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UBC Theses and Dissertations

Biochemical analysis of ABCA4 mutations responsible for Stargardt disease Garces, Fabian


ABCA4 is an ABC transporter encoded by the ABCA4 gene. This transporter is predominantly expressed in the disc membranes of photoreceptor cells in the retina where it plays a crucial role. ABCA4 transports N-retinylidene-phosphatidylethanolamine (N-ret-PE), the Schiff-base adduct formed by all-trans-retinal or 11-cis-retinal with phosphatidylethanolamine, across the disc membranes of photoreceptors. In doing so, ABCA4 acts as an importer that helps to prevent the formation and accumulation of harmful bisretinoids in the disc membranes of photoreceptors. To date, more than 1000 point-mutations in the ABCA4 gene have been linked to the development of Stargardt disease (STGD1) and other retinopathies. Although significant progress has been made in identifying disease-causing mutations in ABCA4, understanding genotype-phenotype relationships remains challenging because most patients are compound heterozygous for disease mutations in ABCA4 and phenotypic variations have been found in individuals with the same genotype. Furthermore, with some exceptions, analysis of STGD1 has relied on clinical and genetic data while lacking any biochemical characterization of disease-causing variants. As a result, it remains to be determined the extent to which the molecular properties of ABCA4 disease variants contribute to the etiology of STGD1 in relation to other factors, such as age, lifestyle, and environmental factors. To address this issue, a major aim of this thesis is to understand how the protein solubilization levels and residual activity of ABCA4 variants influence the disease outcome of STGD1 patients harboring these mutations. Furthermore, to help us delineate the different molecular mechanisms that underpin STGD1, and to help us understand the transport mechanism of ABC transporters in general, this thesis will characterize disease variants found in the transmembrane and the nucleotide binding domains of ABCA4. An in-depth functional analysis of the transmembrane domains (TMDs) will help us identify residues essential for the binding and translocation of N-ret-PE. Likewise, an investigation of common disease variants localized to the Walker-A motif of the nucleotide-binding-domains (NBDs) could help understand how ABCA4 couples binding and hydrolysis of ATP with N-ret-PE transport. Taken together, this information will be useful to develop a full understanding of the mechanism of transport of ABCA4 and similar transporters.

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