Age-related changes in normal and cataractous human lens crystallins, separated by fast-performance liquid chromatography (original) (raw)
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International Journal of Biological Macromolecules, 2016
Total soluble lens proteins (TSPs) and α-crystallin (α-Cry) were individually subjected to the long-term glycation in the presence of D-glucose. The glycated and nonglycated protein counterparts were incubated under different stress conditions and compared according to their structure, stability and aggregation propensity by various spectroscopic techniques and gel mobility shift analyses. Extensive glycation of the lens proteins was accompanied with structural alteration, reduction in their surface hydrophobicity and increment of their surface tension. Our results suggest that glycation causes lens crystallins to partially resist against structural alteration and aggregation/fibrillation under both thermal and thermochemical systems. The conformational stability of lens crystallins was increased upon glycation, showing the reason behind resistance of glycated proteins against stress-induced structural alteration and aggregation. Due to the resistance of glycated lens crystallins against aggregation, the role of this modification in development of senile cataract can be explained with the associated damaging consequences highlighted in this article.
Purpose: The aim of this investigation was to exploit lens-specific glycated crystallins as an immunogen to detect human glycated crystallins and their circulating autoantibodies in human serum during aging in relation to the development of cataract. Methods: Polyclonal antibodies were produced against human total lens proteins (40-80 years) in rabbits. The specificity of the antibodies produced were determined by antibody capture assay using purified human lens crystallins (high molecular weight fraction [HMW]+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat β-, β-glycated, γ-, and γ-glycated lens crystallins was also analyzed. A noncompetitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+α, HMW+α-glycated, β-, and β-glycated crystallins from humans and γ-and γ-glycated crystallins from rats as immobilized antigens. Circulating autoantibodies were also detected in human sera by antibody capture assay. The methodology was validated by evaluating 60 human serum samples collected from cataract patients and 30 human serum samples from apparently normal subjects belonging to the same age group. Results: The polyclonal antibodies raised against human total lens proteins showed 90% and 65% cross-reactivity with rat γ-and β-crystallins, respectively, by ELISA. Further, these polyclonal antibodies were capable of detecting both native and in vitro synthesized glycated crystallins. Their IC50 values were observed to be (i) human total lens proteins (55 ng), (ii) human HMW+α (16.45 ng), (iii) human HMW+α-glycated (273 ng), (iv) human β-(37.82 ng), (v) human β-glycated (260 ng), (vi) rat γ-(105.34 ng), and (vii) rat γ-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating β-glycated and γ-glycated crystallins in the age group of 40-80 years with respect to their control groups. However, there was no statistically significant change in the levels of HMW +α-glycated crystallins in the age group of 40-80 years as compared to their age-matched controls. Notably, the levels of serum γ-glycated crystallins were found to be threefold higher than that of HMW+α-glycated and β-glycated crystallins in the age group of 70-80 years. Circulating autoantibodies to HMW+α-glycated, β-glycated, and γ-glycated crystallins were detected in the serum of both apparently normal and cataract patients in the age group of 40-80 years by antibody capture assay. The levels of these autoantibodies were significantly higher at every time point compared to their respective controls. Autoantibodies to γ-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to β-glycated and HMW+α-glycated crystallins, respectively. Western blot and immunohistochemical analysis substantiated the observations made in non-competitive ELISA. Conclusions: During the course of aging, leakage of lens crystallins (HMW+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) elicit an immune response resulting in the formation of autoantibodies in cataract patients (40-80 years) as compared to age matched controls. This is the first experimental report where polyclonal antibodies raised against lensspecific glycated crystallins were capable of detecting the early leakage of glycated crystallins in human subjects. This immunochemical approach has implications in the early detection of senile cataract.
Effect of chronic hyperglycemia on crystallin levels in rat lens
2014
Crystallins are the major structural proteins in the vertebrate eye lens that contribute to lens transparency. Although cataract, including diabetic cataract, is thought to be a result of the accumulation of crystallins with various modifications, the effect of hyperglycemia on status of crystallin levels has not been investigated. This study evaluated the effect of chronic hyperglycemia on crystallin levels in diabetic cataractous rat lens. Diabetes was induced in rats by injecting streptozotocin and maintained on hyperglycemia for a period of 10 weeks. At the end, levels of a-, b-, c-crystallins and phosphoforms of aB-crystallins (aBC) were analyzed by immunoblotting. Further, solubility of crystallins and phosphoforms of aBC was analyzed by detergent soluble assay. Chronic diabetes significantly decreased the protein levels of a-, band aA-crystallins (aAC) in both soluble and insoluble fraction of lens. Whereas c-crystallin levels were decreased and aBC levels were increased in lens soluble fraction with no change in insoluble fraction in diabetic rat lens. Although, diabetes activated the p38MAPK signaling cascade by increasing the p-p38MAPK in lens, the phosphoforms of aBC were decreased in soluble fraction with a concomitant increase in insoluble fraction of diabetic lens when compared to the controls. Moreover, diabetes strongly enhances the degradation of crystallins and phosphoforms of aBC in lens. Taken together, the decreased levels of crystallins and insolubilization of phosphoforms of aBC under chronic hyperglycemia could be one of the underlying factors in the development of diabetic cataract.
Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 2017
The imbalance of the calcium homeostasis in the lenticular tissues of diabetic patients is an important risk factor for development of cataract diseases. In the current study, the impact of elevated levels of calcium ions were investigated on structure and aggregation propensity of glycated lens crystallins using gel electrophoresis and spectroscopic assessments. The glycated proteins indicated significant resistance against calcium-induced structural insults and aggregation. While, glycated crystallins revealed an increased conformational stability; a slight instability was observed for these proteins upon interaction with calcium ions. Also, in the presence of calcium, the proteolytic pattern of native crystallins was altered and that of glycated protein counterparts remained almost unchanged. According to results of this study it is suggested that the structural alteration of lens crystallins upon glycation may significantly reduce their calcium buffering capacity in eye lenses. ...
Existence of deamidated alphaB-crystallin fragments in normal and cataractous human lenses
Molecular vision, 2003
The aims of this study were to characterize lens crystallin fragments having a molecular mass of <10 kDa, isolated by solubilization in trichloroacetic acid, in order to identify cleavage sites in the parent crystallins for their origin and determine post-translational modifications in the fragments. The water-soluble (WS) and water-insoluble (WI) protein fractions were isolated from normal human lenses of 60 to 80 year old donors and from age-matched cataractous lenses. Both WS and WI protein fractions were treated with TCA at 60 degrees C for 2 h and the TCA-soluble fractions were recovered following centrifugation. The preparations were dialyzed against H2O to remove TCA, concentrated by lyophilization and subjected to two dimensional gel electrophoresis (2D-GE). The spots from 2D-gels were analyzed by western blot analysis, partial N-terminal sequencing, or excised for mass spectrometric analysis. SDS-PAGE analysis showed that TCA solubilized polypeptides having a molecular m...
new method for studying lens protein changes
Human lenses can be separated into concentric layers by dissolution and the fates of various lens constituents in such layers can be studied with appropriately sensitive techniques. These techniques have been applied in a study of the ageing of lens proteins. It was found that insoluble protein increases with progression from periphery to lens centre. This increase is more marked in older lenses. Analysis of soluble protein using High Performance Liquid Chromatography shows that the proportion of a-crystallin decreases towards the centre of the lens, and that this decrease becomes greater with age. j3-Crystallins maintain a constant proportion except in inner layers of older lenses. y-Crystallins show a slight decrease in content from periphery to centre. With cataract formation ageing changes are exaggerated.
Journal of Mass Spectrometry, 1995
High-performance liquid chromatography/mass spectrometry (HPLC/MS) was used to characterize the water-soluble and water-insoluble fractions of human fetal eye lens crystallins. This age group was selected since their crystallins are not expected to have accumulated significant amounts of either age- or cataract-related post-translational modifications owing to their young age. This analysis permitted the identification and determination of the extent of post-translational modifications for a range of crystallins in a single procedure. The excellent agreement between the calculated and experimental molecular weights from HPLC/MS implies that the published sequences for the crystallins αA2, αB2, βA3, βB2 and γC are correct. This agreement also demonstrates that these proteins do not contain any previously undetected post-translational modifications. In addition, the molecular weights for many crystallins with unknown sequences and/or post-translational modifications were determined to within several mass units. This appears to be the first reported analysis of the post-translational modifications occurring in crystallins from this age group. By providing the molecular weights of the native human crystallins, this study provides a baseline for future comparisons of the age- and cataract-related post-translational modifications of human lens crystallins.
Experimental Eye Research, 2000
This investigation of the water-insoluble crystallins from human lenses has used multiple chromatographic separations to obtain proteins of suf®cient purity for mass spectrometric analysis. Each fraction was analysed to determine the molecular masses of the constituent proteins as well as peptides in tryptic digests of these proteins. The major components of the water-insoluble crystallins were identi®ed as aAand aB-crystallins. In addition, gS-, bB1-, gD-, bA3/A1-and bB2-crystallins were found, in order of decreasing abundance. Although there was evidence of some backbone cleavage, the predominant forms of aA-, aB, bB2-, gS-and gD-crystallins were the intact polypeptide chains. The major modi®cations distinguishing the water-soluble crystallins were increased disul®de bonding, oxidation of Met, deamidation of Gln and Asn and backbone cleavage. Of the many reactions hypothesized to lead to crystallin insolubility and cataract, these results most strongly support metal-catalysed oxidation, deamidation and truncation as initiators of conformational changes that favor aggregation.