Influence of Protein-Glutathione Mixed Disulfide on the Chaperone-like Function of α-Crystallin

Abstract

In an earlier report we showed that incubation of ␣-crystallin with oxidized glutathione results in significant loss of its chaperone-like activity. In the present study, we determined the effect of protein-glutathione mixed disulfides (PSSG), formed at Cys-131 in bovine ␣A-crystallin, and Cys-131 and Cys-142 in human ␣A-crystallin, on the function of ␣-crystallin as a molecular chaperone. After incubation of calf and young human ␣ L -crystallin fractions with oxidized glutathione, levels of PSSG were determined by performic acid oxidation of the mixed disulfides followed by reversed-phase high pressure liquid chromatography separation of phenylisothiocyanate-derivatized glutathione sulfonic acid. Levels of PSSG increased from 0.01 to 0.14 nmol/nmol (20 kDa) in bovine ␣ L -crystallin and from 0.022 to 0.25 nmol/ nmol in human ␣ L -crystallin. The presence of glutathione adducts at Cys-131 and Cys-142 were confirmed by mass spectral analysis. The chaperone-like activity was determined by the heat denaturation assay using ␤ Lcrystallin as the target protein. To examine the reversibility of the effect of mixed disulfides on chaperone activity, studies were done before and after reduction with the glutathione reductase system. Increased levels of PSSG resulted in lower chaperone activities. Treatment with the glutathione reductase system led to 80% reduction in PSSG levels with a concomitant recovery of the chaperone activity. These results suggest that cysteine(s) in the ␣A-crystallin subunit play an important role in the function of ␣-crystallin as a molecular chaperone.

Glutaredoxin 2 (Grx2) Gene Deletion Induces Early Onset of Age-dependent Cataracts in Mice

“…Previously, we (7) have proposed that the initiation of oxidative stress-induced cataract may, in part, involve the oxidationinduced PSSG formation in the lens enzymes/proteins, resulting in loss of enzyme activity, such as observed with glyc- eraldehyde-3-dehydrogenase (20) or protein function, as with the chaperone-like function of ␣A-crystallin (54). The extra charge and mass of the -SG group that S-conjugates onto a protein may cause conformational change (55) and/or destabilization of lens crystallin proteins (56,57).…”

Shotgun identification of protein modifications from protein complexes and lens tissue

“…This appears to be especially true for oxidation of tryptophan and tyrosine residues that were not observed in either the test mixture or the cdc2 mixture experiments. Oxidation of methionine residues has been observed when lens crystallins are exposed to hydroxyl radicals, for example in a reaction with Fe ϩ3 and H 2 O 2 (54), and this exposure strongly inhibits chaperone activity (55). Also, it has been suggested that oxidation of tryptophan and tyrosine is initiated by UV radiation (56,57) and can contribute to changes in lens crystallins.…”

Proteomics Approach to Identify Dehydration Responsive Nuclear Proteins from Chickpea (Cicer arietinum L.)

“…Changes in protein levels alone do not imply changes in function as many proteins are known to be regulated by post-translational modifications. Proteins can resolve into multiple spots due to proteolytic processing, multiple isoforms, or changes in charge state resulting from posttranslational modifications, including phosphorylation, deamidation, acetylation, glycation, and glutathionylation (23)(24)(25)(26). It is possible that post-translational modifications may affect in-gel migration of a protein such that a protein classified as repressed may be present elsewhere on the gel or that a putative de novo protein may be found in a new place in the gel.…”