Age-Related Changes in Human Lens Crystallins Identified by HPLC and Mass Spectrometry

Ma, Zijuan; Hanson, Stacy R.A.; Lampi, Kirsten J.; David, Larry L.; Smith, David L.; Smith, Jean B.

doi:10.1006/exer.1998.0482

Cited by 161 publications

(158 citation statements)

References 37 publications

(51 reference statements)

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“…The fundamental goal then is to demonstrate that all advanced cataracts of the common type, i.e., age-related nuclear, have similar features and accumulate evidence for the hypothesis regarding the loss and/or redistribution of cytoplasmic protein. A large body of biochemical evidence points to modifications of the crystallins (Zigler, 1994;Lampi et al, 1998;Ma et al, 1998;Hanson et al, 2000;Truscott, 2005) and some ultrastructural evidence supports the redistribution of cytoplasmic material, most likely soluble crystallins and crystallin fragments, through damaged membranes into extracellular spaces where deposits are formed (Costello et al, 1992;Costello et al, 2007a). Biochemical results support the hypothesis that aggregation of crystallins is favored by oxidation, deamidation and truncation leading to conformational changes that promote crystallin insolubility and cataract (Hanson et al, 2000).…”

Section: Discussionmentioning

confidence: 91%

Analysis of nuclear fiber cell cytoplasmic texture in advanced cataractous lenses from Indian subjects using Debye–Bueche theory

Metlapally

Costello

Gilliland

et al. 2008

Experimental Eye Research

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Alterations in ultrastructural features of the lens fiber cells lead to scattering and opacity typical of cataracts. The organelle-free cytoplasm of the lens nuclear fiber cell is one such component that contains vital information about the packing and organization of crystallins critical to lens transparency. The current work has extended analysis of the cytoplasmic texture to transparent and advanced cataractous lenses from India and related the extent of texturing to the nuclear scattering observed using the Debye-Bueche theory for inhomogeneous materials. Advanced age-related nuclear cataracts (age-range 38-75 years) and transparent lenses (age-range 48-78 years) were obtained following extracapsular cataract removal or from the eye bank, at the L. V. Prasad Eye Institute. Lens nuclei were Vibratome-sectioned, fixed and prepared for transmission electron microscopy using established techniques. Electron micrographs of the unstained thin sections of the cytoplasm were acquired at 6500X and percent scattering for wavelengths 400-700 nm was calculated using the Debye-Bueche theory. Electron micrographs from comparable areas in an oxidative-damage sensitive (OXYS) rat model and normal rat lenses preserved from an earlier study were used, as they have extremely textured and smooth cytoplasms, respectively. The Debye-Bueche theoretical approach produces plots that vary smoothly with wavelength and are sensitive to spatial fluctuations in density. The central lens fiber cells from advanced cataractous lenses from India and the OXYS rat, representing opaque lens nuclei, produced the greatest texture and scattering. The transparent human lenses from India had a smoother texture and less predicted scattering, similar to early cataracts from previous studies. The normal rat lens had a homogeneous cytoplasm and little scattering. The data indicate that this method allowed easy comparison of small variations in cytoplasmic texture and robustly detected differences between transparent and advanced cataractous human lenses. This may relate directly to the proportion of opacification contributed by the packing of crystallins. The percent scattering calculated using this method may thus be used to generate a range of curves with which to compare and quantify the relative contribution of the packing of crystallins to the loss of transparency and scattering observed.

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Section: Discussionmentioning

confidence: 91%

Analysis of nuclear fiber cell cytoplasmic texture in advanced cataractous lenses from Indian subjects using Debye–Bueche theory

Metlapally

Costello

Gilliland

et al. 2008

Experimental Eye Research

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“…A recent study with the WT/R49C neo mouse model showed disruption of normal fiber cell organization and their structure in the deep cortex (18). These reports suggest that cytoskeletal proteins and crystallins interact with membranes and play a structural role in maintaining the lens transparency.Several PTMs in crystallins have been reported that cause their structural and functional instability (19,20). Deamidation has been identified as the most prevalent among PTMs in aging human lenses (10,20).…”

mentioning

confidence: 99%

The Common Modification in αA-Crystallin in the Lens, N101D, Is Associated with Increased Opacity in a Mouse Model

Gupta

Asomugha

Srivastava

2011

Journal of Biological Chemistry

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To elucidate the morphological and cellular changes due to introduction of a charge during development and the possible mechanism that underlies cataract development in humans as a consequence of an additional charge, we generated a transgenic mouse model mimicking deamidation of Asn at position 101. The mouse model expresses a human ␣A-crystallin gene in which Asn-101 was replaced with Asp, which is referred to as ␣AN101D-transgene and is considered to be "deamidated" in this study. Mice expressing ␣AN101D-transgene are referred to here CRYAA N101D mice. All of the lines showed the expression of ␣AN101D-transgene. Compared with the lenses of mice expressing wild-type (WT) ␣A-transgene (referred to as CRYAA WT mice), the lenses of CRYAA N101D mice showed (a) altered ␣A-crystallin membrane protein (aquaporin-0 (AQP0), a specific lens membrane protein) interaction, (b) extracellular spaces between outer cortical fiber cells, (c) attenuated denucleation during confocal microscopic examination, (d) disrupted normal fiber cell organization and structure during scanning electron microscopic examination, (e) distorted posterior suture lines by bright field microscopy, and (f) development of a mild anterior lens opacity in the superior cortical region during the optical coherence tomography scan analysis. Relative to lenses with WT ␣A-crystallin, the lenses containing the deamidated ␣A-crystallin also showed an aggregation of ␣A-crystallin and a higher level of water-insoluble proteins, suggesting that the morphological and cellular changes in these lenses are due to the N101D mutation. This study provides evidence for the first time that expression of deamidated ␣A-crystallin caused disruption of fiber cell structural integrity, protein aggregation, insolubilization, and mild cortical lens opacity.The ocular lens has a unique cellular architecture consisting of a single layer of cuboidal epithelial cells, which divide and differentiate at the equator into fiber cells (1). The fiber cells elongate, and they synthesize fiber cell-specific proteins, such as AQP0 (aquaporin-0)/MIP (main intrinsic protein), cytoskeletal proteins, and crystallins (1-4). As the new fiber cells are laid down at the lens equator, the older fiber cells are pushed toward the lens core and simultaneously lose their nuclei and organelles while exhibiting very little protein turnover. Among the three classes of the vertebrate lens crystallins (␣-, ␤-, and ␥-crystallins), ␣-crystallins are composed of two primary gene products, A and B, known as ␣A-and ␣B-crystallins, that show ϳ60% amino acid homology and constitute up to 50% of the total lens proteins. Both ␣A-and ␣B-crystallins belong to a family of small heat shock proteins with "chaperone" activity (5, 6). These crystallins are constitutively expressed in both lens epithelial and fiber cells (7) and undergo numerous age-related post-translational modifications (PTMs) 2 that lead to their unfolding, aggregation, and insolubilization. These PTMs eventually lead to accumulation of damaged crystal...

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“…Furthermore, specific Gln residues may be deamidated by transglutaminase that catalyzes crosslinking between Lys and Gln, but can also catalyze deamidation of the Gln (16). At a few sites, an Asn or Gln in one β-crystallin subunit is the corresponding acid at the homologous site in another subunit dictated by the gene sequence.The accumulation of modified crystallins is associated with changes in their aggregation properties (7,(17)(18)(19)(20). The seven β-crystallin subunits form dimers and complex heterooligomers that change in size and relative amounts during aging (7,18,(21)(22)(23).…”

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confidence: 99%

Deamidation Alters the Structure and Decreases the Stability of Human Lens βΑ3-Crystallin

et al. 2007

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According to the World Health Organization, cataracts account for half of the blindness in the world, with the majority occurring in developing countries. A cataract is a clouding of the lens of the eye due to light scattering of precipitated lens proteins or aberrant cellular debris. The major proteins in the lens are crystallins and they are extensively deamidated during aging and cataracts. Deamidation has been detected at the domain and monomer interfaces of several crystallins during aging.The purpose of this study was to determine the effects of two potential deamidation sites at the predicted interface of the βA3-crystallin dimer on its structure and stability. The glutamine residues at the reported in vivo deamidation sites of Q180 in the C-terminal domain and at the homologous site Q85 in the N-terminal domain were substituted with glutamic acid residues by site-directed mutagenesis. Far UV and near UV circular dichroism spectroscopy indicated that there were subtle differences in the secondary structure and more notable differences in the tertiary structure of the mutant proteins compared to wild type βA3-crystallin. The Q85E/Q180E mutant also was more susceptible to enzymatic digestion, suggesting increased solvent accessibility. These structural changes in the deamidated mutants led to decreased stability during unfolding in urea and increased precipitation during heat-denaturation. When simulating deamidation at both residues, there was a further decrease in stability and loss of cooperativity. However, multiangle-light scattering and quasielastic light scattering experiments showed that dimer formation was not disrupted, nor did higherorder oligomers form. These results suggest that introducing charges at the predicted domain interface in the βA3 homodimer may contribute to the insolubilization of lens crystallins or favor other, more stable, crystallin subunit interactions.Cataracts account for half of all blindness according to the World Health Organization (1). The greatest incidence is in developing countries. A cataract is opacity within the lens of the eye due to scattering of light by precipitated proteins or by aberrant cellular debris. The major proteins within the lens belong to the α and β/γ-crystallin families. Protein concentrations can reach 400 mg/mL and above in the center of the lens, and it is their ordered packing that is necessary for transparency (2). There is an extremely low rate of turnover of crystallins in differentiated lens cells. Thus, crystallins accumulate modifications due to environmental and metabolic damage during an individual's entire lifetime. This makes the lens an easily accessible tissue to study the effects of post-translational modifications on protein unfolding and aggregation.The major post-translational modifications in lenses are truncation, methylation, oxidation, disulfide bond formation, advanced glycation end-products and deamidation (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Of these AUTHOR EMAIL ADDRESS lampik@ohsu.edu. NIH Public Access Author M...

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Age-Related Changes in Human Lens Crystallins Identified by HPLC and Mass Spectrometry

Cited by 161 publications

References 37 publications

Analysis of nuclear fiber cell cytoplasmic texture in advanced cataractous lenses from Indian subjects using Debye–Bueche theory

Analysis of nuclear fiber cell cytoplasmic texture in advanced cataractous lenses from Indian subjects using Debye–Bueche theory

The Common Modification in αA-Crystallin in the Lens, N101D, Is Associated with Increased Opacity in a Mouse Model

Deamidation Alters the Structure and Decreases the Stability of Human Lens βΑ3-Crystallin

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