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D. Balasubramanian, V. Talla, N. Srinivasan; Human -Crystallin Mutants Truncated at the C-Terminal End: Lose a Tail, Lose Transparency. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4085.
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© ARVO (1962-2015); The Authors (2016-present)
To understand the molecular features underlying the autosomal dominant congenital cataracts caused by the deletion mutations W156X in human γD-crystallin and W157X in human γC-crystallin.
Normal and mutant cDNAs (with the EGFP tag in the front) were cloned into the pEGFP-C1 vector and transfected into various cell lines and observed using a confocal microscope for EGFP fluorescence. Normal and W156X γD cDNAs were also cloned into the pET21a(+) vector and the recombinant proteins were over-expressed in the BL-21(DE3)pLysS strain of E. coli, purified and isolated. The conformational features, structural stability, and solubility in aqueous solution of the mutant protein were compared with those of the wild type using spectroscopic methods. Comparative molecular modeling was done to provide additional structural information.
Transfection of the EGFP-tagged mutant cDNAs into several cell lines led to the visualization of scattering bodies while that of wild type cDNAs did not. Turning to the properties of the expressed proteins, the mutant molecules show remarkable reduction in solubility. They have a more open structure, and a greater degree of surface hydrophobicity than the wild type molecules, accounting for self-aggregation. Molecular modeling studies confirm these features.
The deletion of C-terminal 18 residues of human γC- and γD-crystallins exposes the side chains of several hydrophobic residues in the sequence to the solvent, causing the molecule to self-aggregate. This feature appears to be reflected in-situ, upon introduction of the mutants in human lens epithelial cells. These results are consistent with the observed cataract.
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