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pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants.

Murugan E, Venkatraman A, Lei Z, Mouvet V, Rui Yi Lim R, Muruganantham N, Goh E, Swee Lim Peh G, Beuerman RW, Chaurasia SS, Rajamani L, Mehta JS - Sci Rep (2016)

Bottom Line: 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.Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.

ABSTRACT
Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4(th)_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 4(th)_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.

No MeSH data available.


Related in: MedlinePlus

Biochemical and biophysical properties of the native 4th_FAS1 domains of the wild-type and mutant TGFβIp.(a) Schematic representation of the domain arrangement and boundaries of the full-length TGFβIp and 4th_FAS1 domains of the wild-type, non-amyloidogenic (R555W) and amyloidogenic (H572R) mutants used in the study. (b) SDS-PAGE gel showing the purified fractions of the 4th_FAS1 domains of the WT, R555W and H572R mutants. (c–e) Far UV CD spectra of the 4th_FAS1 domains of WT (d), R555W (e) and H572R (f) incubated for 16 hours at acidic pH conditions (pH 3, 4.5, 5.5 and 7). The R555W mutant displayed clear changes in the CD spectra at 222 nm and 207 nm with decrease in pH (f). The CD intensity at 222 nm decreased with decrease in pH confirming the unfolding of the secondary structures. The CD spectra for the WT (c) and H572R (e) mutant remained almost unchanged. Plotting the intensities at 222 nm at varying pH (g) showed that the non-amyloidogenic phenotype, R555W, was more sensitive to pH and the amyloidogenic phenotype, H572R, remained more stable to pH changes at room temperature.
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f1: Biochemical and biophysical properties of the native 4th_FAS1 domains of the wild-type and mutant TGFβIp.(a) Schematic representation of the domain arrangement and boundaries of the full-length TGFβIp and 4th_FAS1 domains of the wild-type, non-amyloidogenic (R555W) and amyloidogenic (H572R) mutants used in the study. (b) SDS-PAGE gel showing the purified fractions of the 4th_FAS1 domains of the WT, R555W and H572R mutants. (c–e) Far UV CD spectra of the 4th_FAS1 domains of WT (d), R555W (e) and H572R (f) incubated for 16 hours at acidic pH conditions (pH 3, 4.5, 5.5 and 7). The R555W mutant displayed clear changes in the CD spectra at 222 nm and 207 nm with decrease in pH (f). The CD intensity at 222 nm decreased with decrease in pH confirming the unfolding of the secondary structures. The CD spectra for the WT (c) and H572R (e) mutant remained almost unchanged. Plotting the intensities at 222 nm at varying pH (g) showed that the non-amyloidogenic phenotype, R555W, was more sensitive to pH and the amyloidogenic phenotype, H572R, remained more stable to pH changes at room temperature.

Mentions: The native 4th_FAS1 domain (Fig. 1a) of TGFβIp (Genbank_ID-NM_000358; Protein_ID:-NP_000349) and the mutants R555W and H572R were cloned and purified (Fig. 1b) as described previously21. The estimated pI values of the WT, R555W and H572R domains were 6.53, 6.32 and 6.65 respectively. The amino acid substitutions (R→W and H→R) were associated with changes in charge and hydrophobicity as listed in the table (Table 1).


pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants.

Murugan E, Venkatraman A, Lei Z, Mouvet V, Rui Yi Lim R, Muruganantham N, Goh E, Swee Lim Peh G, Beuerman RW, Chaurasia SS, Rajamani L, Mehta JS - Sci Rep (2016)

Biochemical and biophysical properties of the native 4th_FAS1 domains of the wild-type and mutant TGFβIp.(a) Schematic representation of the domain arrangement and boundaries of the full-length TGFβIp and 4th_FAS1 domains of the wild-type, non-amyloidogenic (R555W) and amyloidogenic (H572R) mutants used in the study. (b) SDS-PAGE gel showing the purified fractions of the 4th_FAS1 domains of the WT, R555W and H572R mutants. (c–e) Far UV CD spectra of the 4th_FAS1 domains of WT (d), R555W (e) and H572R (f) incubated for 16 hours at acidic pH conditions (pH 3, 4.5, 5.5 and 7). The R555W mutant displayed clear changes in the CD spectra at 222 nm and 207 nm with decrease in pH (f). The CD intensity at 222 nm decreased with decrease in pH confirming the unfolding of the secondary structures. The CD spectra for the WT (c) and H572R (e) mutant remained almost unchanged. Plotting the intensities at 222 nm at varying pH (g) showed that the non-amyloidogenic phenotype, R555W, was more sensitive to pH and the amyloidogenic phenotype, H572R, remained more stable to pH changes at room temperature.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814907&req=5

f1: Biochemical and biophysical properties of the native 4th_FAS1 domains of the wild-type and mutant TGFβIp.(a) Schematic representation of the domain arrangement and boundaries of the full-length TGFβIp and 4th_FAS1 domains of the wild-type, non-amyloidogenic (R555W) and amyloidogenic (H572R) mutants used in the study. (b) SDS-PAGE gel showing the purified fractions of the 4th_FAS1 domains of the WT, R555W and H572R mutants. (c–e) Far UV CD spectra of the 4th_FAS1 domains of WT (d), R555W (e) and H572R (f) incubated for 16 hours at acidic pH conditions (pH 3, 4.5, 5.5 and 7). The R555W mutant displayed clear changes in the CD spectra at 222 nm and 207 nm with decrease in pH (f). The CD intensity at 222 nm decreased with decrease in pH confirming the unfolding of the secondary structures. The CD spectra for the WT (c) and H572R (e) mutant remained almost unchanged. Plotting the intensities at 222 nm at varying pH (g) showed that the non-amyloidogenic phenotype, R555W, was more sensitive to pH and the amyloidogenic phenotype, H572R, remained more stable to pH changes at room temperature.
Mentions: The native 4th_FAS1 domain (Fig. 1a) of TGFβIp (Genbank_ID-NM_000358; Protein_ID:-NP_000349) and the mutants R555W and H572R were cloned and purified (Fig. 1b) as described previously21. The estimated pI values of the WT, R555W and H572R domains were 6.53, 6.32 and 6.65 respectively. The amino acid substitutions (R→W and H→R) were associated with changes in charge and hydrophobicity as listed in the table (Table 1).

Bottom Line: 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.Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.

ABSTRACT
Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4(th)_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 4(th)_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.

No MeSH data available.


Related in: MedlinePlus