Characterization of the functional roles of ABCA4 in the pathology of inherited visual disease

Abstract

The retina-specific ABC transporter, ABCA4, is localized in the rod and cone photoreceptor outer segment discs and is essential for the proper functioning of the visual cycle. ABCA4 is a key player in the continuous recycling of retinoid substrates required for vision through its transport of vitamin A derivatives across the rod outer segment disc membranes. Mutations in the ABCA4 gene lead to a wide variety of blinding inherited visual diseases, including Stargardt disease, Cone-Rod Dystrophy, autosomal recessive Retinitis Pigmentosa, and Age-Related Macular Degeneration. More than 1,000 variants have been identified in the ABCA4 gene, yet there is no clear correlation between specific genetic variants and the wide ranges in age of onset and degree of progression of ABCA4-linked diseases. This is in part due to the lack of a facile approach to evaluate the association of a given genetic variation, and the consequences in terms of patient phenotype and protein function. ☐ Recombinant full-length ABCA4 protein is difficult to analyze due to its complex transmembrane nature and instability upon purification. Thus, a stable, uniform, and high-throughput expression platform in a biological membrane-like setting is needed to holistically understand the function of ABCA4 and its role in the pathophysiology of visual disease. In the work presented in this dissertation, I have tested the hypothesis that expression of human ABCA4 protein in virus-like particles (VLPs) will lead to the production of stable recombinant protein of uniform topology. Using the baculovirus expression vector system (BEVS), I have developed a novel platform for efficient expression and characterization of the full-length ABCA4 protein and its disease-associated variants in virus-like particles. We have physically, functionally, and topologically characterized ABCA4 VLPs and, similarly, investigated variant VLPs for their enzymatic function. For a comparative analysis, the recombinant NBD2 polypeptide and its variants purified from E. coli were assessed for ATP binding, ATP hydrolysis, and subdomain interactions. Our key findings indicate that expression of ABCA4 in VLPs produces proteins that are biologically active, stable and of uniform membrane topology. Furthermore, I have demonstrated that VLPs are an efficient and robust platform to functionally characterize ABCA4 disease-associated variants. Using this platform, I have interrogated the functional significance of the C-terminal domain in ABCA4. We have demonstrated that the C-terminal VFVNFA motif is essential for both ATP hydrolysis and retinal binding, thereby elucidating the significance of this domain in ABCA4 associated retinopathies. Conclusively, our established platform is ideal for the high-throughput investigation of various ABCA4 disease-causing mutations of unknown significance, which may aid in patient prognoses and the delivery of novel therapies.