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

Ratna Gupta,Chinwe O Asomugha,Om P Srivastava

doi:10.1074/jbc.m110.148627

Ratna Gupta, Chinwe O Asomugha + Show 1 more

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https://doi.org/10.1074/jbc.m110.148627

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Abstract

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 αA-crystallin gene in which Asn-101 was replaced with Asp, which is referred to as αAN101D-transgene and is considered to be "deamidated" in this study. Mice expressing αAN101D-transgene are referred to here CRYAA(N101D) mice. All of the lines showed the expression of αAN101D-transgene. Compared with the lenses of mice expressing wild-type (WT) αA-transgene (referred to as CRYAA(WT) mice), the lenses of CRYAA(N101D) mice showed (a) altered αA-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 αA-crystallin, the lenses containing the deamidated αA-crystallin also showed an aggregation of αA-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 αA-crystallin caused disruption of fiber cell structural integrity, protein aggregation, insolubilization, and mild cortical lens opacity.

Highlights

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
Our results show that compared with wild type (WT), the introduction of a charge during development in ␣A-crystallin showed altered interaction with fiber cell membrane, altered packing of fiber cells, persistence of nuclei in the deeper cortical region, increased water insolubilization of the crystallin, and mild cortical lens opacity
CRYAAN101D Transgenic Mice—To determine the effect of introduction of a charge during development (Asn-101 3 Asp) in ␣A-crystallin in vivo, the expression of deamidated ␣A-crystallin was targeted in the lens fiber cells by employing a mouse ␣A-crystallin promoter fragment, which was previously used to induce lens fiber cell-specific expression in a transgenic mouse model by Dr Mark Petrash [33]

Summary

Introduction

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. A recent study with the WT/R49Cneo 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. Because deamidation of crystallins alters their structure and function, it is likely that interaction of the deamidated crystallins with other crystallins, membranes, and fiber cell-specific protein(s) are affected and might lead to morphological changes and abnormalities in fiber cells [18, 28] Despite these reports, the in vivo effects of deamidated ␣A-crystallin are presently not known. Our results show that compared with wild type (WT), the introduction of a charge during development in ␣A-crystallin showed altered interaction with fiber cell membrane, altered packing of fiber cells, persistence of nuclei in the deeper cortical region, increased water insolubilization of the crystallin, and mild cortical lens opacity

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