Identification and localization of methionine sulfoxide reductase A in the human lens and its direct protection of lens cells against oxidative stress damage

Abstract

Abstract: : Purpose: Up to 60% of membrane associated methionine residues are found oxidized to methionine sulfoxide in age–related cataract lenses (Garner and Spector, 1980). Methionine oxidation causes loss of protein function, however, reduction of oxidized methionines and thus restoration of protein activity can occur by the action of methionine sulfoxide reductase A (MsrA) enzyme which has recently been shown to be an essential regulator of lifespan in species ranging from flies to mammals. Here, we examined the levels and spatial patterning of MsrA in the human lens and we tested the ability of MsrA to directly protect lens cells against oxidative stress. Methods: MsrA levels were compared between whole and microdissected human lenses and multiple other human tissues by semi–quantitative RT–PCR, western analysis, and immunohistochemistry. Stably over–expressing MsrA human lens epithelial cell lines (SRA01/04) were established using retroviral–mediated gene transfer. Endogenously expressed MsrA was selectively deleted from lens cells using siRNA–mediated gene silencing. H₂O₂–induced loss of cell viability was monitored in the MsrA–over–expressing and MsrA–deleted cells by examining mitochondrial function using MTS metabolic assays. Results: MsrA is expressed at the fourth highest level in the lens compared to other human tissues indicating a major role for MsrA in the human lens. MsrA mRNA and protein are expressed at high levels in lens epithelia, cortical, and nuclear fibers where the enzyme could repair proteins damaged by oxidation. Over–expression of MsrA in lens epithelial cells results in as much as a 40% increase, while deletion of endogenous MsrA from lens epithelial cells results in as much as a 30% decrease in the ability of lens epithelial cells to resist H₂O₂–induced oxidative stress indicating a major role for MsrA in oxidative stress protection. Conclusions: The high abundance of oxidized methionine residues in human cataract combined with the present data establishing high levels of MsrA throughout the lens and dramatic protection of lens cells by MsrA against oxidative stress provides evidence that MsrA activity is essential for lens function and that MsrA is a key player in lens maintenance, aging and defense against cataract.

Keywords: oxidation/oxidative or free radical damage • aging • cataract