Molecular approaches provide novel insights into the role of ABCA4 in bisretinoid clearance.

Abstract

Many visual disorders including Stargardt's disease, an early onset macular dystrophy, are caused or contributed to by mutations in the gene encoding the human ATP-binding Cassette (ABC) transporter ABCA4. ABCA4 is expressed in the photoreceptor and retinal pigment epithelial cells of the eye and is thought to act as a lipid translocase of retinal-phospholipid conjugates involved in vision. Molecular investigations on ABCA4 will require its purification from a heterologous expression system capable of providing conditions for the expression of membrane proteins in a functional form. In this thesis I sought to validate a novel expression system for ABCA4 (Drosophila melanogaster eyes) and to expand on our understanding of the protein's function by investigating its interaction with a putative substrate (A2E).

To validate the novel system, ABCA4 was compared to the established expression system in Spodoptera frugiperda insect cells. The protein from both systems was extracted, purified, and reconstituted into liposomes, and different biophysical characteristics were assessed. ABCA4 from both sources was structurally and functionally intact. However, there were marked differences in the ease of solubilisation, protein purity, product yield, oligomeric state, and the level of ATPase activity. This highlights the need to assess the products of different expression systems to ensure the fidelity of subsequent protein characterisation.

Previous research has determined that ABCA4 can transport retinal, phosphotidylethanolamine, and the retinal-phospholipid conjugate N-retinylidene-PE (NRPE). In this thesis, I assessed whether the retinal-phospholipid conjugate A2E can also interact with ABCA4. Using a known substrate (retinal) as a control, I measured the binding of A2E to ABCA4 and its effect on the ATPase activity. Using molecular docking, I also defined the location for A2E binding to ABCA4. The binding and docking data both demonstrate that A2E can interact with ABCA4 and that it binds to the protein in the same substrate binding pocket as NRPE. This interaction hints at an expanded role for ABCA4 in the clearance of toxic products in the retinal pigment epithelial cells of the retina where A2E is formed.