Abstract
Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit–subunit interaction sites in cataract-causing mutant αA-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker–containing peptides. In this study, we cross-linked wild-type (αA-WT) and mutant (αA-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d0 and d4) cross-linker–BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88 — K99 in the β5 strand of the mutant αA-G98R crystallin that is not found in wild-type αA-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly.
Highlights
Crystallins (a, b and c) are the major water-soluble proteins of the lens and are responsible for its transparency
G98R-mutated aA-crystallin exhibits structural and functional differences from aA-WT crystallins [9,10]. These include secondary and tertiary structural perturbations resulting in larger oligomeric size, decreased stability, altered chaperone ability and folding defects in the mutant protein, as reported earlier [9,10]
Increased aggregation propensity of the mutant protein underlies the molecular basis for the lens turbidity and cataract formation
Summary
Crystallins (a, b and c) are the major water-soluble proteins of the lens and are responsible for its transparency. The structural interactions of crystallins and a-crystallin chaperone activity are critical to lens transparency. Chaperone activity of acrystallin prevents the aggregation of lens proteins [1,2,3]. Loss of protein stability and a-crystallin chaperone activity causes changes in the lens architecture, leading to protein aggregation, increasing lens opacity and, cataract development. Hereditary, or congenital, cataract results from mutations in crystallin genes. G98R mutation in the aA-crystallin subunit is associated with a dominant, progressive total cataract that starts in the teenage years [7]. The mutation results in misfolded and destabilized protein, with altered secondary and tertiary structure, increased oligomeric size and a propensity to aggregate [8,9,10,11]
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