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A. Hamze, W. Lee, M. Kantorow, K. Brew; Molecular Characterization of a Sorsby Fundus Dystrophy (SFD) Mutants of TIMP-3. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2153.
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SFD is an autosomal dominant disease characterized by early onset macular degeneration. It is caused by mutations in the C-terminal domain of the TIMP-3 resulting in the production of a protein containing an unpaired cysteine. Here, these mutations were examined for their abilities to effect the viability and apoptotic regulation of retinal pigmented epithelium cells (RPEs) towards understanding the molecular pathology of SFD and, by implication, AMD.
The truncated E139X mutant of TIMP-3 was initially selected as a model. The coding sequence was cloned into pET42b vector and the protein expressed in E.coli as inclusion bodies. After extraction, the mutant was folded and purified by cation exchange chromatography with CM-52 cellulose. The inhibitory activity of this mutant was compared with that of N-TIMP-3 against matrix metalloproteinases (MMPs) -1, -2, -7, -13 and ADAM17 (TACE) by assays with fluorogenic substrates. To examine the effects of this and other TIMP-3 mutants on RPE cell function, mutants were overexpressed in D407 and RPE19 RPE cells and examined for changes in viability, apoptosis and other properties.
Recombinant E139X mutant expressed in bacteria contained a 16KDa, monomeric form, and an apparent dimer. The inhibition constant (Ki) of the mutant for MMPs and TACE, were similar to those of WT N-TIMP-3. This supports the hypothesis that SFD does not arise from loss of inhibitory activity in TIMP-3 for MMPs and TACE. Both WT and E139X mutant were expressed in mammalian cells as both monomer and dimer; the mutant protein was entirely cell-associated while the WT was partly secreted into the medium.
SFD mutations do not affect the metalloproteinase inhibitory activity of TIMP-3 but may affect processing and localization. These effects are likely to significantly alter the function of RPE cells and therefore could underly those mechanisms that contribute to SFD.
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