title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants.
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.
authors.affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.|Duke-NUS Graduate Medical School, Singapore.
authors.forename: Elavazhagan
authors.initials: E
authors.lastname: Murugan
authors.affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.
authors.forename: Anandalakshmi
authors.initials: A
authors.lastname: Venkatraman
authors.affiliation: Proteomics and Microanalysis laboratory, Singapore Eye Research Institute, Singapore.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Lei
authors.affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.
authors.forename: Victoria
authors.initials: V
authors.lastname: Mouvet
authors.affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.
authors.forename: Rayne
authors.initials: R
authors.lastname: Rui Yi Lim
authors.affiliation: Ocular Chemistry and Anti-Infectives, Singapore Eye Research Institute, Singapore.
authors.forename: Nandhakumar
authors.initials: N
authors.lastname: Muruganantham
authors.affiliation: Ocular Chemistry and Anti-Infectives, Singapore Eye Research Institute, Singapore.
authors.forename: Eunice
authors.initials: E
authors.lastname: Goh
authors.affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.|Duke-NUS Graduate Medical School, Singapore.
authors.forename: Gary
authors.initials: G
authors.lastname: Swee Lim Peh
authors.affiliation: Duke-NUS Graduate Medical School, Singapore.|Ocular Chemistry and Anti-Infectives, Singapore Eye Research Institute, Singapore.|Department of Ophthalmology, Yong Loo Lin School of Medicine, NUS, Singapore.|Singapore National Eye Centre, Singapore.
authors.forename: Roger W
authors.initials: RW
authors.lastname: Beuerman
authors.affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.|Duke-NUS Graduate Medical School, Singapore.|Department of Ophthalmology, Yong Loo Lin School of Medicine, NUS, Singapore.
authors.forename: Shyam S
authors.initials: SS
authors.lastname: Chaurasia
authors.affiliation: Duke-NUS Graduate Medical School, Singapore.|Ocular Chemistry and Anti-Infectives, Singapore Eye Research Institute, Singapore.|Department of Ophthalmology, Yong Loo Lin School of Medicine, NUS, Singapore.
authors.forename: Lakshminarayanan
authors.initials: L
authors.lastname: Rajamani
authors.affiliation: Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.|Duke-NUS Graduate Medical School, Singapore.|Department of Ophthalmology, Yong Loo Lin School of Medicine, NUS, Singapore.|Singapore National Eye Centre, Singapore.
authors.forename: Jodhbir S
authors.initials: JS
authors.lastname: Mehta
mesh_terms: D000595:Amino Acid Sequence; D058227:Amyloidogenic Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D003317:Corneal Dystrophies, Hereditary / Q000378:metabolism / Q000473:pathology; D003319:Corneal Stroma / Q000166:cytology / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects*; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D061251:Primary Cell Culture; D011489:Protein Denaturation; D000072417:Protein Domains; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D000099222:betaIG-H3 Protein
chemical_list: D058227:Amyloidogenic Proteins; D016326:Extracellular Matrix Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein