Development of a quantitative assay to distinguish glaucoma-causing and benign olfactomedin variants

Show full item record

Please use this identifier to cite or link to this item: http://hdl.handle.net/1853/42931

Title: Development of a quantitative assay to distinguish glaucoma-causing and benign olfactomedin variants
Author: Burns, Joyce Nicole
Abstract: Myocilin, expressed in the trabecular meshwork of the eye, has been linked to inherited primary open-angle glaucoma (POAG). The biological function of myocilin is unknown, but mutant myocilin exhibits a gain-of-function mechanism, aggregating within the endoplasmic reticulum of human trabecular meshwork cells, causing cell stress and eventually apoptosis. After apoptosis occurs, the trabecular meshwork is compromised, leading to an increase in intraocular pressure, a symptom of glaucoma. In this thesis, I have expressed and purified the wild-type olfactomedin (OLF) domain and 24 reported disease-causing variants. I developed a facile thermal stability assay using differential scanning fluorimetry, which follows the unfolding of a protein through the fluorescence of a dye sensitive to hydrophobic regions of a protein. Also in this thesis I have determined melting temperatures for the wild-type and for each of the disease-causing mutants. I have tested the stability of the mutants in the presence of seven osmolytes, with sarcosine and trimethylamine-N-oxide restoring the melting temperature closest to wild-type. Additionally, I expressed and purified three reported single nucleotide polymorphisms (SNPs) (E352Q, E396D, K398R), which are considered benign variants. Variants were also compared by circular dichroism, revealing high b-sheet content and wild-type structure. When compared to previous studies, there is a positive correlation between the melting temperature, and previously reported qualitative assays, which measure the mutant myocilin solubility in detergent, secretion from mammalian cells, and aggregation propensity. Taken together, these data give insight into the relationship between glaucoma genotypes and phenotypes.
Type: Thesis
URI: http://hdl.handle.net/1853/42931
Date: 2010-11-18
Publisher: Georgia Institute of Technology
Subject: Mutant protein
Myocilin
Glaucoma
Protein expression and purification
Thermal stability
Osmolytes
Open-angle glaucoma
Blindness
Vision disorders
Department: Chemistry and Biochemistry
Advisor: Committee Chair: Lieberman, Raquel; Committee Member: Kelly, Wendy; Committee Member: Kroger, Nils
Degree: MS

All materials in SMARTech are protected under U.S. Copyright Law and all rights are reserved, unless otherwise specifically indicated on or in the materials.

Files in this item

Files Size Format View
burns_joyce_n_201012_mast.pdf 1.271Mb PDF View/ Open

This item appears in the following Collection(s)

Show full item record