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Elavazhagan Murugan, Rajamani Lakshminarayanan, Anandalakshmi Venkatraman, Victoria Mouvet, Roger W Beuerman, Jodhbir S Mehta; Expression and characterization of the proline mutants in the 4th FAS1 domains of TGFβIP associated stromal corneal dystrophies. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1014.
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© ARVO (1962-2015); The Authors (2016-present)
Various mutations associated with the TGFBI gene cause different stromal corneal dystrophies. We studied 6 pathological mutations - L509P, L550P (GCD II) and L518P, T538P, L558P and H626P (LCD) involving substitution of the native residue by proline that could have a significant effect on its secondary (2°) and tertiary (3°) structures. We wanted to test the effect of these proline substitutions on the aggregation of TGFβIP mutants associated with corneal dystrophies.
The relative increase in the rate of amyloid formation (ln(vmut/vwt)) of the 4th FAS1 domains of the TGFβIP proline mutants was calculated using the modified Chiti-Dobson equation. The genes encoding the mutant 4th FAS1 domains were cloned, expressed and purified. The proteins were examined using CD spectropolarimetry.
The proline mutants exhibited positive ln(vmut/vwt) values suggesting that proline substitution accelerated amyloid formation. The ln(vmut/vwt) values for the GCDII mutations (L509P 2.32, L550P 2.32) were lower than the LCD mutations (L518P 3.02, T538P 2.54, L558P 2.75, H626P 2.5). The GCDII L509P mutation was at the end of an α-helix and GCDII L550P was in a coil suggesting lesser implications of the proline substitutions on the 2° structural elements. However, in the LCD mutations the residues were either positioned inside an α-helix (L518P, L558P) or a β-sheet (T538P, H626P), thereby having a direct effect on the 2° structure and hence the folding of the domains. We optimized the expression conditions to enhance the solubility of the recombinant proteins. The LCD mutants showed relatively lower levels of expression compared to the GCDII mutants. While the GCDII mutants displayed the characteristic CD spectra corresponding to native TGFβIP, the LCD mutations revealed minor perturbations in their 2° structure. The GCDII and LCD mutants also exhibited differences in their stabilities showing that the proline substitution has direct implications on the proper folding of the domains.
The proline mutants exhibit a clear propensity to form amyloids. It is possible that proline substitution in the 4th FAS1 domain could increase the amyloid aggregation propensity by destabilizing the 2° structure. Initial studies on the mutants showed their distinct biophysical and biochemical properties invitro which could explain their different modes of aggregation.
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