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Kirsten J Lampi, Phillip A Wilmarth, Larry L David; Deamidation of asparagines destabilizes crystallins by promoting isomerization at nearby aspartic acid residues.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5595. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The significance of isomerization of aspartic acid residues in β-crystallins is largely unknown. This study examined the relationship between Asn deamidation and isomerization of nearby Asp residues and their role in insolubilization of β-crystallins in aged lens.
Proteins from the nuclear region of aged human lenses were trypsinized and analyzed by LC/MS using an Orbitrap Fusion mass spectrometer to detect deamidation using the 19 mDa mass difference of deamidated peptides compared to the isotopic peaks of their non-deamidated forms, and electron transfer dissociation to identify peptide forms containing isoasparate.
Structurally homologous regions of βA3 (96-WDAWSGSNAYHIER-109) and βA4 (71-GEYPSWDAWGGNTAYPAER-89) both contain single N residues that became 46%, and 55% deamidated in the nuclear insoluble protein of a cataractous lens by 85 years of age, respectively. These N residues are also 5-6 amino acids from adjacent D residues in the same peptide. The crystal structure of βA4 (PDB:3LWK) predicts that the N residues are in surface exposed unstructured regions, while the adjacent D residues lay between β-strands and short α-helical regions. In the non-deamidated form of these peptides, there was no significant isomerization or racemization of βA3 D97 or βA4 D77. However, upon deamidation at each peptide’s N residue, 50% of the insoluble deamidated peptide βA3 96-109 contained isomerized D97, and 86% of the insoluble deamidated peptide βA4 71-89 contained isomerized D77.
The lack of isomerization at βA3 D97 or βA4 D77, unless deamidation was present at residues βA3 N103 and βA4 N82, suggested that deamidation is required before isomerization at the adjacent D residues in both crystallins can occur. This likely results from increased conformational flexibility caused by deamidation and isomerization at the N residues, which in turn frees the adjacent D residues to form succinimide ring intermediates that hydrolyze to isomerized forms. Thus, deamidation promotes additional modifications at nearby sites, potentially triggering a cascade of events that lead to insolubilization and cataract.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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