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Lydia Ann, Ricardo F Frausto, Anthony J Aldave; Functional impact of ZEB1 mutations associated with Posterior Polymorphous Corneal Dystrophy 3. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1007.
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© ARVO (1962-2015); The Authors (2016-present)
To assess the impact of mutations in the zinc finger E-box binding homeobox 1 (ZEB1) gene that are associated with posterior polymorphous corneal dystrophy 3 (PPCD3) on the function and regulation of the encoded protein.
13 of 25 reported ZEB1 mutations associated with PPCD3 were generated by a standard mutagenesis protocol. Oligonucleotides containing the mutant sequence were used to introduce mutations into the ZEB1 cDNA sequence, previously cloned into the pReceiver-M49(a,x,y) expression vector. Each mutation was confirmed by Sanger sequencing. Western blotting for the Halo tag upstream of ZEB1 cDNA was performed to confirm the expected molecular weight of each mutant protein. To determine cellular localization of the encoded mutant ZEB1 proteins, constructs containing the wild type (WT) and mutant ZEB1 cDNA sequences were transiently transfected into the HCEnC-21T corneal endothelial cell line. Cells were fixed, immunostained for Halo tag, and mounted with DAPI. Images were acquired using confocal microscopy.
All mutant proteins demonstrated molecular weights as predicted by each truncating mutation. Cellular localization of mutant proteins with a predicted molecular weight of ≤ 58 kilodaltons was primarily in the nucleus, but also showed diffuse cytoplasmic localization, while the WT protein was exclusively in the nucleus. The p.(Ser638Cysfs*5) and p.(Gln884Argfs*37) proteins were primarily localized to the cytoplasm due to absence of the putative nuclear localization signal (NLS; amino acid residues 893-898) while the p.(Glu997Alafs*7) and p.(Glu1039Glyfs*6) mutants, containing an intact putative NLS sequence, were primarily localized to the nucleus.
The majority of the ZEB1 mutations associated with PPCD3 demonstrate differential protein regulation, resulting in decreased protein levels. ZEB1 mutations lead to differential intracellular localization of the encoded protein, while the presence of the putative NLS is necessary to mediate translocation of the full-length ZEB1 to the nucleus. These data suggest that PPCD3 is a result of truncating mutations conferring pathologic defects to ZEB1 processing and/or function.
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