are analyzing the function of optineurin (OPTN) to elucidate the onset mechanism of glaucoma. It was reported that the physiologically existing form of OPTN in cultured cells is oligomer, which is dissociated into monomers when detected by western blotting (WB) using denatured gels under reducing condition. We recently found an OPTN-containing high molecular weight complex (HMC) induced by H₂O₂ treatment of cells using the same detection method and named it covalent HMC. The aim of this study is to analyze the covalent HMC and its relationship with glaucoma pathogenesis.

Methods: Plasmids to express wild-type (Wt) and a series of mutant OPTNs were constructed and transfected into HeLaS3 or NIH3T3 cells. The mutants include the glaucoma causative ones, H26D, E50K, M98K, H486R, and R545Q, as well as a designed mutation D474N which was reported to abolish the binding with ubiquitin (Ub) and a truncated mutant LcUBD (lacking Ub-Binding Domain). The HMC formed in the cells transfected with these constructs with/without stimuli was detected by WB using denatured gels under reducing condition. Dual-luciferase reporter assay was applied for measuring TNFα-induced NF-κB activity (TINA) to examine if the inhibitory effect of OPTN on TINA is abolished by LcUBD and D474N.

Results: 1. The covalent HMC induced by H₂O₂ treatment of cells was probably the covalent trimer of OPTN (cOPTN₃) according to the estimated molecular weight from the WB results; 2. The formation of cOPTN₃ was detected in not only Wt but also all of the point mutants including D474N when cells were stimulated with H₂O₂; 3. Only in the case of E50K, the formation of cOPTN₃ was induced without H₂O₂ addition; 4. cOPTN₃ was not detected in LcUBD with/without H₂O₂ treatment; 5. TNFα did not induce the formation of cOPTN₃.

Conclusions: 1. Our results suggested that physiologically existing non-covalent OPTN oligomer is cross-linked each other with some covalent bond under oxidative environment with unknown mechanism. 2. E50K is a distinct mutation which possibly produces the similar intracellular condition as H₂O₂ treatment, suggesting a possible relationship of E50K with the severity of glaucoma phenotype. 3. The UBD is necessary for cOPTN₃ formation, but the site essential for Ub binding seems unnecessary. 4. cOPTN₃ is not involved in TNFα-induced NF-κB signaling pathway.

Keywords: 660 proteins encoded by disease genes • 659 protein structure/function • 634 oxidation/oxidative or free radical damage