Abstract
Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides 52LFRTVLDSGISEVR65 and 89VQDDFVEIH98, identified within this study, with the l-isoaspartate modification introduced at Asp58 and Asp91, respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native 52LFRTVLDSGISEVR65 peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin 66SDRDKFVIFL(isoAsp)VKHF80, results in enhanced amyloid formation in vitro The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.
Highlights
Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles
Purified recombinant L-isoaspartyl/D-aspartyl protein methyltransferase PCMT1 was used as an analytical probe with [3H]AdoMet to label L-isoAsp residues and to quantitate the extent of isomerization by detecting [3H]methanol in a volatility assay after base hydrolysis of the [3H]methyl esters formed in the incubation
We aimed to establish the extent and potential repercussions of the isomerization of asparaginyl and aspartyl residues across the human lens proteome using highly sensitive and specific radiolabeling of modified residues by PCMT1 with [3H]AdoMet. This method revealed that the L-isoAsp modification in the aged lens localizes to aggregates that elute above 670 kDa on native gel filtration but is found there in low-molecular weight peptide species after SDS-PAGE
Summary
Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. The resulting fiber cells are largely devoid of protein turnover machinery yet contain protein concentrations upwards of 450 mg/ml in the human lens These high protein concentrations provide lens transparency via short-range order interactions that minimize errors in refraction by destructive interference [3, 4]. These proteins, many of which have been synthesized by the time of birth, are not protected by the same turnover mechanisms present in normal somatic cells and are susceptible to spontaneous, age-related covalent modifications and aggregation. Deamidation can affect the structural integrity of proteins through the introduction of a negative charge, but L-isoAsp is harmful at both asparagine and aspartate sites within proteins due to the
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