Dataset Information

Increased Association of Deamidated αA-_N101D with Lens membrane of transgenic αA_N101D vs. wild type αA mice: potential effects on intracellular ionic imbalance and membrane disorganization.

ABSTRACT: We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαA_N101D mice, and in the other by inserting human wild-type αA-transgene in CRYαA_WT mice. The CRYαA_N101D mice developed cortical cataract at about 7-months of age relative to CRYαA_WT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαA_N101D- vs. CRYαA_WT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αA_N101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization.

Methods

Lenses of varying ages from CRYαA_WT and CRYαA_N101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αA_N101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca²⁺ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.

Results

Compared to the lenses of CRYαA_WT, the lenses of CRYαA_N101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαA_N101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca²⁺ uptake were observed in cultured lens epithelial cells of CRYαA_N101D- than those of CRYαA_WT mice.

Conclusions

The results show that an increased lens membrane association of αA_N101D-_-relative WTαA protein in CRYαA_N101D mice than CRYαA_WT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

SUBMITTER: Srivastava O

PROVIDER: S-EPMC7726915 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Increased Association of Deamidated αA-N101D with Lens membrane of transgenic αAN101D vs. wild type αA mice: potential effects on intracellular ionic imbalance and membrane disorganization.

Srivastava Om O Srivastava Kiran K Joseph Roy R Wilson Landon L

BMC ophthalmology 20201210 1

We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with spe ...[more]

PMID: 33302904

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Project description:Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and β-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with βA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with βA3-crystallin. The deamidated mutants of αA-crystallin (αA N101D and αA N123D) displayed lower adherence propensity for βA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for βA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with βA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with βA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with βA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor βA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.