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R.S. Molday, W.W. Wu; Structural Analysis of Retinoschisin and its Role in X-Linked Juvenile Retinoschisis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3572.
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Purpose: Retinoschisin, the protein encoded by the gene responsible for X-linked Juvenile Retinoschisis, is a 24 kDa discoidin-containing polypeptide that is secreted as a disulfide-linked oligomeric complex. The purpose of this study was to identify cysteine residues responsible for inter- and intra-molecular disulfide bonding and determine the effect of cysteine and disease-linked mutations on protein expression, localization, folding, disulfide-linked oligomeric assembly, and secretion from cells. Methods: Wild-type retinoschisin, 10 cysteine (C) to serine (S) mutants, 4 double cysteine mutants and 12 disease-linked missense mutants were constructed by site-directed mutagenesis and individually expressed in 293 cells. Protein expression and disulfide-linked oligomerization were examined on Western blots of intracellular and secreted fractions resolved on SDS gels run under disulfide-reducing and nonreducing conditions. Localization of wild-type and mutant retinoschisin was visualized by confocal microscopy. Results: Retinoschisin and 4 of the 10 C to S mutants were present in both the intracellular and secreted fractions as disulfide-linked oligomers. The 6 other C to S mutants and all the disease mutants were present in the intracellular fraction as misfolded, aggregated protein and either absent in the secreted fraction or present as monomers. Analysis of double cysteine mutants led to the identification of two cysteine residues that are responsible for disulfide-linked oligomerization. All mutants, like WT retinoschisin, localized primarily to the endoplasmic reticulum when immunolabeled 293 cells were visualized by confocal microscopy. Conclusions: Our studies indicate that disease associated missense mutations in retinoschisin cause protein misfolding and defective disulfide-linked oligomerization resulting in X-linked Juvenile Retinoschisis. Importantly, our results allow for the construction of a 3-dimensional model for the folding of the discoidin domain of retinoschisin and the prediction of cysteine residues responsible for intramolecular disulfide bonds and intermolecular disulfide-linked bonds responsible for multimeric assembly of retinoschisin.
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