Dataset Information

Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

ABSTRACT: We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+58 Da), carboxyethyl lysine (+72 Da), and an arginine modification of +55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.

SUBMITTER: Wilmarth PA

PROVIDER: S-EPMC2536618 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

Wilmarth P A PA Tanner S S Dasari S S Nagalla S R SR Riviere M A MA Bafna V V Pevzner P A PA David L L LL

Journal of proteome research 20061001 10

We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 7 ...[more]

PMID: 17022627

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Project description:BackgroundLens transparency is due to the ordered arrangement of the major structural proteins, called crystallins. βB2 crystallin in the lens of the eye readily forms dimers with other β-crystallin subunits, but the resulting heterodimer structures are not known and were investigated in this study.MethodsStructures of βA3 and βB2 crystallin homodimers and the βA3/βB2 crystallin heterodimers were probed by measuring changes in solvent accessibility using hydrogen-deuterium exchange with mass spectrometry. We further mimicked deamidation in βB2 and probed the effect on the βA3/βB2 heterodimer. Results were confirmed with chemical crosslinking and NMR.ResultsBoth βA3 and βB2 had significantly decreased deuterium levels in the heterodimer compared to their respective homodimers, suggesting that they had less solvent accessibility and were more compact in the heterodimer. The compact structure of βB2 was supported by the identification of chemical crosslinks between lysines in βB2 within the heterodimer that were inconsistent with βB2's extended homodimeric structure. The compact structure of βA3 was supported by an overall decrease in mobility of βA3 in the heterodimer detected by NMR. In βB2, peptides 70-84 and 121-134 were exposed in the homodimer, but buried in the heterodimer with ≥50% decreases in deuterium levels. Homologous peptides in βA3, 97-109 and 134-149, had 25-50% decreases in deuterium levels in the heterodimer. These peptides are probable sites of interaction between βB2 and βA3 and are located at the predicted interface between subunits with bent linkers. Deamidation at Q184 in βB2 at this predicted interface led to a less compact βB2 in the heterodimer. The more compact structure of the βA3/βB2 heterodimer was also more heat stable than either of the homodimers.ConclusionsThe major structural proteins in the lens, the β-crystallins, are not static, but dynamic in solution, with differences in accessibility between the homo-and hetero-dimers. This structural flexibility, particularly of βB2, may facilitate formation of different size higher-ordered structures found in the transparent lens.General significanceUnderstanding complex hetero-oligomer interactions between β-crystallins in normal lens and how these interactions change during aging is fundamental to understanding the cause of cataracts. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Project description:Crystallins are transparent, refractive proteins that contribute to the focusing power of the vertebrate eye lens. These proteins are extremely soluble and resist aggregation for decades, even under crowded conditions. Crystallins have evolved to avoid strong interprotein interactions and have unusual hydration properties. Crystallin aggregation resulting from mutation, damage, or aging can lead to cataract, a disease state characterized by opacity of the lens.Different aggregation mechanisms can occur, following multiple pathways and leading to aggregates with varied morphologies. Studies of variant proteins found in individuals with childhood-onset cataract have provided insight into the molecular factors underlying crystallin stability and solubility. Modulation of exposed hydrophobic surface is critical, as is preventing specific intermolecular interactions that could provide nucleation sites for aggregation. Biophysical measurements and structural biology techniques are beginning to provide a detailed picture of how crystallins crowd into the lens, providing high refractivity while avoiding excessively tight binding that would lead to aggregation.Despite the central biological importance of refractivity, relatively few experimental measurements have been made for lens crystallins. Our work and that of others have shown that hydration is important to the high refractive index of crystallin proteins, as are interactions between pairs of aromatic residues and potentially other specific structural features.This Account describes our efforts to understand both the functional and disease states of vertebrate eye lens crystallins, particularly the ?-crystallins. We use a variety of biophysical techniques, notably NMR spectroscopy, to investigate crystallin stability and solubility. In the first section, we describe efforts to understand the relative stability and aggregation propensity of different ?S-crystallin variants. The second section focuses on interactions of these proteins with the holdase chaperone ?B-crystallin. The third, fourth, and fifth sections explore different modes of aggregation available to crystallin proteins, and the final section highlights the importance of refractive index and the sometimes conflicting demands of selection for refractivity and solubility.

Dataset Information

Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

Publications

Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

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