Abstract

Mutations in the gene encoding the human ATP-binding Cassette (ABC) transporter ABCA4 are implicated in several visual disorders including Stargardt’s disease, an early onset macular dystrophy. Expressed in the photoreceptor and retinal pigment epithelial cells of the eye, ABCA4 is thought to act as a lipid translocase of retinal-phospholipid conjugates involved in vision. The investigation of complex membrane proteins such as ABCA4 requires protein purification from a heterologous expression system. This enables the acquisition of detailed molecular information on a protein’s structure and function without the contaminating factors of its native membrane environment. Although there are many different expression systems available, no single one is appropriate for all proteins. In my PhD 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 new system, ABCA4 was expressed in both the new and an established expression system (Spodoptera frugiperda insect cells). The protein from both systems was extracted, purified, and reconstituted into liposomes, and compared in terms of solubility, purity, yield, homogeneity, vesicle size, and structural and functional integrity. The protein from both sources was found to be structurally and functionally intact. However, there were marked differences in the ease of solubilisation, the purity, the oligomeric state, as well as in the level of ATPase activity. This work shows that although multiple expression systems may permit the expression of a protein, they will each have different advantages and limitations that may affect downstream experiments.

From a functional point of view, previous studies have determined that ABCA4 can transport retinal, phosphotidylethanolamine, and the retinal-phospholipid conjugate N-retinylidene-PE (NRPE). In the current work, I tested if a different retinal-phospholipid conjugate (A2E) might interact with ABCA4. Using a known substrate (retinal) as a control, I measured A2E binding to ABCA4 and its effect on the protein’s ATPase activity. Using molecular docking, I also determined where A2E was likely to interact with ABCA4 and what residues could be involved. The binding and docking data both showed that A2E can bind to ABCA4. However, no stimulation of the protein’s ATPase activity was seen in the presence of the compound. Although this work does not conclusively show if A2E is a substrate of ABCA4, it does demonstrate that A2E can interact with the protein. This may suggest a new function for ABCA4 in the retinal pigment epithelial cells of the retina where A2E is formed.