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D. A. Patel, S. Vora, A. J. W. Huang; Mutation-Dependent TGFBI Aggregation in Corneal Stromal Dystrophies. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4310.
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© ARVO (1962-2015); The Authors (2016-present)
Avellino corneal dystrophy (ACD) is known to have lattice and granular stromal deposits, both presumably caused by aggregation of mutant transforming growth factor ß-induced protein (TGFBI). This study further investigates the in vitro aggregation of recombinant TGFBI R124H (associated with ACD) in comparison with WT, R124C (lattice corneal dystrophy, LCD) and R555W (granular corneal dystrophy, GCD), to distinguish the aggregation mechanism of various mutant TGFBI in hereditary corneal dystrophies.
WT, R124C, R124H and R555W recombinant TGFBI proteins purified from HEK 293 cells were allowed to aggregate in vitro from unfolded states with and without urea. Aggregation kinetics were studied with Congo red binding. Changes in secondary structure, protein hydrophobicity and morphology were monitored by circular dichroism, 4,4'-bis(1-anilinonaphthalene 8-sulfonate) (Bis-ANS) fluorescence and TEM respectively. Adhesion of human corneal epithelial (HCE) cells to unaggregated and aggregated TGFBI was also tested.
With respect to Congo red binding, both WT and mutant TGFBI followed nucleation-dependent amyloidogenic aggregation with lag, growth and elongation phases. Fibril formation was slower in the presence of urea, although this effect was less severe for WT. The increase in Congo red binding with time correlated with a transformation from -helix to ß-strand, which was fastest for WT and slowest for R555W. Bis-ANS fluorescence indicated a decrease in hydrophobicity with time for all TGFBI, among which R555W was most hydrophobic. WT TGFBI fibrils were shorter than those of mutant TGFBI. Binding characteristics of HCE cells to R124H were similar to those to R555W, whereas those of R124C were similar to WT.
Although both WT and all mutant TGFBI studied undergo amyloid formation in vitro, the kinetics of aggregation are mutation-dependent, with R124H displaying an intermediate profile between R124C and R55W, consistent with their respective clinical phenotypes. Adhesion of HCE cells with TGFBI is also aggregation-dependent. These in vitro findings may help in elucidating the role of mutant TGFBI aggregation in clinical phenotypes of various corneal stromal dystrophies.
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