IDENTIFICATION OF CRITICAL CYSTEINE RESIDUES INVOLVED IN CRYSTALLIN AGGREGATION IN AGING AND CATARACT FORMATION

Abstract

Purpose : Oxidation of thiol residues in the crystallin is a key feature associated with the onset of age-related nuclear cataract (ARNC). We recently identified cysteine sites involved in crystallin disulfide cross-linking in vitro and in vivo from human cataract and glutathione depleted LEGSKO mouse lens. We found that β-crystallin disulfide linkage is highly conserved in human nuclear cataract and LEGSKO mouse lenses, and reproducible by in vitro oxidation. Some of the disulfide formation sites in γ-crystallin necessitate prior conformational changes. In continuation of the previous findings, we are interested to know if mutation of critical cysteine sites involved in the disulfide cross-linking of human γD-crystallin into non-oxidizable ALA residues would prevent protein polymerization and ultimately cataract formation.

Methods : Recombinant human γD-crystallin was cloned in pET21A vector and Overexpression by IPTG induction and purified by ion exchange and size exclusion chromatograph. This purified protein, at the concentration of 1mg/ml in 20mM Tris and 1mM EDTA, pH.8, was incubated with 1mM, 2mM and 5mM of H₂O₂ at 37^○C for in vitro oxidation and disulfide bond formation. The cysteine residue involved in disulfide bond formation will be revealed by the ICAT labelling method. Similar experiment were carried out in cysteine mutants C19A, C33A, C42A site involve in disulphide cross linking in aged and cataract lens.

Results : Recombinant human γD-crystallin oxidized with H₂0₂ resulted in formation of disulphide-linked dimers, trimers and tetramers analysed by SDS-PAGE similar with those observed in cataract. Surprisingly, the mutation of single cysteine residues into alanine at oxidation sites previously detected in human and LEGSKO mice, i.e. positions 19, 33 and 42 position in γD-crystallin so far did not abolish formation of disulfide-linked dimers, trimers and tetramers upon oxidation with H₂0_2, suggesting involvement of multiple residues. To understand this discrepancy the cysteine sites responsible for dimerization are currently being analysed by ICAT method

Conclusions : The above results reveal that multiple cysteine residues must be involved in the formation γ crystallin multimers. Mutation of these reactive cysteines into inert residues might be an important step for the generation of oxidation resistant lens crystallins and inhibition of catarctogenesis

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.