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Elavazhagan Murugan, Rajamani Lakshminarayanan, Roger Beuerman, Shyam Chaurasia, Jodhbir Mehta; Exploring the mechanism underlying the protein aggregation in stromal corneal dystrophies caused by amyloidogenic and non-amyloidogenic mutants of TGFBIp. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4737.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal dystrophies (CD) are a group of inherited disorders caused by the deposition of proteins in various layers of the cornea. Most of the CDs in the stromal layer of the cornea have been attributed to the mutations in the transforming growth factor induced (TGFBI) gene with a high propensity in the 4thFAS1 (fasciclin-like) domain of the protein (TGFBIp). Though these mutants exhibit distinct clinical phenotypes as as fibrillar, non-fibrillar and combined forms, little or no information is available on their structural and functional differences. To understand the mechanism underlying the pathology, we chose representatives from an amyloid (H572R) and a non-amyloid (R555W) phenotype, expressed and purified the 4th FAS1 domains of these proteins and examined them alongwith the wild-type (WT) TGFBIp under various biophysical and biochemical conditions.
The 4th FAS1 domains of the mutants and the WT TGFBIp were expressed and purified. The ability of the mutants to form oligomers was examined at various conditions of pH and temperature using circular dichroism spectroscopy. The structural stabilities of the mutants were analyzed using Urea denaturation studies. The morphologies of the oligomers formed by heating the mutants were studied using electron microscopy.
While the WT TGFBIp did not show any conversion in secondary structure, the mutant proteins R555W and H572R exhibited a clear pH dependent irreversible conversion to oligomers in acidic conditions when heated. While there is a clear conversion at pH 5.5, there is no conversion at pH 7.0. The oligomers formed at pH 5.5 were also stable when they were resuspended in pH 7.0. The stability of the WT and the mutants were tested using Urea denaturation studies at various pH conditions. Examination using electron microscopy also showed that the oligomers of the amyloidogenic H572R were bigger in size with different morphologies compared to the non-amyloidogenic R555W oligomers.
The mutants clearly show that there is a pH dependent conversion in their secondary structures which leads to their different modes of aggregation and hence their distinct pathologies. The oligomers of the amyloidogenic and non-amyloidogenic mutants show differences in their morphologies.
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