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Raquel L. Lieberman, J. N. Burns, Susan D. Orwig, Chandler A. Walker, Katherine C. Turnage; Quantitative Stability Assessment Of Glaucoma-causing And Single Nucleotide Polymorphism Variants Of The Olfactomedin Domain Of Myocilin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2427.
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© ARVO (1962-2015); The Authors (2016-present)
Myocilin variants, localized to the olfactomedin (OLF) domain, are linked to early onset glaucoma. Disease-causing myocilin variants accumulate in the endoplasmic reticulum (ER) of trabecular meshwork cells instead of being secreted to the extracellular matrix involved in regulating aqueous humor flow. Cell secretion studies at variable temperature support the hypothesis that myocilin glaucoma pathogenesis originates from a trafficking defect in which compromised biophysical properties of myocilin variants are detected by the ER quality control network, and inefficient clearing leads to aggregates that are toxic to TM cells. To directly evaluate the biophysical stability of disease-causing variants, we compared the melting temperature of nineteen purified OLF variants.
The OLF domain of myocilin was expressed recombinantly and purified to homogeneity. The stability was measured with a facile fluorescence thermal stability assay developed in the lab. The stability was measured with and without trimethylamine-N-oxide, which was previously shown to stabilize OLF. For the stable variants, circular dichroism spectra were obtained to identify structural changes compared to wild-type OLF. All data were compared in reference to previously published solubility and secretion data by other groups, as well as age of glaucoma diagnosis.
Variants with lower melting temperatures are correlated with more severe glaucoma phenotypes, and the trimethylamine-N-oxide is capable of restoring stability of all but the least stable variants to wild-type levels. Interestingly, three reported disease lesions, A427T, G246R, and A445V, exhibited both stability and circular dichroism spectra comparable to those of wild-type OLF, suggesting that these are ambiguous variants. Similarly, three annotated single nucleotide polymorphism variants K398R, E396D and E352Q, are also indistinguishable from wild-type OLF and are likely benign variants.
Our work provides the first quantitative demonstration of compromised stability among many identified OLF mutants to strengthen the proposed disease mechanisms for myocilin glaucoma. Stability assessment could represent a more general approach to the evaluation of different missense mutant proteins implicated in other genetic disorders.
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