6 research outputs found

    Western blot analysis of CRYGD over-expression in HEK293T cells.

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    <p>(A) The truncated CRYGD showed decreased solubility. In the supernatant, the mutant protein was truncated and was present in much lower amounts than the wildtype. In the precipitant, there was more mutant protein and the wildtype was not detected. (B) Quantification by band densitometry indicated the prominent reduction of mutant CRYGD in the supernatant (p<0.05).</p

    A Novel Insertion Variant of <i>CRYGD</i> Is Associated with Congenital Nuclear Cataract in a Chinese Family

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    <div><p>Objective</p><p>To investigate a novel insertion variant of <i>CRYGD</i> identified in a Chinese family with nuclear congenital cataract.</p><p>Methods</p><p>A Chinese family with congenital nuclear cataract was recruited for the mutational screening of candidate genes by direct sequencing. Recombinant N-terminal Myc tagged wildtype or mutant CRYGD was expressed in HEK293T cells. The expression pattern, protein solubility and subcellular distribution were analyzed by western blotting and immunofluorescence.</p><p>Principal Findings</p><p>A novel insertion variant, c.451_452insGACT, in <i>CRYGD</i> was identified in the patients. It causes a frameshift and a premature termination of the polypeptide to become Y151*. A significantly reduced solubility was observed for this mutant. Unlike wildtype CRYGD, which existed mainly in the cytoplasm, Y151* was mis-located in the nucleus.</p><p>Conclusions</p><p>We have identified a novel mutation, c.451_452insGACT, in <i>CRYGD</i>, which is associated with nuclear cataract. This is the first insertion mutation of <i>CRYGD</i> found to cause autosomal dominant congenital cataract. The mutant protein, with loss of solubility and localization to the nucleus, is hypothesized to be the major cause of cataract in these patients.</p></div

    Confirmation of the c.451_452insGACT (p.Tyr151*) insertion mutation of <i>CRYGD</i>.

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    <p>(A) Sequence chromatogram showing the heterozygous c.451_452insGACT insertion mutation of <i>CRYGD</i> in the proband. The mutation was numbered according to GenBank NM_006891.3. (B) Polyacrylamide gel electrophoresis showing different sizes of the PCR fragments in the pedigree. The 134 bp and 130 bp fragments were amplified from affected and unaffected chromosome, respectively. (C) Protein alignment of mammalian samples showing that the regions around the mutation are highly conserved. Numbers on left and right indicate the position of this fragment. The position of the mutant is marked by a black triangle.</p

    Comparison of CRYGD wildtype and mutant.

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    <p>The physical and chemical parameters of the protein were analyzed by ProtParam.</p><p>Comparison of CRYGD wildtype and mutant.</p

    Bioinformatics analysis of the mutant CRYGD Y151*.

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    <p>(A) The predicted secondary structure showing the reduced extended strand and random coil in the mutant. (B) The C-terminal domain of the truncated CRYGD favors the unfolded state. The residue-specific stability constant (K<sub>f</sub>) for each residue of the protein was predicted by the BEST/COREX server.</p

    Localization of Myc-tagged wildtype or Y151* CRYGD in HEK293T cells.

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    <p>Immunofluorescence of Myc (green fluorescence) showed the distribution of wildtype CRYGD in both cytomembrane and cytoplasm. However, the mutant CRYGD was mainly localized in the nucleus, in the form of granular deposits. Scale bar: 10 μm.</p