Abstract
Purpose: :
The binding of glutathione to proteins (protein S–glutathiolation) is a major process occurring in the lens. Here we have used SDS–PAGE, immunostaining and mass spectrometry to identify glutathiolated crystallins in lenses of young and old guinea pigs, and old guinea pigs exposed to hyperbaric oxygen (HBO) in vivo.
Methods: :
Guinea pigs, initially 18 months–old, were treated 3x per week for 7 months with HBO, producing an increase in the level of lens nuclear light scattering. O2–treated and age–matched control lenses (25 months–old), as well as normal 1 month–old guinea pig lenses, were divided into cortex and nucleus, and homogenized under N2 in the presence of 2mM EDTA and 50mM iodoacetamide (to prevent artifactual –SS– formation). Water–soluble (WS) proteins were analyzed by SDS–PAGE under non–reducing conditions and immunoblotted with a monoclonal antibody for protein–bound glutathione (PSSG) (ViroGen). A selected number of Coomassie–stained protein bands were excised, digested with trypsin and identified by LC–MS/MS using sequences of guinea pig crystallins recently made available in NEI Bank.
Results: :
Western blotting showed a surprising number of PSSG bands for both the cortex and nucleus of 1 month–old guinea pig lenses, as well as lenses of control and O2–treated animals. In general, it appeared that the level of staining was increased in 25 month–old compared to 1 month–old lenses, in the nucleus compared to the cortex, and in lenses of O2–treated animals, compared to controls. The highest level of staining was seen for the O2–treated nucleus. Major PSSG bands, present between 40–45 kDa and at 27 kDa, were found primarily in the O2–treated nucleus. The 40–45 kDa bands appeared to be a heterogeneous mixture of crosslinked α–, ß– and γ–crystallin dimers, which also formed GSH adducts. The bands also contained ζ–crystallin, possibly as a result of crosslinking and truncation. ßB–1 crystallin at 27 kDa appeared to undergo preferential formation of glutathione adducts in the lens nucleus of the O2–treated guinea pig.
Conclusions: :
Glutathiolation of WS crystallins appears to be an active, dynamic process taking place in the cortex and nucleus of young and old lenses, possibly as a means of inhibiting disulfide–crosslinking of the proteins. Under conditions of O2–induced stress in vivo, the rate of glutathiolation of a variety of crystallins increases dramatically, as a precursor to an increased level of nuclear light scattering.
Keywords: crystalline lens • oxidation/oxidative or free radical damage • cataract