Abstract
Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4th_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4th_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.
Highlights
We have investigated the effects of acidic pH, denaturants and temperature on the secondary structure and conformational stability of the domains
The non-amyloidogenic R555W was more sensitive to pH compared to the WT and amyloidogenic H572R
We incubated the mutants in acidic pH for 1 week and followed their aggregation/ oligomerization by thioflavin T (ThT) fluorescence (Supplementary Fig. S1)
Summary
In granular corneal dystrophies (GCD), they appear as granular, non-amyloidogenic deposits[11,12,13,14,15,16]. Both phenotypes display significant differences in morphology, aggregation and tinctorial properties. Homology-based modelling studies have shown that the 4th_FAS1 domain displays the properties of the full-length TGFβ Ip20. We chose to examine the effects of factors like temperature and pH on the properties of the 4th_ FAS1 domains of TGFβ Ip harboring mutations of the amyloidogenic and non-amyloidogenic phenotypes. We aimed to examine the effects of these charge modifying mutations on the domains, their aggregation, their sensitivity to pH and temperature. The cytotoxicities of the aggregates were studied in primary human corneal stromal fibroblasts (pHCSF)
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