Quaternary structural parameters of the congenital cataract causing mutants of αA-crystallin

Kore, Rajshekhar A.; Hedges, Rebecca A.; Oonthonpan, Lalita; Santhoshkumar, Puttur; Sharma, K. Krishna; Abraham, E.C.

doi:10.1007/s11010-011-1131-8

Cited by 16 publications

(19 citation statements)

References 26 publications

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“…The level of purity was ∼99.9% and the results of SDS-PAGE were published in our previous study [20]. The same stocks of the purified proteins were used for the present study.…”

Section: Resultsmentioning

confidence: 97%

Mutations in Human αA-Crystallin/sHSP Affect Subunit Exchange Interaction with αB-Crystallin

2012

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BackgroundMutation in αA-crystallin contributes to the development of congenital cataract in humans. Heterooligomerization of αA-crystallin and αB-crystallin is essential for maintaining transparency in the eye lens. The effect of congenital cataract causing mutants of αA-crystallin on subunit exchange and interaction with αB-crystallin is unknown. In the present study, interaction of the mutants of αA-crystallin with αB-crystallin was studied both in vitro and in situ by the fluorescence resonance energy transfer (FRET) technique.Methodology/Principal Findings In vitro FRET technique was used to demonstrate the rates of subunit exchange of αB-wt with the following αA-crystallin mutants: R12C, R21L, R21W, R49C, R54C, and R116C. The subunit exchange rates (k values) of R21W and R116C with αB-wt decreased drastically as compared to αA-wt interacting with αB-wt. Moderately decreased k values were seen with R12C, R49C and R54C while R21L showed nearly normal k value. The interaction of αA- mutants with αB-wt was also assessed by in situ FRET. YFP-tagged αA mutants were co-expressed with CFP-tagged αB-wt in HeLa cells and the spectral signals were captured with a confocal microscope before and after acceptor laser photobleaching. The interaction of R21W and R116C with αB-wt was decreased nearly 50% as compared to αA-wt while the rest of the mutants showed slightly decreased interaction. Thus, there is good agreement between the in vitro and in situ FRET data.Conclusions/SignificanceStructural changes occurring in these mutants, as reported earlier, could be the underlying cause for the decreased interaction with αB may contribute to development of congenital cataract.

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“…The level of purity was ∼99.9% and the results of SDS-PAGE were published in our previous study [20]. The same stocks of the purified proteins were used for the present study.…”

Section: Resultsmentioning

confidence: 97%

Mutations in Human αA-Crystallin/sHSP Affect Subunit Exchange Interaction with αB-Crystallin

2012

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“…In contrast, in mutant αA-G98R crystallin, the major interaction point shifts to K99, and only one linking site is seen at K88. This difference in linkage sites is significant as the structural arrangement and conformation of WT and mutant crystallins are different [10], [29], and therefore different cross-linked products are expected. Cross-linked peptide ion pairs were subjected to MS/MS for identification of cross-linked sites.…”

Section: Resultsmentioning

confidence: 99%

Identification of Subunit-Subunit Interaction Sites in αA-WT Crystallin and Mutant αA-G98R Crystallin Using Isotope-Labeled Cross-Linker and Mass Spectrometry

et al. 2013

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Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit–subunit interaction sites in cataract-causing mutant αA-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker–containing peptides. In this study, we cross-linked wild-type (αA-WT) and mutant (αA-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d0 and d4) cross-linker–BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88 — K99 in the β5 strand of the mutant αA-G98R crystallin that is not found in wild-type αA-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly.

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“…Chaperone activity assay revealed that among all these mutations, R12C and R21W mutations caused decrease in chaperone function of αA-crystallin (Fig. 4) [63]. This was accompanied by an increase in the α-helical content, perturbation in tertiary structure, and enhancement in oligomeric size of αA-crystallin.…”

Section: In Vitro Studies Associated With Arginine Mutations In Humentioning

confidence: 99%

“…Secondary structural elements of the mutants were found to be associated with increased α-helical content with R21W having the maximum α-helical content, followed by R21L, R12C, R54C, and R49C [63]. Near UV-CD spectra of mutant proteins revealed that all these mutations perturbed the tertiary structure of αA-crystallin.…”

Section: In Vitro Studies Associated With Arginine Mutations In Humentioning

confidence: 99%

“…Analysis of quaternary structure of the mutants revealed that R21L and R49C had intact oligomeric assembly as that of wild-type protein, while R12C, R21W, and R54C mutations moderately increased the oligomeric size of αA-crystallin. Assessment of the available surface hydrophobic patches of these mutant proteins showed a drastic increase in the surface hydrophobicity for R21W and R21L, while other mutants had lowered surface hydrophobicity as compared to the wild-type protein [63]. Another independent study revealed that the subunit exchange rates of these αA-crystallin mutants, R21W and R116C, with wild-type αB-crystallin decreased considerably as compared to when wild-type αA-crystallin interacted with wild-type αB-crystallin [64].…”

Section: In Vitro Studies Associated With Arginine Mutations In Humentioning

confidence: 99%

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Differential role of arginine mutations on the structure and functions of α-crystallin

Panda¹,

Nandi²,

Chakraborty³

et al. 2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background α-Crystallin is a major protein of the eye lens in vertebrates. It is composed of two subunits, αA- and αB-crystallin. α-Crystallin is an oligomeric protein having these two subunits in 3:1 ratio. It belongs to small heat shock protein family and exhibits molecular chaperone function, which plays an important role in maintaining the lens transparency. Apart from chaperone function, both subunits also exhibit anti-apoptotic property. Comparison of their primary sequences reveals that αA- and αB-crystallin posses 13 and 14 arginine residues, respectively. Several of them undergo mutations which eventually lead to various eye diseases such as congenital cataract, juvenile cataract, and retinal degeneration. Interestingly, many arginine residues of these subunits are modified during glycation and even some are truncated during aging. All these facts indicate the importance of arginine residues in α-crystallin. Scope of review In this review, we will emphasize the recent in vitro and in vivo findings related to congenital cataract causing arginine mutations in α-crystallin. Major conclusions Congenital cataract causing arginine mutations alters the structure and decreases the chaperone function of α-crystallin. These mutations also affect the lens morphology and phenotypes. Interestingly, non-natural arginine mutations (generated for mimicking the glycation and truncation environment) improve the chaperone function of α-crystallin which may play an important role in maintaining the eye lens transparency during aging. General significance The neutralization of positive charge on the guanidino group of arginine residues is not always detrimental to the functionality of α-crystallin.

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Quaternary structural parameters of the congenital cataract causing mutants of αA-crystallin

Cited by 16 publications

References 26 publications

Mutations in Human αA-Crystallin/sHSP Affect Subunit Exchange Interaction with αB-Crystallin

Mutations in Human αA-Crystallin/sHSP Affect Subunit Exchange Interaction with αB-Crystallin

Identification of Subunit-Subunit Interaction Sites in αA-WT Crystallin and Mutant αA-G98R Crystallin Using Isotope-Labeled Cross-Linker and Mass Spectrometry

Differential role of arginine mutations on the structure and functions of α-crystallin

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